EPA/600/R-17/159 | September 2016
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
oEPA
Development of a Sample
Processing Approach for Post
Bleach-Decontamination Ricin
Sample Analysis
Office of Research and Development
Homeland Security Research Program
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EPA/600/R-17/159
September 2016
FINAL REPORT
Development of a Sample Processing Approach for
Post Bleach-Decontamination Ricin Sample
Analysis
by
Sanjiv R. Shah, Ph.D.
Threat and Consequence Assessment Division
National Homeland Security Research Center
Cincinnati, Ohio 45268
Interagency Agreement EPA IA DW-89-92328201-0
U.S. Environmental Protection Agency Project Officer
Office of Research and Development
Homeland Security Research Program
Cincinnati, Ohio 45268
and
Lawrence Livermore National Laboratory
United States Department of Energy
Livermore, California 94551
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Disclaimer
U.S. Environmental Protection Agency
The United States Environmental Protection Agency (EPA) through its Office of Research
and Development funded and managed the research described here under an Interagency
Agreement (EPA IA DW-89-92328201-0). It has been subjected to the Agency's review
and has been approved for publication. Note that approval does not signify that the contents
necessarily reflect the views of the Agency. Mention of trade names, products, or services
does not convey official EPA approval, endorsement, or recommendation.
Lawrence Livermore National Laboratory
This document was prepared as an account of work sponsored by the Environmental
Protection Agency of the United States government under Contract DE-AC52-07NA27344.
Neither the United States government nor Lawrence Livermore National Security, LLC,
nor any of their employees makes any warranty, expressed or implied, or assumes any legal
liability or responsibility for the accuracy, completeness, or usefulness of any information,
apparatus, product, or process disclosed, or represents that its use would not infringe
privately owned rights. Reference herein to any specific commercial product, process, or
service by trade name, trademark, manufacturer, or otherwise does not constitute or imply
its endorsement, recommendation, or favoring by the United States government or
Lawrence Livermore National Security, LLC. The views and opinions expressed herein do
not necessarily state or reflect those of the United States government or Lawrence
Livermore National Security, LLC, and shall not be used for advertising or product
endorsement purposes.
The work presented in this report was performed within a Quality Assurance Project Plan
agreed upon by EPA and Lawrence Livermore National Laboratory. Questions concerning
this document or its application should be addressed to:
Sanjiv R. Shah, Ph.D.
National Homeland Security Research Center
U.S. Environmental Protection Agency
1300 Pennsylvania Avenue, NW
USEPA-8801RR
Washington, DC 20460
(202) 564-9522
shah.sanj iv@epa. gov
If you have difficulty accessing these PDF documents, please contact
Nickel.Kathy@epa.gov or McCall.Amelia@epa.gov for assistance.
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Table of Contents
Disclaimer ii
U.S. Environmental Protection Agency ii
Lawrence Livermore National Laboratory ii
Table of Contents iii
List of Tables v
List of Figures vi
List of Acronyms and Abbreviations: viii
Trademarked Products x
Acknowledgments xi
Executive Summary xii
1 Introduction 1
2 Project Objectives and Experimental Plan 4
3 Materials and Methods 6
3.1 Antibodies and Labeling 6
3.2 Preparation of Ricin Holotoxin and Ricin A-Chain 7
3.3 Preparation of Anti-Ricin Capture and Detector Antibodies 8
3.4 Preparation of Surface Coupons 8
3.5 Swab Sample Preparation and Processing 8
3.6 Sponge-Stick Sample Preparation and Processing 9
3.7 Sample Processing Using Centrifugal UF Devices 9
3.8 Preparation of Arizona Test Dust 10
3.9 Time-Resolved Fluorescence (TRF) Assay 10
3.10 Data Analysis, Interpretation, and Presentation 12
4 Quality Control, Quality Assurance, and Data Quality Objectives 13
4.1 Laboratory Inspections 13
4.2 Calibration 13
4.3 Storage Conditions 13
4.4 Replication 13
4.5 Controls 14
4.6 Qualification of New Antibody Lots 14
4.7 Data Quality Objectives/Data Quality Indicators 18
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5 Results and Discussion 20
5.1 Task 1 Establish the TRF assay dynamic range and the appropriate ricin concentration for use in the
study 20
5.1.1 Preliminary Experiments to Establish the TRF Assay Dynamic Range 20
5.1.2 Evaluation of the Ricin Holotoxin Dilution Method and TRF Assay Dynamic Range 22
5.2 Task 2 Investigation and Characterization of TRF Assay Interferences 27
5.2.1 Experimental Approach for Testing TRF Assay Interferences from Environmental Sample
Matrices - Bleach Residue, Sampling Materials, Wetting Buffer 27
5.2.2 Evaluation of TRF Assay Interferences from Environmental Sample Matrices - Swabs and
Sponge-Stick Samples 30
5.3 Task 3. Determination of Ricin Recovery/Loss for Samples Processed by UF Devices 35
5.3.1 Evaluation of 0.5-mL 3OK UF Devices for Ricin Recovery 35
5.3.2 Evaluation of 0.5-mL 10K UF Devices for Ricin Recovery 38
5.3.3 Evaluation of 0.5-mL 10K UF Devices for Ricin Recovery - Comparison of Lots of Ricin
Holotoxin 40
5.3.4 Evaluation of 0.5-mL 10K UF Devices for Ricin Recovery and Concentration 41
5.4 Task 4 Evaluation of Sample Processing Procedure Using UF for Complex Environmental Samples 45
5.4.1 Evaluation of 2-mL 10K UF Devices for Ricin Purification and Concentration from Samples
Containing ATD 45
5.4.2 Evaluation of 2-mL 10K UF Devices for Ricin Purification and Concentration from Samples
Containing ATD - Replicate Experiment 47
6 Conclusions 49
7 References 51
Appendix A: Protocol Used for Ricin Detection 53
Appendix B: Sample Processing Procedure for Post-Decontamination Ricin Samples
using 0.5 mL 10K UF Devices 61
Appendix C: Sample Processing Procedure for Post-Decontamination Ricin Samples
using 2 mL 10K UF Devices 65
IV
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List of Tables
Table 1 TRF Assay Results for Dilutions of Ricin With Different Concentrations of
Capture Antibody 21
Table 2 Evaluation of Dilution Buffer Effects on Detection of Ricin Holotoxin Using the
TRF Assay 24
Table 3 Evaluation of Dilution Buffer Effects on Detection of Ricin Holotoxin Using the
TRF Assay 25
Table 4 Analysis of Ricin from Dilutions Prepared in PBS/BSA Buffer - Replicate
Experiment 26
Table 5 Testing of the Sample Matrix (Sampling Device, Bleach Residue, and Neutralizing
Buffer) for Background Fluorescence Interference in the TRF Assay Using Swabs ....32
Table 6 Testing of the Sample Matrix (Sampling Device, Bleach Residue, and Neutralizing
Buffer) for Background Fluorescence Interference in the TRF Assay Using
Sponge-Sticks 34
Table 7 Testing of the Sample Matrix (Sampling Device, Bleach Residue, and Neutralizing
Buffer) for Background Fluorescence Interference in the TRF Assay Using Swabs
and Sponge-Sticks 35
Table 8 Evaluation of the 0.5-mL 30K UF Device on Relative Percent Recovery of Ricin 37
Table 9 Evaluation of the 0.5-mL 10K UF Device for Ricin Recovery 39
Table 10 Evaluation of the 0.5-mL 10K UF Device for Ricin Recovery and Concentration 43
Table 11 Evaluation of the 0.5-mL 10K UF Deviceon Ricin Recovery and Concentration -
Replicate Experiment 45
Table 12 Evaluation of 2-mL 10K UF Device for Ricin Recovery and Concentration from
Samples With or Without ATD 47
Table 13 Evaluation of 2-mL 10K UF Devices for Ricin Recovery and Concentration from
Samples With or Without ATD—Replicate Experiment 48
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List of Figures
Figure 1 Schematic of the DELFIA® (Dissociation-Enhanced Lanthanide Fluorescence
Immunoassay; PerkinElmer [PE], Inc.), a time-resolved fluorescence (TRF)
intensity technology 2
Figure 2 Tasks coordination for the effort including project tasks, subtasks, descriptions,
and deliverables 6
Figure 3 Schematic diagram of the TRF immunoassay steps from the perspective of a
single well 11
Figure 4 TRF assay results with 1 ng ricin A-chain and PBS (negative control) using
different concentrations of detector antibody (Lot #2) 15
Figure 5 TRF assay results with 10 ng ricin A-chain using different concentrations of
detector antibody (Lot #2) 16
Figure 6 TRF assay results with 100 pg ricin holotoxin and PBS (negative control) using
different concentrations of detector antibody (Lot #3) 17
Figure 7 TRF assay results for 10 ng ricin A-chain (per well) using different concentrations
of detector antibody (Lot #3) 18
Figure 8 Evaluation of toxin dilution methods comparing dilutions made in the plate with
assay buffer to dilutions made outside the plate (in tubes) with PBS 23
Figure 9 Photo of a 10 x 10 inch stainless steel coupon with 10% chlorine bleach applied
for 10 min, with re-spraying every 2 min, to mimic conditions for ricin
decontamination 28
Figure 10 Photo of a 10 x 10 inch stainless steel coupon after application of 10% chlorine
bleach and drying overnight 28
Figure 11 Photo of 4 x 4 inch sections on a stainless steel coupon for which 10% chlorine
bleach was applied by hand sprayer for 10 min and allowed to dry overnight 29
Figure 12 Photo of 4 x 4 inch sections on a stainless steel coupon for which water was
applied by hand sprayer for 10 min (as a control) and allowed to dry overnight 29
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Figure 13 Effect of Surface Sample Matrix (Bleach Residue, Neutralizing Buffer) on TRF
Assay Performance for Swabs 31
Figure 14 Effect of Surface Sample Matrix (Bleach Residue, Neutralizing Buffer) on TRF
Assay Performance for Sponge-Stick Samples (SS) 33
Figure 15 TRF assay results for 100 pg/mL ricin solutions processed by 10K UF devices
compared to untreated 100 pg/mL solutions (ricin concentration per sample was
~1 ng) for ricin holotoxin lots from Sigma and Vector Labs 41
Figure 16 TRF assay results for 1-mL ricin solutions (10 ng/mL) processed by 0.5-mL 10K
UF devices with 100 [j,L retentate compared to untreated 100 ng/mL solutions
(ricin mass per well in each case was ~1 ng for 10 [xL analyzed) 42
Figure 17 TRF assay results for 1-mL ricin solutions (10 ng/mL) processed by 0.5-mL 10K
UF devices with 100 [j,L retentate compared to untreated 100 ng/mL solutions
(ricin mass per well in each case was ~1 ng for 10 [xL analyzed) 44
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List of Acronyms and Abbreviations:
Ab: antibody
ATD: Arizona Test Dust
Avg: average
BR: bleach residue
BSA: bovine serum albumin
BSC: biosafety cabinet
CDC: Centers for Disease Control and Prevention
CRP: Critical Reagents Program
DBPAO: Defense Biological Product Assurance Office
DELFIA: Dissociation-Enhanced Lanthanide Fluorescent Immunoassay
DOD: U.S. Department of Defense
DOE: U.S. Department of Energy
DOH: Department of Health
DQO: data quality objective
EDTA: Ethylenediaminetetraacetic acid
EPA: U.S. Environmental Protection Agency
ERLN: Environmental Response Laboratory Network
ETF: endotoxin-free
Eu: europium
HABA: 4 -hydroxyazobenzene-2-carboxylic acid
HEPA: high-efficiency particulate air
hr: hour(s)
kDa: kilodalton
kg: kilogram
L: liter
LD50: median lethal dose
LLNL: Lawrence Livermore National Laboratory
LRN: Laboratory Response Network
mL: milliliter
MS: Mississippi
NB: Neutralizing Buffer
ng: nanogram
NHSRC: National Homeland Security Research Center
NIOSH: National Institute for Occupational Safety and Health
NIST: National Institute of Standards and Technology
nm: nanometer
NMWL: Nominal Molecular Weight Limit
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PBS: phosphate-buffered saline
PBST: phosphate-buffered saline with Tween-80
PCR: polymerase chain reaction
PE: PerkinElmer
pg: picogram
QA: quality assurance
QC: quality control
R. communis: Ricinus communis
RCF: relative centrifugal force
RIP: ribosome-inactivating protein
rpm: revolutions per minute
SS: sponge-stick
TRF: time-resolved fluorescence
UF: ultrafiltration
Hg: microgram
|j,L: microliter
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Trademarked Products
Trademark
Holder
Location
Ami con®
EMD Millipore Corp.
Billerica, MA
BD®
Becton, Dickinson and
Company
Franklin Lakes, NJ
BD Falcon®
Becton, Dickinson and
Company
Franklin Lakes, NJ
Beckmann Coulter®
Beckmann Coulter, Inc.
Brea, CA
Clorox®
The Clorox Company
Oakland, CA
Cole Parmer®
Cole Parmer
Vernon Hills, IL
DELFIA®
PerkinElmer, Inc.
Waltham, MA
Durapore®
EMD Millipore Corp.
Billerica, MA
Eppendorf®
Eppendorf North America
Hauppauge, NY
EZ-Link®
Thermo Fisher Scientific
Waltham, MA
Gibco®
Thermo Fisher Scientific
Waltham, MA
Hardy Diagnostics®
Hardy Diagnostics
Santa Maria, CA
Life Technologies®
Thermo Fisher Scientific
Waltham, MA
Millex®
EMD Millipore Corp.
Billerica, MA
Millipore®
EMD Millipore Corp.
Billerica, MA
Sigma-Aldrich®
Sigma-Aldrich Corp.
St. Louis, MO
Solar-Cult®
Solar Biologicals, Inc.
Ogdensburg, NY
Teknova®
Teknova, Inc.
Hollister, CA
T ween®-80
Sigma-Aldrich Corp.
St. Louis, MO
Ultracel®
EMD Millipore Corp.
Billerica, MA
Ultrafree®
EMD Millipore Corp.
Billerica, MA
VICTOR®
PerkinElmer, Inc.
Waltham, MA
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Acknowledgments
This work was performed under the auspices of the U.S. Department of Energy by
Lawrence Livermore National Laboratory (LLNL) under Contract DE-AC52-07NA27344.
Funding for this research was provided by the U.S. Environmental Protection Agency's
National Homeland Security Research Center (NHSRC).
We thank scientists, Brad Bowzard, Todd Parker, and Laura Rose from the Centers for
Disease Control and Prevention, and Dee Pettit and Christina Browne from the North
Carolina State Laboratory of Public Health for technical advice concerning the ricin time-
resolved fluorescence immunoassay.
We acknowledge Mike Nalipinski and Terry Smith of the EPA Office of Emergency
Management, Benjamin Franco, EPA-On Scene Coordinator, and Worth Calfee of EPA
NHSRC for technical advice on ricin decontamination and sampling.
We acknowledge Bruce Goodwin and his team (Kim Williams, Eric Thompson, Melody
Zacharko, and Bryan Necciai) at the Defense Biological Products Assurance Office
(DBPAO), formerly known as the Critical Reagents Program (CRP) of the Department of
Defense for kindly providing affinity-purified anti-ricin antibody.
Research Team
Lawrence Livermore National Laboratory
Staci Kane, Anne Marie Erler, Teneile Alfaro, and Tuijauna Mitchell-Hall (Quality
Assurance)
EPA Technical Lead
Sanjiv Shah, EPA National Homeland Security Research Center
Technical Reviewers
Matthew Magnuson, EPA National Homeland Security Research Center
Francisco Cruz, EPA Office of Emergency Management
Jafrul Hasan, EPA Office of Chemical Safety Pollution Prevention - Office of
Pesticide Programs Microbiology Laboratory
Quality Assurance Reviewers
Eletha Brady-Roberts and Ramona Sherman, EPA National Homeland Security
Research Center
Edit Reviewer
Marti Sinclair, Alion Science and Technology, NHSRC Contract GS35F4594G
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Executive Summary
There have been several ricin contamination incidents since the 2001 anthrax bioterrorism
attacks. The time-resolved fluorescence (TRF) immunoassay is a primary screening
methods to determine the presence of ricin, the Ricinus communis (castor bean) toxin, in
environmental samples. However, during the EPA response to the Tupelo, Mississippi,
ricin incident in June 2013, unsatisfactory results were obtained due to high fluorescence
backgrounds such that the TRF method could not be used for samples for ricin analysis
collected from surfaces to which chlorine bleach had been applied for decontamination.
The assay interferences were attributed to various potential factors including bleach
residue, sampling material, and wetting buffer. To mitigate the TRF immunoassay
interference issue for post-decontamination phase ricin sample analysis, the EPA's
National Homeland Security Research Center (NHSRC), in partnership with the Lawrence
Livermore National Laboratory (LLNL), executed a research project and developed a
sample processing approach.
In the absence of appropriate sample processing prior to analysis, bleach residue and/or
other factors associated with environmental samples could cause assay interferences that
could ultimately lead to false negative or false positive results. False negative results could
occur if high background fluorescence masked actual ricin presence, possibly leading to
human exposure if facilities were re-opened prematurely. False positives could occur if
controls were within range, but samples showed elevated fluorescence, thereby triggering
additional, unwarranted decontamination activities. Furthermore, without sample
concentration, ricin could be present below the assay's ability to detect it, while still being
present at potentially hazardous levels. The current effort was conducted to resolve these
issues and provide more confidence in using the TRF assay for analysis of environmental
samples and for post-decontamination clearance decisions.
The TRF assay is essentially a sandwich immunoassay that uses two different ricin-specific
antibodies that bind ricin, forming an antibody-ricin-antibody complex (i.e., "sandwich").
The biotin-labeled capture antibody attaches to a streptavidin-coated support, and the
europium-labeled detector antibody acts as a reporter for bound ricin. Upon excitation, the
europium ions emit fluorescence with a longer lifetime (ideally) than non-specific
background fluorescence, thereby producing a more selective and sensitive assay.
Prior to developing a sample processing approach, the TRF immunoassay dynamic range
for ricin detection was evaluated. In the current effort, the dynamic range varied with the
quality of the labeled detector antibodies. The lower limit of the dynamic range was 10-
100 pg ricin. With regard to the upper limit of the dynamic range, up to 10 ng ricin was
tested and consistently detected. The lower limit was dependent on the production lot of
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europium-labeled detector antibody produced by the vendor. Therefore, the amount of
antibody used in the assay needed to be optimized for each production lot, such that
background fluorescence counts (in the absence of ricin) were low while counts for ricin-
containing samples were high. For detector antibody with high background fluorescence, a
lower antibody concentration was required, thus negatively impacting the assay sensitivity.
In addition, use of affinity-purified capture antibody (antibody bound to, and later eluted
from a ricin-containing purification column) was shown to be necessary for proper
performance of the TRF immunoassay in this application.
In this study, post-bleach decontamination samples (swabs and sponge-sticks) were
prepared following typical operating procedures; however, even with a worst-case bleach
residue present, no elevated background fluorescence was observed. Both swab and
sponge-stick samples contained levels of bleach residue sufficient to cause a pH of-10 in
the expressed liquid, yellow-color, and visible dried crystals from surfaces after sampling
the surface. Regardless, the TRF assay buffer essentially maintained a pH ~8 upon addition
of the sample (pH -10). Furthermore, no elevated fluorescence levels were observed for
any of the post-decontamination samples suggesting that bleach residues were not
responsible for elevated fluorescence. These results along with high fluorescence counts
seen for poor quality Eu-labeled detector antibody in this study suggest that reagents, rather
than the sample matrix, could have caused the reported high fluorescence backgrounds in
the MS ricin incident. In this effort, high counts (> 10,000 counts) were observed in the
absence of ricin when certain detector antibody lots were used at higher concentrations
(e.g., used as a 200-fold dilution rather than a 1,000-fold dilution). The neutralizing buffer
usually used to pre-wet the swab or sponge for sample collection also did not show any
assay interference.
To mitigate the TRF immunoassay interference by other potential interferents, a sample
processing procedure, which included sample clean up and concentration, was developed
using 10-kilodalton (kDa) ultrafiltration (UF) devices, which are readily commercially
available. The procedure yielded 10- to 20-fold-plus ricin concentration (for 0.5-mL and 2-
mL devices, respectively) based on increased fluorescence counts, thus enhancing detection
of ricin in the samples. Sample processing procedures were effective for sponge-stick
samples containing as much as 250 mg of a reference test dust that is thought to be typical
of dust found on potentially sampled surfaces.
In total, this study demonstrated that for the environmental surface type tested, inclusion of
the sample processing procedure can improve performance of the TRF assay by elevating
the fluorescence response above the background (negative control) by sample
concentration (10 to 20-fold), which essentially improves the assay's signal-to-noise ratio.
The combined outcome of sample cleanup and toxin concentration may enable detection of
ricin at lower concentrations in environmental TRF assays, thus helping to prevent false
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negative results. A broader range of environmental sample types and potential interferences
should be tested to confirm that the cleanup procedure adequately maintains TRF assay
performance. Since the sample processing procedure developed in the current effort is
intended for use after sample extraction steps, it has the potential to be used with other ricin
analytical methods, although performance should be verified due to unanticipated
interfering processes potentially inherent in other methods.
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1 Introduction
The U. S. Environmental Protection Agency (EPA) has a need for rapid methods for accurate
analysis of ricin from environmental samples because EPA is the lead federal agency to respond,
decontaminate, and restore facilities/sites in case of a contamination incident that falls within its
jurisdiction. Ricin analytical methods must be reproducible, sensitive, and specific, even in
complex environmental backgrounds. Furthermore, the EPA through its Environmental
Response Laboratory Network (ERLN), EPA's national network of laboratories that can be
accessed as needed to support large scale environmental responses, may be called on to help
conduct sample analysis to determine the extent of ricin contamination and whether facilities and
areas have been restored to safe conditions after decontamination. Decision-makers at local,
state, federal, and tribal levels require rapid, high-confidence results that are not unduly impacted
by false positives or false negatives for safely reopening facilities or for clearing areas.
Therefore, appropriate analytical protocols are needed.
Ricin is a toxin found in castor beans from the plant, Ricinus communis (R. communis). It is
present in the waste material when castor oil is made for legitimate purposes. The toxin can also
be developed into a bio-weapon by partial purification or refinement of the castor bean pulp.
Ricin toxicity may occur from inhalation, ingestion, dermal penetration, or injection. The median
lethal dose (LD50) of ricin is approximately 21-42 (J,g/kg for inhalation, 1-20 (J,g/kg (8 castor
beans) for ingestion, and 1-1.75 (J,g/kg for injection (Grundmann and Tebbett, 2008). For
inhalation, symptoms including respiratory distress, fever, cough, nausea, and chest tightness
may appear as early as 4-8 hr, and symptoms from ingestion (nausea, vomiting, and diarrhea)
typically develop in less than 10 hr. Death usually occurs after 36-72 hr depending on the
exposure route and the dose received.
The ricin holotoxin is a Type 2 ribosome-inactivating protein (RIP) consisting of two different
protein chains, A- and B- chains, linked by a disulfide bond (heterodimer). The 34 kDa A-chain
has N-glycoside hydrolase activity, which de-purinates a key adenine residue in the 28S rRNA
(ribosome), disrupts binding of elongation factors, and, thus, stops protein synthesis, causing cell
death. The 32 kDa B-chain is catalytically inactive, but it is a lectin that mediates specific
binding (to carbohydrates) and transport of the holotoxin into host cells. Outside the cell, the A-
chain has extremely low toxicity without the B-chain. After transport of the holotoxin to the
endoplasmic reticulum of the cell, the disulfide bond is cleaved, and the A-chain is fully
functional.
In order to detect ricin, several analytical methods are used, including immunoassays, in vitro
cytotoxicity assays, cell-based activity assays (e.g., Rastogi et al., 2010), mass-spectrometric
proteomic analysis, and real-time PCR for R. communis deoxyribonucleic acid (DNA) present in
the ricin preparation (U.S. Department of Health and Human Services, 2006). As part of a public
health investigation of a white powder incident, many of these approaches were used, as well as
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a novel mass spectrometric based activity assay that detected enzymatically active ricin (Schieltz
etal.,2011).
This effort focused on a time-resolved fluorescence (TRF) immunoassay for ricin, which is
similar to solid-state "sandwich" immunoassay approaches. The TRF assay uses a capture
antibody specific to ricin bound to a solid support. The capture antibody is allowed to react and
bind with ricin from a sample. A detector antibody that is also specific to ricin, binds to the ricin,
thus making an antibody-ricin-antibody complex, i.e., "sandwich". Instead of linking the detector
antibody with an enzyme, it is labeled with the lanthanide europium, Eu (See Figure 1). The use
of europium rather than an enzyme enables more sensitive fluorescence-based detection and a
wider dynamic range (as opposed to colorimetric detection).
Enhancement So ution
Detector Antibody
Ricin <
Capture Antibody
340 nm
Figure 1 Schematic of the DELFIA® (Dissociation-Enhanced Lanthanide Fluorescence
Immunoassay; PerkinElmer [PE], Inc.), a time-resolved fluorescence (TRF) intensity
technology.
The DELFIA® is essentially a sandwich-based TRF immunoassay. It uses a stable europium (Eu)
label that exhibits a long-lived emission such that it can be potentially distinguished from short-
lived auto-fluorescence background. The schematic was adapted from PerkinElmer (2015).
Lanthanide chelates such are europium are extremely stable and have long fluorescence lifetimes
(usually over several hundred microseconds to greater than one millisecond for europium) (Yuan
and Wang, 2005). The specific fluorescence from the long-lived label can be potentially
distinguished from short-lived background fluorescence. The other favorable fluorescence
properties of europium include a large difference between excitation and emission wavelengths,
340 nm and 615 nm, respectively, along with a sharp emission fluorescence peak (full width at
half maximum of approximately 10 nm). In the current effort, the Dissociation-Enhanced
Lanthanide Fluorescence Immunoassay (DELFIA®) system was used for ricin TRF analysis.
DELFIA® was developed by PerkinElmer (PE) and has been used by several groups to
standardize the reagents and procedural steps. DELFIA® reagents include an Assay Buffer used
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to dilute antibody solutions, Wash Concentrate used to make wash buffer for removal of
unbound reagents, and an Enhancement Solution, which is an acidic chelating detergent solution.
While the TRF immunoassay has many desirable characteristics, the performance of the assay
for ricin detection could potentially be negatively impacted by the presence of substances found
in environmental decontamination samples, such as bleach, as well as by the presence of
particulates. For example, the Mississippi (MS) Department of Health (DOH) Laboratory, which
used the TRF assay as part of a Tupelo, MS ricin response in 2013 (U.S. EPA, 2013a), reported
the assay to be "unsatisfactory for ricin toxin detection by fluoroimmunoassay due to high
background fluorescence" with post bleach-decontamination samples. One possible cause of
background fluorescence was thought to be disruption of the antibody coating on the plate by
bleach residues, which could lead to non-specific binding of the Eu-labeled detector antibody,
and thus an elevated fluorescence signal in the absence of ricin.
To determine whether bleach residue caused unsatisfactory TRF results, EPA scientists shipped
samples mimicking field samples to the MS DOH Laboratory, which included swabs and
sponges (used to sample bleach-disinfected non-porous surfaces) without ricin present
(essentially negative controls). The MS DOH Laboratory reported elevated TRF values of
approximately 8,600 to 10,400 counts (i.e., false positive results) for these samples, when
negative controls are typically < 2,000 counts. It is possible that the analytical protocol used by
the MS DOH Laboratory was not validated for bleach-treated environmental samples.
In order to meet the need for improved methodology for ricin detection from post-
decontamination clearance samples, EPA National Homeland Security Research Center
(NHSRC) partnered with Lawrence Livermore National Laboratory (LLNL) to develop protocols
to mitigate interferences and improve the robustness on the TRF immunoassay. In addition, since
the cause(s) of the assay interferences reported by the DOH Laboratory was not clear, one task of
this effort was to recreate the problem and investigate possible causes of the reported background
fluorescence and false positive test results by separately examining the individual components
(i.e., sample matrix, wetting buffer, bleach residue). Other tasks were focused on development of
a sample processing procedure that was compatible with TRF immunoassay analysis, as well as
subsequent testing with relevant environmental samples to confirm the procedure's utility. The
TRF assay reported by Schieltz et al. (2011) was used although ricin dilutions and samples
processed by ultrafiltration (UF) were analyzed in triplicate without serial 10-fold dilutions
performed in the plate (except where noted). The sample processing procedure developed in this
effort could (in principle) be usable as a front-end to other analytical methods because, in
addition to cleaning-up the sample, it enables a better assay sensitivity of detection in complex
environmental samples.
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2
Project Objectives and Experimental Plan
For environmental ricin samples, especially after decontamination, sample cleanup is needed
prior to TRF analysis; therefore, sample-processing protocols are needed for a range of sample
types such as sponge-sticks and swabs. The premise of the current effort is that sample cleanup
and concentration will help alleviate false positive results caused from interferences in the
sample matrix and/or associated liquid or arising from the presence of bleach used in (and other
activities performed during) decontamination operations. A further goal of the current effort is to
reduce the possibility that false negative results occur if the sample extract is not concentrated
prior to analysis resulting in ricin levels below limits of detection.
This project included investigation of approaches and development of protocols for processing
post bleach-decontamination samples for ricin analysis by TRF in light of the current challenges.
The proposed sample processing approach was targeted towards not only physically separating
the interference from the ricin but also toward concentrating the ricin to improve the assay
sensitivity. It should be noted that the same sample processing procedures might be useful for
analysis of DNA from R. communis by real-time PCR or analysis by mass spectrometry,
although additional verification studies would be required.
At the project onset, it was not known which of the potential sources of sample interferents—
bleach residue (BR), sponge-stick (SS), Neutralizing Buffer (NB) individually or in
combination—were contributing to background fluorescence; therefore, the plan was to evaluate
potential sources of interferents, alone and in combination. In order to separately test the sample
material and the wetting buffer, the plan was to use gauze wipes in place of SS and to use
phosphate-buffered saline with Tween®-80 (PBST) in place of NB as wetting buffer. Prior to
investigating the cause(s), one objective was to recreate the interference problem following the
sample preparation procedure and sample processing and analysis procedures that presumably
led to the interferences, via the use of protocols from the EPA scientists (not the authors of this
study) who prepared the aforementioned test samples for the MS DOH Laboratory. The EPA
scientists used both macrofoam swabs (Cat. No. 25-1607 1PF SC, Puritan Medical Products,
Guilford, ME) and Solar-Cult® sponges (Cat. No. SH10NB, Solar Biologicals, Inc., Nepean, ON,
Canada) to sample surfaces containing BR. Swabs and sponges were extracted following the
same procedure as the protocol reported in Appendix A for the current study. This protocol uses
a minimum volume of PBS with 3% Bovine Serum Albumin (BSA), namely 1-2 mL, to remove
ricin (and microorganisms for other analysis methods) by vortex mixing.
For sample processing, centrifugal UF devices with different nominal molecular weight limits
(NMWL) were used. These devices are designed for protein and other macromolecule
concentration and purification such as DNA (i.e., Amicon® Ultra centrifugal filter devices,
Millipore, Inc., Billerica, MA). Basically, the devices allow washing out or removal of soluble
materials that could interfere with analysis while also enabling analyte concentration.
4
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Specifically, a 1-mL sample can be concentrated to 100 [j,L (10-fold concentration) with a 0.5-
mL device (by using multiple loadings on the same device), and a 2-mL sample can be
concentrated to 100 [j,L (20-fold concentration) with a 2-mL device. The goal was to minimize
losses of the analyte (ricin) to the devices resulting from toxin precipitation and/or adherence.
Different devices including 10 kDa (10K) and 30 kDa (3OK) NMWL membranes were tested.
Because the ricin holotoxin is about 66 kDa (made up of A-chain at -32 kDa and B-chain at -34
kDa), any device could in principle be effective. It was considered possible that the 10K device
could have better performance characteristics for the A-chain or both chains from partially
denatured holotoxin. However, there were concerns that the 10K device could be more
susceptible to clogging and/or excessively long filtration times. In order to remove potential
assay interferences by particulate matter that could be present in the sample and lead to
background fluorescence, a pre-filtration (0.22 micron) step was used in the sample processing
procedure. Such pre-filtration and UF treatment leading to ricin sample cleanup and
concentration could lead to better performance for other assays.
For different starting ricin concentrations, recovery efficiencies or fold-differences for UF-
treated solutions were determined by comparison with ricin solutions not treated by UF. In some
cases (where noted), the untreated ricin solutions were at a higher initial concentration that was
equivalent to the expected concentration after UF treatment. For these cases, statistical analysis
included T-tests (paired, two-tailed) comparing individual UF-treated (concentrated) samples
with untreated ricin samples, with a confidence level of 95%. For other cases (where noted), the
same ricin solution used for UF treatment was analyzed directly by TRF without UF treatment to
determine the fold-increase from UF treatment.
Experiments were conducted using ricin at different concentrations without sample material,
with clean sample materials, and with samples containing potential interferents (bleach residue,
SS, NB wetting buffer, particulates) sampled from surfaces or added to sample extracts (i.e.,
liquid expressed from pre-wet swabs or sponges). The holotoxin (A/B complex) was used for the
testing TRF assay performance, because it is most relevant to actual scenarios. Ricin A-chain
was also included as a positive control. Negative controls (lacking ricin) were used in triplicate
in each experiment. In addition, matrix controls containing all components (including 10 |iL
sample) except the detector antibody were included for each sample.
A systematic approach was taken to address protocol development, with the following tasks:
• Establishment of the TRF assay dynamic range and the appropriate ricin
concentration for use in the study (Task 1)
• Investigation and characterization of TRF assay interference (Task 2)
• Determination of ricin recovery/loss for samples processed by ultrafiltration
devices (Task 3)
5
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• Evaluation of a sample processing procedure using UF for complex
environmental samples (Task 4)
A flow-chart outlining project tasks, subtasks, and experiment descriptions is shown in Figure 2.
Deliverables
E stablish TRF Assav
Dynamic Range
(Task 1)
Investigate and
Characterize TRF
Assav Interferences
(Task2)
V
Determine Ricin
Recoverv/Loss With
<
Sample Processing
(Task3)
J,
Evaluate Sample
Processing Procedure
with Complex Samples
(T ask 4)
Project SubTasks and Description
Obtain aud QC-test reagents for TRF assay
Determine the dynamic range for the TRF assay and riciu concentrations
to use for subsequent tasks
Evaluate the effect of bleach residue, sponge-sticks, and Neutralizing
Buffer on TRF assay performance
Down-select interfereuces'conditions to use for Task 4
Evaluate ultrafiltration devices in sample processingprocedures
Determine ricin recovery/losses with aud without sample processing
• Include lower concentrations in assay dynamic range (Task 1)
Evaluate TRF assay performancewith and without sample processing,
combining information from Tasks 1-3
• Use riciu concentrations determined from Task 1
• Use interferences/conditions from Task 2 and test with reference dust
• Use sample processing procedure from Task3
Deliverable; Sample
Processing SOP for
TRF Analysis
Figure 2 Tasks coordination for the effort including project tasks, subtasks, descriptions,
and deliverables.
3 Materials and Methods
3.1 Antibodies and Labeling
Anti-ricin antibodies were obtained from BEI Resources, Inc. (Manassas, VA) and from the U.S.
Department of Defense (DOD) Critical Reagents Program (CRP) with proper permits and non-
disclosure agreements. The CRP has recently been disestablished and replaced with the Defense
Biological Product Assurance Office (DBPAO). Initially, polyclonal anti-ricin toxin (immune
globin G from rabbit; BEI Cat. No. NR-862; BEI Resources, Manassas, VA) was used as capture
antibody, although based on poor performance, this was replaced with affinity-purified (antibody
bound to, and later eluted from a riein-containing purification column) polyclonal goat anti-ricin
6
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(CRP, Cat. No. AB-AGRIC; CRP, Fort Detrick, MD). Monoclonal anti-ricin toxin (BEI
Resources Inc., Cat. No. NR-9571 [IgG2aK antibody class]) was used for the detector antibody.
The capture antibody was biotin-labeled using an EZ-Link™ NHS-PEG4 Biotinylation kit (Life
Technologies, Cat. No. 21455) following manufacturer's instructions. Absorbance measurements
(500 nm) were used with the 4 -hydroxyazobenzene-2-carboxylic acid (HABA) displacement
assay to estimate biotin incorporation following the manufacturer's procedure. Using this method
(microtiter plate version), the first lot of labeled capture antibody had approximately seven
biotins per antibody while the second lot had approximately two biotins per antibody.
PE Custom Labeling Service performed Eu-labeling of the detector antibody because the vendor
was experienced with potential issues such as over-labeling, which can lead to aggregation and
an elevated background. PE noted in product literature that polyclonal antibodies tend to have
lower labeling efficiencies, and a labeling ratio of 3:1-6:1 Eu:protein was considered optimal.
The ratios of labeled antibodies prepared by PE for this effort were 3.5:1, 3.9:1, and 1.64:1 for
Lot #1, Lot #2 and Lot #3, respectively. A discussion of different reagent lots is included in
Section 4.6.
3.2 Preparation of Ricin Holotoxin and Ricin A-Chain
All toxin samples were processed in a Class 2 biosafety cabinet (BSC) that was externally ducted
and equipped with high-efficiency particulate air (HEPA) filters. Unconjugated Ricinus
communis Agglutinin II (RCA 60, Ricin) obtained from Vector Laboratories (Cat. No. L-1090;
Burlingame, CA) was used. Another ricin lot obtained from Sigma (Lectin from Ricinus
communis, Toxin RCA6o, Cat. No. L8508; Sigma-Aldrich, St. Louis, MO) was also used for
some experiments, as noted in the report. Dilutions of ricin holotoxin were made in endotoxin-
free lX-phosphate buffered saline (PBS) (Teknova, Cat. No. P0300; Hollister, CA).
Ricin A-chain (Sigma-Aldrich, Cat. No. L9514-1MG, St. Louis, MO) was used as a positive
control. The A-chain was suspended in 40% glycerol containing 10 mM phosphate, pH 6.0, 0.15
M NaCl, 10 mM galactose, and 0.5 mM dithioerythritol. The A-chain concentration was
estimated at -850 ng/mL based on the measured volume, and assuming the vial contained 1 mg
A-chain as stated. For initial experiments, A-chain was diluted in PBS; however, poor stability
was noted because counts significantly decreased upon storage even for 1 day. After consultation
with state public health laboratory scientists (D. Pettit and C. Browne, personal communication),
A-chain dilutions were made in DELFIA® Assay Buffer (Cat. No. 1244-111) just prior to use
and used within one hr of preparation. Specifically, 5.9 [xL (at -850 [xg/mL) were added to 5 mL
assay buffer to make a -1 ng/mL stock. Each plate had at least three wells containing ricin A-
chain including 10 [xL of the stock diluted in assay buffer (1 [xg/mL) added directly to the plate
(10 [j,L added to 100 [xL detector antibody in assay buffer), and two serial ten-fold dilutions were
performed directly in the plate. This was a -10-fold dilution since 10 [xL were added to 100 [xL
in the plate (i.e., actually the dilution was -11-fold).
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3.3 Preparation of Anti-Ricin Capture and Detector Antibodies
Polyclonal biotin-labeled capture antibody (Section 3.1) stock was diluted appropriately
(typically, 200-fold [denoted as 200X] to 400-fold [denoted as 400X]) in DELFIA® Assay Buffer
just prior to use as described in Section 4.6). Antibody concentrations that produced less than
2,000 fluorescence counts for the negative control but still allowed sufficient counts from the
positive control (>100,000 for 10 ng concentration per well) were selected. For the reported data,
the capture antibody concentration was -200 ng per well.
Monoclonal Anti-Ricin Toxin A-Chain, Clone RAC18 antibody (BEI Resources, Cat. No. NR-
9571) was used as detector antibody. Detector antibody was europium-labeled and quantified by
PE Custom Labeling Service (Waltham, MA). Once received from the vendor (on dry ice), the
labeled detector antibody was thawed and QC-tested at different concentrations (diluted in assay
buffer 100-fold [denoted as 100X] to 1000-fold [denoted as 1000X]) to determine optimal
fluorescent signal in the TRF assay. The optimal dilution was determined as described in Section
4.6. The detector antibody was then transferred into screw-cap tubes as -25-40 [j,L aliquots
which were sufficient for use in one experiment (for up to 5-6 strips of 12 wells each), in order
to avoid excessive freezing and thawing. Aliquots were stored at -80°C. For individual
experiments, detector antibody aliquots were thawed, diluted appropriately in assay buffer, and
filtered through a 0.22-[j,m Millex-GV (Millipore, Cat. No. SLGV033RS) filter unit just prior to
use. For the reported data, the detector antibody concentrations ranged from 37 to 100 ng per
well, depending on the reagent lot.
3.4 Preparation of Surface Coupons
Fresh 10% bleach was prepared using Ultra Clorox® Germicidal Bleach (one part) and
autoclaved double distilled water (nine parts). Each stainless steel coupon (excised sample of a
material) was wiped down with 10% bleach, rinsed with double distilled water, and then rinsed
with 70%) isopropyl alcohol and wiped dry prior to use in an experiment. Each 10 x 10 inch
(25.4 x 25.4 cm) stainless steel coupon (20 Gauge 304 - 2B; Alro Steel, Cat. No. 14812194) was
labeled and set on a clean surface. For composite swab samples, three 4x4 inch (10.2 x 10.2
cm) sections were taped off on the 10 x 10 inch squares, and then labeled and placed on a clean
surface. All coupons received 10%> bleach applied by hand sprayer and allowed to remain wet for
10 min. Additional bleach was added during the 10 min when areas on coupons appeared to dry.
All coupons were dried overnight prior to sampling. Bleach application was similar to that
conducted for the Tupelo MS incident (EPA, 2013a; 2013b). Ricin was not applied to the
coupons in any of the testing.
3.5 Swab Sample Preparation and Processing
Foam-tipped swab samples (Puritan Medical Products, Cat. No. 25-1607 1PF SC; Guilford, ME)
were pre-wet with NB from Hardy Diagnostics (10-mL, Cat. No. K105; Santa Maria, CA). The
NB was composed of the following per L: 5 g aryl sulfonate complex, 160 mg sodium
8
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thiosulfate, 42.5 mg potassium phosphate, and 8 mg sodium hydroxide. The swab was pre-wet
by immersing into the solution and expressing excess fluid prior to wiping the coupon surface.
Foam-tipped swab samples were used to sample coupon surfaces according to the Centers for
Disease Control and Prevention (CDC) National Institute for Occupational Safety and Health
(NIOSH) Sampling Procedure (CDC-NIOSH, 2010). S-strokes were used to sample the entire
surface. Once sampling was complete, each swab was placed in a sterile 15-mL conical tube and
the stick was cut with sterile scissors. A 1-mL aliquot of IX PBS (Teknova Cat. No. P0300) with
3% BSA (Fraction V, VWR, Radnor, PA, Cat. No. EM2930) was added to each swab head. The
IX PBS was composed of the following: 137 mM sodium chloride, 1.4 mM potassium phosphate
monobasic, 4.3 mM sodium phosphate dibasic, and 2.7 mM potassium chloride. The tube was
vortex-mixed at -3,200 rpm in 15 sec bursts for 2 min. Using a sterile 1-mL serological pipette,
the liquid sample was transferred to a new pre-labeled 15-mL tube. The original sample tube
containing the swab was re-vortexed in 15 sec bursts for 1 min. The remaining liquid was
removed with a sterile transfer pipette and added to the appropriate 15-mL tube. The swab was
pressed against the tube wall with the transfer pipette to express as much liquid as possible. The
remaining liquid was transferred to the same pre-labeled 15-mL tube, and care was taken to not
transfer any debris. The 15-mL tube containing the swab was briefly centrifuged for up to 1 min
at -3,000 x g to collect the buffer to the bottom of the tube; any fluid was then transferred to the
same pre-labeled 15-mL tube.
3.6 Sponge-Stick Sample Preparation and Processing
Cellulose Sponge-Sticks (SS) samples were obtained pre-wet with 10-mL neutralization buffer
(Solar Biologicals Cat. No. SH10NB; Ogdensburg, NY) with a different recipe than that used for
the swabs. In this case, the neutralization buffer (NB) was composed of the following as a weight
percent: 0.7 lecithin, 0.12 sodium bisulfite, 0.1 sodium thioglycolate, 0.6 sodium thiosulfate,
1.25 potassium phosphate dibasic, 0.39 potassium phosphate monobasic, and 0.5 Tween 80
(polysorbate). The SS samples were used to sample coupon surfaces according to the CDC-
NIOSH Sampling Procedure (CDC-NIOSH, 2010). As for the swab samples, S-strokes were
used. After sampling, the head of the sponge was placed directly into a sterile specimen cup
(Mountainside Medical Equipment, Cat. No. P250400) using the release mechanism and 1-2 mL
of sterile IX PBS with 3% BSA were added (sufficient volume to obtain up to 2-mL expressed
liquid). The cups were vortexed at -3,200 rpm in 15 sec bursts for 3 min. A 2-mL serological
pipette was used to retrieve the liquid and used to push against the sponge to express sufficient
liquid. The recovered liquid sample was transferred to a 15-mL conical tube.
3.7 Sample Processing Using Centrifugal UF Devices
Different Amicon® Ultra centrifugal filter devices were used for sample processing to purify and
concentrate ricin from the matrix prior to analysis. The devices were Amicon® Ultra-0.5
Centrifugal Filter Concentrator with Ultracel® 10 Membrane for 0.5 mL sample volume
9
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(Millipore®, Cat. No. UFC501024) and Amicon® Ultra-2 Centrifugal Filter Concentrator with
Ultracel® 10 Membrane for 2.0 mL sample volume (Millipore Cat. No. UFC201024), both with a
10 kilodalton (kDa) cut off nominal molecular weight limit (NMWL). These devices contain UF
membranes and thus, allow for rapid UF, high concentration factors, and easy recovery of
retentates from dilute and complex sample matrices. For convenience, in this report, the
Amicon® Ultra-0.5 10K and the Amicon® Ultra-2 10K centrifugal filter devices are referred to as
0.5-mL 10K UF devices and 2 mL 10K UF devices, respectively. The 2 mL 10K UF devices
were used for some experiments (Task 4) to enable processing larger volume samples (up to 2-
mL) and providing up to 20-fold concentration (with 100 [jL retentate). The procedure for using
the Amicon® UF devices is included in Appendices B and C for 0.5-mL and 2-mL UF devices,
respectively. Initial testing was done with 0.5-mL 30K UF devices (Millipore Cat. No.
UFC503024).
For ricin solutions (in PBS) and for swab and sponge extracts (in PBS with 3% BSA) from
Sections 3.5 and 3.6, respectively, and with ricin added after extraction, a 400-500 [xL aliquot
was first filtered through a 0.22-|im Ultrafree® MC GV 0.5 mL Filter Unit (Millipore® Cat. No.
UFC30GV0S), followed by processing using the 0.5-mL 10K UF device as described in
Appendix B. For both UF device types, the sample retentate after wash steps was measured and
adjusted to 100 [jL with PBS. Triplicate samples from bleach-disinfected surfaces, along with
one sample from a water-treated (control) surface, were processed and analyzed by TRF.
3.8 Preparation of Arizona Test Dust
Arizona Test Dust (ATD; Powder Technology Inc., 2006; ISO 12103-1, A3 Medium Test Dust)
was used to as a source of debris to evaluate performance of 10K UF devices for cleanup of
samples containing particulates. Based on the manufacturer's analysis, the material consisted of:
Si02 (68-76%), A1203 (10-15%), Fe203 (2-5%), Na20 (2-4%), CaO (2-5%), MgO (1-2%),
TiC>2 (0.5-1.0%)), and K20 (2—5%). The dust usually contained background microbes including
fungi and bacterial spores (Rose et al., 2011). Dust was not sterilized, and a slurry was prepared
in NB that was expressed from a SS sample (Solar-Cult® Cat. No. SH10NB, pre-wet with 10 mL
NB). The slurry was prepared at 1.0 g/mL and 250 [xL were added to yield 250 mg ATD per
sponge (approx. 80 mg ATD/mL).
3.9 Time-Resolved Fluorescence (TRF) Assay
For the current effort, the TRF immunoassay adapted from that reported by Schieltz et al. (2011)
was used with the exception that serial 10-fold ricin dilutions were not performed in the assay
plate. The TRF assay is summarized below. Additional detail with step-by-step instructions is
included in Appendix A. DELFIA® reagents and equipment were used for TRF analysis (PE,
Waltham, MA). A schematic of the major steps of the TRF immunoassay is shown in Figure 3.
10
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Start with
Streptavidin-
Coated Plates
Add Biotinylated
Capture Ab and
Wash Out
Unbound Ab
Add Sample and
Eu-labeled Ab;
Wash to Remove
Non-Specifically
Bound Material
Add
Enhancement
Solution and
Measure TRF
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S = Streptavidin
B = Biotin
Q= Ricin
Eu = Europium
= Eu Chelate
Figure 3 Schematic diagram of the TRF immunoassay steps from the perspective of a single
well. Streptavidin-coated wells enable binding of biotin-labeled capture antibody. After binding
the capture antibody, the sample and Eu-labeled detector antibody are added. If the sample
contains ricin, it binds to both antibodies and forms a "sandwich". The fluorescence signal is
enhanced when europium is released from the detector antibody by addition of enhancement
solution.
Microtitration strips (streptavidin-coated clear plate, 8 x 12 strips, Cat. No. 4009-0010) were
prewashed with 750 [xL Wash Buffer (prepared from Wash Concentrate [Cat. No. 1244-114] by
diluting 1:25 with sterile endotoxin-free water) using the DELFIA® PlateWash (Cat. No. 1296-
026). Next, 100 |iL of biotinylated anti-ricin capture antibody solution (Section 3.3) were added
to each well. The plate was covered loosely using a plastic lid (from the original packaging for
strip plates) and then covered with aluminum foil and incubated at room temperature with the
PlateShake shaker (PerkinElmer, Cat. No. 1296-004) set to "high" for 2 hr. Microtitration strips
were then washed two times with 750 [xL Wash Buffer on the plate washer to remove unbound
capture antibody. After tamping off excess liquid, sample wells received 100 |iL of Eu-labeled
detector antibody solution in assay buffer (Section 3.3). Matrix control wells for samples and
positive and negative controls received 100 |iL of assay buffer instead of detection antibody
solution because this tested for potential Eu-contamination. Triplicate negative controls each
contained 10 |iL PBS buffer. The positive control wells contained 10 |iL ricin A-chain (5.9 [j,L of
850 [j,g/mL into 5-mL assay buffer; final 1 |ig/mL) and two serial dilutions using 10 |iL into 100
|iL assay buffer for each. The fluorescence counts for all three A-chain concentrations were
11
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expected to be greater than 1.5 times the negative control average; controls met these
requirements for all experiments. To each sample well, 10 |iL of the appropriate sample or ricin
standard were added after vortex mixing for 3-5 seconds. The plate was then covered as
described above, and incubated at room temperature with the shaker set to "high" for 1 hr. After
this step, wells were washed eight times with 750 [j.L Wash Buffer and tamped off to remove
excess liquid. Then, 200 |iL Enhancement Solution (PerkinElmer, Cat. No. 1244-105) were
added to each well and the plate was covered as described above, followed by shaking at "low"
setting for 10 min at room temperature. Fluorescence counts were then measured on a Victor X4
plate reader (PerkinElmer, Cat. No. 2030-0040) with the following settings: 400 j_is delay, 400 j_is
window, and 1,000 j_is cycle time. The dynamic range on the Victor X4 instrument was stated to
be linear to 15 x 106 counts with saturation at 25 x 106 counts (PerkinElmer, personal
communication).
3.10 Data Analysis, Interpretation, and Presentation
Typically, the average and standard deviation for fluorescence counts are reported for triplicate
TRF analysis per sample. Cases where duplicates were used for controls are noted. In addition,
the overall average and standard deviation were calculated in order to compare treatments with
one another (i.e., UF-treated vs. untreated sample extracts).
The overall standard deviation from all sample replicates was calculated using the following
equation,
Overall or joint SD = VflTn-i — 1 )si2 + (n2 — 1 )s22 + (n3 — l)s32 + (ni x \X1 — X]2) +
(n2 x [X2 ~Xf) + (n3 x [X3 -X]2)]/^ + n2 + n3 - 1)}
where np « and «3 = the number of TRF analyses per sample for sample replicates 1, 2, and 3;
sp .s2, and .v3 = the standard deviation of the fluorescence counts for the individual samples; Xy
X2, and X3 = the average fluorescence counts for the individual samples; X = the overall average
fluorescence counts for the samples. In some cases, data is shown in both graph and table
formats. This was intended in that the graphs would show the general trend of the data, whereas
exact values would be provided in the tables. Sufficient significant figures are included in the
tables to allow for quality assurance review. Statistical analysis included two-tailed, paired or
unpaired T-tests using a 95% confidence level; calculated p-values are reported in each case.
Comparison was made between non-UF treated and UF-treated individual replicates. In some
cases, where noted, the untreated replicates used ricin concentrations equivalent to those
expected after UF-treatment and concentration (i.e., 10-fold concentration when starting with 1
mL and recovering 100 |iL retentate).
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4 Quality Control, Quality Assurance, and Data Quality Objectives
Prior to initiating experimental work, the data quality objectives (DQO) were reviewed by the
EPA Technical Lead and the EPA quality assurance (QA) manager. In addition, the work
reported here followed the quality guidelines. Monthly data reports were submitted and
discussions were conducted with EPA's Technical Lead to review progress and adherence to the
DQOs. The key quality control (QC)/QA provisions are described below.
4.1 Laboratory Inspections
Monthly laboratory inspections were conducted by the project principal investigator to comply
with U.S. Department of Energy (DOE) and LLNL safety and security policies. In addition, the
LLNL responsible official and/or biosafety officer conducted annual laboratory inspections.
Inspections included the following:
• Documenting laboratory cleanliness
• certifying laboratory safety equipment, including the BSCs and autoclave
• reviewing ricin toxin inventories
• reviewing waste handling procedures
• reviewing personnel training
4.2 Calibration
The Victor X4 plate reader was calibrated by the vendor and set-up and inspected by a PE
service technician prior to use. All work was conducted within the first six months of instrument
procurement. Micropipettors were inspected and calibrated by the vendor annually. In addition,
LLNL conducted quarterly pipettor calibration using a gravimetric method. Balances were
calibrated annually using National Institute of Standards and Technology (NIST)-traceable
standard weights. Records from these calibration activities were documented and reviewed by
the project principal investigator.
4.3 Storage Conditions
An alarm system was used for refrigerators and freezers to ensure storage conditions were within
acceptable ranges. The temperature on the monitoring device was also noted when reagents were
removed or returned to storage locations to ensure the proper range was maintained. NIST-
traceable thermometers were placed in storage units as well to provide temperature monitoring.
4.4 Replication
In general, for each treatment in an experiment a minimum of three replicate samples was
analyzed. In some cases, where specified, duplicate controls were used. Replicate samples were
prepared at the same time using the same ricin holotoxin or A-chain concentration (or same
13
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buffer type and preparation method for negative controls) and processed at the same time
following the same laboratory processes. Results are presented as average (Avg) fluorescence
counts, with corresponding standard deviation (SD).
4.5 Controls
Negative controls included in the experiments used the same matrix as the test samples with no
ricin holotoxin or A-chain added. These controls served as a check on cross-contamination and
issues with assay components (such as the capture or detector antibodies). If the negative control
showed high fluorescence counts, extra care was taken to prepare new negative control solutions
(or TRF assay reagents) followed by repeating the TRF analysis. In addition, only endotoxin-free
buffer was used, and buffers were passed through 0.22-micron filters prior to use. Buffers were
also prepared fresh for each experiment. Matrix controls, identical to the sample except lacking
detector antibody, were included for each sample. Values typically ranged from -400-600
counts, showing an absence of europium carryover or cross contamination. The plate washer was
disinfected as directed by the instrument vendor after each use to prevent salt build-up and
potential cross-contamination issues.
4.6 Qualification of New Antibody Lots
Prior to using either capture or detector antibody in the TRF immunoassay, the concentrated
antibody stocks were diluted in assay buffer and dilution factors for working concentrations were
determined by performing the assay with set amounts of ricin holotoxin or A-chain and different
dilutions of antibodies. Initially, the detector antibody concentration was held constant, and the
capture antibody concentration was varied. For example, the detector antibody can be used at
200-fold dilution (denoted as 200X), while the capture antibody can be tested at both 200-fold
dilution (denoted as 200X) and 400-fold dilution (denoted as 400X). Once the capture antibody
working concentration was determined, this level was used to qualify a new lot of labeled
detector antibody. The selection of the concentration of antibody required analysis of both the
negative controls and the samples containing analyte (ricin). As fluorescence counts were
enhanced with higher antibody concentration, this selection requirement ensured that the
fluorescence counts for negative controls remained in the proper range (i.e., < 2,000) to prevent
false positive and false negative results for the assay.
In this section, qualification data from new, labeled detector antibody lots are presented. For the
majority of the experiments, a single lot of detector antibody (referred to as Lot #1) was used;
however, because all experiments could not be completed with this lot, a new lot was procured
and tested (referred to as Lot #2). The results from testing with negative controls and 1 ng ricin
A-chain (Figure 4) showed high levels of background fluorescence counts (-12,000-13,000)
when Lot #2 was used as 200X diluted (the working concentration used for Lot #1; 100 ng per
well). In order to achieve < 2,000 fluorescence counts for the negative control with this antibody
lot, the antibody needed to be used as 1,000X diluted; however, when diluted to this extent, the
14
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fluorescence counts for 1 ng ricin A-chain were reduced to about 50%. Likewise, fluorescence
counts for 10 ng ricin A-chain were reduced from about 150,000 to about 65,000 counts (-57%
reduction) when this lot was used as 200X diluted and 1,000X diluted, respectively (Figure 5).
Based on this poor performance, Lot #2 did not meet the requirements for use in the TRF
immunoassay.
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Negative Control (PBS)
Ricin A-Chain (1 ng)
200X 300X 400X 500X 750X 1,000X
Detector Antibody Dilution Factor
Figure 4 TRF assay results with 1 ng ricin A-chain and PBS (negative control) using
different concentrations of detector antibody (Lot #2). The fluorescence counts data were
from two separate experiments with the detector antibody used as 200X to 500X dilutions for the
first experiment, and used as 750X and 1,000X dilutions for the second experiment. For the
negative control, the bar graphs and error bars represent the average and ± one standard deviation
of three replicates, respectively. The bar graphs for ricin A-chain represent single replicates.
15
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Detector Antibody Dilution Factor
Figure 7 TRF assay results for 10 ng ricin A-chain (per well) using different concentrations
of detector antibody (Lot #3). Bar graphs represent single replicates.
In addition, a new lot of biotin-labeled capture antibody was qualified for use in the remaining
experiments. This lot was tested with Lot #1 of detector antibody, and tested as 100X, 200X, and
400X dilutions. Results showed optimal assay performance including low backgrounds for the
negative control (average 700 ± 50 fluorescence counts) and sufficient counts with ricin
holotoxin (average 1.4 ± 0.03 x io4 fluorescence counts) when used at 200X (-200 ng per well),
so this working concentration was selected for subsequent experiments (Task 4). These average
fluorescence counts were similar to those for the original lot used at 400X dilution (-200 ng per
well). These results were expected since only 0.25 mg protein was labeled for this lot of capture
antibody compared to 0.5 mg labeled for the first lot.
4.7 Data Quality Objectives/Data Quality Indicators
The balance was calibrated annually and the pipettors were calibrated quarterly; the equipment
was found to be within < 0.01% of the expected values. Calibration of the plate reader, plate
washer, and plate shaker were not required and therefore were not performed. According to the
vendor (PerkinElmer), the reader self-calibrates and does not need to be calibrated by the user.
In addition, PerkinElmer stated that for the plate washer there were no maintenance requirements
for data quality but only for preventative measures (to prevent carryover and cross-
contamination), which were adhered to during the study. These included decontamination of the
plate washer after each use by flushing with a solution of 50 mM potassium hydrogen phthalate
and 0.01% EDTA. The washer also automatically conducted washing while in standby mode
after given intervals to prevent drying of sample materials during inactivity. Likewise, the plate
18
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shaker was configured with appropriate speed settings by the manufacturer (PerkinElmer) and
did not require calibration. In addition, all equipment was used within six months of receipt.
Equipment initially planned for use in reagent preparation and TRF analysis (e.g., autoclave for
reagent preparation and incubators with calibrated thermometers for temperature-controlled
incubation) was not required. Reagents and materials were stored under conditions specified by
the vendor(s) and used within listed expiration dates. Refrigerators and freezers were on
emergency (backup) power and had high and low temperature alarms; the temperature limits
were not exceeded during the study. Finally, replicate experiments showed consistent trends; any
deviations as well as potential explanations for any discrepancies are included in the relevant
sections of the report.
A key data quality objective (DQO) of this effort was to produce data with positive, negative,
and matrix control results in the range of expected values for the TRF assay, namely ~ 1 x io5
fluorescence counts for the undiluted positive control, and < 2,000 fluorescence counts for
negative and matrix controls (Schieltz et al., 2011; PerkinElmer, personal communication). In
this study, samples either consisted of ricin holotoxin or A-chain dilutions for which the
concentration was known, or they were filtrates from UF treatment for which the expected
concentrations were known. The samples were analyzed in triplicate without dilution in the plate
(except where this dilution procedure was being tested), and triplicate negative controls (without
dilution) were included per plate. The fluorescence counts data were reported as averages with
the standard deviation for both samples and negative controls.
For ricin A-chain positive controls, one replicate set was included per plate, which consisted of
10 |iL of the initial 1 -|ig/mL ricin A-chain solution (10 ng ricin A-chain) and two serial 10-fold
dilutions prepared in the plate (in assay buffer). The ricin A-chain fluorescence counts for the
three dilutions were used to compare assay performance across different experiments and used
qualitatively to ensure that positive values were obtained (i.e., the assay was conducted
correctly). There was no requirement for all three dilutions to exceed a minimum number of
counts to be called "positive" for the purposes of this study. Likewise, for the negative control,
results were qualitatively assessed and used as a general indicator of data quality. A negative cut-
off value for fluorescence counts was determined by multiplying the average fluorescence counts
value for the negative control by 1.5 for data comparison purposes (between experiments);
however, this value was not used as a criterion for determining positive or negative results for
the samples but simply to provide a point of reference.
The data typically met the DQOs when appropriate antibody sources and quality metrics were
obtained. It was determined that only affinity-purified anti-ricin polyclonal antibody could be
used as capture antibody for the TRF immunoassay since a non-affinity-purified version did not
produce data in the expected range. This is discussed in more detail in Section 5.1. Furthermore,
the quality of europium-labeled monoclonal antibody used as detector antibody clearly impacted
the TRF assay performance as discussed in Section 4.6. TRF analysis with titrated amounts of
19
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both antibodies—where a new lot of capture antibody is tested at different concentrations with
already qualified detector antibody at a prescribed concentration, and vice versa—led to
selection of optimal concentrations for the experiments. These variations in antibody
preparations (based on specificity toward the analyte, labeling efficiency, etc.) contributed to
variability in TRF assay performance. However, in this effort, variations in antibody quality from
lot to lot did not negatively impact the ability to determine relative differences between
conditions within an experiment. The data reported were generated from two different lots of
labeled capture antibody that originated from the same lot of affinity-purified polyclonal anti-
ricin antibody. Likewise, the data were from two different lots of labeled detector antibody that
originated from the same lot of monoclonal antibody. Overall, the data met the requirement for
less than 20% coefficient of variation (CV or the ratio of the standard deviation to the mean),
with typically < 20% CV observed between replicates.
5 Results and Discussion
5.1 Task 1 Establish the TRF assay dynamic range and the appropriate ricin
concentration for use in the study
5.1.1 Preliminary Experiments to Establish the TRF Assay Dynamic Range
The initial experiments were designed to establish the TRF assay dynamic range for detection
using ricin dilutions from 1 ng to 100 ng per mL. Since 10 |iL were added per well, the ricin
mass per TRF assay replicate was 10 pg to 1 ng. Originally, non-affinity purified anti-ricin
polyclonal antibody was used as capture antibody because this was the only version available
(through BEI Resources, Inc.) at the project start. However, a consistent titration was not
observed with ricin concentration, and fluorescence counts for negative controls were similar to
samples containing ricin (up to 1 ng), showing < 2,000 counts for all. However, when the
detector antibody was analyzed in the presence of enhancement solution as a check on the
europium labeling, values of ~106 counts were measured. In addition, europium standards
showed expected values with the instrument. After data review and discussion, it was determined
that the issue was likely with the capture antibody quality and that an affinity-purified version of
the capture antibody should be used. Without affinity-purification, antibody specific to the
analyte may be < 5% of the total antibody (Life Technologies, personal communication). The
Critical Reagents Program (CRP) supplied affinity-purified anti-ricin polyclonal antibody
(produced in goat) that was used for all subsequent experiments.
The TRF assay was then evaluated using 10 pg to 10 ng ricin A-Chain per well, along with PBS
as a negative control. TRF assay results showed titration with ricin concentration using the
affinity-purified capture antibody (Table 1), confirming the issue with low fluorescence counts
was due to use of non-affinity purified antibody. Two different levels of capture antibody (400X
20
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and 200X dilutions) were used to determine which level provided better signal-to-noise
discrimination (i.e., high fluorescence counts for ricin samples and low counts for negative
controls). Fluorescence counts of approximately 1.0 x io5 were obtained for both capture
antibody levels for 10 ng ricin A-chain. Although the counts were slightly reduced with 400X
dilution of capture antibody at 10 ng ricin A-chain (p = 0.01), the counts for 1 ng ricin A-chain
were not significantly different for different antibody dilutions (p = 0.14) using a 95%
confidence level. Ricin could not be detected down to 100 pg per well since the background
counts were high and the 100 pg concentration was not significantly different from the negative
control.
Table 1 TRF Assay Results for Dilutions of Ricin With Different Concentrations of
Capture Antibody
Ricin A-Chain
Level
Fluorescence Counts*
Capture Ab Diluted 400X
Capture Ab Diluted 200X
Average
SD
Average
SD
10 ng
1.028 x io5
4.981 x 103
1.290 x 105
9.034 x 103
1 ng
1.269 x 104
4.888 x 102
1.545 x 104
1.253 x 103
100 pg
3.845 x 103
1.785 x 102
4.107 x 103
1.045 x 102
10 pg
3.525 x 103
8.886 x 102
3.000 x 103
2.332 x 102
PBS
ND
ND
2.969 x 103
2.177 x 102
PBS 1:10 dilution
ND
ND
2.850 x 103
1.684 x 102
* Average and standard deviation (SD) from triplicate analyses. ND = not determined; Ab = antibody; PBS =
phosphate-buffered saline.
Additional effort was then focused on reducing the background fluorescence counts for the
negative control by using endotoxin-free PBS buffer with filtration through 0.22-micron filters,
as well as filtration of the diluted detector antibody solution. Use of a 0.22-micron filter resulted
in lower counts compared to a 0.45-micron filter.
A subsequent experiment tested both ricin A-chain and negative controls (PBS) with capture
antibody dilutions used as 200X (-400 ng/well) and 400X (-200 ng/well). For both antibody
levels, endotoxin-free PBS was used after 0.22-micron filtration in place of unfiltered, standard
PBS buffer. For PBS controls, the fluorescence counts averaged 1,700 ± 100 and 2,700 ± 500
with capture antibody dilutions used as 400X and 200X, respectively. The counts for the
positive control also decreased when using more dilute capture antibody (400X dilution), about
32 ± 9% on average (approximately 127,000 counts for 10 ng A-chain); however, this signal was
still sufficient and provided data that met the criterion that negative controls should be < 2,000
counts. While 400X antibody dilution led to the largest improvement in assay performance,
21
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filtering the PBS and the capture antibody solution (using 0.22-micron filters) also contributed to
more consistent and lower fluorescence counts for the negative control.
5.1.2 Evaluation of the Ricin Holotoxin Dilution Method and TRF Assay Dynamic Range
An experiment was conducted to determine the optimal buffer for preparing ricin dilutions to
maintain stability. Ricin A-chain was diluted in assay buffer in the plate to make ~ 10-fold and
~100-fold dilutions as a quality control check on assay performance. Ricin dilutions were also
prepared in this manner (referred to as dilution in plates) and compared with ricin dilutions
prepared in PBS (referred to as dilution in tubes) and added to the well for analysis. The Sigma
ricin lot was used for this experiment. The results are shown in Figure 8 for triplicate TRF
analyses for each treatment and concentration. Data points represent the average count per
treatment with error bars for the standard deviation. The positive (ricin A-chain) and negative
(endotoxin-free water) controls were also analyzed in triplicate.
The data showed that ricin dilutions made in assay buffer within the analysis plate produced
significantly higher fluorescence counts compared to dilution in tubes (in PBS). The highest
concentrations between treatments showed similar counts since these were essentially the same
treatment. Dilution in the plate showed about 2.6-fold higher counts for the first dilution (~1 ng
ricin) and about 6-fold higher counts for the second dilution (-100 pg ricin) compared with
dilution in tubes. The counts for 10 ng ricin A-chain were about 3.8 x 104 which were about 4-
fold lower than later experiments where A-chain dilutions were performed directly into assay
buffer instead of in PBS first. In this case, the A-chain dilution had been prepared in advance and
stored at 4°C. Based on the data, ricin A-chain and holotoxin stability in PBS was of concern.
After discussion with technical experts from NC Department of Health and Human Services (D.
Pettit and C. Browne, personal communication), in subsequent experiments A-chain dilutions
were freshly prepared in assay buffer. Furthermore, the background fluorescence for the
experiment was relatively high compared to later experiments. As mentioned above, the
background counts were later reduced by filtration of the detector antibody solution through a
0.22-micron filter as opposed to a 0.45-micron filter used in this case.
22
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assay buffer to dilutions made outside the plate (in tubes) with PBS. Data points represent
the average fluorescence counts from three replicate TRF analyses. Error bars represent ± one
standard deviation. A red line shows the negative (endotoxin-free water) cut-off value (-4,070
counts). Ricin A-chain controls showed lower counts possibly due to degradation since the 1-
|ig/mL stock was not freshly prepared in assay buffer as for later experiments.
According to the vendor, preparations made in assay buffer must be analyzed within 1 hr;
therefore, assay buffer per se could not be used for dilution to prepare solutions with different
ricin concentrations for testing purposes. The assay buffer composition is proprietary (vendor-
supplied), however, it is known to contain a surfactant and BSA, often used to stabilize proteins
and prevent adherence to surfaces. Therefore, in this effort, different buffers were tested to
determine the effect on TRF assay performance, including PBS with 0.05% Tween-80 (PBST),
PBS with 3% BSA, and PBST with 3% BSA. These buffers were compared with PBS by making
10-fold dilutions in the appropriate buffer from the original stock solution (in PBS) at 100
[j,g/mL. Dilutions were performed to generate 1 |ig/mL to 10 ng/mL concentrations in 1-mL
dilutions (i.e., 100 |iL ricin solution added to 900 |iL buffer), and because 10 |iL were added per
well, the final concentrations were approximately 10 ng to 100 pg ricin. Results for the first
replicate experiment are shown in Table 2.
23
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Table 2 Evaluation of Dilution Buffer Effects on Detection of Ricin Holotoxin Using the
TRF Assay
Buffer Type
TRF
Fluorescence Counts by Holotoxin Level*
Replicate
10 ng
1 ng
100 pg
1
1.190 x 106
1.370 x io5
1.587 x 104
2
1.134 x 106
1.382 x 105
1.472 x 104
PBS
3
1.039 x 106
1.372 x 105
1.445 x 104
Average (SD)
1.121 (0.0760) x
106
1.375 (0.00647) x 10s
1.502 (0.0754) x
104
1
1.497 x 106
2.089 x 105
2.544 x 104
2
1.513 x 106
2.100 x 105
2.544 x 104
PBST
3
1.455 x 106
2.208 x 105
2.511 x 104
Average (SD)
1.489 (0.0288) x
106
2.132 (0.0661) x 10s
2.533 (0.0187) x
104
1
1.614 x 106
2.735 x 105
3.170 x 104
2
1.530 x 106
2.605 x 105
3.331 x 104
PBS/BSA
3
1.548 x 106
2.668 x 105
3.214 x 104
Average (SD)
1.563 (0.0442) x
106
2.669 (0.0651) x 10s
3.238 (0.0831) x
104
1
1.365 x 106
2.038 x 105
2.308 x 104
2
1.347 x 106
1.991 x 105
2.666 x 104
PBST/BSA
3
1.447 x 106
2.033 x 105
2.560 x 104
Average (SD)
1.386 (0.0531) x
106
2.021 (0.0257) x 10s
2.511 (0.184) x 104
Negative Control
Average (SD)
1.5 (0.1) x 103
* Average and standard deviation (SD) from triplicate analyses. The negative cut-off value was -2,270 counts.
The data showed the highest fluorescence counts using PBS with BSA, followed by PBST/BSA
and PBST with similar counts, and PBS. The lower ricin concentrations showed greater
improvements, with up to 2.1-fold higher counts for PBS/BSA compared to PBS for 100 pg ricin.
A replicate experiment was conducted using the same conditions as those described for the first
replicate experiment; however, in this case, the ricin dilutions ranged from 100 ng/mL to 1
ng/mL, equivalent to 1 ng to 10 pg per well. This change was made in order to investigate the
dynamic range of the assay further. The other concentrations were replicated in a subsequent
experiment. The results showed the same trends with up to 2.2-fold improvement with
PBS/BSA relative to PBS for 100 pg ricin (Table 3). For 10 pg ricin, fluorescence counts ranged
from 2,800 for PBS to about 5,640 for PBS/BSA (~2-fold higher counts). Similar fold-increases
24
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were noted for the other ricin concentrations (dilutions). Based on these findings, use of PBS
with 3% BSA is recommended to increase the stability or solubility of ricin and, subsequently,
TRF assay performance. It is understood that dilutions of ricin solutions are only prepared for
testing purposes and that actual environmental samples would either be analyzed only undiluted
or could also be diluted in assay buffer in the plate.
Table 3 Evaluation of Dilution Buffer Effects on Detection of Ricin Holotoxin Using the
TRF Assay
Buffer Type
TRF
Replicate
Fluorescence Counts by Holotoxin Level*
1 ng
100 pg
10 pg
PBS
1
1.317 x 105
1.340 x 104
2.799 x 103
2
1.187 x 105
1.308 x 104
3.033 x 103
3
1.129 x 105
1.261 x 104
2.568 x 103
Average (SD)
1.211 (0.0961) x 10s
1.303 (0.0395) x 104
2.800 (0.233) x 103
PBST
1
1.795 x 105
2.024 x 104
4.012 x 103
2
1.777 x 105
2.114 x 104
4.095 x 103
3
1.743 x 105
1.987 x 104
4.554 x 103
Average (SD)
1.772 (0.0262) x 10s
2.042 (0.0653) x 104
4.220 (0.292) x 103
PBS/BSA
1
2.446 x 105
2.836 x 104
5.679 x 103
2
2.446 x 105
2.932 x 104
6.001 x 103
3
2.311 x 105
2.827 x 104
5.263 x 103
Average (SD)
2.401 (0.0781) x 10s
2.865 (0.0584) x 104
5.648 (0.370) x 103
PBST/BSA
1
1.862 x 105
1.965 x 104
4.261 x 103
2
1.777 x 105
2.003 x 104
4.511 x 103
3
1.701 x 105
2.085 x 104
4.422 x 103
Average (SD)
1.780 (0.0806) x 10s
2.018 (0.0616) x 104
4.398 (0.127) x 103
Negative Control
Average (SD)
1.60 (0.202) x
103
* Average and standard deviation (SD) from triplicate analyses. The negative cut-off value was 2,300 counts.
An additional replicate experiment was conducted to obtain data for the entire range of ricin
concentrations tested in the initial replicate experiments (Table 2 and Table 3) and provide data
on reproducibility. In this case, only the PBS/BSA treatment was tested with 10 ng, 100 pg, and
10 pg final concentration per well, because this provided the PBS/BSA buffer provided the best
assay performance in the previous replicate experiments. The results are shown in Table 4.
25
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Table 4 Analysis of Ricin from Dilutions Prepared in PBS/BSA Buffer - Replicate
Experiment
Buffer Type
TRF Replicate
Fluorescence Counts by Holotoxin Level*
10 ng
100 pg
10 pg
PBS/BSA
1
ND
1.379 x 104
ND
2
1.423 x 104
3
1.390 x 104
Average (SD)
1.397 (0.0228) x
104
Negative
Control
Average (SD)
7.00 (0.47) x 102
Buffer Type
TRF Replicate
Fluorescence Counts by Holotoxin Level**
10 ng
100 pg
10 pg
PBS/BSA
1
8.750 x 105
1.403 x 104
2.261 x 103
2
8.746 x 105
1.332 x 104
2.144 x 103
3
8.814 x 105
1.328 x 104
2.290 x 103
Average (SD)
8.770 (0.0380) x
10s
1.354 (0.0421) x
104
2.232 (0.077) x
103
Negative
Control
Average (SD)
9.16 (0.67) x 102
* Average and standard deviation (SD) from triplicate analyses. A new lot of biotin-labeled capture antibody was
used at 200X dilution (same antibody lot from vendor).
** The original lot of biotin-labeled antibody was used at 400X (same antibody lot from vendor).
ND = not determined. The negative cut-off value was 1,340 counts.
In this case, the fluorescence counts were a bit lower likely representing the normal variability
due to different lots of labeled capture antibody, pipetting variation, and assay set-up differences.
However, the one ricin level, 100 pg, where both the original and the new lot of labeled capture
antibody were tested (with all other components the same), showed comparable results, -1.4 x
104 counts. The replicate analyses showed about a 44% decrease for 10 ng and a 60% decrease
for 10-pg compared with the other replicate experiments using these ricin concentrations, Table 2
and Table 3, respectively.
26
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5.2 Task 2 Investigation and Characterization of TRF Assay Interferences
5.2.1 Experimental Approach for Testing TRF Assay Interferences from Environmental
Sample Matrices - Bleach Residue, Sampling Materials, Wetting Buffer
While Task 3 was focused on development of a sample processing protocol for swabs and
sponges, the goal of this task was to reproduce the elevated fluorescence values observed in
previous ricin sampling and TRF analysis efforts, and to evaluate mechanistically what
interferents could contribute to false positive results (from high background fluorescence).
During the 2013 Tupelo MS ricin incident, the TRF assay was shown to produce unsatisfactory
results due to high background fluorescence (presumed associated with bleach residue or other
substances extracted from the surface) such that the assay could not be used for reliable analysis
(U.S. EPA, 2013a; U.S. EPA, 2013b). Furthermore, high backgrounds (~ 8,600 to 10,400
fluorescence counts) were reported from test samples (lacking ricin). These test samples were
prepared by EPA scientists who applied bleach, allowed the surfaces to dry overnight, and used
sponge-sticks to sample surfaces following the CDC-NIOSH protocol (CDC-NIOSH, 2012).
When these samples were analyzed by the MS DOH, the reported values exceeded typical
background fluorescence values of < 2,000 counts. While these samples lacked ricin, they
contained bleach residue from following established ricin decontamination and sampling
methods for swabs and sponges. Elevated fluorescence values (in the same range) were also
observed for surfaces that were rinsed with water prior to sampling.
Following the methods described in Sections 3.4 through 3.6, pre-wet macrofoam swabs and
Solar-Cult sponges were used to wipe stainless steel surfaces that were treated with 10% bleach
with a hand sprayer and kept wet for 10 min, followed by overnight drying, to mimic ricin
decontamination conditions used for the Tupelo MS incident. An example 10x10 inch surface
sprayed with 10% bleach is shown in Figure 9, and a similar surface allowed to dry overnight is
shown in Figure 10. The 4x4 inch areas sprayed with 10% bleach and allowed to dry are shown
in Figure 11, and these are compared with other 4x4 inch areas treated with water and allowed
to dry (Figure 12). Surfaces contained dried bleach solutions showed a large amount of salt
crystals that were largely removed upon sampling efforts (image not shown).
27
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Figure 9 Photo of a 10 x 10 inch stainless steel coupon with 10% chlorine bleach applied for
10 min, with re-spraying every 2 min, to mimic conditions for ricin decontamination.
Figure 10 Photo of a 10 x 10 inch stainless steel coupon after application of 10% chlorine
bleach and drying overnight.
Surfaces were sampled following the procedures used by CDC and EPA (outlined in Section 3.5
for swabs and 3.6 for sponges) for post-decontamination sampling (CDC-NIOSH, 2012). One
swab was used to sample three 4 x 4 inch areas, and one sponge was used to sample one 10 s 10
inch area. Samples were then processed as described in Section 3.5 for swabs and 3.6 for
sponges. Typically, 0.5-mL to 1 mL sample extract volumes were obtained from swabs and
sponges for analysis by TRF. The sample extracts with bleach residue were yellow in color and
had a pIT of about 10 (as measured by pH paper). Based on these features of the extract, it was
assumed that extracts contained high levels of bleach residual, and in fact ricin spiked into these
extracts was degraded and not detected by TRF analysis (data not shown). In this experiment,
replicate samples were prepared with and without bleach residual for processing using the
protocol for 0.5-mL 10K UF devices (Appendix B), with pre-filtration through a 0,22-urn
28
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Ultrafree; MC GV filter unit. Swab and sponge extract volumes of 400 pL were applied to UF
devices and after four wash steps, 100 11L were recovered. This served to streamline the effort by
including treatments with and without UF processing in one experiment rather than sequential
experiments.
Figure 11 Photo of 4 x 4 inch sections on a stainless steel coupon for which 10% chlorine
bleach was applied by hand sprayer for 10 min and allowed to dry overnight.
Figure 12 Photo of 4 x 4 inch sections on a stainless steel coupon for which water was
applied by hand sprayer for 10 min (as a control) and allowed to dry overnight.
29
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5.2.2 Evaluation of TRF Assay Interferences from Environmental Sample Matrices - Swabs
and Sponge-Stick Samples
Results from TRF analysis of swab samples from bleach-applied surfaces are shown in Figure 13
and Table 5. Similarly, results from TRF analysis of sponge samples are shown in Figure 14 and
Table 6. These experiments were also repeated, although the replicate experiment did not include
any additional replicates processed by UF prior to TRF analysis (Table 7). In all cases, samples
with bleach residue either with or without UF treatment showed fluorescence counts similar to
the samples from water-treated surfaces and from negative controls, < 2,000 counts. Although
the sample extracts had high levels of bleach as evidenced by a yellow color and measured pH
values of -10, once the extracts were added to assay buffer (10 [j.L to 100 [j,L assay buffer and
detector antibody), the pH was maintained at 8 (measured by pH paper on replicate samples). In
some cases, particulates from the sponges could also be observed in the samples. Although only
smooth surfaces were sampled, the vortex-mixing step may have led to disintegration of the
sponge. However, these particulates did not appear to negatively impact the TRF assay.
These data were from multiple attempts to reproduce the post-decontamination sample
generation and analysis that led to the reported elevated fluorescence backgrounds. As
mentioned, these efforts produced worst-case bleach residuals and used the same sampling
materials, reagents, and protocols that led to the problem reported by the MS DOH Laboratory;
however, no elevated backgrounds were observed in any of the testing performed for this effort,
for either swabs or sponges. Data from positive and negative controls was not included along
with the sample data showing elevated fluorescence values from the MS DOH Laboratory;
therefore, it was difficult to trouble-shoot what other factor may have led to elevated
fluorescence.
However, the data obtained from QC-testing new europium-labeled detector antibody lots in this
study pointed to other possible causes for the elevated fluorescence levels, either improper
dilution or improper quality control testing of antibody stock solutions. If too much detector
antibody was used in the assay, this would also show elevated fluorescence counts in the
negative controls, but unfortunately the data from controls for the MS DOH Laboratory was not
available. Likewise, if poor quality detector antibody was used (i.e., sub-optimal ratio of
Eu:protein, improper antibody purification, etc.) this could also lead to elevated fluorescence
values in negative controls. Although elevated background fluorescence could not be attributed
to sample matrix effects in this study, a sample processing protocol was still needed to remove
any potential interferences from complex environmental samples, as well as to concentrate ricin
for improved detection.
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Assay Performance for Swabs. Data points represent the average fluorescence counts from
three replicate TRF analyses (except for A-chain data points which are single measurements).
Error bars represent ± one standard deviation. Samples were processed by 0.5-mL 10K UF
devices (Appendix B). A red line shows the negati ve (PBS) cut-off value (~2,050 counts).
31
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Table 5 Testing of the Sample Matrix (Sampling Device, Bleach Residue, and Neutralizing
Buffer) for Background Fluorescence Interference in the TRF Assay Using Swabs
Swab
Treatment
Sample
Replicate -
TRF
Replicate
Fluorescence Counts*
No UF
UF
Swabs with
Bleach
Residue
1-1
1497
1389
1-2
1934
1076
1-3
1572
1548
Average (SD)
1668 (234)
1338 (240)
2-1
1280
1122
2-2
1753
1057
2-3
1425
1033
Average (SD)
1486 (242)
1071 (46)
3-1
1245
1310
3-2
1205
1165
3-3
1340
1366
Average (SD)
1263 (69)
1280 (104)
Swab with
Water
1-1
1537
1163
1-2
1306
1200
Average (SD)
1422 (163)
1182 (26)
PBS
(Control)
1
1442
ND
2
1320
ND
3
1342
ND
Average (SD)
1368 (65)
NA
* Average and standard deviation (SD) from triplicate analyses, except for water controls where duplicate analyses
were conducted. UF = Ultrafiltration; ND = not determined; NA = not applicable.
32
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Figure 14 Effect of Surface Sample Matrix (Bleach Residue, Neutralizing Buffer) on TRF
Assay Performance for Sponge-Stick Samples (SS). Data points represent the average
fluorescence counts from three replicate TRF analyses (except for A-chain data points which are
single measurements). Error bars represent ± one standard deviation. "10K UF" samples were
processed by 0.5-mL 10K UF devices (Appendix B). A red line shows the negative (PBS) cut-off
value (-2,000 counts).
33
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Table 6 Testing of the Sample Matrix (Sampling Device, Bleach Residue, and Neutralizing
Buffer) for Background Fluorescence Interference in the TRF Assay Using Sponge-Sticks
SS
Treatment*
Sample
Replicate -
TRF
Replicate
Fluorescence Counts
No UF
UF
SS with
Bleach
Residue
1-1
1232
1632
1-2
1282
1522
1-3
990
1680
Average (SD)
1168 (156)
1611 (81)
2-1
1174
1018
2-2
1104
1207
2-3
1166
1120
Average (SD)
1148 (38)
1115 (95)
3-1
1143
1079
3-2
1092
1044
3-3
1260
1254
Average (SD)
1165 (86)
1126(113)
SS with
Water
1-1
1571
1332
1-2
1519
1047
Average (SD)
1545 (37)
1190 (202)
PBS
(Control)
1
1451
ND
2
1356
ND
3
1187
ND
Average (SD)
1331 (134)
NA
* Average and standard deviation (SD) from triplicate analyses, except for water controls where duplicate analyses
were conducted. SS = Sponge-stick; ND = not determined; NA = not applicable.
34
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Table 7 Testing of the Sample Matrix (Sampling Device, Bleach Residue, and Neutralizing
Buffer) for Background Fluorescence Interference in the TRF Assay Using Swabs and
Sponge-Sticks
Sample
Replicate -
TRF
Replicate
Fluorescence Counts
Treatment
Swab
Sponge-Stick
1-1
1254
1508
1-2
1568
1764
1-3
1276
1272
Average (SD)
1366 (175)
1515 (246)
2-1
1477
2337
Bleach
2-2
1177
1210
Residue
2-3
1387
1280
Average (SD)
1347 (154)
1609 (631)
3-1
1336
1174
3-2
1340
1320
3-3
1213
1215
Average (SD)
1296 (72)
1236 (75)
1-1
1382
1450
Water
1-2
1209
1322
Average (SD)
1296 (122)
1386 (91)
1
1478
PBS
2
1220
(Control)
3
1250
Average (SD)
1316(141)
* Average and standard deviation (SD) from triplicate analyses, except for water controls where duplicate analyses
were conducted.
5.3 Task 3. Determination of Ricin Recovery/Loss for Samples Processed by UF Devices
5.3.1 Evaluation of 0.5-mL 3 OK UF Devices for Ricin Recovery
A preliminary experiment was conducted using 0.5-mL 30K UF devices with 1 ng/mL and 100
ng/mL solutions of ricin to assess ricin recovery efficiency. As mentioned, these devices were
used with multiple wash steps to remove interferents resulting from bleach decontamination
protocols. In order to compare with and without UF directly for ricin recovery, the 30K device
was not used to concentrate the samples. Rather, 100 [j,L of each ricin solution were applied to
35
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the UF devices (in triplicate) and 300 [xL PBS were added to bring the total volume to 400 [j,L.
The UF procedure was followed (see Appendix B) except that four washes instead of two were
conducted, and 100 [j,L retentate was recovered. These UF-treated samples were analyzed in
triplicate by the TRF assay and compared with the original dilution—1 [j.g/mL or 100 ng/mL—
for which nine replicates were analyzed.
The results showed high background fluorescence for the negative control PBS (Table 8), with
an average 5,500 fluorescence counts making the negative cut-off value at -8,200 counts, such
that only the 1 ng/mL solution data could be used to compare ricin recovery efficiencies from
30K-UF. PBS that was filtered through a 0.22-micron filter was also analyzed, although the
filtered buffer also showed high counts (avg. 4,200 counts). The detector antibody (diluted to IX
in assay buffer) was passed through a 0.45-micron filter instead of a 0.22-micron filter; it was
thought that the larger pore filter could have contributed to the high counts since more detector
antibody aggregates could be present. In addition, data for 1 [j,g/mL replicates (corresponding to
10 ng per well since 10 [j,L were analyzed) which were UF-treated showed about 72% loss
relative to the untreated solution. It should also be noted that the counts for 10 ng ricin were
lower compared to subsequent experiments with this ricin concentration because assay
conditions (i.e., antibody concentrations, reagent preparation) were not optimal in this case.
Regardless, the poor recovery observed by processing through the 0.5 mL 30K UF device
required testing a different device, namely the lower NMWL device, 0.5 mL 10K UF device.
Therefore, no additional testing was done using the 0.5 mL 30K UF devices.
36
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Table 8 Evaluation of the 0.5-mL 30K UF Device on Relative Percent Recovery of Ricin
Treatment*
Sample Replicate - TRF
Replicate
Fluorescence Counts
1-1
1.535 x 104
1-2
1.509 x 104
1-3
1.493 x 104
Average (SD)
1.512 (0.0212) x 104
2-1
1.751 x 104
10 ng Ricin
30KUF
2-2
1.781 x 104
2-3
1.775 x 104
Average (SD)
1.769 (0.0156) x 104
3-1
1.546 x 104
3-2
1.716 x 104
3-3
1.558 x 104
Average (SD)
1.607 (0.0946) x 104
Overall Average (SD)
1.630 (0.124) x 104
Treatment*
TRF Replicate
Fluorescence Counts
1
5.509 x 104
2
5.688 x 104
3
5.592 x 104
4
5.755 x 104
10 ng Ricin
5
5.633 x 104
No UF
6
5.453 x 104
7
5.828 x 104
8
6.282 x 104
9
5.824 x 104
Average (SD)
5.729 (0.245) x 104
PBS
(Control)
30KUF
1
3.624 x 103
2
4.442 x 103
3
5.842 x 103
Average (SD)
4.636 (1.122) x 103
1
9.491 x 103
2
4.070 x 103
PBS
3
3.233 x 103
(Control)
4
4.247 x 103
No UF
5
5.418 x 103
6
6.448 x 103
Average (SD)
5.485 (2.262) x 103
* Ricin samples were approximately 10 ng per sample replicate (10 |iL) for UF treatment or per TRF replicate for
the untreated ricin solution (1 (ig/mL). Ricin solutions were prepared in unfiltered PBS using the Sigma ricin lot.
UF = Ultrafiltration. Results are averages and standard deviations (SD) for three or more replicate analyses (as
shown).
37
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5.3.2 Evaluation of 0.5-mL 1 OK UF Devices for Ricin Recovery
An experiment was conducted using 0.5-mL 10K UF devices to determine ricin recovery. As for
the data shown in Table 8, this experiment was not designed to test the ability to concentrate
ricin but rather to determine losses by comparing solutions that should have the same ricin
concentration if there were no losses to the UF device. Ricin was tested at 1 [j,g/mL as 100 [xL
added to the UF device and 300 [xL PBS added to bring the volume to 400 [j,L. Triplicate samples
were processed through 0.5-mL 10K UF devices (with four wash steps as for Section 5.3.1) with
100 [jL retentate, and then analyzed by the TRF assay to allow comparison with the original ricin
solution that was untreated.
In this case, the data did not show loss of ricin, but rather showed 100%+ recovery relative to the
untreated solution (Table 9). The average fold-difference between UF-treated and untreated
samples ranged from 1.1 to 1.5 with an average of 1.3 ± 0.2. Using unpaired T-tests (2-tailed) on
individual UF-treated replicates (sample and TRF replicates) compared with the untreated
control, the UF-treated replicates had statistically higher counts than the control at a 95%
confidence level but not at a 99% confidence level (p-values were 0.012). The slightly higher
fluorescence values for UF-treated samples would not affect the results interpretation for the
assay. The 10K UF device did not show loss of ricin, even with four wash steps, such that this
device showed promise for processing complex environmental samples prior to TRF analysis.
However, it was also important to evaluate performance of the UF devices for lower ricin
concentrations as well (as was done in subsequent experiments), especially because, for lower
ricin levels, the UF devices could enable detection where there would otherwise be no positive
detection without ricin concentration and purification. In addition, impact of the UF devices on
ricin recovery could be masked by high levels of ricin.
38
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Table 9 Evaluation of the 0.5-mL 10K UF Device for Ricin Recovery
Avg. Fold-
Treatment*
Sample Replicate
Fluorescence
Difference From
- TRF Replicate
Counts
No UF
Treatment**
1-1
6.370 x 105
1-2
6.296 x 105
1.3
1-3
6.232 x 105
Average (SD)
6.299 (0.0692) x 10s
2-1
5.360 x 105
10 ng Ricin
10K UF
2-2
5.306 x 105
1.1
2-3
5.346 x 105
Average (SD)
5.337 (0.0283) x 10s
3-1
7.163 x io5
3-2
7.061 x 105
1.5
3-3
7.167 x 105
Average (SD)
7.13 (0.0600) x 10s
Overall Avg (SD)
6.256 (0.779) x 10s* *
1.3 (0.2)
Treatment*
TRF Replicate
Fluorescence
Counts
1
4.844 x 105
10 ng Ricin
No UF
2
4.910 x 105
NA
3
4.755 x 105
Average (SD)
4.836 (0.0774) x
J05* *
PBS
(Control)
10K UF
1
1.607 x 103
2
1.516 x 103
NA
3
1.150x 103
Average (SD)
1.424 (0.242) x 103
PBS
(Control)
No UF
1
1.468 x 103
2
1.276 x 103
NA
3
1.464 x 103
Average (SD)
1.403 (0.110) x 103
* Ricin samples were approximately 10 ng per sample replicate (10 |iL) for UF treatment or per TRF replicate for
the untreated ricin solution (1 (ig/mL). Ricin solutions were prepared in PBS using the Sigma ricin lot. 10K UF =
ultrafiltration with 10K device. Average (Avg) and standard deviation (SD) are from triplicate TRF analyses.
NA = not applicable.
** Denotes no statistically significant difference compared to the "No UF" control at the 95% confidence level (p =
0.012).
39
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5.3.3 Evaluation of 0.5-mL 1 OK UF Devices for Ricin Recovery - Comparison of Lots of
Ricin Holotoxin
An experiment was conducted using the 0.5-mL 10K UF devices to confirm that no loss of ricin
occurred and furthermore, to evaluate a new Vector Labs ricin lot in comparison with the Sigma
ricin lot to investigate whether similar trends were observed. A new lot was procured from
Vector Labs, Inc. (Unconjugated Ricinus communis Agglutinin II [RCA60, ricin]; Section 3.2)
since this form of ricin holotoxin was no longer available from Sigma, who originally supplied
the material. The new lot was obtained to confirm there were no differences in assay
performance compared with the Sigma lot (in case there was some degradation over time). As for
the previous experiment, the 0.5-mL 10K UF devices were not used to concentrate the ricin but
rather to mimic purification through the wash steps and subsequent recovery of retentate in the
protocol; namely, 100 [jL aliquots were loaded onto UF devices, and the final retentate volume
was also 100 [jL. The experiment used 100 [xL of 100 ng/mL plus 300 [xL PBS added to the UF
device, and processed with 100 [xL retentate recovered. This was compared with the original
solution of 100 ng/mL (1 ng final concentration since 10 [xL were loaded per well). In this case,
only two wash steps on the 10K UF devices were used (Appendix B).
The results showed that there were no significant differences between average fluorescence
counts between the Sigma and Vector Labs ricin lots for untreated samples (Figure 15), with p-
value = 0.46 (two-tailed, unpaired T-test); however, the Sigma ricin lot had significantly higher
counts than the new lot for UF-treated samples, p-value = 2 x 10"4. It was possible that the Sigma
ricin lot was more degraded such that essentially a larger number of epitopes were exposed
enabling different binding. It is also possible that differences in ricin quality (other than
degradation) or quantity between the two lots led to the differences. Regardless, for both ricin
lots, the average counts for UF-treated samples were similar or had slightly higher counts
relative to those for untreated samples showing that ricin was not lost to the UF devices. While
UF-treated samples showed slightly higher average counts for some replicates relative to the
unfiltered controls (on average -30% higher counts for the Vector Labs ricin lot and -48%
higher counts for the Sigma ricin lot), these differences are not expected to affect the
interpretation of results for this qualitative analysis. The positive and negative controls (A-chain
and PBS, respectively) gave expected results. The majority of the experiments were conducted
with the Vector Labs ricin lot and where noted the Sigma ricin lot was used.
40
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Figure 15 TRF assay results for 100 pg/mL ricin solutions processed by 10K UF devices
compared to untreated 100 pg/mL solutions (ricin concentration per sample was ~1 ng) for
ricin holotoxin lots from Sigma and Vector Labs. Data points represent the average
fluorescence counts from three replicate TRF analyses (except for A-chain data points which are
single measurements). Error bars represent ± one standard deviation. A red line shows the
negative (PBS) cut-off value (-2,190 counts).
5.3.4 Evaluation of 0.5-mL 10K UF Devices for Ricin Recovery and Concentration
An experiment was used to test whether the 0.5-mL 10K UF devices could concentrate ricin
from a more dilute solution. In this effort, a protocol for processing 1-mL samples was
developed and evaluated. In this case, 1-mL samples at 10-ng/mL ricin were processed and
compared with untreated samples at 100-ng/mL ricin. Ten-fold different concentrations were
used such that comparable fluorescence counts would be obtained after 10-fold concentration of
the sample volume using the UF device (1 mL concentrated to 100 [xL).
The results are shown in Figure 16 for UF-treated ("10K UF") and untreated extracts ("No UF")
showing that a 10-fold concentration factor (based on the fluorescence counts) was achieved
with the protocol and thus, ricin losses were not observed. The UF-treated samples actually
41
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showed more than 10-fold concentration ranging from -10.9 to 12.1-fold concentration. Positive
(A-chain) and negative (PBS buffer) controls provided expected results. The results are also
shown in Table 10 where fold differences between sample treatments are included. In each case,
10 [j,L of the resulting solution were analyzed by TRF. Because 10-fold lower ricin
concentrations were used for the 1-mL aliquot concentrated down to 100 [j,L (10-fold
concentration by volume) than for the ricin solution not processed by UF, the average fold
difference between UF-treated and untreated sample counts was actually -11- to 12-fold (or 11-
to 12-fold concentration).
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Figure 16 TRF assay results for 1-mL ricin solutions (10 ng/mL) processed by 0.5-mL 10K
UF devices with 100 jiL retentate compared to untreated 100 ng/mL solutions (ricin mass
per well in each case was ~1 ng for 10 jiL analyzed). Results are from the Vector Lab
holotoxin lot. Data points represent the average fluorescence counts from three replicate TRF
analyses (except for A-chain data points which are single measurements). Error bars represent ±
one standard deviation. A red line shows the negative (PBS) cut-off value (-1,400 counts).
42
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Table 10 Evaluation of the 0.5-mL 10K UF Device for Ricin Recovery and Concentration
Sample
Avg F old-
Treatment*
Replicate -
TRF Replicate
Fluorescence Counts
Difference From No
UF Treatment**
1-1
3.987 x 104
1-2
3.856 x 104
1.19f
1-3
3.767 x 104
Average (SD)
3.870 (0.111) x 104
2-1
3.581 x 104
10 ng/mL
Ricin
10KUF
2-2
3.499 x 104
1.09t
2-3
3.518 x 104
Average (SD)
3.533 (0.0427) x 104
3-1
3.923 x 104
3-2
4.032 x 104
1.21f
3-3
3.862 x 104
Average (SD)
3.939 (0.0860) x 104
Overall Avg
(SD)
3.781 (0.202) x 104
1.16 ±0.07
100 ng/mL
Ricin
No UF
1
3.295 x 104
2
3.191 x 104
NA
3
3.277 x 104
Average (SD)
3.254 (0.0558) x 104
PBS
(Control)
No UF
1
1.024 x 103
2
9.48 x 102
NA
3
8.36 x 102
Average (SD)
9.36 (0.95) x 102
* The negative cut-off value was 1,400 counts. 10K UF = ultrafiltration with 10K device. Average (Avg) and
standard deviation (SD) are from triplicate TRF analyses. NA = not applicable. UF = ultrafiltration.
** Since 10-fold lower ricin concentrations were used for the 1-inL aliquot concentrated down to 100 |iL. than for
the ricin solution not processed by UF, the average fold difference between UF-treated and untreated was actually
~12-fold.
denotes a statistically significant difference compared to the "No UF" control (95% confidence level).
A replicate experiment was conducted using the sample parameters as that for Table 10 with
results listed in Table 11 and plotted in Figure 17. Similar results were obtained showing about
14-fold average ricin concentration based on differences in fluorescence counts between UF-
treated (1 mL of 10 ng/mL concentrated to 100 |iL) and untreated ricin solutions (100 ng/mL). In
each case 10 [xL of the resulting solution were analyzed by TRF. Statistical analysis showed that
UF-treated samples had significantly higher fluorescence counts based on a theoretical
43
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concentration factor of 10-fold, with fold-differences of 14-fold. As for the previous experiment,
the positive and negative controls gave expected results although the negative controls showed
higher background counts.
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Table 11 Evaluation of the 0.5-mL 10K UF Device on Ricin Recovery and Concentration -
Replicate Experiment
Sample
Avg F old-
Treatment*
Replicate -
TRF
Replicate
Fluorescence Counts
Difference
From No UF
Treatment**
1-1
4.866 x 104
1-2
4.893 x 104
1.41
1-3
4.838 x 104
Average (SD)
4.865 (0.0276) x 104
2-1
4.927 x 104
10 ng/mL
Ricin
10K UF
2-2
4.902 x 104
1.43
2-3
4.905 x 104
Average (SD)
4.911 (0.0138) x 104
3-1
4.726 x 104
3-2
4.971 x 104
1.42
3-3
4.991 x 104
Average (SD)
4.896 (0.147) x 104
Overall Avg
(SD)
4.891 (0.0779) x 104
1.42 (0.007)
100 ng/mL
Ricin
No UF
1
3.523 x 104
2
3.398 x 104
NA
3
3.396 x 104
Average (SD)
3.439 (0.0726) x 104
PBS
(Control)
No UF
1
1.776 x 103
2
1.690 x 103
NA
3
1.502 x 103
Average (SD)
1.656 (0.140) x 103
* The negative cut-off value was 2, 480 counts. 10K UF = ultrafiltration with 10K device. Average and standard
deviation (SD) are from triplicate TRF analyses. Avg = Average.
** Since 10-fold lower ricin concentrations were used for the 1-inL aliquot concentrated down to 100 |iL. than for
the ricin solution not processed by UF, the average fold difference between UF-treated and untreated was actually
~14-fold. UF = ultrafiltration.
5.4 Task 4 Evaluation of Sample Processing Procedure Using UF for Complex
Environmental Samples
5.4.1 Evaluation of 2-mL 10K UF Devices for Ricin Purification and Concentration from
Samples Containing ATD
As mentioned, bleach residue, sample materials (swab, sponge), and wetting buffer did not
contribute to elevated fluorescence counts in our assessment; however, post-decontamination
45
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samples could contain other interferents such as particulate substances (referred to as "debris")
that could lead to elevated fluorescence backgrounds and/or inaccurate results. This task focused
on evaluating whether sample processing with UF devices could be used to purify ricin from
complex sample matrices; therefore, Arizona Test Dust (ATD) was used as a source of debris to
challenge the sample processing procedure developed in Task 3. ATD represented a challenge
material that has been used by the CDC and others, although it should be noted that it is largely
inorganic, containing metal oxides, with some fungal and bacterial spores or cells present (Rose
et al., 2011). ATD was used in the current study in a limited effort was conducted with debris to
gain some information about robustness of the UF-treatment and the TRF assay while it is
understood that other types of environmental backgrounds including soluble organic materials
could also affect sample processing and analysis.
An ATD slurry was prepared in the NB expressed from SS samples (as 1 g/mL) in order to use
the same recipe of NB as that used for sampling. The ATD was included at 250 mg per SS
sample (by addition of 250 |iL slurry per SS). The pre-wet sponge samples were then extracted
with 1-mL PBS containing 3% BSA (Section 3.6). In each case, 3 mL of extract were obtained.
A 2-mL aliquot of this extract was spiked with ricin holotoxin to a final concentration of 10
pg/|iL (10 ng/mL) by adding 20 |iL of 1 |ig/mL ricin to 1980 |iL SS extract. Ricin added to SS
extract without ATD was included as a control. The resulting extracts were analyzed both with
and without purification and concentration with 10K UF devices.
In addition, since the TRF assay results could be affected by debris particulates in the sample, the
procedure included an initial 0.22-micron pre-filtration step. Specifically, four 500-|iL aliquots
of extract/toxin solution were filtered through four separate 0.22-micron filter units. The filtrates
were combined, and the entire 2-mL were then transferred into a single 2-mL 10K UF device.
The manufacturer's protocol was used as guidance, with the actual procedure listed in Appendix
C. Each centrifugation step to reduce 2-mL to ~100-|iL in the device took about 1 hr; therefore,
only two wash steps were included. Interferences from bleach and environmental samples were
not observed (Task 2); consequently, a systematic study of how many wash steps are required to
eliminate the interferences could not be conducted. Aliquots (2 mL) of this extract were spiked
with ricin to a final concentration of 10 pg/|iL (10 ng/mL), by addition of 20 |iL of a 1 -|ig/mL
ricin solution to 1980 |iL SS extract (NB with or without ATD).
The data showed greater than 20-fold concentration of ricin (based on fluorescence counts) by
use of the 2-mL 10K UF devices, with an average ~32-fold concentration factor for clean
extracts and an average ~24-fold concentration factor for ATD extracts (Table 12).
Concentration of ricin in the sample improved the detection limit afforded by the overall method
(by about 24- to 30-fold) since the initial values prior to concentration were only about 1.5 - 2-
fold above the negative control. The difference between average fluorescence counts for UF-
treated clean and ATD-containing samples were significantly different at the 95% confidence
level but not at the 99% confidence level (p = 0.016, two-tailed, unpaired T-test), whereas, the
46
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difference between average values for untreated clean and ATD-containing samples was not
significant at both confidence levels (p = 0.44, two-tailed, unpaired T-test).
Table 12 Evaluation of 2-mL 10K UF Device for Ricin Recovery and Concentration from
Samples With or Without ATD
Fluorescence Counts*
Sample
Type
TRF
Replicate
Without UF -- Sample Replicates
With UF
-- Sample Replicates
1
2
3
1
2
3
1
4.117 x
103
3.509 x 103
3.202 x 103
1.185 x
105
1.128 x
105
1.127 x
105
2
3.90 2x
103
3.609 x 103
3.235 x 103
1.203 x
105
1.109 x
105
1.059 x
105
SS
Solution
3
3.754 x
103
3.624 x 103
3.455 x 103
1.175 x
105
1.160 x
105
1.061 x
105
3.924
3.581
3.297
1.188
1.132
1.082
Avg (SD)
(0.183) x
103
(0.063) x
103
(0.138) x
103
(0.0145) x
10s
(0.0256) x
10s
(0.0386) x
10s
Overall
Avg (SD)
3.601 (0.297) x 103
1.134 (0.0517) x 10s
1
3.860 x
103
3.791 x 103
3.623 x 103
9.848 x
104
8.141 x
104
9.381 x
104
SS
Solution
With 250
mg ATD
2
3.853 x
103
4.081 x 103
3.648 x 103
1.008 x
105
8.145 x
104
9.273 x
104
3
3.690 x
103
3.370 x 103
3.885 x 103
9.343 x
104
8.238 x
104
9.359x 104
3.801
3.747
3.719
9.756
8.175
9.338
Avg (SD)
(0.096) x
103
(0.358) x
103
(0.145) x
103
(0.375) x
104
(0.0546) x
104
(0.0571) x
104
Overall
Avg (SD)
3.756 (0.202) x 103
9.089 (0.735) x 104
* The negative cut-off value was 1,390 counts. Average (Avg) and standard deviation (SD) are from triplicate TRF
analyses. The initial ricin concentration was 10 ng/inL prior to UF treatment. SS = sponge-stick; ATD = Arizona
Test Dust; UF = Ultrafiltration.
5.4.2 Evaluation of 2-mL 10K UF Devices for Ricin Purification and Concentration from
Samples Containing ATD — Replicate Experiment
Similar results were observed for a replicate of this experiment (Table 13), with the same ricin
concentrations and 250 mg concentration of ATD used per SS sample. In this case, there was an
average ~22-fold concentration factor for clean extracts and an average ~18-fold concentration
47
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factor for dirty extracts. In both experiments, the concentration factor (based on counts) for dirty
extracts was lower than the difference for clean extracts. Similar to the first experiment, the
difference between average fluorescence counts for UF-treated clean and ATD-containing
samples were not significantly different at the 95% confidence level (p = 0.052, two-tailed,
unpaired T-test), as was also the case for the difference between average values for untreated
clean and ATD-containing samples (p = 0.52, two-tailed, unpaired T-test). The addition of 250
mg ATD per SS sample did not significantly impact the TRF results; however, these results
cannot be generalized to all environmental surface samples. In other types of samples, different
types of background debris and interferences could affect the assay results, such that additional
sample processing prior to TRF analysis is necessary. However, in principle, the sample
processing procedure that includes 0.22-micron pre-filtration and subsequent washes of the
analyte in the UF device may alleviate TRF assay interferences for other environmental samples.
Table 13 Evaluation of 2-mL 10K UF Devices for Ricin Recovery and Concentration from
Samples With or Without ATD—Replicate Experiment
Sample
Type
TRF
Replicate
Fluorescence Counts*
Without UF -- Sample Replicates
With UF -- Sample Replicates
1
2
3
1
2
3
SS
Solution
1
5.539 x 103
4.705 x 103
4.913 x 103
1.243 x 10s
1.068 x 105
1.019 x 105
2
5.440 x 103
4.826 x 103
5.824 x 103
1.280 x 105
1.040 x 105
1.019 x 105
3
5.342 x 103
4.823 x 103
4.376 x 103
1.174 x 105
1.044 x 105
1.028 x 105
Avg (SD)
5.440
(0.099) x
103
4.785
(0.069) x
103
5.038
(0.732) x
103
1.232
(0.0537) x
10s
1.051
(0.0147) x
10s
1.022
(0.00525) x
10s
Overall
Avg (SD)
5.088 (0.469) x 103
1.102 (0.103) x 10s
SS
Solution
With
250 mg
ATD
1
5.778 x 103
4.814 x 103
4.574 x 103
9.620 x 104
9.484 x 104
8.136 x 104
2
4.965 x 103
5.123 x 103
4.678 x 103
9.297 x 104
9.384 x 104
8.269 x 104
3
4.685 x 103
4.887 x 103
4.794 x 103
8.790 x 104
9.376 x 104
8.387 x 104
Avg (SD)
5.143
(0.568) x
103
4.941
(0.162) x
103
4.682
(0.110) x
103
9.235
(0.418) x
104
9.414
(0.0601) x
104
8.264
(0.125) x
104
Overall
Avg (SD)
4.922 (0.361) x 103
8.971 (0.580) x 104
* The negative cut-off value was 1,530 counts. Average (Avg) and standard deviation (SD) are from triplicate TRF
analyses. The initial ricin concentration was 10 ng/inL prior to UF treatment. SS = sponge-stick; ATD = Arizona
Test Dust; UF = Ultrafiltration.
48
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6 Conclusions
In this effort, the potential causes of previously reported, elevated background fluorescence in
TRF analysis for post-decontamination samples were investigated. It should be noted that the
effort was solely to troubleshoot the TRF assay for post-decontamination applications and was
not meant to directly inform the protocol used for unknown samples. Specifically, since samples
contained known amounts of ricin, the multiple dilutions used for real-world, unknown samples
were not prepared and analyzed. Unknown samples should be prepared in multiple serial
dilutions so the fluorescence counts are in proper range for accurate detection/quantification;
furthermore, there would be more confidence in the results when sequential dilutions were
positive.
The TRF immunoassay reported herein appeared to tolerate high concentrations of bleach
residue, wetting buffer, and materials from sampling devices (sponge-sticks and macrofoam
swabs) since it did not show elevated background fluorescence responses (i.e., > 7,000-8,000
fluorescence counts) with these potential interferents. Furthermore, samples containing
particulates (from a reference test dust) up to -250 mg/Spoge-Stick did not contribute to high
background fluorescence.
However, high fluorescence counts were observed for samples lacking ricin (PBS controls) when
detector antibody preparations with high background fluorescence were used at more
concentrated levels; for example, >10,000 counts were evident for 200-fold dilutions of these
antibody lots, whereas other antibody lots did not show elevated fluorescence backgrounds when
used at similar concentrations. Therefore, it is possible that reported high backgrounds leading to
unsatisfactory results reported for the Tupelo, MS ricin incident, may not have been due to the
post-bleach decontamination samples but rather due to a reagent issue. Because the antibodies
must be appropriately diluted prior to use, elevated backgrounds could also occur if antibodies
are used at too high concentration levels as a result of improper dilution.
This effort led to development of a processing procedure for surface samples (swabs, sponge-
sticks) for both sample cleanup and ricin concentration that might be useful for any assay
including fluorescence-based and electrochemiluminescence immunoassays to minimize false
positive and false negative results. Using 0.5-mL or 2-mL UF devices, 10- to 20-fold or greater
concentration factors were achieved based on fluorescence counts. The 10 kDa UF devices
provided better recovery with 2-4 wash steps compared with 30 kDa UF devices. Incorporation
of the sample processing procedure prior to TRF analysis may enable improved assay sensitivity
of detection and provide greater usability of the TRF data by elevating the fluorescence response
above the background. Therefore, more consistent results are expected for both pre- and post-
decontamination samples, providing high quality data for high consequence decisions concerning
public health.
49
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The combined process of sample clean up and toxin concentration can enable detection of ricin
at low concentrations. Because the sample processing procedure developed in this effort is
intended for use following the sample extraction steps, it might be used with any ricin analytical
method, although further verification and validation may be required, e.g., by expanding the
number of surface types and potential interferences.
50
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7 References
CDC-NIOSH. 2012. Surface sampling procedures for Bacillus anthracis spores from smooth,
non-porous surfaces, http://www.cdc.gov/niosh/topics/emres/surface-sampling-bacillus-
anthracis.html.
Grundmann, O., and I Tebbett. 2008. "Ricin on the Rise: Are we prepared?" Forensic Magazine,
June 1. http://www.forensicmag.eom/articles/2008/06/ricin-rise-are-we-prepared
PerkinElmer, Inc. 2015. Accessed on August 4.
http://www.perkinelmer.com/Resources/TechnicalResources/ApplicationSupportKnowle
dgebase/DELFIA/delfia.xhtml
Rastogi, V., L. Wallace, and S.P. Ryan. 2010. Novel cell-based assay for testing active holo-ricin
and its application in detection following decontamination. EPA 600/R-10/097.
September.
Rose, L.J., L. Hodges, H. O'Connell, and J. Noble-Wang. 2011. National validation study of a
cellulose sponge wipe-processing method for use after sampling Bacillus anthracis
spores from surfaces. Appl. Environ. Microbiol. 77:8355-8359.
Schieltz, D.M., S.C. McGrath, L.G. McWilliams, J. Rees, M.D. Bowen, J.J. Kools, L.A.
Dauphin, E. Gomez-Saladin, B.N. Newton, H.L. Stang, M.J. Vick, J. Thomas, J.L. Pirkle,
and J.R. Barr. 2011. Analysis of active ricin and castor bean proteins in a ricin
preparation, castor bean extract, and surface swabs from a public health investigation.
Forensic Sci. Int. 209:70-79.
U.S. Department of Health and Human Services. 2006. Response to a Ricin Incident: Guidelines
for Federal, State and Local Public Health and Medical Officials. Washington, DC:
Department of Health and Human Services.
U.S. Environmental Protection Agency (EPA), 2013a. Ricin Tupelo, MS Response. RRT
Meeting, Research Triangle Park, NC. August.
http://www.nrt.org/production/nrt/RRTHomeResources.nsf/resources/RRT4Aug2013Me
etingPPT_l/$File/Tupelo_MS_Ricin_Response_%28Spurlin&Franco%29.pdf
U.S. Environmental Protection Agency (EPA), 2013b. Pollution/Situation Report. Tupelo Ricin
Site. August 27.
http://www.epaosc.org/site/sitrep profile.aspx'?site id=8630&counter=20255
51
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Yuan, J., and G. Wang. 2005. Lanthanide complex-based fluorescence label for time-resolved
fluorescence bioassay. J. Fluorescence. 15:559-568.
52
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Appendix A
Appendix A: Protocol Used for Ricin Detection
Materials
• Affinity-purified polyclonal anti-ricin antibody (Critical Reagents Program, CRP, Ft.
Detrick, MD, Cat. No. AB-AG RIC)
• EZ-link® NHS-PEG4 Biotintylation Kit (Life Technologies, Waltham, MA, Cat. No.
21455)
• Monoclonal Anti-ricin Toxin A-Chain, Clone RAC18 antibody (BEI Resources,
Manassas, VA, Cat. No. NR-9571)
• 0.22 Micron Syringe Filter for Detector Antibody filtration (MilliporeR MillexR GV,
Billerica, MA, Cat. No. SLGV033RS)
• Polypropylene bioblock (Beckmann Coulter® Square well PP Plate, Cat. No. 609681 or
equivalent reservior for transferring antibody solutions to strip plates
• Ricin A-chain used as positive control (Sigma-Aldrich®, St. Louis, MO, Cat. No. L9514-
1MG)
• PerkinElmer PET FT A® 25X Wash Concentrate (PerkinElmer, Inc., Waltham, MA, Cat.
No. 1244-114)
• PerkinElmer DELFIA® Assay Buffer (Cat. No. 1244-111)
• PerkinElmer DELFIA® Enhancement Solution (Cat. No. 1244-105)
• PerkinElmer PET FT A® Streptavidin-coated clear plate, 8 x 12 strips, 10 plates (Cat.
No. 4009-0010)
• Tubes, sterile 2 mL DNase- and RNasefree, gasketed, screw caps (National Scientific,
Rockwood, TN, Cat. No. BC20NA-PS)
• Gibco® Water for Injection (WFI) for cell culture, VSP, EP Antimicrobial Water (Life
Technologies, Cat. No. A12873-02)
• IX PBS (TeknovaR, Hollister, CA, Cat. No. P0300; endotoxin free filtered through
0.22-micron filter sterilization unit)
• Amicon® Ultra- 0.5 10K Centrifugal UF Devices with IUtraceU 10 Membrane
(Millipore®, Cat. No. UFC501024)
• Amicon® Ultra collection tubes for use with Amicon® Ultra Centrifugal Filters
(Amicon®/Millipore®, Cat. No. UFC50VL96)
• 0.22 um Ultrafree® MC GV Sterile 0.5 mL Centrifugal Filter Unit with Durapore®
PVDFMembrane, Yellow Cap (Millipore®, Cat. No. UFC30GV0S)
• Bovine Serum Albumin, Fraction V (VWR, Radnor, PA, Cat. No. VWR Cat. No.
EM2930)
53
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• Amicon® Ultra-2 10K Centrifugal UF Devices with Ultrace/® 10 Membrane
(Millipore®, Cat. No. UFC201024)
• Neutralizing Buffer (Hardy Diagnostics®, Santa Maria, CA, Cat. No. K105)
• Cellulose Sponge-Stick samples pre-wet with Neutralizing Buffer (Solar Biologicals,
Ogdensburg, NY, Cat. No. SH10NB)
• Foam-tipped swab samples (Puritan® Medical Products, Guilford, ME, Cat. No. 25-
1607 1PFSC)
• Victor® X4 Plate Reader (PerkinElmer®, Cat. No. 2030-0040)
• Disposable sterile polystyrene forceps, individually wrapped (Cole Parmer®, Vernon
Hills, IL, Cat. No. YO-06443-20 or equivalent)
• BI)K Falcon, Polypropylene 15 mL conical tubes, Sterile, (Becton, Dickinson and Co.,
Franklin Lakes, NJ, Cat. No. 352098 or equivalent)
• DELFIA® Plate Wash (PerkinElmer, Cat. No. 1296-026)
• Pro-Advantage®, Sterile specimen cup, 4 ounces, Polyethylene, Mountainside Medical
Equipment, Marcy, NY, Cat. No. P250400
• DELFIA® PLATESHAKE (PerkinElmer, Cat. No. 1296-004)
• Potassium Hydrogen Phthalate 100 g (Fisher Scientific, Waltham, MA, Cat. No. P243-
100 or equivalent)
• 0.5 MEDTA, pH 8.0 (Ambion®, Fisher Scientific, Cat. No. AM9260G)
• Bleach Wipes, Dispatch® (Clorox, Oakland, CA, Cat. No. 69150 or equivalent)
• 50 mL serological pipettes, sterile, individually wrapped (Corning®, Corning Inc.,
Corning, NY, Cat. No. 29442-440)
• 10 mL serological pipettes, sterile, individually wrapped (Corning® Cat. No. 29442-
430)
• 25 mL serological pipettes, sterile, individually wrapped (Corning® Cat. No. 29442-
436)
• 5 mL serological pipettes, sterile, individually wrapped (Corning® Cat. No. 29442-422)
• Table top centrifuge with adapters for 15-mL tubes (EppendorJ®, Eppendorf North
America, Hauppauge, NY, 581 OR or equivent)
Reagent Preparation for Processing Sponges and Swabs
1. Prepare phosphate buffered saline (PBS) with 3% bovine serum albumin (BSA) (Fraction
V, VWR Cat. No. EM2930).
2. Use sterile IXPBS, pH 7.2-7.4 (does not need to be endotoxin-free).
3. Make 10 mL solution with 0.3 g BSA (or prepare 100 mL with 3 g BSA and store at 2-
8°C for up to 1 month)
4. Filter sterilize through a 0.22 micron filter.
5. This solution is used to process the swab and sponge samples after surface sampling.
54
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Swab Surface Sampling and Sample Processing Procedure to Collect Bleach Residue and
Other Matrices in the Absence of Ricin
Note: The procedure was used to generate a realistic sample matrix for testing the UF protocol
and time-resolved fluorescence (TRF) assay performance. Ricin was not applied to surfaces and
ricin recovery from surfaces was not evaluated.
1. Prepare stainless steel coupons by wiping them with isopropanol and air drying in the
biosafety cabinet (BSC).
2. Tape off 4x4 inch (10.2 x 10.2 cm) squares, 3 total squares per plate. Use 3 plates for
bleach treatment and one plate for the negative control (water sprayed on—same water
used to make the 10% bleach solution).
3. Prepare 10% bleach just before use (9 parts sterile filtered water, 1 part Ultra Clorox®
Germicidal Bleach). Autoclaved, double distilled water can be used.
4. Apply 10% bleach by hand sprayer, completely covering surface with layer of aqueous
solution. Monitor plates and reapply bleach if any drying is observed within 10 min
period. Allow samples to air dry overnight.
5. On the next day, sample coupons by wiping with swabs pre-wet by dipping into 10 mL NB
(Hardy Diagnostics® Cat. No. K105) according to Macrofoam Swab Procedure (CDC-
NIOSH source http://www.cdc.gov/niosh/topics/emres/surface-sampling-bacillus-
anthracis.html), and pressing against the tube to express liquid from swab head. Make
sure there is sufficient liquid to keep wet for all three 4x4 inch areas. Record weight of
tube with liquid before and after dipping the swab and expressing the liquid.
6. Use S-strokes to sample the entire surface.
7. After sampling, break off swab stick by bending at the notch so that the swab head is
inside the 15-mL conical tube.
8. Add 1 mL of IXPBS with 3% BSA to each swab head in 15-mL conical tube.
9. Vortex the tube at the highest setting (-3,200 rpm) in 15 sec bursts for 2 min.
10. Using a sterile transfer pipette (or serological pipette), transfer the liquid sample to a
new pre-labeled 15 mL conical tube.
11. Re-vortex the original sample tube containing the swab in 15 sec bursts for 1 min.
12. Using a sterile transfer pipette, draw up the remaining liquid. Use the bottom of the
transfer pipette to press the head of the swab against the tube wall to express as much
liquid as possible.
13. Transfer the remaining liquid to the same pre-labeled 15 mL conical tube that contains
liquid from step 10. Avoid transferring any debris from the bottom of the tube. Briefly
centrifuge the 15 mL tube with the swab for up to 1 min at approximately 3,000 RCF to
collect the liquid to the bottom of the tube. Transfer any fluid to the appropriate conical
tube.
55
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14. Avoiding any debris at bottom of tube, transfer liquid into 2 mL screw-cap tube. Proceed
to the UF Sample Processing Procedure for sample clean-up and concentration
depending on the desired sample volume to treat (Appendix B or C).
Sponge-stick Surface Sampling and Sample Processing Procedure to Collect Bleach
Residue and Other Matrices in the Absence of Ricin
Note: The procedure was used to generate a realistic sample matrix for testing the UF protocol
and TRF assay performance. Ricin was not applied to surfaces and ricin recovery from surfaces
was not evaluated.
1. Prepare stainless steel coupons by wiping them with isopropanol and air drying in
BSC.
2. Use 10 x 10 inch (25.4 x 25.4 cm) stainless steel plates. Use 3 plates for bleach
treatment and one plate for the negative control (water sprayed on—same water used
to make the 10% bleach solution).
3. Prepare 10% bleach just before use (9 parts sterile filtered water, 1 part Ultra Clorox®
germicidal bleach). Autoclaved, double distilled water can be used.
4. Apply 10% bleach by hand sprayer, completely covering surface with layer of aqueous
liquid. Monitor plates and reapply bleach if any drying is observed. If possible, apply
more bleach even if significant drying is not observed. Allow samples to air dry
overnight.
5. Next day, sample coupons by wiping with pre-wet Sponge-sticks according to Cellulose
Sponge Procedure (CDC/NIOSH source)
http://www.cdc.gov/niosh/topics/emres/surface-sampling-bacillus-anthracis.html).
6. Use S-strokes to sample entire surface.
7. After sampling, place the head of the sponge directly into the sterile specimen cup using
the release mechanism.
8. Add 1 mL of IXPBS with 3% BSA to each sponge head in the specimen cup. Note: Use
up to 2 mL of IX PBS/3% BSA if no fluid can be recoveredfrom sponge. Press against
the sponge with apipet tip or serologicalpipet to express sufficient liquid.
9. Vortex the specimen cup at the highest setting in 15 sec bursts for 2 min.
10. Recover sample extract and process by pre-filtration through yellow-top Ultrafree-MC
filter units (Millipore® Cat. No. UFC30GV0S).
11. To pre-filter 1-mL extract, carefully transfer 0.5 mL each into two yellow-top filter
units with collection tube (follow manufacturer's directions for use). Note: 2 mL may
be pre-filtered if four yellow-top filter units are used.
12. Centrifuge at 7,000 rpm for 3 minutes.
13. Proceed to the UF Sample Processing Procedure for sample cleanup and concentration
(Appendix B for 0.5-1 mL sample volume or Appendix C for up to 2-mL sample
volume).
56
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DELFIA® Time-Resolved Fluorescence Assay:
Note: This protocol was slightly modified from Schieltz et al. (2011) 1 since the purpose in the
present study was to evaluate the sample processing approach and determine the cause of high
background fluorescence rather than for TRF analysis of unknown samples. Therefore, for
actual ricin sample analysis, additional steps may be requiredfor the TRF assay.
1. Waste set up: All tip waste must be rinsed with bleach prior to placement into sharps
container. Set up sharps and freshly prepared 2.5% bleach reservoir in secondary
container/tray inside BSC. Ensure that items such as counter tops, pipets, tubes, gloves,
etc. that may be used in TRF assays are free of bleach residue and bleach reservoirs are
not located in close proximity to sample processing activities.
2. Equipment preparation
a. Decontaminate the plate washer by flushing with 50 mM Potassium Hydrogen
Phthalate, 0.01% EDTA in distilled endotoxin-free (ETF) water (Prepared with
0.2 g potassium hydrogen phthalate, 1.4 mL 5 mM EDTA, 198.6 mL ETF H2O)
b. Rinse the plate washer with distilled, ETF water.
c. Wipe the plate reader internal plate platform with 95% ethanol to remove dust.
d. Start the plate reader instrument prior to starting the associated computer.
3. Reagent Preparation
a. Bring DELFIA® reagents to room temperature; Assay Buffer (PerkinElmer® Cat.
No. 1244-111), Wash Buffer (PerkinElmer® Cat. No. 1244-114) and
Enhancement Solution (PerkinElmer® Cat. No. 1244-105).
b. Place an aliquot of each antibody (capture and detector), and positive control
materials on ice.
c. Prepare wash buffer by 1:25 dilution in sterile distilled ETF water. (150 mL per
strip of 12 wells)
4. Positive Control Preparation (Ricin A-chain)
a. Just before use (no more than 1 hr), prepare a fresh dilution of Ricin A chain in
Assay Buffer (add 5.9 [j,L of stock at -850 [j,g/mL into 5 mL Assay Buffer for a
final concentration of-10 ng/10 |iL), Discard any unused preparation after use.
5. Antibody Preparation
1 Schieltz, D.M., S.C. McGrath, L.G. McWilliams, J. Rees, M.D. Bowen, J.J. Kools, L.A. Dauphin, E. Gomez-
Saladin, B.N. Newton, H.L. Stang, M.J. Vick, J. Thomas, J.L. Pirkle, and J.R. Barr. 2011. Analysis of
active ricin and castor bean proteins in a ricin preparation, castor bean extract, and surface swabs from a
public health investigation. Forensic Sci. Int. 209:70-79.
57
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a. Thaw aliquots of each antibody at 4°C (detector and capture). Calculate the
number of concentrated capture and detector antibody needed based on the sample
number (-1.4 mL of antibody in assay buffers per 12 samples).
b. Make working dilutions of each antibody in DELFIA® Assay Buffer.
Note: Dilutions must be used within 1 hr.
o Capture Antibody
o Detector antibody
Note: when calculating detector antibody amount to use, the matrix control
samples do not receive detector antibody so omit those samples from the
calculation.
6. Sample Preparation (Ricin holotoxin)
a. Just before use (no more than 1 hr), prepare fresh dilution of ricin holotoxin.
Discard any unused preparation after use.
7. Coating the assay plate with capture antibody
a. Determine the number of strips needed to test all samples and controls according to
the plate layout. Remove excess 12-well strips (DELFIA® Streptavidin
Microtitration Strips; PerkinElmer® Cat. No. 4009-0010) from the microtiter tray,
leaving enough in the plastic frame to perform the assay. The extra strips may be
stored in a sealed plastic bag, or a spare frame, with the desiccant that is included
with each plate. Refrigerate the extra strips until needed.
b. Centrifuge antibody aliquots in a microcentrifuge for 10 sec to collect the solution
to the bottom of the vial.
c. Dilute the capture antibody with Assay Buffer in a polypropylene tube. Since the
assay requires 100 [j,L of capture antibody in Assay Buffer per well, prepare 10%
more of the antibody dilution than needed to compensate for loss during
dispensing (i.e., 1.3 mL per 12-well test strip or 10.5 mL per plate).
d. Pre-wash all strips once (IX) with 750 [j,L Wash Buffer per well using the plate
washer. Invert plate over an absorbent pad and tamp vigorously 1-2 times to
remove any residual fluid.
e. Add 100 [jL of the capture antibody (at working dilution in Assay Buffer) to each
well using a calibrated multichannel pipette. NOTE: Avoid contact between pipette
tips and the microtiter strips since touching of the tips to the plate may cause cross-
contamination of adjacent wells, loss of reagent or improper dilution of controls.
f. Place the plate on the PlateShake and cover it with the bottom portion of the plastic
container in which the plate and strips came. Cover strips/plate and plastic cover
with aluminum foil before starting plate shaker.
g. Incubate for 2 hr at room temperature on the PlateShake set to "high".
8. Addition of detector antibody
a. Ten minutes before the end of the above incubation, centrifuge the detector
antibody in a microcentrifuge for 10 sec to collect the solution to the bottom of the
vial.
b. Dilute the detector antibody with Assay Buffer in a polypropylene tube. Prepare
extra detector antibody working solution (1.4 mL per 12-well test strip or 11 mL
per plate) to cover any loss of volume during filtering and dispensing.
c. Filter the detector antibody-buffer solution through a 0.22-micron low-protein
binding filter syringe to remove any particulates.
58
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Note: Make additional antibody-buffer solution to account for losses during
filtration (~1 mL extra volume).
d. Wash all wells twice with the plate washer using 750 [xL Wash Buffer per well.
Invert plate over absorbent pad and tamp vigorously 1-2 times to remove residual
fluid.
e. Add 100 [jL of Assay Buffer to matrix control wells. Note: Do not add detector
antibody to matrix control wells.
f. Add 100 |iL of the detector antibody in Assay Buffer to all wells used for negative
controls, positive controls and samples.
9. Addition of sample and controls
a. Following the plate layout, add 10 [xL of sample to each well on the assay plate,
excepting the Ricin A-chain with will be direct addition of 10 [xL into first well,
then two serial dilutions on the plate.
b. Once all samples and controls are added to the plate, cover strips/ plate and plastic
cover with aluminum foil before starting the plate shaker. Incubate for 1 hr at room
temperature on the PlateShake set on "high".
10. Addition of Enhancement Solution
a. Wash strips eight times, followed by two times with 750 [xL Wash Buffer per well.
Invert plate and tamp vigorously 1-2 times to remove residual fluid.
b. Using a multichannel pipette dedicated solely for the addition of Enhancement
Solution, add 200 [xL of Enhancement Solution per well.
c. Incubate for 5 min at room temperature with the PlateShake set to "low".
d. Turn on the Victor® X4 plate reader during this incubation and load the appropriate
reading procedure for the europium TRF assay.
e. Read the plate on the Victor® X4 plate reader. See manufacturer's instructions for
setting up the europium procedure.
Data Analysis and Interpretation
1. Review the counts for the negative control replicates; counts should be less than 2,000.
Higher readings may indicate inadequate washing, contamination of washer with
unbound europium, or improper detector antibody concentrations used (i.e., too high
concentration).
a. Calculate the negative cut-off value by averaging the negative control well values
and multiplying by 1.5
2. Review the data for the sample matrix control(s). Values should be less than those for the
negative control wells, < 2,000 counts. If elevated values are consistently observed, this
could indicate europium contamination. Corrective actions include additional rinses on
the plate washer. In addition, phthalate buffer (50 mM potassium hydrogen phthalate,
0.01% EDTA in distilled water) could be run through the plate washer followed by
additional rinses. Analysis of samples showing elevated matrix control values should be
repeated.
3. The positive control (ricin A-chain) is expected to be positive in all three dilutions. If the
proper concentration of ricin A-chain was used, failure to give a positive result for the
three dilutions of the positive control may indicate an assay component is not functional.
In this case, implement corrective actions and repeat the test.
59
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a. When all controls are acceptable, a sample is considered negative for this assay if
the average count values are below the higher of the plate average negative control
value or the matrix control value (i.e., referred to as the negative cut-off value).
b. When all controls are acceptable, a sample is considered "positive" (reactive) if the
average counts are above the negative cut-off value.
4. Sample values must be reproducible across the sample replicates with no more than 20%
variability (from mean value or < 20% CV) between wells. Variability greater than this
may indicate inadequate washing of the plate or cross-contamination of wells during
pipetting.
5. The estimated detection limit for this assay is -10-100 pg total protein (1-10 ng ricin
toxin/mL) which is dependent on the quality of Europium labeling for the anti-ricin
antibody lot and resultant background fluorescence.
6. Environmental samples (e.g., soil extracts or metal containing materials) may have
europium present (i.e., elevated counts for matrix controls without detector antibody),
which may interfere with the TRF assay. In this case, dilution of the sample would be
expected to reduce the signal caused by europium contamination.
60
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Appendix B
Appendix B: Sample Processing Procedure for Post-Decontamination Ricin
Samples using 0.5 mL I OK UF Devices
This procedure uses commercially available UF devices typically used for protein or DNA
purification and concentration, namely Amicon® Ultra-0.5 10K Centrifugal UF Devices (EMD
Millipore, Billerica, MA; Cat. No. UFC501024). Schematic diagrams are from the Amicon®
Ultra-0.5 Centrifugal Filter Devices (for volumes up to 500 |iL) User Guide .
This procedure can be used for environmental sample cleanup and concentration prior to ricin
analysis.
Recommendation for Sample Collection: The post-decontamination SS samples after
collection should be placed in a specimen cup containing 1-2 mL IX PBS (Teknova Inc.,
Hollister, CA, Teknova® Cat. No. P0300) with 3% BSA (Fraction V, VWR, Radnor, PA, Cat.
No. EM2930). Alternatively, if the sponge-stick was pre-wet with 10 mL Neutralizing Buffer
(NB) (from the vendor), liquid remaining in the bag holding the sponge after excess liquid is
expelled, prior to sampling, could then be used to extract ricin from the sponge after sampling
and used for ricin analysis; however, addition of 3% BSA could enhance ricin stability and thus,
detection.
Equipment and Supplies:
1. Millipore® 0.22 |im Ultrafree® MC GV Sterile 0.5 mL Centrifugal Filter Unit with
Durapore®PVDF Membrane: EMD Millipore, Billerica, MA, Cat. No. UFC30GV00
2. Amicon® Ultra- 0.5 10K Centrifugal UF Devices: EMD Millipore, Billerica, MA, Cat. No.
UFC501024
3. Amicon® Ultra- 0.5 Collection Tubes: EMD Millipore, Billerica, MA, Cat No.
UFC50VL96
4. Eppendorf® centrifuge: 5424/5424R (formerly, 5415/5415R) (Eppendorf North America,
Hauppauge, NY) or equivalent
2 User Guide: Amicon® Ultra-0.5 Centrifugal Filter Devices;
http://www.emdmillipore.com/US/en/product/Amicon-Ultra-0.5%C2%A0niL-Centrifugal-Filters-for-DNA-and-
Protein-Purification-and-Concentration,MM_NF-C82301#documentation.
61
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5. IX PBS, endotoxin-free, pH 7.3 ± 0.2: Teknova, Hollister, CA, Cat. No. P0300 or
equivalent
6. 1.0 mL micropipettor and 1.0 mL pipet tips
7. 2.0 mL microcentrifuge screw cap tubes
Sample Pre-filtration
Note: For environmental samples, it is recommended to pre-filter the sample through a 0.22
micron filter prior to concentration by ultrafiltration.
1. Using a micropipettor, carefully transfer 500 |iL of sample into a 0.22 |im Ultrafree® MC
GV Sterile 0.5 mL Centrifugal Filter Unit with Durapore® PVDF Membrane (Millipore®
Cat. No. UFC30GV00) with collection tube (for 1 mL sample use 2 units, each with a
collection tube).
2. Centrifuge tubes at 5,200 RCF for 9 minutes.
Note: Ensure that the supernatant has been completely filtered. Clean samples may take
less than 9 minutes to complete filtration. Centrifuge for an additional 2 minutes if there
is any liquid remaining in the filter.
3. Remove the filter unit using sterile disposable forceps, gripping only on the sides, and
dispose to waste. Cap the tube and proceed to the next section.
Sample Processing Using the Millipore Amicon® Ultra-0.5 Centrifugal UF Devices
I 1QK
I 4=
1 Filter device
rt -ii
1 —
]
:
3
Filtrate
Concentrate
collection tube
collection tube
Amicon Ultra-0.5 Centrifugal Filter Device Parts
1. In a biosafety cabinet (BSC), set up for each sample one Amicon® Ultra-0.5 10K
Centrifugal UF Device (Millipore®, Cat. No. UFC501024) with Filtrate and Concentrate
Collection microcentrifuge tubes. Label each UF device and concentrate collection tube.
Labeling the filtrate collection tubes is optional as long as the UF device is clearly labeled.
62
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2. In a BSC, briefly vortex the sample tubes (3-5 sec at -3,200 rpm).
3. Allow the particulate matter to settle for 1 min.
4. Process each sample as described in the following steps.
5. Insert the Amicon® Ultra-0.5 Centrifugal filter device into the filtrate collection
microcentrifuge tubes making sure that the device is fully seated in the tube.
6. Using a 1.0 mL micropipettor and respective pipet tip, carefully transfer the sample (up to
0.45 mL at a time) to the Amicon® Ultra-0.5 Centrifugal filter device (try to avoid
particulate matter).
7. Cap the Amicon® Ultra-0.5 Centrifugal filter device. This step generates a filter assembly.
8. Place the filter assembly in a fixed angle microcentrifuge (Eppendorf® 5424/5424R or
equivalent), aligning the cap strap toward the center of the centrifuge rotor. Counter
balance the centrifuge rotor with a similar assembly.
9. Centrifuge at 11,200 x g for 11 min at 4°C. If a refrigerated microcentrifuge is not
available, this step could be performed at room/ambient temperature (no more than
25°C).
10. If the sample volume is more than 0.45 mL, remove the filter assembly from the
centrifuge and transfer the Amicon® Ultra-0.5 Centrifugal filter device to a new filtrate
collection tube. Dispose of the used filtrate collection tube with filtrate. Transfer the
remainder of the sample (up to 0.45 mL) to the respective Amicon® Ultra-0.5 Centrifugal
filter device and repeat steps 12 to 15. For a 1 mL sample, perform two centrifugation
W
Add Sample
Cap
Centrifuge
63
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steps with 0.45 mL and one with the remaining volume, -0.1 mL (the last centrifugation
step with less volume can be completed in ~5 min).
Note: Ensure that the sample has been filtered to <0.1 mL. If more than 0.1 mL retentate is
left, spin for additional time depending upon the volume left in the device.
11. Remove the filter assembly from the centrifuge and transfer the Amicon® Ultra-0.5
Centrifugal filter device to a new filtrate collection tube. Dispose of used filtrate collection
tube with filtrate.
12. Carefully pipet 0.45 mL PBS into the filter device for the first wash.
13. Cap the device and centrifuge at 11,200 RCF for 11 min at 4°C.
Note: The manufacturer states the device should be centrifuged at 14,000 RCF for 10 - 30
min dependent on the NMWL of the device.
14. Remove the filter assembly from the centrifuge and transfer the filter device to a new
filtrate collection tube. Dispose of used filtrate collection tube with filtrate.
15. Repeat steps 12 and 13 for second wash. Ensure that -100 |iL remains after centrifugation.
Note: While two wash steps are described, additional wash steps may be used as
described, for samples containing large quantities of potential interferences.
16. Remove the filter assembly from the centrifuge.
17. Using a 1.0 mL micropipettor and respective pipet tip, carefully transfer the sample to a
2.0 mL microcentrifuge screw cap tube. (Try to avoid particulate matter at the bottom of
the filter).
18. Measure the sample retentate volume and adjust the volume to 0.1 mL with PBS for
replicate sample analysis.
19. Store the processed sample at 4°C until the time-resolved fluorescence (TRF)
immunoassay analysis is initiated.
64
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Appendix C
Appendix C: Sample Processing Procedure for Post-Decontamination Ricin
Samples using 2 mL I OK UF Devices
This procedure uses commercially available UF devices typically used for protein or DNA
purification and concentration, namely Amicon® Ultra-2 Centrifugal Devices (EMD Millipore,
Cat. No. UFC201024). Schematic diagrams are from the Amicon® Ultra-2 10K Centrifugal
"3
Filter Devices (for volumes up to 2 mL) User Guide .
This procedure can be used for environmental sample cleanup and concentration prior to ricin
analysis.
Recommendation for Sample Collection: The post-decontamination sponge-stick (SS) samples
after collection should be placed in a specimen cup containing 1-2 mL IX Phosphate Buffered
Saline (PBS) (Teknova® Cat. No. P300) with 3% BSA (Fraction V, VWR, Radnor, PA, Cat. No.
EM2930). Alternatively, if the sponge-stick was pre-wet with 10 mL Neutralizing Buffer (NB)
(from the vendor), liquid remaining the bag holding the SS after excess liquid is expelled, prior
to sampling could then be used to extract ricin from the SS after sampling and used for ricin
analysis; however, addition of 3% BSA could enhance ricin stability and thus, detection.
Equipment and Supplies:
1. Millipore® 0.22 |im Ultrafree® MC GV Sterile 0.5 mL Centrifugal Filter Unit with
Durapore®PVDF Membrane: EMD Millipore, Billerica, MA, Cat. No. UFC30GV00
2. Amicon® Ultra-2 10K Centrifugal Filter UF Device: EMD Millipore, Billerica, MA, Cat.
No. UFC201024
3. Eppendorf Centrifuge (Eppendorf North America, Hauppauge, NY): 5430/5430R with
fixed angle rotor, or equivalent that can accommodate 15 mL conical centrifuge tubes.
An Eppendorf centrifuge 5810/5810R with swinging bucket rotor may also be used.
4. 15-mL conical tubes
3User Guide: Amicon® Ultra-2 Centrifugal Filter Devices;
https://www.emdmillipore.com/US/en/product/Amicon-Ultra-2-niL-Centrifugal-Filters-for-DNA-and-Protein-
Purification-and-Concentration,MM_NF-C86533#documentation
65
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5. IX PBS, endotoxin-free, pH 7.3 ± 0.2: Teknova, Hollister, CA, Cat. No. P0300 or
equivalent
6. 1.0 mL micropipettor and respective 1.0 mL pipet tips
7. 2.0 mL microcentrifuge screw cap tube
Sample Pre-filtration
Note: For environmental samples, it is recommended to pre-filter the sample through a 0.22
micron filter prior to concentration by ultrafiltration.
1. Using a micropipettor, carefully transfer 500 |iL of sample into a 0.22 |im Ultrafree® MC
GV Sterile 0.5 mL Centrifugal Filter Unit with Durapore® PVDF Membrane, Yellow
Color Coded (Millipore® Cat. No. UFC30GV00) with collection tube (for 2 mL sample
use 4 units each with a collection tube).
2. Centrifuge tubes at 7,000 rpm for 9 minutes.
Note: Ensure that the supernatant has been completely filtered. Clean samples may take
less than 9 minutes to complete filtration. Centrifuge for an additional 2 minutes if there
is any liquid remaining in the filter.
3. Remove the filter unit using sterile disposable forceps, gripping only on the sides, and
dispose to waste. Cap the tube and proceed to the next section.
4. The filtered sample aliquots will be combined (up to 2.0 mL) into the Amicon Ultra-2
10K Centrifugal device as described below.
Sample Processing using the Millipore Amicon Ultra-2 Centrifugal Devices
Filter dev ce
3'
1
filtrate
collection-
tube
n-
V
Concentrate
collection
tube
Amicon Ultra-2 Centrifugal Filter Device Parts
1. In a biosafety cabinet (BSC), set up for each sample, one Amicon® Ultra-2 10K
Centrifugal Filter UF Device (Millipore®, Cat. No. UFC201024) and one 1.5 mL
microcentrifuge tube. Label each UF device, concentrate collection tube, and one 1.5 mL
66
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microcentrifuge tube. Labeling the filtrate collection tubes is optional as long as the UF
device is clearly labeled.
Note: It may not be necessary to label all the collection tubes as long as the Amicotf
Ultra filter insert is clearly labeled.
2. In a BSC, briefly vortex-mix the samples in sample tubes (3-5 sec at -3,200 rpm).
3. Allow the particulate matter to settle at the bottom of the tube for 1 min.
4. Process each sample as described in the following steps.
5. Insert the Amicon® Ultra-2 Centrifugal filter device into the filtrate collection tube
making sure that the device is fully seated in the tube.
6. Using a 1.0 mL micropipettor and respective pipet tip(s), carefully transfer the sample
(up to 2.0 mL) to the Amicon® Ultra-2 Centrifugal filter device (try to avoid particulate
matter at bottom of the sample tube).
7. Using the concentrate collection tube, cap/cover the top of the Amicon® Ultra-2
Centrifugal filter device. This step generates a filter assembly.
1
t
Jul
ii
Add ample
Make sure both tubes are fully seated onto device
Amicon Ultra-2 Centrifugal Filter Device Assembly
8. Place the filter assembly in a fixed angle microcentrifuge (Eppendorf® 5430/543OR or
equivalent) or place into a 15-mL conical tube for a swinging bucket tabletop centrifuge
(Eppendorf® 5810/5810R or equivalent) with 15-mL tube adapter. Counter balance the
centrifuge rotor with a similar assembly. Make sure the device is seated on the bottom of
the rotor well and that the rim of the concentrate collection tube is completely inside the
well for the fixed angle rotor, or the device is completely within the 15-mL conical for
the swinging bucket rotor/adapter.
67
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White membrane
panel facing
center of rotor
Concentrate tube
should be Inside
the rotor well
Rim of tube is
abow the well
:>
a
LI
Orient dev'ce correctly in rotor
Seat device fully in *vell
9. For a fixed angle rotor, centrifuge at a maximum of 7,500 RCF for 10-60 min at 4°C, to
leave 100 [xL in the device. For a swinging bucket rotor, centrifuge at a maximum of
4,000 RCF for -60 min at 4°C, to leave 100 [xL in the device. Note: For the 5810 581OR
the maximum RCF is -3,200, therefore 60 min was requiredfor centrifngation.
10. If the sample volume is more than 2.0 mL, remove the filter assembly from the centrifuge
and transfer the Amicon® Ultra-2 Centrifugal filter device with the concentrate collection
tube on top to new filtrate collection tube. Dispose of the used filtrate collection tube with
filtrate. Transfer the remainder of the sample to the respective Amicon® Ultra-2
Centrifugal filter device and repeat steps 6 to 9.
Note: Ensure that the sample has been filtered, but the filter device is not dry. If there is
more than 0.2 mL retentate is left, spin for additional time depending upon the volume
left in the device.
11. Remove the filter assembly from the centrifuge and transfer the Amicon® Ultra-2
Centrifugal filter device with the concentrate collection tube on top to new filtrate
collection tube. Dispose of used filtrate collection tube with filtrate.
12. Open the Amicon® Ultra-2 Centrifugal filter device and carefully pipet 1.5 mL of PBS
for the first wash. Try to rinse the wall of the device while dispensing the buffer.
13. Cap/cover the Amicon® Ultra-2 Centrifugal filter device again with the respective
concentrate collection tube and centrifuge as described in step 9. Note: if a lower rpm
must be used, longer centrifngation times may be required.
14. Remove the filter assembly from the centrifuge and transfer the Amicon® Ultra-2
Centrifugal filter device with the concentrate collection tube on top and transfer to new
filtrate collection tube. Dispose of used filtrate collection tube with filtrate.
15. Repeat steps 12 and 13 for second wash.
68
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Note: While two wash steps are described, additional wash steps may be used as
described, for samples containing large quantities of potential interferences.
16. Remove the filter assembly from the centrifuge.
17. Remove the filtrate collection tube with filtrate and discard.
18. Invert the Amicon® Ultra-2 Centrifugal filter device with the concentrate collection tube
and place back in the centrifuge. Counter balance the centrifuge rotor with similar
19. Centrifuge at 1000 RCF for 2 min at 4°C to collect the sample retentate in the concentrate
collection tube.
20. Carefully, remove the Amicon® Ultra-2 Centrifugal filter device with the concentrate
collection tube from the centrifuge.
21. Separate the concentrate collection tube containing the sample retentate from the filter
device and discard the filter device.
22. Using a 1.0 mL micropipettor and respective pipet tip, transfer the sample retentate from
the concentrate collection tube to a labeled 2.0 mL screw cap tube. Cap the tube.
23. Measure the sample retentate volume and adjust the volume to 0.1 mL with PBS for
replicate sample analysis.
24. Store the processed sample at 4°C until the time-resolved fluorescence (TRF)
immunoassay analysis is initiated.
assembly.
Filtrate Concentrate
Separate device
from filtrate Cube
Inwrt device and
concentrate tube
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vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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