c/EPA
United States
Environmental Protection
Agency
tPA 600/R-10/097 | September 2010 | www.epa.gov/ord
Novel Cell-Based Assay
for Testing Active Holo-Ricin
and Its Application in Detection
Following Decontamination
Office of Research and Development
National Homeland Security Research Center

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Novel Cell-Based Assay for
Testing Active Holo-Ricin
and Its Application in Detection
Following Decontamination
VIPIN K. RASTOGI AND LALENA WALLACE
UNITED STATES ARMY - EDGEWOOD CHEMICAL
BIOLOGICAL CENTER (ECBC)
RESEARCH AND TECHNOLOGY DIRECTORATE
BIODEFENSE TEAM
ABERDEEN PROVING GROUND, EDGEWOOD, MD
21005
SHAWN P. RYAN
NATIONAL HOMELAND SECURITY
RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NC 27711
vvEPA

Office of Research and Development
National Homeland Security Research Center

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Disclaimer
The U.S. Environmental Protection Agency, through its Office of Research and Development,
funded and managed this investigation through a collaborative Interagency Agreement (DVV-21-
92224001-2) with the United States Army - Edgewood Chemical Biological Center. This document
has been subjected to the Agency's review and has been approved for publication. Note that approval
docs not signify that the contents necessarily reflect the views of the Agency.
Mention of trade names or commercial products in this document or in the methods referenced in
this document docs not constitute endorsement or recommendation for use.
If you have difficulty assessing these PDF documents, please contact Nickel.Kathv@epa.gov or
McCa 11. Ainelia@.epa.gov for assistance.

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iv

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Contents
Disclaimer	iii
Tabic of Contents	v
List of Figures	vii
List of Tables	viii
List of Acronyms	ix
1.0 Introduction	1
2.0 Background	3
2.1	Ricin Toxin	3
2.2	Ricin Detection	3
2.2.1	Antibody-based Detection	3
2.2.2	Cytotoxic MTT Viability Assay	4
2.2.3	I nhibition of In vitro Protein Synthesis	4
2.2.4	Assay-based on Engineered Cells with a Reporter Gene	5
2.3	Project Goals and Specific Objectives	5
3.0 Materials and Method	7
3.1	Cell line and Plasmid Specifications	7
3.2	Plasmid DNA Isolation	8
3.3	Transfcction and Selection of Recombinant Cell Line	8
3.4	Routine Maintenance of the Recombinant Cell Line	9
3.5	Assay Protocol and Optimization Parameters	9
3.6	Determination of Cell Line Sensitivity	10
3.7	Primary Data Acquisition and Reduction	10
3.8	Coupon Inoculation and Sample Preparation	10
3.9	Decontamination Assay	10
3.10	Ricin Limit of Detection Estimations	11
3.11	Coupon Preparation	11
V

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4.0 Quality Assurance / Quality Control	13
4.1	Performance Evaluation Audit	13
4.2	Data Quality Audit	13
4.3	QA/QC Reporting	13
4.4	Deviations from the Test/QA Plan	13
5.0 Results	15
5.1	Selection of a Recombinant HeLa Cell Line	15
5.2	Assay Optimization	15
5.2.1	Cell Concentration and Incubation Time	15
5.2.2	Sensitivity of the Recombinant Cell Line to Pure Ricin Standards	17
5.3	Decontamination Study	21
5.3.1	Decontamination Sample Preparation	21
5.3.2	Ricin Decontamination with Bleach. Hydrogen Peroxide, and
Chlorine Dioxide Solutions	22
6.0 Summary and Conclusions	23
7.0 References	25
Appendix A	27
Appendix B	29

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List of Figures
Figure 1: Mode of Action for Cytotoxicity of Ricin Toxin	4
Figure 2: Effect of Cell Number and Incubation Period on Lucifcra.sc Expression	17
Figure 3: Sensitivity of Two Cell Lines to Varying Amounts of Holo-ricin	18
Figure 4: Effect of Low Levels of Holo-ricin on Lucifcrase Expression	18
Figure 5: Optimization of Ricin Addition	19
Figure 6: Response Reproducibility of LVVVR-2 Cells to Ricin	20
Figure 7: Linear Relationship between LVVVR-2 and Ricin Amount	20

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List of Tables
Tabic 1: List of Cell Lines and Plasmids	7
Table 2: List of Supplies	8
Table 3: Clonal Screening for Lucifcrase Expression	16
Table 4: Effect of Neutralized Disinfectant on Lucifcrase Induction in Recombinant
LVVVR-2 Cell Line	21
Table 5: Decontamination of Crude Ricin with Diluted Bleach. Chlorine Dioxide,
and Hydrogen Peroxide	22

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List
of Acronyms
BSC-2
Biological Safety Cabinet. Level 2
BSL
Biological Safety Level
BW
Biological Warfare
C/BW
Chemical / Biological Warfare
CDC
Center for Disease Control
CHO
Chinese Hamster Ovary
CMV
Cytomegalovirus
COA
Certificate of Analysis
DMEM
Dulbecco's Modified Eagle's Medium
DNA
Deoxyribonucleic Acid
ECBC
Edgevvood Chemical Biological Center
ECL
Electrochemiluminscence
EDTA
Ethylenediamine Tetraaceticacid
ER
Endoplasmic Reticulum
FBS
Fetal Bovine Serum
GC-MS
Gas Chromatography Mass Spectrometry
GOI
Gene of Interest
HPLC
High-Performance Liquid Chromatography
IAG
Interagency Agreement
IOP
Internal Operating Procedure
ld50
Lethal Dose that kills 50% of a sample population
LWVR-2
Final selected double transfectant cell line
NADH
Nicotinamide Adenine Dinucleotide
MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
NHSRC
National Homeland Security Research Center
NIST
National Institute of Standards and Technology
ORD
Office of Research & Development
PCR
Polymerase Chain Reaction
R&D
Research and Development
R&T
Research and Technology Directorate
RLU
Relative Light Units
RNA
Ribonucleic Acid
RU
Ruthenium trisbipyridine chelate
SOP
Standard Operating Procedure
tTA
Tet-Off Advanced transactivator
Tct
Tetracycline
TRE
Tetracycline Responsive Element
ix

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1.0
Introduction
Since 2004, the U.S. EPA's National Homeland Security
Research Center and U.S. Army's Edgewood Chemical
Biological Center (ECBC) have been partnering in an
effort to advance the science and technology behind the
nation's ability to recover from the potential adverse
impacts related to the intentional release of chemical
or biological agents. As part of this collaboration, one
goal was to detect functional holo-ricin and to better
understand the efficacy of decontamination options
for surfaces contaminated with this toxin. This effort
required the ability to detect biologically active ricin
toxin on surfaces both before and after decontamination
application, in order to determine the effectiveness
of the treatment. While several methods of detection
approaches have been used in other studies of this nature,
each had their drawbacks in terms of their relationship to
the "gold standard" analysis (in vitro toxicity studies in
mice). However, this ultimate confirmation of ricin toxin
activity is not practical for decontamination studies.
Based upon an understanding of the state of the science
and technology related to protein toxin detection
and analysis, and the requirements for systematic
decontamination investigations, the EPA and ECBC
initiated an effort to address current decontamination-
related data gaps specifically for ricin toxin. The work
plan was jointly developed between ECBC and EPA, and
was executed by ECBC's Bio-decon Group members
of the BioDcfcn.se Branch. The overall goals of the
project were to 1) to develop a novel cell-based assay
for functional ricin; and 2) to investigate the utility
and application of the newly-developed bioassay in
confirming the absence or presence of functional toxin
in post-decontaminated samples from building interior
surfaces.
This report discusses the currently available approaches
for halo-ricin detection, documents the development
and results of the novel bioassay. and reports the
effectiveness of three decontamination methods based
upon use of this newly developed bioassay.
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2

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2.0
Background
In this section, background about ricin toxin and its
detection is presented. Additionally, the purpose and
objectives arc provided.
2.1	Ricin Toxin
Ricin is a potent protein toxin making up 1-5% by seed
weight of the castor bean plant. Ricinus communis. The
R. communis plant is cultivated in the southern United
States but also grows as a weed and an ornamental plant.
The whole plant is poisonous, containing the toxin ricin.
which reaches the highest levels in the seeds. Each fruit
contains three mottled seeds. 5-15 mm long. One to
three seeds may be fatal to a child; two to four seeds
may be poisonous to an adult, while eight may be fatal.
In laboratory mice, the LD,;i (dose of a chemical which
kills 50% of a sample population) and time to death
respectively are: 3 - 5 fxg/kg and 60 hours by inhalation.
5 nig/kg and 90 hours by intravenous injection, and 20
nig/kg and 85 hours via intra-gastric ingestion (Stripe
and Barbieri. 1986).
Ricin is a hetcrodinicric ribosonic-inactivating protein
(66 kD in si/c) with, Chain A (267 amino acids in
length. 32kD) and Chain B (267 amino acids in length,
34kD), linked via disulfide bonds. Both, Chain A and B
re glycoproteins and must be linked for the holo-ricin
to be toxic to mammalian cells. The Chain B is lectin
specific for binding two galactose sugars and can bind to
the mammalian cell surface. The mode of ricin cytotoxic
action is summarized in Figure 1. Upon binding to the
cell surface, the ricin toxin is internalized. Ricin Chain
A cn/yniatically cleaves 28S ribosonial RNA at adenine
nucleotide (A4324) near the 3' end of the polynucleotide
chain (Barbieri, 2004). This deletion results in the failure
of elongation factor-2 to bind to the ribosonie and.
tliercby. inhibits protein synthesis, resulting in cell death.
Ricin Chain A has a Km (binding affinity) of 0.1 (.uiiol/L
and an enzymatic constant of 1,500/min for ribosonics
(Franz, 1997).
2.2	Ricin Detection
Detection of ricin toxin is based on one of the four
general approaches: a) detection of surface antigen on
the protein toxin; b) general loss in mammalian cell
viability due to inhibition of protein synthesis; c) in vitro
protein synthesis inhibition of a reporter gene. Incite rase;
and d) cell-based assay, engineered with a reporter
gene. A brief description of each of these approaches is
detailed below.
2.2.1 Antibody-based Detection
Historically, one of the most common approaches for
detecting protein toxins is based on detection of antigens
using antibodies. The clcctroclicniiluniinsccncc (ECL)
based M1M Analyzer is a commercially available
instrument marketed by BioVeris/Roche. ECL uses
a sandwich immunoassay format, in which anti-ricin
antibodies (capture antibody) arc biotinylated and
prc-bound to Strcpavidin-coated paramagnetic beads.
Detection of ricin antigen is facilitated by use of a
second anti-ricin antibody labeled with ruthenium
trisbipy ridine chelate (RU) as a reporter antibody.
Presence of target antigen bridges the reporter antibody
to the capture antibody on the paramagnetic beads.
The ECL analy/cr collects the beads on the surface
of an electrode using a magnet and rids the sample of
unbound non-specific material with a washing step.
The coniplcxcd ricin antigen labeled with RU and
tripolaniine present in the reaction buffer arc activated
by oxidation at the electrode. The tripolaniine loses a
proton and becomes a powerful reducer for the RU,
resulting in excited-state RU. The excited-state RU then
returns to its ground-state after emission of a photon at
620 inn. which is detected by the analy/cr. Even though
the ECL detection limit is low (< l ng/niL), antibody-
bascd detection provides no information with regards to
whether the ricin is biologically active.

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Chain A
32k°^
Chain B
34 kDa
Ricin 60-65 kDa
two chains held
by S-S bridge
Holo-ricin, i.e. both chains
necessary for binding and
cytotoxicity. Chain B binds to the
cell surface glycoproteins and
glycolipids
Mamma nan
After attachment, the holo-ricin internalizes,
membrane vesicles transport this to ER, where
the two chains dissociate and Chain A
translocates to the cytosol
Chain A catalytically depurinates
the adenine at position 4324 of the
28S ribosomal RNA
A single chain A molecule in the cytosol can
inactivate approximately 1,500 ribosomes
per minute, leading to rapid inhibition of
protein synthesis and cell death
Ricin may also mediate other cytotoxic
effects, such as programmed cell death, i.e.
Apopotosis
Figure 1: Mode of Action for Cytotoxicity of Ricin Toxin
2.2.2 Cytotoxic MTT Viability Assay
Mammalian cell lines (monkey Vero or HEK 253)
viability and proliferation can be readily assayed
by use of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide], which is converted to
a purple fonnazan dye by cellular reductive power,
including the pyridine nucleotide cofactors, NADH/
NADPH. Production of the reductive cofactors and
MTT reduction can only be carried out in metabolically
active cells. Cessation of protein synthesis resulting by
catalytic cleavage of ribosomal RNA in the presence of
functional ricin leads to a general decay in cell viability.
Loss in cell viability is detected as loss in absorbance of
fonnazan at 550-620 mn, resulting from the reduction of
MTT by NADH/NADPH in the cell. Since generation of
NADH/NADPH is dependent on a functional respiratory
pathway and intact mitochondria, any shock or treatment
impacting this metabolic machinery is expected to give
false-positive results even in the absence of functional
ricin. Cytotoxicity to mammalian cells, therefore, is
unrelated to the specific mode of action by ricin.
2.2.3 Inhibition of In vitro Protein
Synthesis
Since ricin Chain A catalytically depurinates A4324
from 28S ribosomal RNA, a rapid loss of protein
synthesis is the mode of action for active toxin.
Luciferase gene expression in nuclease treated cell-free
rabbit reticulocyte lysate and monitoring of its activity,
provides a rapid approach for determining the presence
of active ricin toxin. A micro titer plate based assay lias
been developed (Hale, 2001) to monitor the presence of
active ricin toxin in a sample. Inhibition of luciferase
activity was observed in the presence of both, single
Chain A and holo-ricin. A semi-linear relationship
between luciferase activity was reported to be 0.001-0.15
nM and 0.15-0.6 nM (1 nM = 66 |_ig/mL). for Chain A
and holo-ricin, respectively. Due to partial or complete
loss of ricin Chain B, which affords initial binding,
the entry, and internalization of holo-ricin within the
cells; detection based on such assays is relevant to only
catalytic Chain A. Presence of active or functional holo-
ricin cannot be ascertained by such an approach.

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2.2.4 Assay-based on Engineered Cells with a
Reporter Gene
Recently. Halter et al (2009) reported development of a
mechanistic bioassay by engineering a Vera cell line with
a reporter gene, green fluorescent protein (GFP). A stable
transfectant Vera cell line expressed the reporter gene
in a constitutive manner, and responded to the presence
of ricin within 6-8 hours of contact. Quantitative
microscopy in conjunction with flow cytometry was used
to measure ricin response. This is a functional assay, but
requires sophisticated microscopy and specially trained
personnel. Furthermore, such an assay is not scalable and
has not been adapted for post-decon analy sis of residual
ricin.
2.3 Project Goals and Specific
Objectives
The purpose of this project was to advance the current
science of detection of functional ricin based on its
binding to the cell surface by functional ricin Chain
B and consequent cessation of protein sy nthesis via
cataly tic depurination of the 28S ribosonial subunit
of ribosonies by functional ricin Chain A. The overall
goal included providing guidance on the selection
of decontamination technologies by confirming the
presence of functional ricin in environmental samples.
Specific objectives were: 1) to develop a novel cell-
based assay for functional ricin; and 2) to investigate the
utility and application of the newly-developed bioassay
in confirming the absence or presence of functional toxin
in post-decontaminated samples from building interior
surfaces.
The strategy for developing the cell assay included the
use of an engineered human-derived cell line harboring
a reporter gene that yields a quantifiable and dosimetric
response in the presence of functional ricin. For this
effort, a lucifcrase gene was stably transfccted into
the cell line, and the resulting cell clone was used to
develop an optimized lucifcrase assay for the detection
of functional ricin. To avoid cy totoxic effects of
constitutive lucifcrase expression, the reporter gene was
placed under the control of a reprcssible promoter (Tet-
OIT). Ultimately, a cell line was constructed that could
be used for the detection of low levels of functional ricin.
5

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6

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3.0
Materials and Method
Development of the bioassay required setting up of a
dedicated mammalian culture laboratory. The cell lines,
plasmid containing the Incite rase gene TRE promoter,
and the culture media were procured from commercial
sources. A description of the cell lines and genetic
markers used to construct the double stable cell line
are listed in Table 1. Table 2 lists suppliers and catalog
numbers for the materials used in this project.
3.1 Cell Line and Plasmid Specifications
Tabic 1: List of Cell Lines and Plasniids
Cell Line or
Plasmid
Source
Description
HeLaTet-Off
Advanced Cell Line
Clontech
Cell line used as recipient,
produces Tet repressor
and has a G418 resistance
marker
pTRE-Tight-Luc
Plasmid + linear
hygromycin marker
Clontech
Recombinant plasmid
containing luciferase
gene under Tet responsive
element used as donor DNA
for double-transfectant
Double
Transfectant
This
Project
Stable double transfectant
resulting from
introduction of pTRE-
Tight-Luc plasmid into
HeLa Tet-Off cells
CHO-AA8-Luc Cell
Line
Clontech
Positive control cell line,
derived from Chinese
Hamster ovary, stably
transfected with luciferase
gene under control of Tet-
Off system
The HeLa cells used to construct the double-stable cell
line were derived from a human cervical epitheloid
carcinoma and arc commonly used for bioassays. The
HeLa Tet-OIT cells used for this project have been
transfccted with the tctracyclinc-controlled transactivator
gene, producing a tctR-fusion repressor protein. This
cell line has been well characterized by the commercial
supplier, Clontech (Mountain View, CA). Clontech
has determined the doubling time for these cells to
be approximately 20 hours during log phase. The cell
morphology is reported to be adherent and elongated
with 2 or 3 filopodia. The cells were maintained in
complete media containing 90% Dulbecco's Modified
Eagle's Medium (DMEM), supplemented with 10%
Tet System approved Fetal Bovine Serum (FBS), 4 mM
L-glutamine. 100 ng/mL G4IX. 100 units/mL penicillin
G sodium and 100 (.ig/mL streptomycin sulfate. Trypsin-
EDTAand phosphate buffered saline (PBS) were used
for routine cell passage.
The positive control cell line used for this project
was CHO-AAX-Luc. It was maintained in 90% Eagle
Minimum Essential Medium supplemented with 10%
FBS. 4 mM L-glutamine. 100 (.ig/mL G41X. 100 units/
iuL penicillin G sodium, 100 (.ig/mL streptomycin
sulfate, and 100 ng/mL hygromycin B. This cell line is
very similar to the stable double transfectant developed
in this project. However, this cell line was derived from
a Chinese hamster ovarian (CHO) cell line. Although it
expresses high levels of luciferase upon induction, it is
not a human derived cell line. Consequently, sensitivity
of this cell line to ricin will likely differ from that of the
HeLa cell line. Data presented in the Results section
(Section 5) of this report suggest that these cells arc less
sensitive to ricin toxicity .

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Tabic 2: List of Supplies
3.2 Plasmid DNA Isolation
8
Reagent
Supplier
Catalog
number
HeLa Tet-Off Advanced Cell
Line which includes the CHO
AA8-Luc Tet-Off Control Cell
Line
Clontech
632111
Tet-Off Advanced Inducible
Gene Expression System which
includes the pTRE-Tight-Luc
plasmid
Clontech
630934
Tet System Approved Fetal
Bovine Serum
Clontech
631101
Qiagen midi-prep kit
Qiagen
12643
BamHI restriction endonuclease
Invitrogen
15201023
Nhe I restriction endonuclease
Invitrogen
15444011
Xba I restriction endonuclease
Invitrogen
15226012
2% agarose E gel
Invitrogen
G5018-02
CLONfectin Transfection
Reagent
Clontech
631301
Hygromycin B
Clontech
631309
Doxycycline
Clontech
631311
G418
Clontech
631307
ONE-Glo™ Luciferase Assay
Promega
E6110
D-PBS
Invitrogen
14040141
L-glutamine
Invitrogen
35050
Dulbecco's Minimum Essential
Medium (DMEM)
Invitrogen
11995-065
Eagle Minimum Essential
Medium (EMEM)
ATCC
30-2003
Trypsin-EDTA
Invitrogen
25200-106
Penicillin Streptomycin
Invitrogen
15140-155
Trypan Blue Stain
Invitrogen
15250-061
Cell Freezing Medium-DMSO
Sigma-Aldrich
C6295
Pure ricin
Vector Labs
L-1090
Buffered Peptone Water
Becton
Dickinson
212367
Tween 80
Sigma-Aldrich
P1754
In order to propagate the plasmid used for transfection.
pTRE-Tight-Luc. chemically competent Escherichia
coli DH5a cells were used for heat-shock transformation
of the plasmid and subsequent growth. The plasmid
was then purified using a Qiagen midi-prep kit. The
plasmid prep was quantified and a quality assessment
was conducted using spectrophotometric readings at
wavelengths 260 and 280 nm. Further confirmation
was performed on the plasmid prep by restriction
mapping. The clone of interest was subjected to an
enzymatic double digestion with BaniH l/Nlie I, and
a single digestion of Xba I. The expected sizes of
digested fragments were verified following separation by
electrophoresis on a 2% agarose/tris-acetic acid-EDTA
gel and visualization under UV light following staining
with ethidiuni bromide (data not shown).
3.3 Transfection and Selection of
Recombinant Cell Line
A transfection procedure was used to develop a double-
stable Tet-OIT cell line expressing the lucifcra.se gene.
The liposomal reagent Clonfectin was used to transfect
the pTRE-Tight-Luc vector into the HeLa Tet-OIT
cells. This procedure results in stable transfectants
when plasmid DNA inserts randomly into the host
chromosome. Lucifcrase expression is dependent on
both the number of copies inserted and the insertion
location. To obtain stable transfectants. pTRE-Luc
plasmid DNA was cotransfected with a hygroniycin
linear selection marker. Clones that grew on media
containing hygromycin were then screened for lucifcrase
expression to determine if the lucifcrase gene had been
stably inserted as well. This strategy required screening
many hygroniycin-rcsistant colonics, as some were not
transfccted with both genetic elements, the selection
marker and the plasmid.
For the transfection procedure, HeLa cells were seeded
in a 6-well plate at a density of approximately 2 x 105
cells/well. The cells were grown for 18-24 hours to 60-
90% conllucncy. The pTRE-Tight-Luc plasmid was then
cotransfected with the linear hygroniycin marker. The
Clonfectin reagent was added to the plasmid DNA, and
then added to the cells, following the vendor's protocol.
After a four hour incubation period, the transfection
cocktail was removed, cells were washed, and fresh
complete media was added. The transfccted cells were
incubated at 37°C for 24 h prior to hygroniycin selection.
Complete media containing 200 (.ig/niL hygroniycin B
was used for selection of clones successfully transfccted
with both plasmids. Hygroniycin-rcsistant clones were
analyzed microscopically and the location of viable

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clone clusters was marked on the dish, and removed
using cloning cylinders. The media was removed from
the dish, the cloning cylinder was placed on the clone,
and trypsin was added to the cloning cylinder to remove
the cells. The cells were pipetted out of the cylinder
and placed in a 24-well dish with fresh media. Isolated
clones were moved to larger growth vessels as necessary.
All clones that survived the selection process and
grew to sufficient numbers were grown in the absence
or presence of 100 iig/niL doxycycline. resulting in
induction or repression of lucifcra.sc. respectively. Either
doxycycline or tetracycline can be used for suppression
of lucifcra.sc induction, as the system responds equally
well to both. However, doxycycline was used instead of
tetracycline for maintenance of the stable cell line, since
it has a much longer half-life. Induction of lucifcra.sc
activity was measured using a standard lucifcra.sc assay.
Initially, lucifcra.sc was assayed following incubation
for up to 72 h in the presence or absence of doxycycline.
Induction of enzyme expression was assayed by
adding lucifcra.sc assay reagent to the cells, which
results in complete lysis of the cells and generation
of a luminescent signal, which was measured using a
luminomcter (Promega GLOMAX 96 well luminomcter.
Pro mega. Madison, WI). Clones expressing the highest
levels of lucifcra.sc upon induction, and the lowest level
of background expression under uninduccd conditions
(resulting in a high signal to noise ratio), were selected
and further passaged. All selected clones were expanded
to multiple flasks, and then two flasks were used to make
fro/en cell stocks. Cells were fro/en in freezing media
using the vendor recommended protocol.
3.4 Routine Maintenance of the
Recombinant CeSS Line
Clonal cell lines were split in the presence of
doxycycline (uninduccd) twice a week to maintain the
desired cell conllucncy needed for healthy cell growth.
The typical split ratio (how much the cell culture was
diluted) was 1:5, but varied depending on the si/c of
the culture vessel and the age of the culture. Cells were
washed with sterile Dulbecco's phosphate buffer saline
solution (D-PBS). before detaching the cells from the
llask surface using trypsin-EDTA solution. Once cells
detached from the vessel surface, the trypsin-EDTA was
neutrali/cd with complete DMEM media containing
doxycycline. All but 20 % of the suspended cell volume
was discarded, and fresh complete DMEM media
containing doxycycline was added to the remaining cells.
For inducing the lucifcra.se gene expression, the cells
were trypsini/ed from the lla.sk and collected by
ccntrifugation at 100 x g for 5 minutes in a 50 niL
conical tube. The supernatant was removed and the
cells were washed by rcsuspension in D-PBS. and then
collected again by ccntrifugation. This wash procedure
was repeated a total of three times in complete DMEM
media lacking doxycycline. Cell titer was estimated by
mixing an aliquot of cells with trypan blue in a 1:10
ratio. Cells were enumerated in a 10 j.iL aliquot using a
hemacytometer. Based on the cell count, the cells were
then diluted to a concentration of 105 cclls/niL using
fresh complete DMEM without doxycycline, and a 100
fxL aliquot of cell suspension was seeded in each well of
a micro titer plate.
3.5 Assay Protocol and Optimization
Parameters
The general protocol for the lucifcra.sc assay was as
follows (flow chart is provided in Appendix A):
1.	Cells were seeded in a volume of 100 fiL/well in
a 96-well micro titer plate at an optimal density
(lOMOVwell). The optimal density was determined
during the assay optimization and was based on
incubation requirements and expression levels.
2.	Ricin toxin samples or dccon samples in a volume
of 25 fxL/well were added to the cells and incubated
for a predetermined amount of time. Time of ricin
addition after cell seeding was determined during
the assay optimization and was based on the time
required for cells to adhere to the plate surface,
and time required for toxin entry into the cell and
subsequent detection by inhibition in lucifcrase
expression. In addition, preliminary experiments
were conducted to determine the extent of dilution
of post-dccon samples necessary for eliminating
toxic effects of the decontamination by-product! s) or
residual decontamination agents.
3.	Cells were lyscd by adding an equal volume (125
fiL) of Promega ONE-Glo™ reagent (ONE-Glo™
Lucifcrase Assay E6110, Promega. Madison. WI).
4.	Light output was then measured within five minutes
on the Promega Gloniax luminomcter.
5.	Typically, each sample was taken through eight-
point 1:2 dilutions, i.e., 1/2, 1/4, 1/8, 1/16, 1/32,
1/64, 1/128, and 1/256. These dilutions were
necessary, as ricin concentration must be >0.02 ng/
niL and <1.5 iig/niL in order for the response to be
in the linear part of the relationship.
As stated. Steps 1 and 2 both required optimization prior
to the analy sis of decontaminated samples. Controls used
for assay development and optimization included both
induced (- dox) and uninduccd (+ dox) cells. Induced cell
controls were included to determine the highest signal
9

-------
levels resulting from lucifcra.se expression. Uninduced
cell controls were used to determine background signal
levels. These control values were used for data analysis.
In order to determine the optimal cell concentration
per well and the appropriate induction time, two cell
concentrations were tested in a time course experiment.
Wells of a 96-well plate were seeded with either 104 or
105 total cells per well. Three assay plates were set up
in order to assess lucifcrase induction levels at three
distinct time points. 24, 48 or 72 hours.
3.6	Determination of CeSS Line
Sensitivity
Following the determination of cell concentration and
assay incubation time, preliminary experiments were
performed to determine the sensitivity of the stably
transfccted cell line to functional ricin exposure relative
to a similar CHO cell line. In initial experiments, pure
ricin procured from Vector Labs was used to prepare
an eight-point 1:5 dilution series, i.e., 0.0256 ng/niL to
2000 ng/niL. Ricin dilutions were prepared in DM EM
media (lacking serum and all additives). However, in
subsequent experiments, eight-point 1:2 dilutions were
made between 0.0256 ng/niL and 40 ng/niL. A typical
assay involved seeding of a 100 fiL aliquot containing
~104 of washed cells, and 25 fiL of dilute ricin. The final
selected double transfectant cell line (LVVVR-2), and
CHO cell lines were seeded in triplicate in a 96-well
plate. In the initial set of experiments, ricin was added
after 24 hours of cell seeding. The cells were lysed and
assayed for lucifcrase activity after 24 hours of seeding.
In subsequent experiments, ricin was added at time zero
(the time of cell seeding).
3.7	Primary Data Acquisition and
Reduction
Luminescence intensity was measured on a Pro mega
GloMax luniinonictcr and expressed as Relative Light
Units (RLU). Output data from the luniinonictcr was
exported in Excel file format. Reduction of data
included subtraction of background (as determined from
uninduced control cells) from each data point. Duplicate
and triplicate data points were averaged, and inhibition
was calculated as a percentage of lucifcrase expression
of fully induced cells.
3.8	Coupon Inoculation and Sample
Preparation
Baseline experiments were performed to determine
ricin recovery efficiency from coupons and appropriate
dilution for neutralized disinfectant samples. Recovery
efficiency was compared using three extraction solutions.
buffered peptone water (BPW), sterile distilled water,
and sterile distilled water containing 0.05% Tween
80. No significant difference in toxin recovery was
observed (data not shown), and therefore, water was
used as the extraction solution. In order to determine
recovery efficiency, coupons were spotted with 25 fxL
of pure or crude ricin (see Section 4.2.3) and allowed to
dry overnight in a bio-safety level 2 cabinet (BSC-2).
Inoculated coupons were dropped in 10 niL extraction
solution and vortcxed for 2 minutes. Serial dilutions of
extracted samples were made in DMEM, then 25 fxL of
diluted samples were added to wells in a 96-well plate
containing 104 seeded cells per well. The appropriate
dilution necessary to avoid cell toxicity from neutralized
disinfectant samples was also determined. This was
done by dropping ricin inoculated coupons in 10 niL of
neutrali/ed disinfectant (see Section 3.9) and vortexing
each sample for 2 minutes. Both an undiluted and 1:50
diluted aliquot from each sample were evaluated as
described for recovery experiments. The appropriate
dilution was chosen based on the dilution level at which
no toxic effects were observed as measured by inhibition
of lucifcrase expression.
3.9 Decontamination Assay
The optimized bioassay was used to determine the
efficacy of decontamination of coupons inoculated with
ricin. Three commercial disinfectants were tested for
their ricin decontamination efficacy: 1:20 diluted bleach
(Clorox* Germicidal Bleach, Clorox); 1% hy drogen
peroxide (35% w/v from Aldrich); and 250 ppni chlorine
dioxide (prepared as below). Hy drochloric Acid (6 N,
Aldrich; 1.1 niL), bleach (Clorox* Germicidal Bleac,
-6%, Clorox; 2.1 niL), Sodium Chlorite (25%, Sabre
Technical Services. LLC. Albany. NY; 2.5 niL), and
deioni/ed water (494 niL) were mixed to prepare a
500 ppni solution of aqueous chlorine dioxide (CD).
All dccontaniinant solutions were diluted using sterile
distilled water.
Steel coupons (lxl cm in size, cut from a filing cabinet
from HON, HON 370 series-4, procured from Office
Depot) were spotted with 25 fxL crude ricin (see Section
4.2.3) containing 35 nig/niL total protein (equivalent
to 0.2 nig pure ricin) and allowed to dry overnight in
a bio-safety level 2 cabinet. Inoculated coupons were
then dropped in 7.5 niL of 1% hydrogen peroxide. 1:20
diluted bleach, or 250 ppni chlorine dioxide. After 30
seconds. 2.5 niL of 2 M sodium thiosulfate was added
as a ncutralizcr. A control for each disinfectant was
conducted by adding 25 fxL of crude ricin extract to 10
niL of neutrali/ed disinfectant. Neutrali/ed samples
were vortcxed for 2 minutes to extract ricin from the

-------
steel coupon. The extracted ricin samples were serially
diluted in cell media, and 25 fxL of diluted samples were
added to a 96-well plate containing 104 cells (in 100 fiL
volume) per well. Plates were incubated for 24 hours at
37°C, and then developed by adding 125 fxL of lucifera.se
reagent. The contents of each well were mixed three
times using a multichannel pipcttor. Plates were assayed
for luminescence immediately after mixing.
3.10	Ricin Limit of Detection
Estimations
The ricin working stock solutions were made using pure
Vector stock. The working stock dilution series were
prepared each time just prior to assay, ranging from
0-80 ng/niL. For the bioassay, an aliquot of 100 jiL of
cells and 25 jiL were mixed per well. Therefore, a total
of 125 fiL volume in each well contained between 0-2
ng ricin. The ricin concentrations presented in this study
were estimated as ng/mL in each well after multiplying
by eight, i.e., 0-16 ng/mL.
3.11	Coupon Preparation
Initially, three coupon types, filing cabinet stainless
steel (from HON, HON 370 scries-4. procured from
Office Depot), keyboard plastic (DataCal. Lexan FR 700
film), and white Formica laminate - grade 10 (Stock
number 949, matte finish - 5'x8', procured from Wurth
Wood Group) were included in the study. All materials
were cut to 2 cm x 2 cm. The coupons were washed
with sterile water, rinsed with 70% ethanol, and dried
overnight. In the baseline studies, ricin was found to
be decontaminated in less than 1 minute with 250 ppm
bleach. The coupon material was found to play no
role in the decontamination kinetics. Hence, only steel
coupons were selected for use in this effort. The steel
coupons were stcrili/cd by autoclaving before use.
11

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12

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4.0
Quality Assurance / Quality Control
Quality assurance/quality control (QC) procedures
were performed in accordance with the QA/test plan.
Quality assurance/quality control procedures arc listed in
Appendix B.
4.1	Performance Evaluation Audit
Sub-system performance evaluation audits were
performed during the study. These included cell
maintenance, cell sub-culture, ricin standard curve,
and decon assay development. No major findings were
found and corrective steps were taken to address the
observations.
4.2	Data Quality Audit
Nearly 100% of the data acquired during the
investigation was audited by the ECBC QA Manager
or a designee. An ECBC Q A auditor traced the data
from the initial acquisition, through reduction and
analysis, to final reporting to ensure the integrity of the
reported results. All calculations performed on the data
undergoing the audit were checked.
4.3	QA/QC Reporting
Each assessment and audit was documented in
accordance with the test/QA plan. For these tests, no
significant findings were noted in any assessment or
audit, and no follow-up corrective action was necessary.
QA/QC procedures were performed in accordance with
the test/QA plan (see Appendix B).
4.4	Deviations from the Test/QA Plan
Deviations from the test/QA plan included a few minor
changes: a) revising the ricin standard curve range from
1 ng/niL - 66 ng/niL to 0.02 - 2 ng/mL; b) choosing
only one coupon type, i.e., steel; and c) the addition of
ricin at the time of cell seeding. These deviations were
made because of the sensitive nature of the bioassay.
rapid decontamination of ricin toxin by disinfectants,
and minimal to no effect of coupon material on ricin
decontamination, respectively.
13

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14

-------
5.0
Results
In this section, the results of the development and
optimization of the novel ricin toxin bioassay arc
presented in detail. Additionally, the results of a study
using the newly developed bioassay for pre- and post-
decontamination ricin toxin detection arc documented.
These results arc meant to the show the potential utility
of the bioassay and present comparative results for three
surface decontamination chemicals.
5.1	Selection of a Recombinant HeLa
Cell Line
Initially, a total of 33 clones were selected on the
basis of their growth in the presence of hygroniycin.
However, eight of these clones did not grow well, and
consequently, were not carried further. The remaining
25 clones were screened for induction of lucifcra.se
expression in the absence of doxycycline. In addition,
each clone was assessed for any background expression
present under conditions that suppress expression. As a
positive control, a CHO cell line stably transfcctcd with
the lucifcrase gene under control of the tctracyclinc-
rcsponsivc element was also included. The results arc
summarized below in Table 3. As seen in the table,
many of the clones show only l to 2 fold induction of
lucifcrase activity. The highest level of expression, with
the lowest background, was seen with Clone 1-2, which
showed 1 (K)O-fold induction. Clone 1 -2, henceforth,
referred to as LVVVR-2, was selected based on growth
rate, high expression of induced lucifcrase, and low
background in the uninduccd state. This clone was used
for optimization of the ricin bioassay.
5.2	Assay Optimization
Several parameters were evaluated in order to optimize
the ricin bioassay. These parameters included general
issues associated with the assay setup such as cell
concentration, incubation period, time of addition for
ricin, ricin standard curve concentration and preparation,
and processing of decontamination samples. Initial
optimization experiments also included a comparison
with a CHO control cell line.
5.2.1 Cell Concentration and Incubation Time
In order to determine the optimal cell number for the
assay and the optimal time to incubate between cell
seeding and assay development, cells were seeded at
two concentrations. 104 or 105 per well, and the cells
were allowed to grow for 24, 48, or 72 hours before the
plates were developed. Both, clone LVVVR-2 and the
CHO control cell line were included in this scries of
experiments. The results arc summarized in Figure 2.
As seen in Figure 2, the highest levels of lucifcrase
induction for LVVVR-2 were seen for 104 cells at 48 and
72 hours. However, sufficient levels of induction were
seen as early as 24 hours. The fold induction levels at 24,
48, and 72 hours for 104 cells ranged from 800 to 2300
RLU. Between 0 hours and 48 hours, the LVVVR-2 cells
seeded at an initial concentration of 104 per well showed
a marked increase in lucifcrase production, suggesting
that this cell concentration results in liealthy cell activity,
and hence, is the most optimal cell density for this assay.
Although the levels for lucifcrase induction were higher
for 105 cells at 24 hours, the general trend in this time-
course experiment at this cell density showed a decrease
in lucifcrase expression over time. This suggests that
seeding at the higher density results in overcrowding
of cells which a fleets cell viability and transcription
activity. Based on these results, a 24 hour incubation and
10" cell number/well for LVVVR-2 were determined to
be optimal and selected as experimental conditions for
subsequent work.

-------
Tabic 3: Clonal Screening for Luciferase Expression
Clone #
Uninduced
Induced
Fold Induction
Average RLIJ
SI)
Average RLIJ
SI)
1-1
32
5
56
4
2
1-2
42
5
43434
10239
1040
1-3
36
6
38
11
1
1-4
35
6
37
3
1
1-5
26
3
28
6
1
1-6
29
7
75
102
3
1-7
17
4
30
9
2
2-1
36
5
92
10
3
2-2
31
3
525
464
17
2-3
42
6
33
5
1
2-5
25
4
23
3
1
2-6
32
3
16
5
0
2-7
44
44
23
5
1
2-8
93
132
24
7
0
2-9
23
2
21
5
1
3-1
95
21
6644
492
70
3-3
28
6
168
52
6
3-4
31
5
53
10
2
3-5
210
41
16149
899
77
4-1
34
9
337
47
10
4-2
28
8
6182
1227
221
4-3
30
3
113
19
4
4-5
29
6
45
31
2
4-6
39
18
35
8
1
4-7
29
8
42
18
1

-------
3500
3000
2500
2000
1500
1000
500
0
24 hours
48 hours
72 hours
Time
Figure 2: Effect of Cell Number and Incubation Period on Luciferase Expression (Luciferase induction in
Clone 1-2 (1-2) and CHO Control Cells (CHO) at 104 (10A4) and 105 (10A5) cells per well seeding levels.)
5.2.2 Sensitivity of the Recombinant Cell Line to
Pure Ricin Standards
Sensitivity of both cell types (LWVR-2 and CHO) to
various amounts of ricin is summarized graphically
in Figure 3 and Figure 4. As seen in Figure 3, the
recombinant LWVR-2 cell line is significantly more
sensitive to functional ricin than the control CHO cell
line. In addition, the LWVR-2 cell line demonstrated a
50% inhibition in luciferase activity in the presence of
~3 ng/mL. The limit of holo-ricin detection by LWVR-2
is 0.6-0.8 ng/mL. In comparison, the CHO cell line
showed a similar level of inhibition in the presence of
between 40-50 ng/mL. The same data are re-plotted to
indicate the inhibitory response in the presence of 0 - 16
ng/ml of holo-ricin (Figure 4).
Optimization of the bioassay included determining
the optimal time, following cell seeding, to add ricin
samples. Initially, ricin samples were added 24 hours
after the cells were seeded (Figures 3 and 4). However,
as seen in Figure 5, it was shown that the addition of
ricin at the time of cell seeding lias no effect on assay
sensitivity.
17

-------
	ree-














-~-CHO LWVR-2


	1	1	9-

0.001	0.010	0.100	1.000	10.000	100.000	1000.000
Ricin (ng/mL)
Figure 3: Sensitivity of Two Cell Lines to Varying Amounts of Holo-ricin
100
80
CHO
LWVR-2
60
40
20
0
0
2
4
6
8
10
12
14
16
Ricin (ng/mL)
Figure 4: Effect of Low Levels of Holo-ricin on Luciferase Expression
18

-------
	W0-n
•

~ HeLa (LWVR-2)
¦ CHO

¦





~


¦


~ "0



¦	,	¦	¦ 	0

o.ooi	o.oi	o.i	1	10	ioo
Ricin ng /mL
Figure 5: Optimization of Ricin Addition (Effect of ricin addition at zero time on luciferase
inhibition in recombinant HeLa and CHO cell lines.)
The reproducibility of ricin cytotoxicity to recombinant
luciferase HeLa cells was evaluated over a two month
period with three independent experiments. The change
in luciferase expression in HeLa cells after 24 hours
incubation with ricin concentrations from 0 to 8 ng/
mL is shown in Figure 6. The relative luminescence
at 0 ng/mL ricin concentration was used to normalize
the luminescence values in Figure 6. The average
standard deviation between the three experiments
for each data point was 0.04. The cellular luciferase
activity in LWVR-2 cells significantly decreased at ricin
concentrations >1.6 ng/mL. The linear response of HeLa
cells to ricin cytotoxicity is shown in Figure 7, which
shows that luciferase expression is almost completely
inhibited in the presence of >8 ng/mL of ricin. The
correlation coefficient of the linear portion of the
cytotoxicity curve is 0.91, indicating an adequately linear
relationship between ricin concentration and normalized
cellular luminescence. Cellular luciferase activity
becomes significantly nonlinear at ricin concentrations
greater than >8 ng/mL.
19

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1.2
1.0
w 0.8
c
QJ
4-»
£ 0.6
a;
N
"I 0.4
I 0.2
0.0
A
_±_
< 24-Sep ¦ 14-Oct 24-Nov
0
2	4	6
Ricin ng/mL
Figure 6: Response Reproducibility of LWVR-2 Cells to Ricin
4-
8
10
1.2
1.0
0.8
y= -0.5364X +0.9248
R2 = 0.9174
0.6
0.4
0.2
0.0
0
0.5
1
1.5
2
Ricin ng/mL
Figure 7: Linear Relationship between LWVR-2 and Ricin Amount
20

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5.3 Decontamination Study
The novel bioassay developed and optimized as
discussed in Sections 5.1 and 5.2 was used in a surface
decontamination study. The results of this study arc
presented in the sub-sections below.
5.3.1 Decontamination Sample Preparation
For the decontamination assays, crude ricin was obtained
from ECBC's BioPhysical Chemistry Group. Based
on the protein estimation, the crude ricin sample was
0.5% ricin (0.2-mg ricin and 35-mg total protein per
niL). Based on lucifcra.sc activity measurements of the
crude ricin extract (data not shown) and the linear ricin
response curve generated with known quantities of ricin
(Figure 7), it was estimated that the crude ricin stock
contained approximately 0.56% active ricin.
Prior to analyzing disinfected coupons inoculated with
crude ricin in the optimized bioassay, experiments
were run to determine if neutrali/cd disinfectants have
any toxic effect on the cells and if samples could be
diluted to abate this effect. Table 4 shows that all three
neutral i/cd disinfectants at zero dilution resulted in full
inhibition of luciferase activity. A likely explanation for
this is the high salt concentration which likely results
in hypertonic conditions for the cells, leads to loss of
viability after 24 hour incubation in the neutralized
disinfectant solution. A 1:50 dilution of the neutrali/cd
disinfectants in cell media alleviated the osmotic effects
of the salts on the cells and the cells remained viable
after 24 hours incubation.
Tabic 4: Effect of Neutralized Disinfectant on Luciferase Induction in Recombinant LWVR-2 Cell Line
(Percent inhibition of luciferase activity at zero and 1/50 dilution in cell media.)
Dilution
Control
Chlorine Dioxide
250-ppm
Bleach
1:20 dil.
Hydrogen Peroxide
1%
0
0
100
100
100
1/50
0
0
0
0

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5.3.2 Ricin Decontamination with Bleach,
Hydrogen Peroxide, and Chlorine Dioxide
Solutions
Following thorough optimization of the bioassay, three
disinfectant solutions were tested for their ability to
decontaminate coupons inoculated with crude ricin
samples. The results of the disinfection experiments arc
shown in Table 5. For neutrali/cd disinfectant controls,
crude ricin extract had >92% Incite rase inhibition.
The neutralized solution did not adversely a fleet ricin
toxicity to He La cells. Greater than 80% active ricin
was recovered from the positive control coupons which
were not exposed to disinfectant. The results indicate
that 30 second contact with a 1:20 diluted bleach or 250
ppin chlorine dioxide results in complete inactivation of
ricin toxic effects to HeLa cells. Conversely, a 30 second
contact time with 1% hydrogen peroxide had partial
effect on the ricin toxicity to the cells. This ricin sample
resulted in 62% inhibition of lucifcra.se expression,
indicating that active ricin was still present following
exposure to 1% hydrogen peroxide.
Tabic 5: Decontamination of Crude Ricin with Diluted Bleach, Chlorine Dioxide, and Hydrogen Peroxide
Test Decon Solution
Ricin Recovery (%)
Positive Coupons
Estimated Ricin
Remaining (ng) —
Test Coupons"
Comments
1:20 Diluted bleach solution
80
0
No pH adjustment3
250 ppm Chlorine Dioxide Solution
97
0
No ricin detected
(LOD = <0.2 ng)
1% hydrogen peroxide solution
94
1900
Partial Decon
a.	Average recovered from three coupons.
b.	Average recovered from three coupons. Active ricin remaining after 30 second contact time with disinfectants. Each coupon was con-
taminated with a total 35 mg total protein containing 5 \xg active ricin and exposed to 30 seconds of contact time with each of the listed
disinfectants.

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6.0
Summary and Conclusions
The purpose of this project was to develop a sensitive
and activity based cell-based assay for the detection of
functional ricin following decontamination of building
interior surfaces. The strategy for developing the cell
assay included the stable transfection of a reporter
gene, lucifcrase. into an engineered HcLa cell line. The
stable transfection of the cell line and the subsequent
optimization of the bioassav have successfully been
achieved. The basis for the newly developed assay was
inhibition in the expression of a reporter gene, lucifcrase.
in the presence of functional ricin. Even though, in this
report only testing with ricin has been discussed, this
assay could be used to detect the presence of any toxin or
chemical affecting gene expression. Further work needs
to be performed to substantiate this claim.
A total of 33 stably transfcctcd clones were screened
for inducible expression of lucifcrase with low basal
expression. Clone LVVVR-2 was selected for further
optimization based on these criteria. This clone was used
for optimization experiments to determine optimal cell
seeding concentration, assay incubation time, time of
ricin addition, and decon sample procedures. In addition,
the engineered cell line was determined to be highly
stable, as assessed by no change in expression level over
a six month period.
The bioassav is highly sensitive; it can detect as low as
0.6 - 0.8 ng/niL of active ricin. The assay is easy to set
up and requires two steps: a) two hours for preparation
of cells inducing lucifcrase (by removal of doxycyclinc)
and toxin addition and 18-24 hour incubation; and b)
15 mill for cell lysis and light output readout. For high-
throughput analysis, the assay can be easily automated
while maintaining high precision by using a robotics
system to process multiple plates. The inhibition in
lucifcrase expression is closely related to the presence
of low amounts (<1 ng/niL) of holo-ricin. The limit of
detection (LOD) could be improved by use of fewer cells
(lOVwell in current assay). Assuming a certain number
of ricin molecules arc needed for saturating 10,000 cells
used per well, the amount of ricin molecules required
for saturating l/5ft or 1/10"1 number of cells would be
significantly less. It is, therefore, expected tliat a lower
LOD would result from use of a lower cell density
Following optimization, the efficacy of three
disinfectants in ricin decontamination was determined.
A number of parameters had to be optimized before
a decon protocol was developed, such as complete
neutralization of the active moiety within the decon
solution, 1:50 dilution of the ncutrali/ed decon solution,
and 104cells/well. The data show that two of the three
disinfectants successfully decontaminated ricin-
contaniinatcd steel coupons within 30 seconds. Based
on these results, we conclude that this assay can be used
for the detection of low residual active ricin remaining
on surfaces follow ing decontamination. It will also be
of interest to extend this assay to other building interior
surfaces.

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24

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7.0
References
Barbieri L., et al., Enzymatic activity of toxic and non-
toxic type 2 ribosomc-inactivating proteins. FEBSLet-
ters 2004, 563: 219-222.
DeWet J R.. et al., Firefly Lucifcrase Gene: Structure and
Expression in Mammalian Cells. Mol. Cell. Biol. 1987,
7: 725-737.
Franz D R., et al., Clinical recognition and management
of patients exposed to biological warfare agents. JAMA
1997, 278: 399-411.
Gritilth G.D., et al., Examination of the toxicity of sev-
eral protein toxins of plant origin using bovine pulmo-
nary endothelial cell. Toxicology 1994, 90: 11-27.
Hale M L., Micro liter-Based Assay for Evaluating the
Biological Activity of Ribosomc-inactivating Proteins.
Pharmacology & Toxicology 2001, 88- 255-260.
Halter M„ et al., A Mechanistically Relevant Cytotoxic-
ity Assay Based on the Detection of Cellular GFP. Assay
Drug Devel Tech 2009, 7: 356-65.
Heisler I., et al., A Colorimetric Assay for the Quantita-
tion of Free Adenine Applied to Determine the Enzy-
matic Activity of Ribosomc-inactivating Proteins. Anal.
Biochem. 2002, 302: 114-122.
Lord J.M., et al, Ricin: structure, mode of action, and
some current applications. EASES 1994, 8: 201-208.
Stripe F., Ribosomc-inactivating proteins. Toxicon 2004,
44: 371-383.
Stripe F. and Luigi L., Ribosome-inactivating proteins up
to date. FEBS Letter 1986, 195: 1-8
Zhao L. & Haslam D.B., A quantitative and highly
sensitive lucifcrasc-based assay for bacterial toxins that
inhibit protein synthesis. J. Med. Microbiol. 2005, 54:
1023-103.
25

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26

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Appendix A
RICIN DECON FLOW CHART
25 rr1_ Crude ricin (0.5%; 875 mg total protein or 5 mg ricin) spotted onto coupon
surface and dried over-night in bio-safety level 2 cabinet at Room Temperature
y
I	1
Add 7.5 mL of decon solution	OR 10 ml_ of neutralized decon solution
to test coupons in a 50 mL tube,	to control coupons in a 50 mL tube
and add 2.5 ml of 2M Na2S204
I		I
V
Ricin extracted by vortexing for 2 min and the extracted samples diluted 1:50
4
Performed an eight-series 1:2 serial dilutions and tested 25 rrL aliquot in a micro-titer
plate well containing 100 rrL of induced LWVR-2 cells (-doxycycline)
4
Perform luciferase assay after 24 hours, and read luminescence intensity using a
Promega GloMax luminometer

-------
28

-------
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SEPA
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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