September 2006
Environmental Technology
Verification Report
BlOVERIS
BlOVERIFY™ BOTULINUM TOXIN A AND
RICIN TEST KITS AND
M-SERIES® M1M ANALYZER
Prepared by
Batteiie
Tlic Business o/ Innovation
Under a cooperative agreement with
U.S. Environmental Protection Agency
ET1/ET1/ET1/
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THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM A
v
nEPA Balteiie
' " The Business of Innovation
U.S. Environmental Protection Agency
ETV Joint Verification Statement
TECHNOLOGY TYPE: IMMUNOASSAY TEST KITS
APPLICATION: DETECTING BOTULINUM TOXIN A AND RICIN
TECHNOLOGY NAME: Bio Verify™ Botulinum Toxin A and Ricin Test Kits and
M-SERIES® M1M Analyzer
COMPANY: BioVeris Corporation
ADDRESS: 16020 Industrial Drive PHONE: 800-336-4436
Gaithersburg, Maryland 20877 FAX: 301-230-0158
WEB SITE: www.bioveris.com
E-MAIL: bvcorp@bioveris.com
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology Verification (ETV)
Program to facilitate the deployment of innovative or improved environmental technologies through performance
verification and dissemination of information. The goal of the ETV Program is to further environmental protection
by accelerating the acceptance and use of improved and cost-effective technologies. ETV seeks to achieve this goal
by providing high-quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies. Information and ETV
documents are available at www.epa.gov/etv.
ETV works in partnership with recognized standards and testing organizations, with stakeholder groups (consisting
of buyers, vendor organizations, and permitters), and with individual technology developers. The program evaluates
the performance of innovative technologies by developing test plans that are responsive to the needs of
stakeholders, conducting field or laboratory tests (as appropriate), collecting and analyzing data, and preparing
peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality assurance (QA) protocols
to ensure that data of known and adequate quality are generated and that the results are defensible.
The Advanced Monitoring Systems (AMS) Center, one of six technology areas under ETV, is operated by Battelle
in cooperation with EPA's National Exposure Research Laboratory. The AMS Center evaluated the performance of
immunoassay test kits used to detect botulinum toxin A and ricin in water. This verification statement provides a
summary of the test results for Bio Verify™ Botulinum Toxin A and Ricin Test Kits using the M-SERIES® M1M
analyzer.
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VERIFICATION TEST DESCRIPTION
The verification test for the Bio Verify™ Botulinum Toxin A and Ricin Test Kits using the M-SERIES® M1M
analyzer was conducted at Battelle between December 2005 and August 2006 according to procedures specified in
the Test/QA Plan for Verification oflmmunoassay Test Kits for the following parameters: contaminant
presence/absence; false positive/false negative response to interferents, drinking water (DW) matrix effects, and
cross-reactivity; consistency; lowest detectable concentration; field portability; ease of use; and sample throughput.
The ability of the BioVerify™ Botulinum Toxin A and Ricin Test Kits to detect various concentrations of
botulinum toxin A and ricin using the M-SERIES® M1M analyzer was evaluated by analyzing performance test
(PT) and DW samples. PT samples included American Society for Testing and Materials Type II deionized (DI)
water fortified with the target contaminant, an interferent, both, or only a cross-reactive species. Target analytes
were added to DI water at lethal dose concentrations as well as at several concentrations selected based on the
vendor-stated limit of detection (LOD). The effect of interferents was evaluated by analyzing two types of
interferent solutions. The first type contained both humic and fulvic acids in DI water, and the second type
contained magnesium (Mg) and calcium (Ca) in DI water. Both types of interferent solutions were prepared with
and without the addition of the contaminants at a single concentration level (10 times the vendor-stated LOD). In
addition, specificity was evaluated by exposing the BioVerify™ test kits to lipopolysaccharide, a potentially cross-
reactive compound for botulinum toxin A, and lectin from soybean, a potentially cross-reactive compound for ricin.
PT samples were analyzed in triplicate (with the exception of DI water fortified with target analytes at five times
the vendor-stated LOD, for which ten replicates were analyzed). DW samples were collected from four water
utilities that use a variety of treatment methods. DW samples, both unconcentrated and concentrated by a factor of
400, were analyzed in triplicate with and without the addition of botulinum toxin A and ricin at a concentration of
10 times the vendor-stated LOD. In addition to the PT and DW samples analyzed, method blank samples consisting
of DI water were analyzed to confirm negative responses in the absence of any contaminant and to ensure that no
sources of contamination were introduced during the analysis procedures.
QA oversight of verification testing was provided by Battelle and EPA. Battelle QA staff conducted a technical
systems audit and a data quality audit of 10% of the test data. This verification statement, the full report on which it
is based, and the test/QA plan for this verification are all available at www.epa.gov/etv/centers/centerl .html.
TECHNOLOGY DESCRIPTION
The following description of the BioVerify™ Test Kits and M-SERIES® M1M analyzer was provided by the
vendor and was not verified in this test.
BioVerify™ Test Kits detect biological agents such as bacteria, viruses, and toxins in various matrices, including
food and environmental samples. The test kits use proprietary BioVeris Technology™ based on a process that uses
labels designed to emit light when electrochemically stimulated. The tests use two antibodies specific for the
antigen of interest in a single-tube lyophilized reagent format. One antibody is immobilized on paramagnetic
microparticles, and the other is labeled with BioVeris' BV-TAG™ label. When the antigen of interest is present in
the sample, both antibodies bind to the antigen, effectively linking the microparticle, the antigen, and the BV-
TAG™ label. The electrode stimulates the BV-TAG™ labels bound (via the antibodies and antigen) to the
microparticles, and the emitted light is measured. If the antigen of interest is not present in the sample, the
microparticle and the label are not linked, and no signal is generated.
Sample analysis tubes are arranged in a 96-well format, and tests tubes containing reagents for specific target
analytes are color coded for the operator's convenience. The analyzer provides real time data acquisition using
preset test protocols and includes both audible and visual warnings in the event a positive sample is encountered.
The system allows storage and retrieval of all plate, sample, and quality control data in Microsoft® Excel format.
All reagent information is entered into the system through a bar code, and reagent usage is monitored electronically.
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The analyzer includes an internal shaker and pipetting capability so that once a sample is added to a tube containing
lyophilized reagents and loaded into the analyzer; no further user intervention is required. The analyzer is 38
centimeters (cm) (14.8 inches) wide, 30 cm high (11.7 inches), and 38 cm (14.8 inches) deep and weighs 16
kilograms (35 pounds). The analyzer and computer are contained within an instrument transport case with
dimensions of 65 cm (25.5 inches) by 61 cm (24.1 inches) by 51 cm (20 inches). The total weight of the analyzer,
computer, transport case, and accessories is 36.4 kilograms (80 pounds). The analyzer requires a power source or
use of a battery backup.
Required reagents as well as waste are contained in a second transport case to segregate liquids and electronics
during transport. The transport case dimensions are 48 cm (19 inches) by 38 cm (14.9 inches) by 35 cm (13.7
inches). The transport case, including reagents and liquid waste, weighs 9.5 kilograms (20.9 pounds). A
Bio Verify™ test kit containing 96 tests and the controls to run them is $1,440. The M-SERIES® M1M analyzer is
$69,500. Additional materials that may be purchased include BV-GLO™ Plus ($148 per bottle), BV-CLEAN™
Plus ($148 per bottle), BV-STORE™ ($100 per bottle), BV-DILUENT™ ($100 per bottle), and BV-SANITIZE™
($690 for eight single-use bottles for decontaminating the instrument system).
VERIFICATION OF PERFORMANCE
The tables below summarize the performance of the BioVerify™ test kits using the M-SERIES® M1M analyzer in
detecting botulinum toxin A and ricin, respectively.
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Botulinum Toxin A Summary Table
Parameter
Contaminant-
only PT samples
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Lowest
detectable
concentration
Sample Information
DI water
0.5 milligrams per liter (mg/L)
humic and fulvic
2.5 mg/L humic and fulvic
50 mg/L Ca and Mg
250 mg/L Ca and Mg
Unconcentrated CA
Concentrated CA
Unconcentrated FL
Concentrated FL
Unconcentrated NY
Concentrated NY
Unconcentrated OH
Concentrated OH
0.5 mg/L lipopolysaccharide
Botulinum Toxin A
Concentration (mg/L)
0.00005 (vendor-stated
limit of detection)
0.00025
0.0005
0.0025
0.3 (lethal dose)
unspiked
unspiked
0.0005
0.0005
unspiked
No. of Positive
Results'3'
0
0
3
o
J
3
0
0
0
0
0
0
0
0
0
0
0
0
3
3
3
0
3
3
0
3
3
3
3
3
0
There were no false positive results.
False negatives were observed in the presence of 250 mg/L Ca and Mg and in the
Unconcentrated FL drinking water samples.
Results were consistent (i.e., produced positive or negative results without variation
among replicates) in 29 out of 29 sets of replicates or 100%.
The lowest concentration where at least two-thirds of the replicates generated a
positive response was 0.0005 mg/L.
Other
performance
factors
Test kits require storage at 2-8° C. Analyzer software requires training. The M-
SERIES® M1M analyzer uses electricity or battery backup and includes a rugged
carrying case. Analyzer console weighs approximately 80 pounds. Test kits and
analyzer were used inside and outside a laboratory by a trained operator; one 96
tube sample set can be processed in approximately two hours, provided the analyzer
is primed and system diagnostics have already been performed.
(a) Number of positive results out of three replicates, except for the 0.00025 mg/L contaminant-only PT sample
which is out of 10 replicates.
Shading indicates results for unspiked sample.
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Ricin Summary Table
Parameter
Contaminant-
only PT samples
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Lowest
detectable
concentration
Sample Information
DI water
0.5 mg/L humic and fulvic
2.5 mg/L humic and fulvic
50 mg/L Ca and Mg
250 mg/L Ca and Mg
Unconcentrated CA
Concentrated CA
Unconcentrated FL
Concentrated FL
Unconcentrated NY
Concentrated NY
Unconcentrated OH
Concentrated OH
0.5 mg/L
Lectin from soybean
Ricin Concentration
(mg/L)
0.00005 (vendor-stated
limit of detection)
0.00025
0.0005
0.0025
15 (lethal dose)
unspiked
unspiked
0.0005
0.0005
unspiked
No. of Positive
Results'3'
0
6
3
3
3
0
0
0
0
0
0
0
0
0
0
0
0
3
3
3
0
3
3
3
3
3
3
3
3
0
There were no false positive results.
False negatives were observed only in the 250 mg/L Ca and Mg sample.
Results were consistent (i.e., produced positive or negative results without variation
among replicates) in 28 out of 29 sets of replicates or 97%.
The lowest concentration where at least two-thirds of the replicates generated a
positive response was 0.0005 mg/L, although the 0.00025 mg/L concentration was
detected in 6 out of 10 replicates.
Other
performance
factors
Test kits require storage at 2-8° C. Analyzer software requires training. The M-
SERIES® M1M analyzer uses electricity or battery backup and includes a rugged
carrying case. Analyzer console weighs approximately 80 pounds. Test kits and
analyzer were used inside and outside a laboratory by a trained operator; one 96 tube
sample set can be processed in approximately two hours, provided the analyzer is
primed and system diagnostics have already been performed.
(a) Number of positive results out of three replicates, except for the 0.00025 mg/L contaminant-only PT sample
which is out of 10 replicates.
Shading indicates results for unspiked sample.
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Original signed by Gregory A Mack 10/26/2006 Original signed by Jonathan G. Herrmann 11/12/2006
Gregory A. Mack Date Jonathan G. Herrmann Date
Vice President Director
Energy, Transportation, and Environment Division National Homeland Security Research Center
Battelle U.S. Environmental Protection Agency
NOTICE: ETV verifications are based on an evaluation of technology performance under specific, predetermined
criteria and the appropriate quality assurance procedures. EPA and Battelle make no expressed or implied
warranties as to the performance of the technology and do not certify that a technology will always operate as
verified. The end user is solely responsible for complying with any and all applicable federal, state, and local
requirements. Mention of commercial product names does not imply endorsement.
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September 2006
Environmental Technology Verification
Report
ETV Advanced Monitoring Systems Center
BioVeris
BioVerify™ Botulinum Toxin A and
RicinTest Kits
and
M-SERIES® M1M Analyzer
by
Mary Schrock
Ryan James
Amy Dindal
Zachary Willenberg
Karen Riggs
Battelle
Columbus, Ohio 43201
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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, has financially supported and collaborated in the extramural program described
here. This document has been peer reviewed by the Agency. Mention of trade names or
commercial products does not constitute endorsement or recommendation by the EPA for use.
11
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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the
nation's air, water, and land resources. Under a mandate of national environmental laws, the
Agency strives to formulate and implement actions leading to a compatible balance between
human activities and the ability of natural systems to support and nurture life. To meet this
mandate, the EPA's Office of Research and Development provides data and science support that
can be used to solve environmental problems and to build the scientific knowledge base needed
to manage our ecological resources wisely, to understand how pollutants affect our health, and to
prevent or reduce environmental risks.
The Environmental Technology Verification (ETV) Program has been established by the EPA to
verify the performance characteristics of innovative environmental technology across all media
and to report this objective information to permitters, buyers, and users of the technology, thus
substantially accelerating the entrance of new environmental technologies into the marketplace.
Verification organizations oversee and report verification activities based on testing and quality
assurance protocols developed with input from major stakeholders and customer groups
associated with the technology area. ETV consists of six environmental technology centers.
Information about each of these centers can be found on the Internet at http://www.epa.gov/etv/.
Effective verifications of monitoring technologies are needed to assess environmental quality
and to supply cost and performance data to select the most appropriate technology for that
assessment. Under a cooperative agreement, Battelle has received EPA funding to plan,
coordinate, and conduct such verification tests for "Advanced Monitoring Systems for Air,
Water, and Soil" and report the results to the community at large. Information concerning this
specific environmental technology area can be found on the Internet at
http://www.epa.gov/etv/centers/centerl.html.
in
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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. We sincerely appreciate the
contribution of drinking water samples from the Metropolitan Water District of Southern
California (Paul Rochelle and Melinda Stalvey), the New York Department of Environmental
Protection (Virginia Murray), and Orange County Utilities, Orlando, Florida (Theresa Slifko and
Liza Robles). Also, thank you to the Metropolitan Water District of Southern California for
concentrating each drinking water sample. We would also like to thank Karen Bradham, U.S.
EPA National Exposure Research Laboratory; Steve Allgeier, U.S. EPA Office of Water;
Ricardo DeLeon, Metropolitan Water District of Southern California; and Stanley States,
Pittsburgh Water and Sewer Authority, for their careful review of the test/QA plan and this
verification report.
IV
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Contents
Page
Notice ii
Foreword iii
Acknowledgments iv
List of Abbreviations vii
Chapter 1 Background 1
Chapter 2 Technology Description 2
Chapter 3 Test Design 4
3.1 Test Samples 5
3.1.1 Performance Test Samples 6
3.1.2 Drinking Water Samples 7
3.1.3 Quality Control Samples 8
3.2 Test Procedure 8
3.2.1 Laboratory Testing 8
3.2.2 Non-Laboratory Testing 9
3.2.3 Drinking Water Characterization 9
Chapter 4 Quality Assurance Quality Control 11
4.1 Quality Control of Stock Solutions 11
4.2 Technical Systems Audit 11
4.3 Audit of Data Quality 12
4.4 QA/QC Reporting 12
4.5 Data Review 12
Chapter 5 Statistical Methods and Reported Parameters 14
5.1 Qualitative Contaminant Presence/Absence 14
5.2 False Positive/Negative Responses 14
5.3 Consistency 14
5.4 Lowest Detectable Concentration 14
5.5 Other Performance Factors 15
Chapter 6 Test Results 16
6.1 Qualitative Contaminant Presence/Absence 16
6.1.1 Botulinum Toxin A 16
6.1.2 Ricin 16
6.2 False Positive/Negative Responses 19
6.2.1 Interferent PT Samples 19
6.2.2 DW Samples 21
6.2.3 Cross-Reactivity PT Samples 24
6.3 Consistency 24
6.4 Lowest Detectable Concentration 24
6.5 Other Performance Factors 25
6.5.1 Ease of Use 25
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6.5.2 Field Portability 25
6.5.3 Throughput 26
Chapter 7 Performance Summary 27
Chapter 8 References 30
Figures
Figure 2-1. BioVeris M-SERIES® M1M Analyzer 2
Tables
Table 3-1. Lethal Dose and Source of Contaminants 5
Table 3-2. Performance Test Samples 6
Table 3-3. Drinking Water Samples 6
Table 3-4. Water Quality Characterization of Drinking Water Samples 10
Table 4-1. Summary of Data Recording Process 13
Table 6-la. Botulinum Toxin A Contaminant-Only PT Sample Results-Contaminant
Presence/Absence Evaluation 17
Table 6-lb. Ricin Contaminant-Only PT Sample Results-Contaminant Presence/Absence
Evaluation 18
Table 6-2. Interferent PT Sample Results- False Positive/Negative Evaluation 20
Table 6-3. DW Sample Results-False Positive/Negative Evaluation 22
Table 6-3. DW Sample Results- False Positive/Negative Evaluation (continued) 23
Table 6-4. Potentially Cross-Reactive PT Sample Results 24
Table 7-1. Botulinum Toxin A Summary Table 27
Table 7-1. Botulinum Toxin A Summary Table (Continued) 28
Table 7-2. Ricin Summary Table 29
VI
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List of Abbreviations
AMS Advanced Monitoring Systems
ATEL Aqua Tech Environmental Laboratories, Inc.
Ca calcium
COA certificate of analysis
DI deionized
DW drinking water
EPA U.S. Environmental Protection Agency
ETV Environmental Technology Verification
L liter
LD lethal dose
LOD limit of detection
MB method blank
Mg magnesium
mg/L milligram per liter
jiL microliter
mL milliliter
PT performance test
QA quality assurance
QC quality control
QMP quality management plan
TSA technical systems audit
vn
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental
technologies through performance verification and dissemination of information. The goal of the
ETV Program is to further environmental protection by accelerating the acceptance and use of
improved and cost-effective technologies. ETV seeks to achieve this goal by providing high-
quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies.
ETV works in partnership with recognized testing organizations; with stakeholder groups
consisting of buyers, vendor organizations, and permitters; and with the full participation of
individual technology developers. The program evaluates the performance of innovative
technologies by developing test plans that are responsive to the needs of stakeholders,
conducting field or laboratory tests (as appropriate), collecting and analyzing data, and preparing
peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality
assurance (QA) protocols to ensure that data of known and adequate quality are generated and
that the results are defensible.
The EPA's National Exposure Research Laboratory and its verification organization partner,
Battelle, operate the Advanced Monitoring Systems (AMS) Center under ETV. The AMS Center
recently evaluated the performance of the BioVeris BioVerify™ Botulinum Toxin A and Ricin
Test Kits using the BioVeris M-SERIES® M1M analyzer. Immunoassay test kits were identified
as a priority technology category for verification through the AMS Center stakeholder process.
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Chapter 2
Technology Description
The objective of the ETV AMS Center is to verify the performance characteristics of
environmental monitoring technologies for air, water, and soil. This verification report provides
results for the verification testing of BioVerify™ Botulinum Toxin A and Ricin Test Kits using
the M-SERIES® M1M analyzer. The M-SERTES® M1M analyzer is shown in Figure 2-1.
Following is a description of the system, based on information provided by the vendor. The
information provided below was not subjected to verification in this test.
BioVerify™ Test Kits detect biological agents such as bacteria, viruses, and toxins in various
matrices, including food and environmental samples. The test kits use proprietary BioVeris
Technology™ based on a process that uses labels designed to emit light when electrochemically
stimulated. The tests use two antibodies specific for the antigen of interest in a single-tube
lyophilized reagent format. One antibody is immobilized on paramagnetic microparticles, and
the other is labeled with BioVeris' BV-TAG™ label. When the antigen of interest is present in
the sample, both antibodies bind to the antigen, effectively linking the microparticle, the antigen,
and the BV-TAG™ label. The
electrode stimulates the BV-TAG™
labels bound (via the antibodies and
antigen) to the microparticles, and the
emitted light is measured. If the antigen
of interest is not present in the sample,
the microparticle and the label are not
linked, and no signal is generated.
Sample analysis tubes are arranged in a
96-well format, and tests tubes
containing reagents for specific target
analytes are color coded for the
operator's convenience. The analyzer
provides real time data acquisition
using preset test protocols and includes
both audible and visual warnings in the
event a positive sample is encountered.
Figure 2-1 BioVeris M-SERIESW M1M Analyzer The system allows storage and retrieval
of all plate, sample, and quality control
data in Microsoft® Excel format. All reagent information is entered into the system through a
bar code, and reagent usage is monitored electronically.
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The analyzer includes an internal shaker and pipetting capability so that once a sample is added
to a tube containing lyophilized reagents and loaded into the analyzer; no further user
intervention is required. The analyzer is 38 centimeters (cm) (14.8 inches) wide, 30 cm high
(11.7 inches), and 38 cm (14.8 inches) deep and weighs 16 kilograms (35 pounds). The analyzer
and computer are contained within an instrument transport case with dimensions of 65 cm (25.5
inches) by 61 cm (24.1 inches) by 51 cm (20 inches). The total weight of the analyzer, computer,
transport case, and accessories is 36.4 kilograms (80 pounds). The analyzer requires a power
source or use of a battery backup.
Required reagents as well as waste are contained in a second transport case to segregate liquids
and electronics during transport. The transport case dimensions are 48 cm (19 inches) by 38 cm
(14.9 inches) by 35 cm (13.7 inches). The transport case, including reagents and liquid waste,
weighs 9.5 kilograms (20.9 pounds). A BioVerify™ test kit containing 96 tests and the controls
to run them is $1,440. The M-SERTES® M1M analyzer is $69,500. Additional materials that
may be purchased include BV-GLO™ Plus ($148 per bottle), BV-CLEAN™ Plus ($148 per
bottle), BV-STORE™ ($100 per bottle), BV-DILUENT™ ($100 per bottle), and BV-
SANITIZE™ ($690 for eight single-use bottles for decontaminating the instrument system).
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Chapter 3
Test Design
The objective of this verification test was to evaluate the ability of the BioVerify™ Botulinum
Toxin A and Ricin Test Kits using the M-SERTES® M1M analyzer to detect specific biological
toxins and agents in water samples and to determine whether the test kits are susceptible to
interferents in drinking water (DW).
During this verification test, the BioVerify™ Botulinum Toxin A and Ricin Test Kits were
subjected to various concentrations of their respective target analyte in American Society for
Testing and Materials Type II deionized (DI) water and analyzed with the M-SERIES® M1M
analyzer. Table 3-1 shows the contaminants, the vendor-stated limit of detection (LOD), the
lethal dose (LD) concentrations, and the contaminant source. It should be recognized that there is
a wide range of LD concentrations in the literature. In selecting an LD level for use in
verification testing, literature oral LD50 values were reviewed and included in the test/QA plan
and amendments.(1) In addition to reviewing the LD values in the literature, two factors were
taken into consideration in selecting the final LD concentration for use in testing:
1) Consistency with the LD concentrations used in the first round of ETV immunoassay
technology evaluations.
2) Applicability of the LD concentration level to the participating technologies'
expected limits of detection.
In some instances this resulted in an LD level being selected that was on the high end of the
literature values reported. Given the range of LD concentrations that are available in the
literature, it is recommended that all readers evaluate the LD concentrations used for verification
testing with respect to their particular LD requirements. The lethal dose concentration was
determined using a 250 mL ingestion volume.
The BioVerify™ Botulinum Toxin A and Ricin Test Kits also were used to analyze contaminant-
fortified DW samples that were collected from four water utilities that use a variety of treatment
methods. The effect of interferents was evaluated by analyzing various solutions of interferences.
One type of interference solution contained both humic and fulvic acids in DI water and the
second type contained magnesium (Mg) and calcium (Ca) in DI water. Both types of interferent
solutions were prepared with and without the addition of the contaminants. In addition,
specificity was evaluated by exposing the BioVerify™ Botulinum Toxin A and Ricin Test Kits
to a potentially cross-reactive compound for each target contaminant.
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Table 3-1. Lethal Dose and Source of Contaminants
Contaminant
Botulinum toxin
A
Ricinus communis
Agglutinin II (ricin)
Vendor-Stated
LOD
0.00005
milligrams/liter
(mg/L)
0.00005 mg/L
Lethal Dose
Concentration^
0.3 mg/L
15 mg/L
Source of Contaminant
Metabiologics, Inc. (Madison,
Wisconsin)
Vector Laboratories, Inc.
(Burlingame, California)
The lethal dose of each contaminant was determined by calculating the concentration at which 250 mL of water
would probably cause the death of a 154-pound person, based on human mortality data and as outlined in the
Test/QA Plan for Verification of Immunoassay Test Kits Amendment Number 5 (1).
The verification test for the BioVerify™ Botulinum Toxin A and Ricin Test Kits was conducted
from December 2005 through August 2006, according to procedures specified in the Test/QA
Plan for Verification of Immunoassay Test Kits including amendments 1-5.(1) This test was
conducted at Battelle in West Jefferson, Ohio. Aqua Tech Environmental Laboratories, Inc.
(ATEL) of Marion, Ohio, performed physicochemical characterization for each DW sample to
determine the following parameters: turbidity; concentration of dissolved and total organic
carbon; specific conductivity; alkalinity; concentration of Mg and Ca; pH; hardness; and
concentration of total organic halides, trihalomethanes, and haloacetic acids.
The BioVerify™ Botulinum Toxin A and Ricin Test Kits were evaluated for the following
parameters:
• Contaminant presence/absence
• False positive/false negative response
Interferents
DW matrix effects
Cross-reactivity
• Consistency
• Lowest detectable concentration
• Other performance factors
- Field portability
- Ease of use by technical operators
Sample throughput.
3.1 Test Samples
Tables 3-2 and 3-3 summarize the samples analyzed for each contaminant. The ability of the
BioVerify™ Botulinum Toxin A and Ricin Test Kits to individually detect various
concentrations of botulinum toxin A and ricin was evaluated by analyzing performance test (PT)
and DW samples. PT samples included DI water fortified with either the target contaminant, an
interferent, both, or only a cross-reactive species. DW samples were analyzed using the
BioVerify™ Botulinum Toxin A and Ricin Test Kits with and without the addition of each target
contaminant.
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Table 3-2. Performance Test Samples
Type of PT
Sample
Contaminant
Interferent
Cross-reactive
species
Sample Characteristics
Botulinum toxin A
Ricin
Contaminants in 50 mg/L Ca and
50 mg/L Mg
Contaminants in 250 mg/L Ca
and 250 mg/L Mg
Contaminants in 0.5 mg/L humic
acid and 0.5 mg/L ful vie acid
Contaminants in 2.5 mg/L humic
acid and 2.5 mg/L fill vie acids
Lipopolysaccharide
(botulinum toxin analogue)
Lectin from soybean
(ricin analogue)
Approximate Concentrations
0.00005 to 0.3 mg/L
0.00005 to 15 mg/L
Botulinum toxin A- 0.0005 mg/L
Ricin - 0.0005 mg/L
Botulinum toxin A- 0.0005 mg/L
Ricin - 0.0005 mg/L
Botulinum toxin A- 0.0005 mg/L
Ricin - 0.0005 mg/L
Botulinum toxin A- 0.0005 mg/L
Ricin - 0.0005 mg/L
0.5 mg/L
0.5 mg/L
Table 3-3. Drinking Water Samples
Drinking Water Sample Description
Water Utility
Metropolitan Water
District of Southern
California (CA)
New York City, New
York (NY)
Columbus, Ohio
(OH)
Orlando, Florida
(FL)
Water
Treatment
Filtered
chloraminated
Unfiltered
chlorinated
Filtered
chlorinated
Filtered
chlorinated
Source
Type
surface
surface
surface
ground
Cone. /
Unconc.
both
both
both
both
Botulinum
Toxin A (mg/L)
unspiked
and
0.0005
Ricin
(mg/L)
unspiked
and
0.0005
Approximate Contaminant
Concentrations
3.1.1 Performance Test Samples
The contaminant-only PT samples (shown in Table 3-2) were prepared in DI water using
certified standards of ricin or botulinum toxin A. Reference methods were not available for
quantitative confirmation of botulinum toxin A or ricin test solutions so certificates of analysis
-------
(COA) and QA oversight of solution preparation were used to determine their concentrations.
All test samples were prepared from the standards or stock solutions on the day of analysis.
The interferent PT samples consisted of samples of humic and fulvic acids isolated from Elliott
Soil (obtained from the International Humic Substances Society) and Ca and Mg (prepared from
their chlorides with concentrations based on metals only), each spiked into DI water at two
concentration levels. These samples were analyzed both with and without the target contaminant
and were used to evaluate whether interferences which are commonly found in water have the
potential to cause false positive or negative results with the BioVerify™ Botulinum Toxin A and
Ricin Test Kits. In addition, because the commercially available ricin contained sodium azide as
a preservative, a preservative blank sample consisting of 0.16 mg/L sodium azide was prepared
in DI water and processed with the ricin samples. This solution was prepared as for a
concentrated stock solution, actual samples would contain lower concentrations because of the
dilution of the stock to testing concentrations. However, if there was to be an interference effect,
it would be more likely to occur at higher concentrations. This preservative blank was analyzed
along with the contaminant solutions to ensure that the preservative did not have a significant
effect on the performance of the BioVerify™ Ricin Test Kits during testing.
The last type of PT sample was a cross-reactivity check sample to determine whether the test kits
produced false positive results in response to similar analytes. Lectin from soybean (for ricin)
and lipopolysaccharide (for botulinum toxin) are chemically or biologically similar to the
specified targets. Solutions of these were prepared in DI water.
Three replicates of each PT sample were analyzed, except for the sample concentration five
times greater than the vendor-stated LOD (0.00025 mg/L), for which a total often replicates
were analyzed. The results provided information about how well the BioVerify™ Botulinum
Toxin A and Ricin Test Kits detected the presence of each contaminant at several concentration
levels, the consistency of its responses, and its susceptibility to interferents.
3.1.2 Drinking Water Samples
The DW samples were collected from four geographically distributed municipal sources
(Table 3-3). These samples were unique in terms of their source, treatment, and disinfection
process. All collected samples were finished DW either ready for the distribution system or from
within the distribution system.
Approximately 175 liters (L) of each of the DW samples were collected in pre-cleaned low-
density polyethylene containers. One hundred twenty-five liters of each DW sample were
shipped to the Metropolitan Water District of Southern California and dechlorinated with sodium
thiosulfate. Out of this, 100 L was concentrated using ultra-filtration techniques to a final volume
of 250 mL. This concentration factor was selected because it is the goal of an EPA on-site ultra-
filtration sample concentration method that is being developed to increase the concentration of
insoluble microbiological species in a water sample so they may be detected by available
detection technologies. Twenty-five liters of each water sample was shipped to ATEL for water
quality analysis. The remaining 25 L of each sample was shipped to Battelle where the sample
was dechlorinated with sodium thiosulfate. Each DW sample (unconcentrated and concentrated)
was analyzed without adding any contaminant, as well as after fortification with individual
contaminants at a single concentration level.
-------
3.1.3 Quality Control Samples
In addition to the PT and DW samples analyzed, method blank (MB) samples consisting of DI
water were analyzed to confirm negative responses in the absence of any contaminant and to
ensure that no sources of contamination were introduced during the analysis procedures.
Positive controls included in the BioVerify™ Botulinum Toxin A and Ricin Test Kits were
analyzed to verify that the M-SERIES® M1M analyzer was operating properly.
3.2 Test Procedure
3.2.1 Laboratory Testing
Each day, fresh samples were prepared from standards or stock solutions in either DI water, an
interferent matrix, or a DW matrix. Each sample was prepared in its own container and labeled
with a sample identification number that was recorded on a data sheet. To test a liquid sample for
the presence of botulinum toxin A or ricin the procedure described below was used. Test kits
were specific to either botulinum toxin A or ricin.
Before starting, the M-SERIES® M1M Analyzer was put through system checks and diagnostics
to ensure that sufficient volume of reagents were present in the analyzer, that the proper tubing
connections were in place, and that the dispensing and shaker functions were performing
properly. For each sample and control, test tubes, color coded based on the target analyte, were
removed from the kit and placed in a 96-well tube holder. The first four test tubes in the tube
holder of each analysis set are used as calibrators and are always reserved for two negative
control samples followed by two positive control samples. For this study, the negative controls
consisted of DI water. This was selected at the suggestion of the vendor. However, note that it
is possible that analysis of other sources of water could cause matrix effects requiring that a
different negative control (such as the water matrix being analyzed) be used as the negative
control. The positive control is provided in the test kit and is prepared by adding 125 jiL of DI
water to the positive control tube included in the kit. The kit includes multiple positive control
tubes as one is needed for each positive control sample to be analyzed (possibly several per
sample set). DI water (100 jiL) was added to the first two tubes in the tube holder (slated for
negative controls) and to any other tubes intended for method blank samples. The reconsitituted
positive control solution (100 jiL) was added to the third and fourth tube in the tube holder
(slated for positive controls) and to any other tubes intended for additional positive control
samples. Finally 100 jiL of each test sample to be processed with the set were placed
individually into the remaining test tubes. A description of each sample was entered into the
analysis software. The 96-well tube holder was then loaded in the M-SERIES® M1M Analyzer
and the run sequence was started. Once the 96-well tube holder was placed into the analyzer, the
sample analyses, including reagent additions and electrochemiluminescence measurements, were
performed automatically without additional operator intervention. The M-SERIES® M1M
Analyzer calculates a raw data threshold value based on the response of the first two negative
control samples analyzed (referred to as the negative calibrator by BioVeris). Other samples
were then automatically designated as positive or negative based on whether their resulting raw
data value was above the raw data threshold (positive) or below the raw data threshold
(negative). An audible alarm was also automatically triggered each time the analyzer detected a
-------
sample with a positive result. Note that the raw data threshold value is recalculated with each set
of samples analyzed based on the results of the two negative controls and two positive controls in
the first four tubes of the plate. Upon assay completion, the data were transferred via a
removable drive to a personal computer and imported into Microsoft Excel.
3.2.2 Non-Laboratory Testing
Because of the toxic nature of the contaminants, only positive and negative control samples were
analyzed at a non-laboratory location. The non-laboratory location was a well-lit
shipping/receiving area where there was a power source. The temperature and relative humidity
were ambient (20 +/- 2 °C and 40-50%, respectively). Because the M-SERIES ® M1M Analyzer
requires training to operate, only trained technical staff performed the non-laboratory testing.
3.2.3 Drinking Water Characterization
An aliquot of each DW sample, collected as described in Section 3.1.2, was sent to ATEL.
Table 3-4 lists the methods used to characterize the DW samples, as well as the characterization
data for the four water samples collected as part of this verification test. Water quality
parameters were measured by ATEL in June, 2005. Test kits were analyzed with DW from
December 2005 through August 2006. The time delay between collection and testing was due to
the fact that the water samples were collected for use during a separate ETV test conducted prior
to this one. Because of this, an aliquot of each DW was tested by ATEL again in January 2006
to verify some of the parameters with the most potential to change over time. Note that
dissolved organic carbon was not retested as this result was verified by the total organic carbon
results, additionally the total organic halides and calcium and magnesium were not verified as
there was no reason to expect a change in these parameters. The concentrations of most water
quality parameters were similar; however, there was a decrease in levels of volatile compounds
such as trihalomethanes and haloacetic acids over this time-period.
-------
Table 3-4. Water Quality Characterization of Drinking Water Samples
Parameter
Alkalinity (mg/L)
Specific conductivity
(umho)
Hardness (mg/L)
pH
Total haloacetic acids
(Mg/L)
Total organic carbon
(mg/L)
Dissolved organic
carbon (mg/L)
Total organic halides
(Mg/L)
Total trihalomethanes
(Mg/L)
Turbidity (NTU)
Calcium (mg/L)
Magnesium (mg/L)
Method
SM 2320 B(2)
SM2510B(2)
EPA 130.2(3)
EPA 150.1(3)
EPA 552.2(5)
SM5310B(2)
SM5310B(2)
SM 5320B(2)
EPA 524.2(4)
SM2130B(7)
EPA 200.7 (6)
EPA 200.7 (6)
Columbus,
Ohio
2005
40
572
118
7.6
32.8
2.1
2.1
220
74.9
0.1
33
7.7
2006
44
602
107
7.4
<6.0
2.3
NA
NA
16.6
0.6
NA
NA
Metropolitan
Water District
of Southern
California
2005
71
807
192
8.0
17.4
2.5
2.9
170
39.2
0.1
45
20
2006
97
812
182
7.9
<6.0
2.7
NA
NA
24.1
0.2
NA
NA
New York City,
New York
2005
14
84
20
6.9
39.0
1.6
1.1
82
39.0
1.1
5.6
1.3
2006
12
78
26
6.8
<6.0
4.1
NA
NA
23.1
1.3
NA
NA
Orlando,
Florida
2005
142
322
143
8.5
34.6
1.7
1.6
300
56.4
0.5
8.8
43
2006
125
325
130
7.6
<6.0
2.1
NA
NA
41.8
0.1
NA
NA
NTU = nephelometric turbidity unit
NA = not retested
10
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Chapter 4
Quality Assurance Quality Control
Quality assurance/quality control (QC) procedures were performed in accordance with the
quality management plan (QMP) for the AMS Center(8) and the test/QA plan(1) for this
verification test with the following exception.
The cross-reactivity reagents for ricin (lectin from soybean) and botulinum toxin A
(lipopolysaccharide) were inadvertently prepared at 0.5 mg/L (a concentration 10,000 times the
vendor stated LOD) rather than at the 0.0005 mg/L concentration (ten times the vendor stated
LOD) specified in Section 2.3.1 of the test/QA plan.(1) Because no interference effects were
noted at the higher solution concentration, where they would be more likely to occur than at
lower concentrations, results are reported from the 0.5 mg/L solutions. The cross reactivity
results are presented in Section 6.2.3.
4.1 Quality Control of Stock Solutions
The COAs for botulinum toxin A and ricin were provided by the suppliers. Because standard
reference methods do not exist, the concentrations of botulinum toxin A and ricin were not
independently confirmed. The botulinum toxin A COA (Metabiologics, Inc., Madison,
Wisconsin) indicated that the standard had a concentration of 1000 mg/L and was prepared in
phosphate buffer saline at a pH of 6.2 and had passed Metabiologics' tests for activity, identity
and purity. The ricin COA (Vector Laboratories, Inc.; Burlingame, California) indicated that the
ricin standard had a concentration of 5.0 mg/mL and was prepared in a 0.08% sodium azide
buffer at pH 7.8. Test samples containing these contaminants were prepared by diluting aliquots
of these stock solutions. All records pertaining to stock solution dilutions were reviewed as part
of the TSA review. For the interferent samples, the concentration of calcium and magnesium
was confirmed by EPA Method 200.7.(6) Subsequent preparations of calcium and magnesium at
the same stock concentrations were made following the same preparation procedure.
4.2 Technical Systems Audit
The Battelle Quality Manager conducted a technical systems audit (TSA) to ensure that the
verification test was performed in accordance with the test/QA plan(1) and the AMS Center
SQ\
QMP. ; As part of the audit, the Battelle Quality Manager reviewed the standards and methods
used, compared actual test procedures with those specified in the test/QA plan,(1) and reviewed
data acquisition and handling procedures. Observations and findings from this audit were
documented and submitted to the Battelle Verification Test Coordinator for response. No
11
-------
findings were documented that required any significant action. The records concerning the ISA
are permanently stored with the Battelle Quality Manager.
4.3 Audit of Data Quality
At least 10% of the data acquired during the verification test was audited. Battelle's Quality
Manager or designee traced the data from the initial acquisition, through reduction and statistical
analysis, to final reporting, to ensure the integrity of the reported results. All calculations
performed on the data undergoing the audit were checked.
4.4 QA/QC Reporting
Each internal assessment and audit was documented in accordance with Sections 3.3.4 and 3.3.5
of the QMP for the ETV AMS Center.(8) Once the assessment report was prepared, the Battelle
Verification Test Coordinator responded to each potential problem and implemented any
necessary follow-up corrective action. The Battelle Quality Manager ensured that follow-up
corrective action was taken. The results of the TSA were sent to the EPA.
4.5 Data Review
Records generated in the verification test were reviewed before they were used to calculate,
evaluate, or report verification results. Table 4-1 summarizes the types of data recorded. The
review was performed by a technical staff member involved in the verification test, but not the
staff member who originally generated the record. The person performing the review added
his/her initials and the date to a hard copy of the record being reviewed.
12
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Table 4-1. Summary of Data Recording Process
Data to Be Recorded
Dates and times of
test events
Sample collection and
preparation
information,
including chain-of-
custody
M-SERIES® M1M
analyzer and
BioVerify™
Botulinum Toxin A
and Ricin Test Kit
procedures and
sample results
Reference method
procedures and
sample results
Responsible
Party
Battelle
Battelle and
Water
Utilities
providing
DW samples
Battelle
ATEL
Where
Recorded
ETV data
sheets
ETV data
sheets,
laboratory
record books
and/or chain-
of-custody
forms
ETV data
sheets
Data
acquisition
system, as
appropriate
How Often
Recorded
Start/end of
test, and at each
change of a test
parameter
At time of
sample
collection and
preparation
Throughout test
duration
Throughout
sample analysis
process
Disposition
of Data
Used to
organize/check test
results; manually
incorporated in data
spreadsheets as
necessary
Used to
organize/check test
results; manually
incorporated in data
spreadsheets as
necessary
Manually
incorporated in data
spreadsheets
Transferred to
spreadsheets and
reported to Battelle
13
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Chapter 5
Statistical Methods and Reported Parameters
The methods presented in this chapter were used to verify the performance parameters listed in
Chapter 3. The BioVerify™ Botulinum Toxin A and Ricin Test Kits and M-SERTES® M1M
analyzer produced qualitative results; i.e., they indicated only the presence or absence of the
contaminant and did not measure the concentration present. Therefore, the data evaluation
methods were applied in that context.
5.1 Qualitative Contaminant Presence/Absence
Contaminant presence/absence was assessed by reporting the number of positive results out of
the total number of contaminant-only PT samples tested for botulinum toxin A and ricin. A
positive result was determined automatically by the M-SERIES® M1M analyzer through an
evaluation of the background signal generated by the negative control analyzed as the first two
samples on each 96 tube assay.
5.2 False Positive/Negative Responses
A false positive response was defined as a positive response when the DI water or DW sample
was spiked with a potential interferent, a cross-reactive compound, or not spiked at all. A false
negative response was defined as a negative response when any sample was spiked with ten
times the vendor-stated LOD or more for each analyte. Interferent PT samples, cross-reactivity
PT samples, and DW samples were included in the analysis. The number of false positive and
negative results is reported.
5.3 Consistency
The reproducibility of the results was assessed by calculating the percentage of individual test
samples that produced positive or negative results without variation within replicates.
5.4 Lowest Detectable Concentration
The lowest detectable concentration for each contaminant was determined to be the
concentration level at which at least two-thirds of the replicates generated positive responses.
These concentration levels are determined for each target contaminant in solutions of DI water.
14
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5.5 Other Performance Factors
Aspects of the BioVerify™ Botulinum Toxin A and Ricin Test Kits and M-SERTES® M1M
analyzer performance such as ease of use, field portability, and sample throughput are discussed
in Section 6. Also addressed are qualitative observations of the verification staff pertaining to the
performance of the BioVerify™ Botulinum Toxin A and Ricin Test Kits and M-SERIES® M1M
analyzer.
15
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Chapter 6
Test Results
6.1 Qualitative Contaminant Presence/Absence
The responses for the BioVerify™ Botulinum Toxin A and Ricin Test Kits using the
contaminant-only PT samples containing botulinum toxin A and ricin are discussed in the
following sections. Three replicates of each PT sample were analyzed except for the sample
concentration five times greater than the vendor-stated LOD (0.00025 mg/L) for which a total of
ten replicates were analyzed. A total often replicates were analyzed at this concentration level
because three replicates were contaminant-only PT samples and seven were included in the
test/QA plan as a method detection limit study. Because of the qualitative nature of the
BioVerify™ Botulinum Toxin A and Ricin Test Kits, the results of all ten analyses are reported
as additional contaminant-only PT replicates because a method detection limit cannot be
calculated for a technology that reports a presence/absence result. For the M-SERIES® M1M
analyzer used in this study, results were considered positive if a sample's raw data value
exceeded a threshold raw data value automatically calculated as approximately two times the
average background signal generated by the negative control samples (for this ETV test, DI
water) analyzed at the start of each set of samples. Note that these raw data threshold values will
be unique for every set of samples analyzed and every type of analyte-specific test kit used since
it is based on the negative control sample responses.
6.1.1 Botulinum Toxin A
The results obtained for the PT samples containing botulinum toxin are given in Table 6-la. The
instrument raw data threshold for a positive result generated during testing of these samples was
a raw data value of 945 for all concentration levels except the lethal dose (due to its analysis on a
different day), for which the positive result threshold was 1172. Based on these threshold levels,
positive results were obtained in all solutions at 0.0005 mg/L (10 x LOD) and higher.
6.1.2 Ricin
The results obtained for the PT samples containing ricin are given in Table 6-lb. The instrument
raw data threshold for a positive result generated during testing of these samples was a raw data
value of 676 for all concentration levels except the lethal dose (due to its analysis on a different
day), for which the positive result threshold was 898. Based on these threshold levels, positive
results were obtained in all solutions at 0.0005 mg/L (10 x LOD) and higher and in six out often
solutions at 0.00025 mg/L (5 x LOD).
16
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Table 6-la. Botulinum Toxin A Contaminant-Only PT Sample Results-Contaminant
Presence/Absence Evaluation
Testing
Level
LOD
SxLOD
10 x LOD
50 x LOD
LD
Concentration
(mg/L)
0.00005
0.00025
0.0005
0.0025
0.3
Raw
Data
Result
559
495
495
746
776
774
838
808
844
793
758
781
784
1198
1058
1168
4220
4233
4305
235643
237258
218150
No. of
Positive
Results(a)
0
0
3
3
3
LD = Lethal dose concentration.
LOD = Vendor-stated limit of detection.
(a) Number of positive results in each concentration level. Due to analysis on different days, the positive result raw
data threshold was 1172 for LD concentration and 945 for all other concentrations.
Shaded areas indicate positive results.
17
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Table 6-lb. Ricin Contaminant-Only PT Sample Results-Contaminant Presence/Absence
Evaluation
Testing
Level
LOD
SxLOD
10 x LOD
50 x LOD
LD
Concentration
(mg/L)
0.00005
0.00025
0.0005
0.0025
15
Raw
Data
Result
391
416
408
661
669
111
638
627
722
704
111
772
703
1100
1224
1269
4892
4096
4754
102884
111269
113522
No. of
Positive
Results(a)
0
6
3
3
3
LD = Lethal dose concentration.
LOD = Vendor-stated limit of detection.
(a) Number of positive results in each concentration level. Due to analysis on different days, the positive result raw
data threshold was 898 for LD concentration and 676 for all other concentrations.
Shaded areas indicate positive results.
18
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6.2 False Positive/Negative Responses
Three types of samples were analyzed to evaluate the susceptibility of the BioVerify™
Botulinum Toxin A and Ricin Test Kits to false positive and negative results. These included
interferent PT samples, made up of DI water fortified with Ca and Mg or with humic and fulvic
acids both with and without the addition of target contaminants; cross-reactivity PT samples
made up of DI water fortified with a contaminant similar biologically or chemically with each
specific target contaminant; and DW samples both concentrated and unconcentrated and both
with and without the addition of target contaminants. In addition, a preservative blank containing
sodium azide, which is used as a preservative in commercially available ricin, was analyzed with
the ricin test kit to evaluate the potential for interference from the preservative. A false positive
result was defined as a positive result (i.e., above the instrument generated threshold for positive
values) in the absence of the target contaminant and a false negative result was defined as a
result below the instrument generated threshold for positive values from a DW or interferent
sample containing the target contaminant at levels ten times the vendor-stated LOD.
6.2.1 Interferent PT Samples
The results from the interferent PT samples are given in Table 6-2. There were no false positive
results generated by any of the intereferent solutions for either the BioVerify™ Botulinum Toxin
A or Ricin Test Kit. There were, however, false negative results for both the BioVerify™
Botulinum Toxin A and Ricin Test Kits for all replicates containing the target contaminant in the
presence of 250 mg/L each of calcium and magnesium. A preservative blank (0.16 mg/L sodium
azide) was also analyzed with the Ricin Test Kit to assess possible interference from the
preservative in the commercially available ricin. The preservative did not cause any false
positive responses (average raw data result based on three replicates equaled 489 ± 67).
19
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Table 6-2. Interferent PT Sample Results- False Positive/Negative Evaluation
Interferent
0.5 mg/L
each humic
and fulvic
acids
2.5 mg/L
each humic
and fulvic
acids
50 mg/L
each Ca and
Mg
250 mg/L
each Ca
and Mg
Botulinum Toxin A
Botulinum
Toxin A
(mg/L)
none
0.0005
none
0.0005
none
0.0005
none
0.0005
Raw
Data
Result
468
404
465
1489
1466
1447
457
447
449
1516
1449
1365
486
473
429
1155
1094
1025
486
418
436
715
543
518
No. of
Positive
Results(a)
0
3
0
3
0
3
0
0
Ricin
Ricin
(mg/L)
none
0.0005
None
0.0005
None
0.0005
None
0.0005
Raw
Data
Result
439
490
489
1847
2133
2331
479
492
491
2084
2339
2372
475
484
491
839
956
1041
525
485
492
408
414
409
No. of
Positive
Results(a)
0
3
0
3
0
3
0
0
^Number of positive results in each concentration level. Due to analysis on different days, the botulinum toxin A
positive result raw data threshold was 892 for unspiked samples and 945 for all other samples. Similarly, the ricin
positive result raw data threshold was 925 for unspiked samples and 676 for all other samples.
Shaded areas indicate positive results.
20
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6.2.2 DWSamples
Table 6-3 shows the results of testing with drinking water obtained from four different
geographic locations, both unconcentrated and concentrated and both with and without
contaminant. There were no false positive results generated by any of the drinking waters in the
absence of contaminant for either the BioVerify™ Botulinum Toxin A or Ricin Test Kit. There
were also no false negative results in the presence of ricin for any of the waters, both
concentrated and unconcentrated, from all locations with the BioVerify™ Ricin Test Kit. For
the BioVerify™ Botulinum Toxin A Test Kit there were false negative results only with the
unconcentrated water from Florida.
21
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Table 6-3. DW Sample Results-False Positive/Negative Evaluation
DW Sample
California
California-
Concentrated
Florida
Florida-
Concentrated
Botulinum Toxin A
Botulinum
Toxin A
(mg/L)
none
0.0005
none
0.0005
none
0.0005
none
0.0005
Raw Data
Result
537
508
448
1107
1108
1133
627
626
585
1082
966
1063
540
516
506
826
772
769
563
530
466
1117
1004
1050
No. of
Positive
Results(a)
0
o
6
0
3
0
0
0
3
Ricin
Ricin
(mg/L)
none
0.0005
none
0.0005
none
0.0005
none
0.0005
Raw Data
Result
480
511
466
1448
1396
1391
485
480
468
1826
1949
1838
493
496
476
1345
1391
1381
494
477
472
1382
1411
1370
No. of
Positive
Results(a)
0
3
0
3
0
3
0
3
a) Number of positive results in each concentration level. Due to analysis on different days, the botulinum toxin A
positive result raw data threshold was 1106 for unspiked samples and 945 for all other samples. Similarly, the
ricin positive result raw data threshold was 893 for unspiked samples and 676 for all other samples.
Shaded areas indicate positive results.
22
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Table 6-3. DW Sample Results- False Positive/Negative Evaluation (continued)
DW Sample
New York
New York-
Concentrated
Ohio
Ohio-
Concentrated
Botulinum Toxin A
Botulinum
Toxin A
(mg/L)
none
0.0005
none
0.0005
none
0.0005
none
0.0005
Raw Data
Result
556
509
467
1359
1258
1301
895
882
826
1328
1360
1355
559
554
538
1021
1005
977
540
495
548
1260
1179
1231
No. of
Positive
Results(a)
0
o
6
0
o
6
0
o
J
0
3
Ricin
Botulinum
Toxin A
(mg/L)
none
0.0005
none
0.0005
none
0.0005
none
0.0005
Raw Data
Result
469
479
469
1133
1116
1271
522
516
497
1848
2001
2198
488
483
495
1324
1466
1419
482
463
465
1271
1277
1290
No. of
Positive
Results(a)
0
3
0
3
0
3
0
3
a) Number of positive results in each concentration level. Due to analysis on different days, the botulinum toxin A
positive result raw data threshold was 1106 for unspiked samples and 945 for all other samples. Similarly, the
ricin positive result raw data threshold was 893 for unspiked samples and 676 for all other samples.
Shaded areas indicate positive results.
23
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6.2.3 Cross-Reactivity PT Samples
The results from the cross-reactivity PT samples are given in Table 6-4. A PT sample fortified
with a chemical similar to each target contaminant was analyzed in the absence of any of the
target contaminant. The number of positive results out of the number of replicates is given for
each sample. As noted in Chapter 4, the cross-reactivity PT samples were prepared at a
concentration greater than specified in the test/QA plan. No false positive results were obtained
with the potentially cross-reactive compounds at the concentration prepared. Because there was
no response to the higher concentration, which would be expected to cause greater interference
than a lower concentration, the results of testing with the higher concentration solution are
reported.
Table 6-4. Potentially Cross-Reactive PT Sample Results
Cross-Reactivity Compound
Botulinum Toxin A:
lipopolysaccharide
(0.5 mg/L)
Ricin: Lectin from soybean
(0.5 mg/L)
Raw Data Result
457
407
469
493
487
502
No. of Positive
Results(a)
0
0
(a) Number of positive results out of three replicates. Due to analysis on different days, the positive result raw data
threshold was 892 for botulinum toxin A and 925 for ricin.
6.3 Consistency
Using the BioVerify™ Botulinum Toxin A Test Kit, results were consistent (i.e., produced
positive or negative results without variation among replicates) in 29 out of 29 sets of replicates
or 100%. Using the BioVerify™ Ricin Test Kit, results were consistent in 28 out of 29 sets of
replicates or 97%. Replicates included in the consistency calculation are the contaminant-only
PT samples, the interferent PT samples and the DW samples.
6.4 Lowest Detectable Concentration
The lowest detectable concentration of each target contaminant was defined as the lowest
concentration of contaminant-only PT sample to have at least two-thirds of the replicates
generate positive results. For both botulinum toxin A and ricin, the lowest detectable
concentration was 0.0005 mg/L (10 x LOD). It should be noted that the results for ricin at the
0.00025 mg/L level (5 x LOD) were very close to having two-thirds of the replicates generate a
positive response (six out of 10 replicates were positive) and the four negative results (raw data
values ranging from 627 to 669) were less than 10% different from the 676 threshold value for a
positive result.
24
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6.5 Other Performance Factors
6.5.1 Ease of Use
The BioVerify™ Botulinum Toxin A and Ricin Test Kits came with clearly written and
informative instructions. In addition the M-SERIES® M1M analyzer came with a detailed
operation manual. Contents of the test kits were color-coded for easy identification and storage
requirements were readily available. Overall, the test kit packaging was easy to open. The MI-
SERIES® M1M analyzer required two people to move it into position for analysis due to its
weight (approximately 80 pounds).
The analyzer system reagents used for all of the analyte-specific test kits were supplied ready to
use and could be stored at room temperature. The analyte-specific test kits, however, required
storage between 2-8° C. The shelf-life for analyzer system reagents is four to six months, while
the shelf-life for the analyte-specific kits is one month once they are opened (note that if all kit
reagents are not depleted during testing, they can be resealed and stored). Once a sample tube is
prepared for analysis, it needs to be used immediately. The M-SERIES® M1M analyzer software
is somewhat complex; however, verification testing staff found the software to be intuitive and
were able to operate the system after a four hour training session. In addition to determining a
raw data result value for each sample, the data system automatically identified if the sample
result was positive or negative. An audible alarm was triggered each time a positive result was
detected and needed to be turned off manually; this feature made it difficult for the operator to
multi-task while multiple samples were being analyzed as the operator was continually turning
the alarm off. The operator observed that the complex surface of the M-SERIES® M1M analyzer
and the interior of the carrying case would be difficult to wipe clean; however, cleaning was not
attempted during the course of testing. Pipette tips, sample tubes and spent reagent were
generated as waste with each analysis. The M-SERIES® M1M analyzer contains a sealed waste
container which is electronically monitored to ensure that its capacity is not exceeded.
No formal scientific education would be required for using the BioVerify™ Botulinum Toxin A
and Ricin Test Kits and M-SERIES® M1M analyzer, but general good laboratory skills and
reasonable computer skills are helpful. Because the kits are intended for evaluating biological
and chemical agents, users should know and understand the procedures for safely working with
or near these agents before using this product. Contact information for BioVeris, including
phone, fax, and website address is included in the instruction manual for easy access to the
vendor's contact information.
6.5.2 Field Portability
Field portability testing was accomplished by transporting the BioVerify™ Ricin Test Kits and
M-SERIES® M1M analyzer to a well lit shipping/receiving area. The temperature and relative
humidity were ambient (20 +/- 2 °C and 40-50%, respectively). The equipment packs neatly into
two separate cases for field portability. Due to the weight of the system (approximately 80
pounds) three people were used to transport the system to the field (two to carry the analyzer and
the third to carry the reagents). While fewer people could transport the equipment if aided by a
cart, lifting and orienting the analyzer would be difficult for one person. Once at the field testing
25
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location, the equipment was set up within approximately 15 minutes. All analyzer system
reagents were supplied ready-to-use. At this non-laboratory location, the M-SERIES® M1M
analyzer was operated using a battery back-up. The only equipment needed for field transport
that was not supplied with the BioVerify™ Ricin Test Kit and M-SERIES® M1M analyzer was a
cooler to transport test kits while maintaining a temperature of 2-8 °C. For short-term use a
cooler is sufficient for transporting the test kits which need to be maintained between 2-8°C;
however, for long-term field deployment a refrigerator would be useful to keep the test kits cold.
The technology was field tested with a DI water method blank sample and the vendor-provided
positive control for ricin processed in duplicate. Results in the field were similar to results
obtained in the laboratory with the method blank raw data result values below the positive result
raw data threshold and the positive control raw data result well above the positive result raw data
threshold.
6.5.3 Throughput
Approximately one hour is needed to prime the analyzer plumbing and run system diagnostics.
After this initial set up effort, a 96 tube sample set can be processed in less than two hours. The
test kits are supplied in 96 tube format. Four tubes must be reserved for system-required
controls. The remaining 92 tubes can be used for field samples with the number of samples
being processed depending on the number of replicates the user chooses to process for each
sample.
26
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Chapter 7
Performance Summary
Table 7-1. Botulinum Toxin A Summary Table
Parameter
Contaminant-
only PT samples
Interferent
PT samples
DW samples
Cross-reactivity
Sample Information
DI water
0.5 mg/L humic and fulvic
2.5 mg/L humic and fulvic
50 mg/L Ca and Mg
250 mg/L Ca and Mg
Unconcentrated CA
Concentrated CA
Unconcentrated FL
Concentrated FL
Unconcentrated NY
Concentrated NY
Unconcentrated OH
Concentrated OH
0.5 mg/L lipopolysaccharide
Botulinum Toxin A
Concentration (mg/L)
0.00005 (vendor-stated
limit of detection)
0.00025
0.0005
0.0025
0.3 (lethal dose)
unspiked
unspiked
0.0005
0.0005
unspiked
No. of Positive
Results'3'
0
0
3
3
3
0
0
0
0
0
0
0
0
0
0
0
0
3
3
3
0
3
3
0
3
3
3
3
3
0
(a) Number of positive results out of three replicates, except for the 0.00025 mg/L contaminant-only PT sample
which is out of 10 replicates.
Shading indicates results for unspiked sample.
27
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Table 7-1. Botulinum Toxin A Summary Table (Continued)
False positives
False negatives
Consistency
Lowest
detectable
concentration
Other
performance
factors
There were no false positive results.
False negatives were observed in the presence of 250 mg/L Ca and Mg and in the
unconcentrated FL drinking water samples.
Results were consistent (i.e., produced positive or negative results without variation
among replicates) in 29 out of 29 sets of replicates or 100%.
The lowest concentration where at least two-thirds of the replicates generated a
positive response was 0.0005 mg/L.
Test kits require storage at 2-8° C. Analyzer software requires training. The M-
SERIES® M1M analyzer uses electricity or battery backup and includes a rugged
carrying case. Analyzer console weighs approximately 80 pounds. Test kits and
analyzer were used inside and outside a laboratory by a trained operator; one 96
tube sample set can be processed in approximately two hours, provided the analyzer
is primed and system diagnostics have already been performed.
28
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Table 7-2. Ricin Summary Table
Parameter
Contaminant-
only PT samples
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Lowest
detectable
concentration
Sample Information
DI water
0.5 mg/L humic and fulvic
2.5 mg/L humic and fulvic
50 mg/L Ca and Mg
250 mg/L Ca and Mg
Unconcentrated CA
Concentrated CA
Unconcentrated FL
Concentrated FL
Unconcentrated NY
Concentrated NY
Unconcentrated OH
Concentrated OH
0.5 mg/L
Lectin from soybean
Ricin Concentration
(mg/L)
0.00005 (vendor-stated
limit of detection)
0.00025
0.0005
0.0025
15 (lethal dose)
unspiked
unspiked
0.0005
0.0005
unspiked
No. of Positive
Results'3'
0
6
3
3
3
0
0
0
0
0
0
0
0
0
0
0
0
3
o
6
o
6
0
o
J
3
o
J
3
o
J
3
o
J
3
0
There were no false positive results.
False negatives were observed only in the 250 mg/L Ca and Mg sample.
Results were consistent (i.e., produced positive or negative results without variation
among replicates) in 28 out of 29 sets of replicates or 97%.
The lowest concentration where at least two-thirds of the replicates generated a
positive response was 0.0005 mg/L, although the 0.00025 mg/L concentration was
detected in 6 out of 10 replicates.
Other
performance
factors
Test kits require storage at 2-8° C. Analyzer software requires training. The M-
SERIES® M1M analyzer uses electricity or battery backup and includes a rugged
carrying case. Analyzer console weighs approximately 80 pounds. Test kits and
analyzer were used inside and outside a laboratory by a trained operator; one 96 tube
sample set can be processed in approximately two hours, provided the analyzer is
primed and system diagnostics have already been performed.
(a) Number of positive results out of three replicates, except for the 0.00025 mg/L contaminant-only PT sample
which is out of 10 replicates.
Shading indicates results for unspiked sample.
29
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Chapter 8
References
1. Test/QA Plan for Verification of Immunoassay Test Kits, Battelle, Columbus, Ohio,
January 2004.
2. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 19th Edition, Washington, D.C., 1997.
3. U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020,
March 1983.
4. U.S. EPA Method 524.2, "Purgeable Organic Compounds by Capillary Column GC/Mass
Spectrometry," Methods for the Determination of Organic Compounds in Drinking Water—
Supplement HI, EPA/600/R-95/131, August 1995.
5. U.S. EPA Method 552.2, "Haloacetic Acids and Dalapon by Liquid-Liquid Extraction,
Derivatization and GC with Electron Capture Detector," Methods for the Determination of
Organic Compounds in Drinking Water—Supplement III, EPA/600/R-95/131, August 1995.
6. U.S. EPA Method 200.7, "Trace Elements in Water, Solids, and Biosolids by Inductively
Coupled Plasma—Atomic Emission Spectrometry," EPA-821-R-01-010, January 2001.
7. American Public Health Association, et al. Standard Methods for the Examination of Water
and Wastewater. 20th Edition, Washington, D.C., 1998.
8. Quality Management Plan (QMP) for the ETV Advanced Monitoring Systems Center,
Version 5.0, U.S. EPA Environmental Technology Verification Program, Battelle,
Columbus, Ohio, March 2004.
30
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