THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM A
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«EPA Baireiie
' ™ 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/center 1 .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-
SEPJES® 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
o
J
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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