THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
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ETV Joint Verification Statement
TECHNOLOGY TYPE: IMMUNOASSAY TEST KITS
APPLICATION: DETECTING ANTHRAX, BOTULINUM TOXIN, AND
RICIN
TECHNOLOGY NAME: BioThreat Alert® Test Strips
COMPANY: Tetracore, Inc.
ADDRESS: 11 Firstfield Road, Suite C PHONE: 301-258-7553
Gaithersburg, MD 20878 FAX: 301-258-9740
WEB SITE: www.tetracore.com/
E-MAIL: tobrien@tetracore.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 pre-
paring 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 verification centers under ETV, is operated by
Battelle in cooperation with EPA's National Exposure Research Laboratory. The AMS Center has recently
evaluated the performance of immunoassay test kits used to detect anthrax, botulinum toxin, and ricin. This
verification statement provides a summary of the test results for the Tetracore, Inc., BioThreat Alert® test strips.
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VERIFICATION TEST DESCRIPTION
The ability of the BioThreat Alert® test strips to individually detect various concentrations of anthrax spores,
botulinum toxin, and ricin was evaluated between January 14 and April 23, 2004, by analyzing performance test
(PT) and drinking water (DW) samples. PT samples included deionized (DI) water fortified with either the target
contaminant, an interferent, both, or only a cross-reactive species. In addition to the PT and DW samples analyzed,
method blank (MB) samples consisting of DI water also were analyzed to confirm negative responses in the
absence of contaminants and to ensure that no sources of contamination were introduced during the analysis
procedures. MB samples were analyzed by both a trained technician and a non-technical/untrained, first-time user
at a non-laboratory location to evaluate the BioThreat Alert® performance and ease of use outside of the
laboratory. The test strips generated either positive or negative qualitative results. Verification test results showed
how effective the BioThreat Alert® test strips were at detecting the presence of each contaminant at several
concentration levels, the consistency of the responses, and the susceptibility of the BioThreat Alert® test strips to
selected interferents and cross-reactive species. In most cases, three replicates of each PT and DW sample were
analyzed to evaluate the reproducibility of the BioThreat Alert® test strip results. Approximately 120 liters (L) of
four DW samples were collected from geographically distributed municipal sources located in Florida (FL), New
York (NY), Ohio (OH), and California (CA). These samples were dechlorinated with sodium thiosulfate, and then
100 L of each sample were concentrated using an ultra-filtration technique to a final volume of 250 milliliters
(mL). Each DW sample (non-concentrated and concentrated) was analyzed without adding any contaminant, as
well as after fortification with individual contaminants at a single concentration level to evaluate the effect of the
DW matrix on the performance of the BioThreat Alert® test strips. During the anthrax spore PT sample analysis,
the lowest detectable concentration of the BioThreat Alert® test strips was shown to be much higher than claimed
by the vendor. Therefore, three preparations of spores were analyzed to further investigate these results. The three
preparations included spores prepared at Battelle and preserved in a solution of water and phenol, spores prepared
at Battelle and not preserved in phenol, and spores prepared at Dugway Proving Ground and stored in spent
culture media. Most of the samples analyzed were made from the Battelle-prepared, phenol-preserved spores. The
other two preparations were used to determine if the phenol preservation or the preparation technique was
negatively affecting the sensitivity of the BioThreat Alert® test strips. Solutions of vegetative anthrax cells also
were analyzed to determine the sensitivity of the BioThreat Alert® test strips to vegetative anthrax cells.
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 BioThreat Alert® test strips was provided by the vendor and was not subjected to
verification in this test.
The BioThreat Alert® test strip from Tetracore, Inc., is a lateral flow immunochromatographic device that uses two
antibodies in combination to specifically detect target antigen in solution. One of the specific antibodies is labeled
with a colloidal gold derivative. Samples applied to the BioThreat Alert® test strips mix with the colloidal
gold-labeled antibody and move along the strip membrane by capillary action. The second specific antibody
captures the colloidal gold-labeled antibody and bound target. When a sufficient amount of target antigen is
present, the colloidal gold label accumulates in the sample ("S") window on the test strip, forming a visible
reddish-brown colored line. As an internal control, a second band in the control ("C") window indicates that the
test strip functioned properly. Two bands or colored lines (in the "S" and "C" windows) are required for a positive
result determination. Twenty-five individually packaged BioThreat Alert® test strips (including a disposable
pipette) are provided in a small box. In addition to the test strips, the box contains several small plastic vials,
25 mL of sample buffer, and step-by-step instructions. To complete a test on a liquid sample, the sample is mixed
with the provided buffer, and five or six drops are added to the sample well of the BioThreat Alert® test strip. A
positive result is indicated by the appearance of a colored line in the test window of the test strip and can be read
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visually or with a reader. During this verification test, a reader was used to make the determination of a positive or
negative result. One kit of 25 strips including sample buffer, instruction brochure, and vials needed for sampling
costs approximately $625. The Alexeter strip reader used during this verification test costs approximately $4,000.
VERIFICATION OF PERFORMANCE
The tables below summarize the performance of the BioThreat Alert® test strips in detecting anthrax, botulinum
toxin, and ricin.
Anthrax Summary Table
Parameter
Qualitative
contaminant
results
Contaminant-
only PT samples
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Lowest detectable concentration
Positive
Actual Results Out
Fortified Anthrax of Total
Sample Information Concentration'*0 Replicates
8 x 108 spores/mL 3/3
^ „ , , , , 8x 107spores/mL 3/3
Battelle-prepared, phenol-preserved spores g x IQ6 spores/mL Q/3
8 x 105 spores/mL 0/1
4 x 106 colony -forming units .
(cfu)/mL
Vegetative cells 3xl05cfu/mL 1/1
3xl04cfu/mL 2/3
3xl03cfu/mL 0/1
7x 108spores/mL 2/2
Dugway -prepared spores „ . „-, , T „ ,,
5 y F F F 8 x 107spores/mL 0/1
230 mg/L Calcium (Ca) and 8 ^
r\r\ rr ~\ r ' /"\ r \ L ^ L\J oULJlCo/llLL/ \JI J
90 mg/L Magnesium (Mg)
2.5 mg/L humic acid and , Iri8 / T tw ->n
„ . &. ,, , . . , 1x10 spores/mLw 3/3
2.5 mg/L fulvic acid
Concentrated C A 1 x 108 spores/mL® 2/3
Concentrated NY 1 x 108 spores/mL00 1/3
Unconcentrated DW 1 x 106 spores/mL 0/24
1 x 105 spores/mL Bacillus thuringiensis unspiked 0/3
Two false positives resulted from the analysis of the DW samples. One out of three
replicates for each of the FL DW and concentrated NY DW falsely generated positive
results. Bacillus thuringiensis was prepared at concentrations much lower than the lowest
detectable concentration of Bacillus anthracis. Therefore, negative results with these
samples do not necessarily indicate a lack of cross-reactivity.
None of three results was positive for the 230-mg/L Ca and 90-mg/L Mg spiked with a
detectable concentration of anthrax. In addition, one and two false negative results were
generated for the concentrated CA and concentrated NY DW samples, respectively.
BioThreat Alert® test strips were not able to detect anthrax spores at the vendor-stated
limit of detection (LOD). All of the unconcentrated DW samples were spiked at
concentrations less than detectable by the test strips and, therefore, were, as expected,
negative.
96% (25 of 26 replicates) of the contaminant and interferent PT sample results were
obtained in replicate sets in which all the individual replicates had the same result,
whether positive or negative. This was the case for 78% of the DW samples.
8 x 107 spores/mL - Battelle prep; 7 x 1 08 spores/mL - Dugway prep (vendor-stated LOD:
1 x 105 spores/mL); 3 x 104 cfu/mL - vegetative anthrax (no vendor-stated LOD)
(a) The uncertainty of the enumeration technique is approximately 1 5%.
(b) Battelle-prepared, phenol-preserved spores.
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Botulinum Toxin Summary Table
Parameter
Contaminant-
only PT
samples
Qualitative
contaminant
positive results
Interferent
PT samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Lowest detectable concentration
Botulinum Toxin
Concentration
Sample Information (mg/L)
0.01
0.05
Type A
0.1
0.5
0.01
0.05
TypeB 0.1
0.3
0.5
CaandMg 0.1
Humic acid and fulvic acid 0. 1
Concentrated DW 0.1
Unconcentrated DW 0.1
0.1 mg/L Lipopolysaccharide unspiked
Positive Results Out of
Total Replicates
3/3
3/3
3/3
3/3
1/3
3/3
3/3
3/3
3/3
3/3 Type A
6/6 Type B
3/3 Type A
6/6 Type B
6/6 Type A
12/1 2 TypeB
12/1 2 TypeB
1/3
There was one false positive replicate out of three for the unspiked 2.5-mg/L humic and
fulvic acid interferent PT sample; the unspiked concentrated OH DW sample and the
lipopolysaccharide each generated one false positive result out of three replicates.
No false negatives resulted from the analysis of the interferent and DW samples spiked
with detectable levels of Types A and B botulinum toxin.
92% of the results were obtained in replicate sets in which all the individual replicates
had the same result, whether positive or negative.
0.01 mg/L (Type A); 0.05 mg/L (Type B) (vendor- stated
0.01 mg/L)
LOD for both Types A and B:
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Ricin Summary Table
Parameter
Contaminant-only PT
samples
Qualitative
contaminant
positive results Interferef
PI samples
DW samples
Cross-reactivity
False positives
False negatives
Consistency
Lowest detectable concentration
Ricin Concentration Positive Results Out
(mg/L) of Total Replicates
0.035
0.2
0.4
2
15
Ca and Mg 0.4
Humic acid and fulvic acid 0.4
Concentrated DW 0.4
Unconcentrated DW 0.4
0.4 mg/L
. - . unspiked
Lectm from soybean
The unspiked concentrated FL DW generated two
other DW and cross-reactivity samples resulted in
3/3
3/3
3/3
3/3
3/3
6/6
6/6
12/12
11/12
0/3
false positives out of three. All
correctly negative responses.
There was one false negative out of three for the NY DW sample spiked with a
detectable concentration of ricin. The other spiked interferent and DW samples
were correctly determined to be positive.
100% of the contaminant and interferent PT results were obtained in replicate
sets in which all the individual replicates had the same result, whether positive or
negative. That was the case 88% (14 out of 16) of the time for the DW samples.
0.035 mg/L (Vendor-stated LOD: 0.035 mg/L)
Other Performance Factors for Anthrax, Botulinum Toxin, and Ricin Test Strips: All components for testing
were provided in a box of 25 test strips; the strip reader used during the verification test was powered using
electricity or batteries, was easy to operate, and was contained in a rugged carrying case; test strips and reader were
used easily inside and outside a laboratory with trained operator; non-technical operator performed tests as well as
a trained operator; and sample throughput was 20 samples per hour.
Original signed by Gabor J. Kovacs 9/13/04
Gabor J. Kovacs
Vice President
Energy and Environment Division
Battelle
Date
Original signed by E. Timothy Oppelt
9/21/04
E. Timothy Oppelt Date
Director
National Homeland Security Research Center
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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