THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM ^
ETV
EPA	Baneiie
U.S. Environmental Protection Agency
The B usiness of Innovation
ETV Joint Verification Statement
TECHNOLOGY TYPE:
IMMUNOASSAY TEST KITS
APPLICATION:
DETECTING ANTHRAX, BOTULINUM TOXIN, AND

RICIN

TECHNOLOGY NAME:
BADD™ Test Strips

COMPANY:
ADVNT Biotechnologies

ADDRESS:
P.O. Box 870
PHONE: 928-368-2804

1920 W. Commerce Drive
FAX: 928-368-2808

Lakeside, Arizona 85929

WEB SITE:
www.baddbox.com

E-MAIL:
support@baddbox.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 ADVNT Biotechnologies Biowarfare Agent
Detection Device (BADD™) test strips.

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VERIFICATION TEST DESCRIPTION
The ability of the BADD™ 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 BADD™ 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 BADD™ test strips were at detecting the presence of each contaminant at several concentration levels, the
consistency of the responses, and the susceptibility of the BADD™ 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 BADD™ 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 BADD™ test strips. During the anthrax spore PT sample analysis, the lowest detectable concentration of the
BADD™ 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
BADD™ test strips. Solutions of vegetative anthrax cells also were analyzed to determine the sensitivity of the
BADD™ 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 BADD™ test strips was provided by the vendor and was not subjected to verification
in this test.
BADD™ test strips are self-contained, qualitative assays for screening environmental samples for the presence of
anthrax, botulinum toxin, and ricin. These test strips work on similar principles, but each is single use and can
detect only one contaminant. The BADD™ test strips are stored in resealable packages, which include all the items
necessary to analyze each sample. Each individually packaged test includes approximately 250 microliters (|iL) of
buffer in a small plastic screw-top vial, a sample collection swab, a bulb syringe, the test strip (within its own
sealed package), and step-by-step instructions. This package is approximately 5 inches (12.7 centimeters) by
6 inches (15.2 centimeters) and weighs only a few ounces. The vendor suggests that the resealable package be
used as a sealed waste receptacle for all testing materials. The testing procedure involves dipping the dry collection
swab into a solution suspected of containing anthrax, botulinum toxin, or ricin, followed by eluting (extracting) the
collected sample into a collection tube containing a sample diluent. After the sample is collected, it is transferred
onto the BADD™ test strip where dye-labeled antibodies detect trace amounts of the contaminant collected by the
swab, as indicated by the presence of two bands in the test result window. After 15 minutes, the results are read
visually. BADD™ test strips are sold in boxes of 10 for approximately $250 per box.

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VERIFICATION OF PERFORMANCE
The tables below summarize the performance of the BADD™ test strips in detecting anthrax, botulinum toxin, and
ricin.
Anthrax Summary Table
Parameter
Sample Information
Actual Fortified Anthrax
Concentration'3'
Positive Results Out
of Total Replicates



8 x 10s spores/mL
3/3


Battelle-prepared, phenol-
preserved spores
8 x 107 spores/mL
4 x 107 spores/mL
8 x 106 spores/mL
3/3
2/3
0/3

Contaminant-
only PT samples

8 x 105 spores/mL
0/3

Vegetative cells
4 x 106 colony-forming
units (cfu)/mL
3 x 105 cfu/mL
1/1
2/3
Qualitative
contaminant
results


3 x 104 cfu/mL
0/1

Dugway-prepared spores
8 x 107 spores/mL
8 x 106 spores/mL
3/3
0/1

Interferent
PT samples
230 mg/L Calcium (Ca)
90 mg/L Magnesium (Mg)
2.5 mg/L humic acid
2.5 mg/L fulvic acid
1 x 10s spores/mL®
1 x 108 spores/mL®
3/3
3/3


Concentrated CA
1 x 108 spores/mL®
3/3

DW samples
Concentrated NY
1 x 108 spores/mL®
2/3


Unconcentrated DW
1 x 107 spores/mL®
0/24

Cross-reactivity
5 x 105 spores/mL
Bacillus thuringiensis
un spiked
0/3
False positives
No false positives resulted from the analysis of the interferent, DW, or cross-
reactivity samples. 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.
F alse negatives
One false negative replicate resulted from the analysis of the interferent and DW
samples spiked with detectable levels of anthrax spores (concentrated NY DW); the
BADD™ test strips were not able to detect anthrax spores at the concentration levels
claimed by the vendor, but they were able to detect much higher concentration
levels. All of the unconcentrated DW samples were spiked at concentrations less
than detectable by the test strips and, therefore, were, as expected, negative.
Consistency
90% of the results were obtained in replicate sets in which all the individual
replicates had the same result, whether positive or negative.
Lowest detectable concentration
4 x 107 spores/mL - Battelle prep; 8 x 107 spores/mL - Dugway prep (vendor-stated
limit of detection [LOD]: 1 x 106 spores/mL);
3 x 105 cfu/mL - vegetative anthrax (no vendor-stated LOD)
(a)	The uncertainty of the enumeration technique is approximately 15%.
(b)	Battelle-prepared, phenol-preserved spores.

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Botulinum Toxin Summary Table
Parameter
Sample Information
Botulinum Toxin
Concentration
(mg/L)
Positive Results Out
of Total Replicates



0.5
1/3


Type A
2
5
25
0/3
3/3
3/3

Contaminant-
only PT samples

0.3
0.4
2
0/3
0/3
1/3
Qualitative
contaminant
positive results

Type B
4
20
200
1,000
1/3
0/3
0/3
0/3

Interferent
PT samples(a)
230 mg/L Ca
90 mg/L Mg
2.5 mg/L humic acid
2.5 mg/L fulvic acid
5(a)
5(a)
3/3
3/3


Concentrated CA
5W
3/3

DW samples1-3-1
Concentrated NY
5(a)
3/3


Unconcentrated DW
4(b)
2/24

Cross-reactivity
5 mg/L
Lipopoly saccharide
unspiked
1/3
False positives
No false positives resulted from the analysis of the interferent or unspiked
DW samples. There was one false positive replicate out of three when
lipopoly saccharide was analyzed as a possible cross-reactive compound.
F alse negatives
No false negatives resulted from the analysis of the interferent and DW
samples spiked with detectable levels of Type A botulinum toxin; however,
the BADD™ test strips were not able to reproducibly detect Type B
botulinum toxin when spiked into DW or interferent samples at 4 mg/L or
DI water up to 1,000 mg/L.
Consistency
84% of the results were obtained in replicate sets in which all the individual
replicates had the same result, whether positive or negative.
Lowest detectable concentration
5 mg/L (Type A), Type B was not reproducibly detectable, (vendor-stated
LOD for botulinum toxin [non-specific]: 0.4 mg/L)
(a) Type A botulinum toxin.
^ Type B botulinum toxin.

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Ricin Summary Table
Parameter
Ricin
Concentration Positive Results Out
(mg/L) of Total Replicates
Qualitative contaminant
positive results
Contaminant-only
PT samples
0.4 0/3
2 0/3
5 0/3
15 0/3
20 3/3
200 3/3
2,000 3/3
Interferent PT and
DW Samples
5 0/36
Cross-reactivity
4 mg/L ., , ^ „
T ° j, , unspiked 0/3
Lectin irom soybean
False positives
No false positives resulted from the analysis of the interferent, DW, or
cross-reactivity samples.
F alse negatives
Ricin was not detectable when spiked into DW and interferent samples at
5 mg/L. No expanded testing was done involving the interferent or DW
samples.
Consistency
100% of the results were obtained in replicate sets in which all the
individual replicates had the same result, whether positive or negative.
Lowest detectable concentration
20 mg/L (vendor-stated LOD: 0.4 mg/L)
Other Performance Factors for Anthrax, Botulinum Toxin, and Ricin Test Strips: All components for testing
were provided in a resealable package weighing just a few ounces; strips used easily inside and outside a
laboratory with trained operator; non-technical operator needed minor direction from a trained operator; indicator
line color change for the anthrax test strips was very faint 44% of the time, 29% of the time for botulinum toxin,
and 100% for ricin, increasing the likelihood of false negative results; and sample throughput was 20 to 30
samples per hour.
Original signed by Gabor J. Kovacs 9/13/04
Gabor J. Kovacs	Date
Vice President
Energy and Environment Division
Battelle
Original signed by E. Timothy Qppelt	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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