ETV Joint Verification Statement: Detecting Anthrax Botulinum Toxin and Ricin Tetracore Inc Enzyme-linked Immunosorbent Assay


                 THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
                                         PROGRAM
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          EPA                                                  Bairene
         .mental Protection Agency                                             TljC Business oj Ilinova
                       ETV Joint Verification Statement
      TECHNOLOGY TYPE:  IMMUNOASSAY TEST KITS

      APPLICATION:         DETECTING ANTHRAX, BOTULINUM TOXIN, AND
                                RICIN

      TECHNOLOGY NAME: Enzyme-Linked Immunosorbent Assay

      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., enzyme-linked immunosorbent
assay (ELISA).

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VERIFICATION TEST DESCRIPTION

The ability of the Tetracore ELISA 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. Verification test results showed how effective the Tetracore ELISA was at detecting the presence of
each contaminant at several concentration levels, the consistency of the responses, and the susceptibility of the
Tetracore ELISA 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 Tetracore ELISA 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 Tetracore ELISA. During the anthrax spore PT sample analysis, the lowest
detectable concentration of the Tetracore ELISA was shown to be much higher than claimed by the vendor.
Therefore, two preparations of spores were analyzed to further investigate these results. The two preparations
included spores prepared at Battelle and preserved in a solution of water and 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 preparation was used to determine if the phenol preservation
or the preparation technique was negatively affecting the sensitivity of the Tetracore ELISA. Solutions of
vegetative anthrax cells also were analyzed to determine the sensitivity of the Tetracore ELISA 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 Tetracore ELISA was provided by the vendor and was not subjected to verification in
this test.

The antigen-capture Tetracore ELISA detects antigens in samples by capturing them between a sandwich of
antibodies. The immunosorbent assay uses immunological reagents to identify antibodies. The Tetracore ELISA
can be read qualitatively (visually) and recorded by hand or quantitatively (using a photometer that measures and
prints out the optical density of fluid samples in the microplate). Readings were made qualitatively during this
verification test. To perform a test, positive and negative capture antibody reagents are applied to alternating wells
of a 96-well plate, where they are passively adsorbed. If the target antigen is present in a sample, it will bind to the
reagent. A detector antibody forms the top of the sandwich and binds to any antigen in the sample after it is
captured. The conjugate, to which the enzyme is covalently bound, is the third reagent added; and it binds to the
detector antibody. The substrate, added after the conjugate, contains 2,2'-azinobis(3-ethylbenzthiazoline-6-
sulfonate), which, in the presence of horseradish peroxidase, changes to a bright green. The amount of color
change is directly proportional to the amount of horseradish peroxidase present, which correlates to the amount of
antigen (target contaminant) bound in the sandwich. The color change confirms the "capture" of antigen by the
antibody reagents. For 48 samples, the process takes approximately 5 hours. The Tetracore ELISA includes two
96-well plates, dilution buffer, wash buffer, and the appropriate reagents needed for the analysis. The 96-well
microplate is 12.5 centimeters (cm) by 8 cm. One Tetracore ELISA (positive and negative coated wells) costs
$400.

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VERIFICATION OF PERFORMANCE
The tables below summarize the performance of the Tetracore ELISA 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
Actual Fortified Anthrax Positive Results Out
Sample Information Concentration'3' of Total Replicates
8 x 108 spores/mL 3/3
Battelle-prepared, phenol- 8 x 107 spores/mL 3/3
preserved spores 8 x 106 spores/mL 3/3
8 x 105 spores/mL 0/3
3 x 105 colony -forming ,,,
units (cfu/mL)
Vegetative cells 3xl04cfu/mL 3/3
3xl03cfu/mL 0/3
3xl03cfu/mL 0/3
8 x 106spores/mL 0/3
^ , 8 x 105spores/mL 0/3
Dugway-prepared spores g x 104spores/mL Q/3
8 x 103spores/mL 0/3
230 mg/L Calcium (Ca) 0 , _7 , T fbi *>n
on n n/r • n n \ 8x 107 spores/mL(b) 3/3
90 mg/L Magnesium (Mg)
2.5 mg/L humic acid , ,„» / T rw in
„ ^ „ „ , . . . 1x1 0s spores/mL1 > 3/3
2.5 mg/L fulvic acid
Humic acid and fulvic acid 2 x 106 spores/mL*' 6/6
Ca and Mg 2 x 106 spores/mL 0/6
Concentrated C A 5 x 107 spores/mL® 3/3
Concentrated NY 5 x 107 spores/mL® 3/3
Unconcentrated DW 2xl06 spores/mL 0/24
1 x 104 spores/mL ., , .„
unspiked 0/3
Haciuus thuringiensis
No false positives resulted from the analysis of the interferent, DW, or cross-reactivity
samples. However, two humic and fulvic acid samples, spiked at concentrations below
what was detectable in DI water, 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.
No false negative results were generated for the analysis of interferent or DW samples
spiked with detectable levels of anthrax. Tetracore ELISA was not able to detect
anthrax at the vendor-stated limit of detection (LOD), but was able to at much higher
concentrations. All of the unconcentrated DW samples and six Ca and Mg samples
were spiked at concentrations less than detectable and, therefore, were, as expected,
negative.
100% (47 out of 47) of the results were obtained in replicate sets in which all the
individual replicates had the same result, whether positive or negative.
8 x 106 spores/mL - Battelle prep (vendor- stated LOD: 2 x 104 spores/mL); 3 x 104
cfu/mL - vegetative anthrax (no vendor-stated LOD); the Dugway preparation of spores
was not detectable at concentrations up to 8 x 1 06 spores/mL
(a) The uncertainty of the enumeration technique is approximately 1 5%.
^ Battelle-prepared, phenol-preserved spores.

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Botulinum Toxin Summary Table
Parameter
Qualitative
contaminant
positive results
Contaminant-
only PT
samples
Interferent
PT samples
DW samples
Cross-
reactivity
False positives
False negatives
Consistency
Lowest detectable concentration
Botulinum Toxin Positive Results Out
Sample Information Concentration (mg/L) of Total Replicates
0.004 0/3
Type A °-02 3/3
0.04 3/3
0.2 3/3
0.004 2/3
0.02 0/3
TypeB 0.04 1/3
0.2 3/3
0.3 1/3
CaandMg 0.04 ^ ^ype A
0 6/6 Type B
TT • •, , r , • •, r,r,A 1/3 Type A
Humic acid and tulvic acid 0.04 , ., _ „
3/6 Type B
Concentrated DW 0.04 Z,pe
12/12 TypeB
Unconcentrated DW 0.04 ^n^^^
12/12 TypeB
0.04 mg/L
, . f . . , unspiked 0/3
Lipopolysaccharide
There were no false positive results for the interferent, DW, or cross-reactivity
samples.
Two out of three results were false negative when 0.04 mg/L botulinum toxin
Type A was spiked into 2.5 mg/L humic and fulvic acids, and three out of three
were false negatives when botulinum toxin Type B was spiked into 0.5 mg/L humic
and fulvic acids. There were no false negatives for the spiked DW samples.
With the exception of 2.5 mg/L humic and fulvic acids spiked with 0.04 mg/L
botulinum toxin Type A (1 out of 3 positive), results generated for botulinum toxin
Type A were 1 00% consistent. The D W and interferent samples spiked with
botulinum toxin Type B were equally consistent, but the contaminant PT samples
containing botulinum toxin Type B generated consistent results in just 2 out of 5
sample sets. Overall, 98% of the results were from sample sets that were either all
positive or all negative.
0.02 mg/L (Type A); not clear for Type B because of sporadic results, (vendor-
stated LOD for botulinum toxin [non-specific]: 0.004 mg/L)

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Ricin Summary Table
Parameter
Contaminant-
only PT
samples
contaminant
positive results Interfered PT
samples
DW samples
Cross-
reactivity
False positives
False negatives
Consistency
Lowest detectable concentration
Ricin
Concentration Positive Results Out of Total
Sample Information (mg/L) Replicates
0.0015
0.0075
Ricin PT samples
0.075
15
CaandMg 0.015
Humic acid and fulvic n 0 1 S
acid
Concentrated CW 0.015
Unconcentrated D W 0.015
0.015 mg/L .. ,
, • ,, , unspiked
Lectm from soybean
0/3
3/3
3/3
3/3
3/3
6/6
6/6
12/12
12/12
0/3
No false positive results were generated for ricin in DW or interferent samples.
There were no false negative results for interferent or DW
detectable concentrations of ricin.
samples spiked with
1 00% of the results for ricin were obtained in replicate sets in which all the
individual replicates had the same result, whether positive or negative.
0.0075 mg/L (vendor- stated LOD: 0.0015 mg/L)
Other Performance Factors for Anthrax, Botulinum Toxin, and Ricin: A technically trained operator easily
performed the Tetracore ELISA analysis. Untrained, non-technical, first-time users would not likely be able to
perform the testing because of the need to use a multichannel pipettor, prepare solutions, and read a technical
operating procedure. The Tetracore ELISA could be used outside the laboratory without a problem. At times it was
difficult to determine whether the color of the sample had changed; no reader was used. Sample throughput was 48
samples in 5 hours.
Original signed by Gabor J. Kovacs
Gabor J. Kovacs
Vice President
Energy and Environment Division
Battelle
9/13/04
Date
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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