Etv Joint Verification Statement: Immunoassay Test Kits Detecting Anthrax Botulinum Toxin and Ricin RAMP Test Cartridges

THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
EPA Battelle
.mental Protection Agency 7"/"? Business of Innova

ETV Joint Verification Statement
TECHNOLOGY TYPE: IMMUNOASSAY TEST KITS

APPLICATION: DETECTING ANTHRAX, BOTULINUM TOXIN, AND
RICIN

TECHNOLOGY NAME: RAMP® Test Cartridges

COMPANY: Response Biomedical Corp.
ADDRESS: 8081 Lougheed Highway PHONE: 604-681-4101
Burnaby, British Columbia FAX: 604-412-9830
CANADA V5A 1W9
WEB SITE: www.responsebio.com
E-MAIL: jstephenson@responsebio.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 Response Biomedical Corp. Rapid Analyte
Measurement Platform (RAMP®) test cartridges.

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VERIFICATION TEST DESCRIPTION

The ability of the RAMP® test cartridges 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 RAMP® 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
RAMP® test cartridges were at detecting the presence of each contaminant at several concentration levels, the
consistency of the responses, and the susceptibility of the RAMP® test cartridges 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 RAMP® test cartridge 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 RAMP® test cartridges. During the anthrax spore PT sample analysis, the lowest detectable
concentration of the RAMP® test cartridges 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 RAMP® test cartridges. Solutions of vegetative anthrax cells also were analyzed to determine the
sensitivity of the RAMP® test cartridges 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 RAMP® test cartridges was provided by the vendor and was not subjected to
verification in this test.

RAMP® is a rapid immunochromatographic system for screening environmental samples. The RAMP® system
comprises a portable fluorescence reader and RAMP® test cartridges specific for detecting anthrax, botulinum
toxin, and ricin. Test cartridges specific for detecting smallpox are also available, but were not tested. The RAMP®
reader is a scanning fluorometer and data analysis system used to measure fluorescence from RAMP® test
cartridges. The reader can be operated on built-in battery power or using an alternating current adapter. RAMP®
uses an immunochromatographic strip, housed in the disposable test cartridges. Each test cartridge is single-use,
disposable, and analyte-specific and is used to detect whether an analyte (e.g., anthrax spores) is present in an
aqueous sample. Twenty-five individually packaged RAMP® test cartridges are provided in a small box. In
addition to the test cartridges, the box contains 25 small plastic screw-top vials containing approximately
250 microliters (|J,L) of buffer, a box of sample collection swabs, a 70-|iL micropipette, a lot card for insertion into
the reader, a marking pen, and step-by-step instructions. To perform a test on a liquid sample, a small amount
(10 (iL) of sample is added to the provided buffer, and that solution is mixed and 70 |j,L of sample is pipetted onto
the RAMP® test cartridge. The cartridge is then read using the reader, and a positive or negative result is generated
on the reader's display. Each result, along with the time, date, and sample identification is printed using a printer
provided by Response Biomedical Corp. The reader is also capable of downloading the results to a computer. The

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dimensions of the RAMP® are 10.5 inches wide by 10 inches deep by 6 inches high (27 centimeters wide by 25
centimeters deep by 5 centimeters high), and it weighs 4.6 pounds (2.1 kilograms). A RAMP® system including 25
test cartridges, a reader, a printer, and a carrying case costs approximately $10,000. Regardless of whether the test
strips are specific to anthrax, botulinum toxin, ricin, or smallpox, each additional box of 25 test cartridges costs
approximately $500.
VERIFICATION OF PERFORMANCE
The tables below summarize the performance of the RAMP® test cartridges 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^' of Total Replicates
8 x 108 spores/mL 3/3
Battelle-prepared, phenol- 8 x 1 07 spores/mL 0/3
preserved spores 8 x 106 spores/mL 0/3
3 x 105 spores/mL 0/3
3 x 105 colony -forming units
Vegetative cells (cfu)/mL
3xl04cfu/mL 0/1
7 x 108spores/mL 3/3
Dugway -prepared spores
8 x 107spores/mL 0/1
230 mg/L Calcium (Ca) , , _9 , T rw -,,-,
on n n/r • ///r N 1 x 109 spores/mL(b) 3/3
90 mg/L Magnesium (Mg)
2.5 mg/L humic acid l x IQ9 s/mL(b) 3/3
2.5 mg/L fulvic acid
Concentrated C A 5 x 108 spores/mL(b) 3/3
Concentrated NY 5 x 108 spores/mL(b) 3/3
Unconcentrated DW 4 x 106 spores/mL(b) 0/24
5 x 105 spores/mL ., , .„
n .„ , . . . unspiked 0/3
Bacillus thurmgiensis
No false positives resulted from the analysis of the interferent, DW, or cross-reactivity
samples. Bacillus thuringiemis 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 from the analysis of the interferent and DW
samples spiked with detectable levels of anthrax spores; the RAMP® test cartridges
were not able to detect anthrax spores at the vendor-stated limit of detection (LOD), 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.
96% of the results were obtained in replicate sets in which all the individual replicates
had the same result, whether positive or negative.
8 x 108 spores/mL - Battelle prep ; 7x 108 spores/mL - Dugway prep (vendor-stated
LOD: 4 x 105 spores/mL); 3 x 105 cfu/mL - vegetative anthrax (no vendor-stated LOD)
(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
Sample Information


Type A





TypeB




230 mg/L Ca
90 mg/L Mg
2.5 mg/L humic acid
2.5 mg/L fulvic acid
Concentrated CA
Concentrated NY
Unconcentrated DW
5 mg/L
Lipopolysaccharide
Botulinum Toxin Positive Results Out of
Concentration (mg/L) Total Replicates
0.5
2

5
25
0.3
0.5
2.5

5
200

1,000
5(1)

5(1)

5(1)
5(a)
500
unspiked
0/3
2/3

2/3
3/3
0/3
0/3
0/3

0/3
0/3

0/3
3/3

2/3

3/3
3/3
0/24
0/3
No false positives resulted from the analysis of the interferent, DW, or cross-
reactivity samples.
One false negative replicate resulted from the analysis of the 2.5 mg/L humic and
fulvic acid interferent samples spiked with a detectable leve'
of Type A botulinum
toxin; in addition, the RAMP® test cartridges were not able to detect Type B
botulinum toxin spiked
1,000 mg/L.
95% of the results were
replicates had the same
into DW at 5 mg/L or in DI water at concentrations up to


obtained in replicate sets in which all the individual
result, whether positive or negative.
2 mg/L (Type A), Type B was not detectable up to concentrations of 1,000 mg/L.
(vendor- stated LOD for botulinum toxin [non-specific]: 0.5 mg/L)
(a) Type A botulinum toxin.
(b:i Type B botulinum toxin.

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Ricin 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
Ricin Concentration Positive Results Out
Sample Information

Ricin in DI water

Ca and Mg
Fulvic and humic acid
Concentrated DW

Unconcentrated DW
lOmg/L
Lectin from soybean
(mg/L) of Total Replicates
1
5

15
20
50
10
10
10

10
unspiked
No false positives resulted from the analysis of the interferent,
reactivity samples.
No false negative results were

generated by analyzing DW and
0/3
3/3

3/3
3/3
3/3
6/6
6/6
12/12

12/12
0/3
DW, or cross-

interferent
samples spiked with detectable levels of ricin.
1 00% of the results were obtained in replicate sets in which all
replicates had the same result,
5 mg/L (vendor-stated LOD: 1
whether positive or negative.
mg/L)
the individual

Other Performance Factorfor Anthrax, Botulinum Toxin, and Ricin Test Strips: All components for testing
were provided in a box of 25 test cartridges; the required cartridge reader was operated using electricity or
batteries, was easy to operate, and was contained in a rugged carrying case; test cartridges used easily inside and
outside a laboratory with trained operator; non-technical operator needed minor direction from a trained operator;
"low signal" resulted from highly concentrated anthrax solutions; and sample throughput was 4 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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