Environmental Technology Verification Statement Report Pharmaleads ExyBot A and EzyBot B Test Kits September 2006

THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
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ETV
vvEPA Baltelle
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5. Emironmental Protection Agency
ETV Joint Verification Statement
TECHNOLOGY TYPE: IMMUNOASSAY TEST KITS
APPLICATION: DETECTING BOTULINUM TOXIN
TECHNOLOGY NAME: EzyBot® A and EzyBot® B Test Kits
COMPANY: PharmaLeads
ADDRESS: 11/13 rue Watt
75013 Paris PHONE +33 1 44 85 62 21
FRANCE FAX: +33 1 44 85 62 27
WEB SITE: www.pharmaleads.com
E-MAIL: Jean-pierre.rogala@pharmaleads.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 in water. This verification statement provides a summary of
the test results for the PharmaLeads EzyBot® A and B test kits.

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VERIFICATION TEST DESCRIPTION
The verification test for the EzyBot® test kits was conducted at Battelle between November 2005 and January 2006
according to procedures specified in the Test/QA Plan for Verification of Immunoassay 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 EzyBot® test kits to detect various concentrations of botulinum toxin
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 fiilvic 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 EzyBot® test kits to
lipopolysaccharide, a potentially cross-reactive compound for botulinum toxin. 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 both with and without the addition of botulinum toxin A and B at a concentration of 10 times the vendor-
stated LOD. The EzyBot® A test kit was specific to botulinum toxin A, and the EzyBot® B test kit was specific to
botulinum toxin B. In addition to the PT and DW samples analyzed, method blank (MB) 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/centerl .html.
TECHNOLOGY DESCRIPTION
The following description of EzyBot® was provided by the vendor and was not verified in this test.
EzyBot® test kits provide a means for detecting botulinum toxins A (EzyBot® A) and B (EzyBot® B) in water. The
technology is based on the PharmaLeads internal collision fluorescence quenching technology. A fluorogenic
substance and a quenching substance in the substrate bracket an amino-acid sequence that, in the presence of
botulinum toxin A or B, is cleaved, generating an intense fluorescence. This fluorescence is measured using either a
laboratory or a field fluorimeter. Note that a laboratory fluorimeter is not provided by PharmaLeads with the
EzyBot® kit; however, a field fluorimeter is available for purchase as part of the field case. The type of fluorimeter
used for detection can affect the sensitivity of the analysis obtained with the EzyBot® test kit, therefore users may
want to contact the vendor for recommended fluorimeters in order to achieve optimal sensitivity with the EzyBot®
kits. The fluorescence generated by the EzyBot® test kit increases in intensity with time and with botulinum toxin
concentration. Data can be read from the fluorimeter display or for the PharmaLeads field portable fluorimeter can
be transferred to a computer through a cable provided with the fluorimeter. Note that a computer is not provided by
PharmaLeads.
EzyBot® A and B are available individually in kits of 50 ready-to-use cuvettes containing freeze-dried reagents,
which can be used in the laboratory or in the field. The PharmaLeads field case provides a field incubator which
can be plugged into the auxiliary power outlet of a car to perform the 1-hour incubation at 37°C in the field. The
field case also includes the PharmaLeads field portable fluorimeter. The price of an EzyBot® kit depends on the
quantity ordered. For large quantities, unit price is approximately $30 per ready-to-use cuvette. Cost for the field
case, including the field fluorimeter, the portable incubator, and 100 cuvettes, is less than $12,500.

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VERIFICATION OF PERFORMANCE
The tables below summarize the performance of the EzyBot®test kits in detecting botulinum toxins A and B.
EzyBot® A Summary Table
Parameter
Sample Information
Botulinum Toxin
A (mg/L)
Lab Bench Scale
Fluorimeter(a)
Field Portable
Fluorimeter(a)

30 min.
60 min.
30 min.
60 min.

0.01 (vendor-stated
limit of detection)
0
0
0
0
Contaminant-only
DI water
0.05
0
10
0
0
0.1
0
3
0
0

0.3 (lethal dose)
3
3
0
3

0.5
3
3
1
3
Interferent
0.5 and 2.5 mg/L each
humic/fulvic acids
0.1

50 and 250 mg/L each
Ca/Mg
0.1

DW-all locations
unconcentrated
0.1
NA
3
NA
DW-California
concentrated
0.1
3
DW-Florida
concentrated
0.1

DW-New York
concentrated
0.1

DW-Ohio
concentrated
0.1

Lowest Detectable Concentration® (mg/L)
0.3
0.05
ND
0.3
False positives
There were no false positive results from interferents including a preservative blank, humic and
fulvic acids, and Ca and Mg; DW from four locations using different water treatment
technologies; or the potentially cross-reactive lipopolysaccharide (0.1 mg/L).
False negatives
False negatives were obtained in the presence of both 0.5 and 2.5 mg/L each humic and fulvic
acids. A false negative was also obtained in New York water which was concentrated by a
factor of 400. A total of 3 false negative results were obtained out of the 12 solutions assessed
at 60 minutes. The vendor informed Battelle after testing that the lab bench scale fluorimeter
provided for testing may have had inconsistent functioning which could have caused the false
negative results that were obtained.
Consistency
Using the lab bench scale fluorimeter, results were consistent in 100% of the samples tested.
Using the field portable fluorimeter, results were consistent in 90% of the samples tested.
Other
Performance
Factors
Convenient ready-to use cuvettes. Easy to operate in the lab and easy to transport and operate in
the field. No formal scientific education would be required to use the kit; however, general lab
skills and training on fluorimeter use were helpful. Approximately 12-15 analyses were
completed in one hour in the laboratory. Only five samples could be processed in one hour in the
field due to size limitation of the field portable incubator. Each Ezybot ® kit contains 50 ready-
to-use cuvettes.
NA = Not tested. Testing concentration below detection in the contaminant only PT testing.
ND = not detectable at concentrations tested.
(a) Results out of 3 replicates except for the 0.05 mg/L contaminant only concentration for which results are out of 10 replicates
(b) The lowest concentration of contaminant-only PT samples to have at least two thirds of the measurements generate positive results.

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EzyBot®B Summary Table
Parameter
Sample Information
Botulinum Toxin
B (mg/L)
Lab Bench Scale
Fluorimeter(a)
Field Portable
Fluorimeter(a)
30 min.
60 min.
30 min.
60 min.
Contaminant-only
DI water
0.01 (vendor-stated
limit of detection)
0
3
0
0
0.05
7
10
0
0
0.1
3
3
0
0
0.3 (lethal dose)
3
3
3
3
0.5
3
3
0
3
Interferent
0.5 mg/L each
humic/fulvic acids
0.1
3
3
NA
2.5 mg/L each
humic/fulvic acids
0.1
1
3
50 and 250 mg/L each
Ca/Mg
0.1
0
0
DW- all but New York
unconcentrated
0.1
0
3
DW- New York
unconcentrated
0.1
3
3
DW-California
concentrated
0.1
0
3
DW-Florida
concentrated
0.1
3
3
DW-New York
concentrated
0.1
0
3
DW-Ohio
concentrated
0.1
3
3
Lowest Detectable Concentration(b) (mg/L)
0.05
0.01
ND 0.3
False positives
There were no false positive results from interferents including a preservative blank, humic and
fulvic acids, and Ca and Mg; DW from four locations using different water treatment technologies;
or the potentially cross-reactive lipopolysaccharide (0.1 mg/L).
False negatives
False negative results were obtained in the presence of both 50 and 250 mg/L Ca and Mg using
both a 30 minute and 60 minute incubation time. The 30 minute incubation time also generated
false negative results in unconcentrated water from California, Florida, and Ohio; and in
concentrated water from California and New York. A total of 8 false negative results were
obtained out of the 12 solutions assessed at 30 minutes. A total of 2 false negative results were
obtained out of the 12 solutions assessed at 60 minutes. The vendor informed Battelle after testing
that the lab bench scale fluorimeter provided for testing may have had inconsistent functioning
which could have caused the false negative results that were obtained.
Consistency
For the lab bench scale fluorimeter, results were consistent in 97% of the samples tested. With the
field portable fluorimeter, results were consistent in 100% of the samples tested.
Other Performance
Factors
Convenient ready-to use cuvettes. Easy to operate in the lab and easy to transport and operate in
the field. No formal scientific education would be required to use the kit; however, general lab
skills and training on fluorimeter use were helpful. Approximately 12-15 analyses were completed
in one hour in the laboratory. Only five samples could be processed in one hour in the field due to
size limitation of the field portable incubator. Each Ezybot ® kit contains 50 ready-to-use cuvettes.
NA = Not tested. Testing concentration below detection in the contaminant only PT testing.
ND = not detectable at concentrations tested.
(a) Results out of 3 replicates except for the 0.05 mg/L contaminant only concentration for which results are out of 10 replicates.
(b) The lowest concentration of contaminant-only PT samples to have at least two thirds of the measurements generate positive results.

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Original signed by Gregory A. Mack	10/26/2006
Gregory A. Mack	Date
Vice President
Energy, Transportation, and Environment Division
Battelle
Original signed by Jonathan G. Herrmann 11/12/2006
Jonathan G. Herrmann	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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