&EPA
www.epa.gov/nhsrc
technical B RIEP
Detecting Biological Contaminants in Water, Using
Immunoassay Technologies
Seven immunoassay technologies evaluated for determining the presence of biotoxins
in water
In the past, people in the United States have largely taken
for granted the convenience of potable municipal water. However, the threat of intentional
contamination of our water supplies is becoming a concern because of a rise in the number of terrorist
acts around the world. As a result, there is much interest in technologies that can be used to detect a
contamination event, as well as dispel or confirm the
credibility of a threat. Such technologies include
immunoassay tests that can be used to determine the
presence of biotoxins and pathogens in water. The
immunoassay devices are based on immunological
interactions during which specific antibodies react with
contaminants, or antigens, to produce a response
indicating the presence of the contaminant.
Between 2004 and 2006, EPA evaluated seven
immunoassay technologies:
•	BADD™ Test Strips (ADVNT Biotechnologies)
•	BioVerify Test Kits (BioVeris)
•	EzyBot® A and EzyBot® B Test Kits (Pharmaleads)
•	RAMP® Test Cartridges (Response Biomedical Corp.)
•	BioThreat Alert® Test Strips (Tetracore, Inc.)
•	Enzyme Linked Immunosorbent Assay (Tetracore, Inc.)
•	QTL Biosensor (QTL Biosystems LLC)
EPA tested each immunoassay technology's ability to detect specific biotoxins, as well as
its propensity to register false positive and false negative responses as a result of interfering
compounds, cross-reactive species, or matrix-specific information. Because immunoassay
technologies are expected to serve mainly as screening tools in water monitoring scenarios,
this testing produces only qualitative results (i.e., results indicate only the presence or absence
of a contaminant, not a concentration level). Each of the seven technologies was evaluated for:
•	Contaminant presence/absence (i.e., accuracy of the technology)
•	False positive/false negative response
•	Consistency
•	Lowest detectable concentration
•	Other performance factors
U.S. EPA's Homeland Security Research Program
(HSRP) develops products based on scientific
research and technology evaluations. Our products
and expertise are widely used in preventing, preparing
for, and recovering from public health and
environmental emergencies that arise from terrorist
attacks. Our research and products address
biological, radiological, or chemical contaminants that
could affect indoor areas, outdoor areas, or water
infrastructure. HSRP provides these products,
technical assistance, and expertise to support EPA's
roles and responsibilities under the National
Response Framework, statutory requirements, and
Homeland Security Presidential Directives.
This document does not constitute nor should be construed as an EPA endorsement of any particular product,
service, or technology.

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Test Design
Table 1 identifies the immunoassay technologies tested using various water types fortified (spiked)
separately with contaminants, interfering compounds, and cross-reactive species (i.e., a compound
or spore that is chemically similar to a contaminant of interest).
Table 1. Immunoassay Technologies, Contaminants, Cross-Reactive Species, Interfering Compounds
Technologies
Contaminants
Cross-Reactive
Species
Interfering
Compounds
BADD"'' Test Strips
Anthrax
Botulinum toxins
Ricin
B. thuringiensis
Lipopolysaccharide
Lectin

BioVerify Test Kits
Botulinum toxin A
Ricin
Lipopolysaccharide
Lectin

EzyBot® A and
EzyBot® B Test Kits
Botulinum toxins
A and B
Lipopolysaccharide
Calcium
Magnesium
Humic Acid
Fulvic Acid
RAMP® Test
Cartridges
Anthrax
Botulinum toxins
Ricin
B. thuringiensis
Lipopolysaccharide
Lectin
BioThreat Alert®
Test Strips
Anthrax
Botulinum toxins
Ricin
B. thuringiensis
Lipopolysaccharide
Lectin

Enzyme Linked
Immunosorbent
Assay
Anthrax
Botulinum toxins
Ricin
B. thuringiensis
Lipopolysaccharide
Lectin

QTL Biosensor
Anthrax
Ricin
B. thuringiensis
Lectin

Three types of water samples were tested in these evaluations: performance test (PT), drinking water
(DW), and quality control (QC). PT samples were prepared with deionized (Dl) water and fortified with
the target contaminant, an interferent, both, or only a cross-reactive species. Contaminant-only PT
samples were tested in a series of concentrations that included the accepted lethal/infective dose,
the vendor-stated detection limit, and approximately 5, 10, and 50 times the identified detection limit.
DW samples were tested to determine the effects of matrix-specific characteristics (e.g., location,
filtering) on the technology being evaluated. DW samples were collected from four geographically
diverse municipal sources that varied in source (ground water or surface water), treatment (filtered or
unfiltered), and disinfection process (chlorination or chloramination). In order to evaluate the effect of
a concentrated DW sample, 100 L of DW was dechlorinated and then concentrated to 250 m!_,
using an ultrafiltration sample concentration method. Each DW sample (nonconcentrated and
concentrated) was analyzed without adding any contaminant, as well as after fortification with
individual contaminants at concentration levels approximately 10 times greater than the immunoassay
test kit detection limit, Interferent compounds, cross-reactive species, and DWwere used to
determine the immunoassay's propensity to register false positive and false negative responses.
June 2008
E PA/600/S-08/009
This document does not constitute nor should be construed as an EPA endorsement of any particular product,
service, or technology.

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All PT arid DW samples were analyzed in triplicate when possible. Fewer replicates were analyzed if
vendor-supplied materials were limited. The results of each replicate sample set were reported as a
ratio of the number of positive results to the total number of replicates (e.g., 0/3, 1/3). Method blank
QC samples consisted of at least 10% of all samples.
Performance and Results
The accuracy of the technology was determined by dividing the number of positive responses by the
overall number of analyses of spiked contaminant-only PT samples. A false positive response was
defined as a positive response from DW samples that were either spiked with a potential interferent
or cross-reactive compound, or not spiked at all. A false negative response was defined as a negative
response from any sample that was spiked with a contaminant concentration greater than the lowest
detectable concentration.
Consistency or reproducibility of results was determined by calculating the percentage of individual
test samples that produced positive or negative responses without variation within replicates. The
lowest detectable concentration for each contaminant was determined to be the concentration level
at which at least two of the three replicates generated positive responses. Table 2 summarizes the
results for each evaluation parameter and technology.
Table 2. Summary of Results
Technology	Contaminant _ Contaminant False Positive False Negative Consjst	Lowest Detectable
Presence/ Absence Responses	Responses	J Concentration
BADD™ Test Strips
Anthrax
14/24
0
1
90%
4x10' cfu/mL
Botulinum toxin A
7/12
1
0
84%
5 mg/L
Botulinum toxin B
2/21
ND
Ricin
9/21
0
0
100%
20 mg/L
BioVerify Test Kits
Botulinum toxin A
9/22
0
6
100%
0.0005 mg/L
Ricin
15/22
0
3
97%
0.0005 mg/L
EzyBot® A and
EzyBot® B Test Kits
Botulinum toxin A
19/22
0
9
100%
0.05 mg/L
Botulinum toxin B
22/22
0
6
97%
0.01 mg/L
RAMP® Test
Cartridges
Anthrax
8/20
0
0
96%
8x10" cfu/mL
Botulinum toxin A
7/12
0
1
95%
2 mg/L
Botulinum toxin B
0/18
ND
Ricin
12/15
0
0
100%
5 mg/L
BioThreat Alert®
Test Strips
Anthrax
12/19
2
6
96%
8x10' cfu/mL
Botulinum toxin A
12/12
3
0
92%
0.01 mg/L
Botulinum toxin B
13/15
0.05 mg/L
Ricin
15/15
2
1
100%
0.035 mg/L
Enzyme Linked
Immunosorbent
Assay
Anthrax
15/36
2
0
100%
8x106 cfu/mL
Botulinum toxin A
9/12
0
5
98%
0.02 mg/L
Botulinum toxin B
7/15
ND
Ricin
12/15
0
0
100%
0.0075 mg/L
QTL Biosensor
Anthrax
10/15
22
3
72%
5x105 cfu/mL
Ricin
12/15
2
2
90%
0.25 mg/L
The most accurate results were obtained in three instances, using two separate technologies: the
EzyBot® B test kit accurately detected the presence of the botulinum toxin B in 22/22 tests and the
Bio-Threat® Alert test strips detected 12/12 and 15/15 for botulinum toxin A and ricin, respectively.
June 2008
E PA/600/S-08/009
This document does not constitute nor should be construed as an EPA endorsement of any particular product,
service, or technology.

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The two least accurate were 0/18 and 2/21, both botulinum toxin B results from the RAMP® Test
Cartridges and the BADD™, respectively. Review of the associated QA plan identified that the
vendors did not indicate whether or not their technology was specific to a particular type (A or B)
of botulinum toxin. The results suggested that at least two of the technologies were designed for
botulinum toxin A, and this was confirmed by the vendors.
The maximum number of false positives for anthrax tests was 22 out of 22, using the QTL Biosensor,
with the remaining tests exhibiting 3 or fewer false positives. The maximum number of false negatives
was 9 for the botulinum toxin A tests, using the EzyBot® A Test Kit. Thirteen of the 18 biotoxin tests
achieved 95% consistency or above, while the minimum consistency was 72%. The detection limits
for each immunoassay technology are also indicated in the table for the respective contaminants.
CONTACT INFORMATION
For more information, visit the EPA Web site at www.epa.gov/nhsrc.
Technical Contact: Eric Koglin (koglin.eric@epa.gov)
General Feedback/Questions: Kathy Nickel (nickel.kathy@epa.gov)
June 2008
E PA/600/S-08/009
This document does not constitute nor should be construed as an EPA endorsement of any particular product,
service, or technology.

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