THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
&EPA
tal Protection Agei
Balteiie
The Btisiness o} Innovation
TECHNOLOGY TYPE: MICROCYSTIN TEST KIT
APPLICATION:
RECREATIONAL WATER MICROCYSTIN
DETECTION
TECHNOLOGY NAME: Microcystin ADDA ELISA Test Kit
COMPANY: Abraxis
ADDRESS:
WEB SITE:
54 Steamwhistle Drive
Warminster, PA 18974
http://www.abraxiskits.com/
PHONE: 215-357-3911
ETV Joint Verification Statement
The U.S. Environmental Protection Agency (EPA) has established 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 and 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 verification centers under ETV, is operated by
Battelle in cooperation with EPA's National Risk Management Research Laboratory. The AMS Center
evaluated the performance of microcystin test kits for water monitoring. This verification statement provides a
summary of the test results for the Abraxis Microcystin ADDA enzyme-linked immunosorbent assay (ELISA)
Test Kit.
VERIFICATION TEST DESCRIPTION
This verification test of the Abraxis Microcystin ADDA ELISA Test Kit was conducted from July 26 through
August 12, 2010 at Battelle laboratories in Columbus, OH. Reference analyses by liquid chromatography tandem
-------
mass spectrometry (LC-MS/MS) were performed the week of August 16, 2010 by the University of Nebraska
Water Sciences Laboratory.
The objective of this verification test was to evaluate the performance of the microcystin test kit in analyzing
known concentrations of microcystin in ASTM International Type II deionized (DI) water and in natural
recreational water (RW) samples. The technology was used to analyze a variety of water samples for the variants
microcystin-LR, microcystin-LA, and microcystin-RR. Because the technology cannot specify between the more
than 80 microcystin variants, the samples prepared for this test were spiked with three individual variants. The
Microcystin ADDA ELISA Test Kit provided a quantitative determination of microcystins and was evaluated in
terms of:
• Accuracy - comparison of test kit results (samples prepared in DI water) to results from a reference
method;
• Precision - repeatability of test kit results from three sample replicates analyzed in DI water, matrix
interference, and RW samples;
• Linearity - determination of whether or not the test kit response increases in direct proportion to the
known concentration of microcystin;
• Method detection limit - the lowest quantity of toxin that can be distinguished from the absence of that
toxin (a blank value) at a 95% confidence level;
• Inter-kit lot reproducibility - determination of whether or not the test kit response is significantly different
between two different lots of calibration standards within the kits;
• Matrix Interference - evaluation of the effect of natural RW matrices and chlorophyll-a on the results of
the test kits; and
• Operational and sustainability factors - general operation, data acquisition, setup, consumables, etc.
Each microcystin test kit was operated according to the vendor's instructions by a vendor-trained Battelle
technician. Samples and calibration standards were analyzed in duplicate and positive and negative controls were
analyzed at the vendor-specified frequency.
The ability of the Abraxis ADDA Microcystin Test Kit to determine the concentration of microcystin was
challenged using quality control (QC) samples, performance test (PT) samples and RW samples. QC, PT, and
RW samples were prepared by Battelle technical staff the day before testing began. The test samples were
prepared in glass volumetric flasks and stored in amber glass vials at 4 °C ± 3 °C until use. The reference samples
that were prepared from the test solutions were stored in amber glass bottles at < -10°C. Replicate samples for the
test kits were taken from the same sample bottle. The QC, PT, and RW samples were prepared blindly for the
operator by coding the sample labels to ensure the results were not influenced by the operator's knowledge of the
sample concentration and variant.
Unlike many contaminants, certified microcystin standards are not commercially available. In planning this
verification test, multiple sources of standards were investigated. With agreement from the stakeholders, all
vendors and the EPA project officer, the standards used for this verification were purchased from the most
reputable sources (LR and RR from Canadian National Research Council and LA from Abraxis), based on a
Performance Evaluation Audit, and used for both the testing solutions and the reference method calibration.
QA oversight of verification testing was provided by Battelle and EPA. Battelle QA staff conducted technical
systems audits of both the laboratory and field testing, and Battelle QA staff conducted a data quality audit of at
least 10% of the test data. This verification statement, the full report on which it is based, and the test/QA plan
for this verification test are available at www.epa.gov/etv/centers/center 1 .html.
TECHNOLOGY DESCRIPTION
Following is a description of the Abraxis ADDA Test Kit, based on information provided by the vendor. The
information provided below was not verified in this test.
-------
The ADDA Test Kit is an ELISA for the congener independent determination of microcystins and nodularins in
water samples. The assay utilizes polyclonal antibodies that have been raised against the Microcystin ADDA
ELISA Test Kit moiety of the molecule, allowing for the detection of numerous microcystin and nodularin
variants (over 80 variants are currently known) in drinking, surface, and groundwater at levels below World
Health Organization (WHO) guidelines.
The test is an indirect competitive ELISA and is based on the recognition of microcystins, nodularins and their
variants by a polyclonal sheep antibody. When present in a sample, microcystins and nodularins compete with a
microcystins-protein analog that is immobilized on wells of a microtiter plate for the binding sites of antibodies in
solution. After a washing step, a second antibody(Horseradish Peroxidase) is added and incubated. After a
washing step and addition of a substrate/chromogen solution, a color signal is generated. The intensity of the
color is inversely proportional to the concentration of the microcystins/nodularins present in the sample. The
color reaction is stopped after a specified time and analyzed using a plate photometer to obtain the optical density
(OD) at a wavelength of 450 nanometers (nm).
The Microcystin ADDA ELISA Test Kit is not able to distinguish the difference between two microcystin
variants. Results from the Microcystin ADDA ELISA Test Kit are calibrated with respect to the microcystin-LR
variant. However, other microcystin variants are known (based on information provided by Abraxis) to react to
different extents with the antibodies used for detection; this is referred to as the cross reactivity (CR) of the
variant. For this verification test, Microcystin ADDA ELISA Test Kit results for LR were reported from the
calibration curve, results for the LA variant were reported as 125% of the kit results (based on the CR value of
125%), and the RR variant was reported as 91% of the test kit results.
VERIFICATION RESULTS
The verification of the Abraxis Microcystin ADDA ELISA Test Kit is summarized by the parameters described in
Table 1.
Table 1. Abraxis Microcystin ADDA ELISA Test Kit Performance Summary
Verification Parameters
LR
LA
RR
Accuracy (range of %D)
O.lOppb
0.50 ppb
1.0 ppb
2.0 ppb
4.0 ppb
Precision (range of %RSD)
Precision (RW samples)
Linearity (y=)
Method Detection Limit (ppb)
-13% to 2%
-9% to 40%
19% to 5 8%
-45% to 3 9%
-6% and 3%
5% to 45% (7 of 9
samples < 16%)
27% to 109%
77% to 133%
69% to 113%
105% and 113%
3% to 25%
42% to 64%
98% to 123%
23% to 69%
11% to 50%
4% to 16%
3% to 47%, all except 2 RSDs were < 12%
0.933x + 0.087
r2=0.906
0.137
2.66x - 0.220
r2=0.990
0.218
1.18x + 0.168
r2=0.961
0.047
Inter-kit lot reprodudbility. Calibration standards from two different lots were measured and the relative percent
difference (RPD) of the resulting ODs ranged from 0% to 13%, with eight of the 12 being less than 6%.
Matrix Interference. Matrix interference effects were assessed by using a t-test to compare results from samples
made by spiking undiluted and diluted interference matrices with the PT sample results at 2.0 ppb spiked
concentration. For chlorophyll-a and RW matrices, three out of 16 comparisons resulted in statistically
significant differences (two comparisons could not be performed because there were only two replicate results for
the LR spiked undiluted RW samples): 1) between the 2.0 ppb LA spike into DI water and the 2.0 ppb LA spike
into chlorophyll-a at 1.0 mg/L (p = 0.005); 2) between the 2.0 ppb LA spike into DI water and the 2.0 ppb LA
spike into 10 mg/L chlorophyll-a (p = 0.006); and 3) between the RR spikes into undiluted and diluted RW (p =
0.01). Given that the molecular basis on which the test kits operate is well-characterized and understood from the
-------
literature, these results were unexpected. Two variants (LR and LA) demonstrated an interference effect but the
third variant (RR) did not. This could have been caused by a number of factors, such as chlorophyll-a source and
stability. However, due to the limited number of replicates and low power of this study, additional testing would
be required to provide a better understanding as to whether there is a matrix interference due to chlorophyll-a, or
another variable not investigated in this verification testing.
Recreational Water (RW). Because the reference method did not measure all possible microcystin variants, no
quantitative comparison was made between the Microcystin ADDA ELISA Test Kit and the reference method
results. The reference data were converted into LR-equivalents according to the Microcystin ADDA ELISA Test
Kit cross reactivity for the variants. In general, the samples that were determined to have higher total
concentrations by the Microcystin ADDA ELISA Test Kit had higher total concentrations as determined by the
reference method. All of the Microcystin ADDA ELISA Test Kit total microcystin results were greater than the
reference method results, which was consistent with the likelihood that all of the microcystins were not being
measured by the reference method.
Operational Factors. The test kit operator reported that the Microcystin ADDA ELISA Test Kit was easy to use.
Solution or sample preparation was minimal, mostly involving diluting the samples that were initially above the
quantification range. The procedure included three incubation periods that totaled 2.5 hours. Previous
knowledge or training on the use of micro-pipettes and or multi-channel pipettes with 96-well plates is
recommended for consistent readings. A spectrophotometer plate reader is necessary for obtaining the
spectrophotometric readings that are then analyzed using any commercial ELISA evaluation program (four-
parameter is recommended by the vendor). Once the analysis was complete, the remaining solutions were
disposed in the trash in accordance with local regulations.
The listed price for the Microcystin ADDA ELISA Test Kit at the time of the verification test was $440. The kit
has a 12-month shelf life when received, and should be stored at 4 to 8 °C. Of the 96 wells on one plate, 16 wells
are needed for calibration and control samples. The remaining 80 wells are for sample analyses that are
performed in duplicate. Other consumables not included in the kit are pipettes, pipette tips, and distilled or DI
water that can be supplied by the vendor.
Signed by Tracy Stenner 9/22/10 Signed by Sally Gutierrez 10/25/10
Tracy Stenner Date Sally Gutierrez Date
Manager Director
Environmental Product Line National Risk Management Research Laboratory
National Security Global Business Office of Research and Development
Battelle 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.
------- |