THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
&EPA
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Batreiie
The Business of Innovation
TECHNOLOGY TYPE: MICROCYSTIN TEST KIT
APPLICATION:
RECREATIONAL WATER MICROCYSTIN
DETECTION
TECHNOLOGY NAME: Microcystin Plate Kit
COMPANY: Beacon Analytical Systems
ADDRESS:
WEB SITE:
383 Presumpscot Street
Portland, Maine 04103
http://www.beaconkits.com/
PHONE: 207-571-4302
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 Beacon Analytical Systems' Microcystins Plate Kit.
VERIFICATION TEST DESCRIPTION
This verification test of the Beacon Plate Kit was conducted from July 26 through August 12, 2010 at Battelle
laboratories in Columbus, OH. Reference analyses by liquid chromatography tandem mass spectrometry
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(LC-MS/MS) were performed the week of August 16th, 2010 by the University of Nebraska Water Sciences
Laboratory.
The objective of this verification test was to evaluate the microcystin test kit performance in analyzing known
concentrations of microcystin in ASTM International Type II deionized (DI) water and in natural recreational
water 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 distinguish individual congeners among
the more than 80 microcystin variants, the samples prepared for this test were spiked with individual variants.
The plate kit provided a quantitative determination of microcystins and was evaluated in terms of:
• Accuracy - comparison of test kit results (samples prepared in DI) to results from a reference method;
• Precision - repeatability of test kit results from three sample replicates analyzed in DI water, matrix
interference, and recreational water 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 recreational water matrices and chlorophyll-a on
the results of the test kits; and
• Operational and sustainability factors - such as general operation, data acquisition, set up, and
consumables.
Each microcystin test kit was operated according to the vendor's instructions by a vendor-trained Battelle
technician. The Battelle technician had previous experience with using ELISA test kits. Samples and calibration
standards were analyzed in duplicate and positive and negative controls were analyzed at the vendor-specified
frequency.
The ability of the plate kit to determine the concentration of microcystin was challenged using quality control
(QC) samples, performance test (PT) samples and recreational water (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. The reference samples that were aliquotted from
the test samples 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 all vendors and the EPA
Project Officer, the standards used for this verification were purchased from reputable sources (Canadian
National Research Council and 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 the 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/centerl.html.
TECHNOLOGY DESCRIPTION
The following is a description of the Beacon Plate Kit, based on information provided by the vendor. The
information provided below was not verified in this test.
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The Beacon Plate Kit is an immunological laboratory test for the quantification of microcystins in water. The kit
uses a polyclonal antibody that binds both microcystins and a microcystin-enzyme conjugate. Microcystins in the
sample compete with the microcystin-enzyme conjugate for a limited number of antibody binding sites. The assay
procedure included the following steps:
• Add microcystin-enzyme conjugate and a sample containing microcystins to the wells in the plate,
followed by antibody solution. The conjugate competes with any microcystins in the sample for the same
antibody binding sites. The test well is coated with anti-rabbit IgG to capture the rabbit anti-microcystin
added.
• Wash away any unbound molecules, after a 20-minute incubation.
• Add clear substrate solution to each well in the plate. In the presence of bound microcystin-enzyme
conjugate, the substrate is converted to a blue compound. One enzyme molecule can convert many
substrate molecules.
Since the same number of antibody binding sites that are available in every well, and each well receives the same
number of microcystin-enzyme conjugate molecules, a sample containing a low concentration of microcystins
allows the antibody to bind many microcystin-enzyme conjugate molecules. The result is a dark blue solution.
Conversely, a high concentration of microcystins allows fewer microcystin-enzyme conjugate molecules to be
bound by the antibodies, resulting in a lighter blue solution. The color is analyzed using a colorimeter or
spectrophotometer to obtain optical density (OD) values at 450 nanometers (nm). Reader software or a
spreadsheet is used to generate a standard curve and interpolate the sample values off that curve.
VERIFICATION RESULTS
The verification of the Beacon Plate Kit is summarized by the parameters described in Table 1.
Table 1. Beacon Plate Test Kit Performance Summary"
Verification Parameters
LR
LA
RR
Accuracy (ppb, range of %D)
0.10
0.50
1.0
2.0
4.0
7.0
Precision (range of %RSD)
Precision (RW samples)
Linearity (y=)
Method Detection Limit (ppb)
34% to 81%
16% to 72%
26% to 47%
21% to 3 8%
I%tol5%
270% to 2900% The
LR equivalent values
were closer to the
spiked values
suggesting that the 2%
CR for LA may differ
from those provided by
Beacon.
3% to 16%
49% to 170%
170% to 190%
59% to 100%
4% to 18%
All RSDs < 9%, except one at 59%
1.2x + 0.052
r2 = 0.99
0.15
2.9x + 9.8
r2 = 0.76
0.043
1.6x + 0.29
r2 = 0.91
0.20
Cross reactivity values were used to quantify results for different variants based on the LR calibration. See the full report
for more information.
Inter-kit lot reprodudbility. Calibration standards from two different lots were measured and the RPD of the
resulting optical densities were all less than 14%.
Matrix Interference. Matrix interference effects were assessed by using a t-test to compare the plate kit results
generated from samples made by spiking undiluted and diluted interference matrices with the PT sample results at
the same concentration. For chlorophyll-a and RW matrices, five comparisons resulted in statistically significant
differences: 1) 4.0-ppb LA spike into DI water was significantly different from the 4.0-ppb LA spike into the
tenfold diluted RW samples (p=0.006); 2) the RW matrix was between the RR spikes into undiluted and diluted
RW (p=0.006); 3) 4.0-ppb LR spike into DI water was significantly different from the 4.0-ppb LR spike into the
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10 mg/L (p=0.002); 4) the 1.0-mg/L (p=0.003) chlorophyll-a; 5) for LA, the 1.0-mg/L and 10-mg/L chlorophyll-a
solutions average results were different by 0.01-ppb and when compared to the DI water results, and the 1.0-mg/L
chlorophyll-a solution results were statistically different (p = 0.045).
Recreational Water (RW). Because the reference method did not measure all possible microcystin variants, no
quantitative comparison was made between the plate kit and the reference method results. The reference data
were converted into LR-equivalents according to the plate kit cross reactivity for the variants. In general, the
samples that were determined to have higher total concentrations by the plate kit had higher total concentrations
as determined by the reference method. All of the plate 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, which only measured three variants. However, the results of the plate kit were
usually within 25% of the reference method, indicating that the LR, LA, and RR variants make up a significant
proportion of the microcystins that are measurable by the plate kit.
Operational Factors. The test kit operator reported that the plate kit was easy to use. Solution or sample
preparation was minimal, mostly involving diluting the wash solution or the samples that were above the
quantification range. The procedure included two incubation periods that were 30 minutes each. 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 was necessary for obtaining the
spectrophotometric readings that were then analyzed using any commercial ELISA evaluation program (for
example, 4-parameters, Logit/Log or alternatively point to point). According to the vendor, once the analysis was
complete, the remaining solutions and tube contents could be flushed down the drain with no hazardous waste
generated for disposal. Since waste disposal requirements vary from state-to-state, the reader is encouraged to
consult with the appropriate state government agency for proper waste disposal requirements.
The listed price for the plate kit at the time of the verification test was $275 for a plate kit that will analyze 84
samples. According to the vendor, the kit has a 6-month shelf life as received and should be stored at 4 - 8 °C.
Other consumables not included in the kit are pipettes, pipette tips, and distilled or DI water.
Signed by Tracy Stenner
6/15/2011
Tracy Stenner Date
Manager
Environmental Solutions Product Line
Energy, Environment, and Material Sciences
Battelle
Signed by Sally Gutierrez
6/17/2011
Date
Sally Gutierrez
Director
National Risk Management Research Laboratory
Office of Research and Development
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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