www.epa.gov
TECHBRIEF
invironmental Technology Verification Pro-
Immunoassay Test Kits for Microcystins
Some cyanobacteria, also known as blue-green algae, can
produce toxic compounds called microcystins, that may pose
a significant threat to human and animal health. In 2010 and
2011, the U.S. EPA Environmental Technology Verification
(ETV) Program's Advanced Monitoring Systems (AMS)
Center, operated by Battelle under a cooperative agreement
with EPA, evaluated the performance of six test kits for
microcystins in recreational waters. The Nebraska
Department of Environmental Quality (NDEQ) and the
Suffolk County Department of Health Services (SCDHS) in
New York collaborated in this verification effort.
Technology Description and Verification Testing
In the summer of 2009, Battelle collected recreational water
samples from a total of nine lakes in Nebraska and New
York. Technicians evaluated the test kits against known
microcystin concentrations in deionized (DI) water as well as
unknown concentrations in recreational water samples, and
then compared to the reference method by calculating the
percent difference (%D). Liquid chromatography tandem
mass spectrometry (LC-MS/MS) analysis for the
determination of algal toxins was the proven reference
method of choice. Five of the six kits provided quantitative
results and one kit provided semi-quantitative results;
technology descriptions are provided in Table 1. The kits
were evaluated for concentrations of microcystin-LR, -LA,
and -RR. The results were expressed as microcystin-LR
equivalent concentrations.
ETV evaluated the test kits for accuracy, precision, linearity,
method detection limit, inter-kit lot reproducibility, matrix
interference, and operational and sustainability factors.
Battelle completed the recreational water analysis at their
laboratory in Columbus, Ohio, while the reference method
analysis was performed at the University of Nebraska Water
Center in Lincoln, Nebraska. Table 2 provides selected
performance results for the six verified kits. Additional
information is available in the verification reports and
statements on ETV's website at http://www.epa.gov/nrmrl/
std/etv/vt-ams.html#itkm.
Contact Information
ETV Advanced Monitoring Systems Center
John McKernan, EPA Project Officer
Phone:(513)569-7415
Email: mckernan.john@epa.gov
Amy Dindal, Battelle
Phone: (561)422-0113
Environmental, Health, and Regulatory Background
of Microcystins at a Glance
Cyanobacteria, also known as blue-green algae, are a
family of single-celled organisms that proliferate in warm,
nutrient-rich fresh waters and estuaries. Many
cyanobacteria species produce toxic compounds called
microcystins. Microcystins are nonribosomal peptides that
are naturally produced within the cell wall of living
cyanobacteria. When a cell dies or ruptures through a
process called lysis, microcystins are released into the
water. Human and animal exposures to microcystins
have the potential to result in skin rashes, eye irritations,
respiratory symptoms, and liver damage.
Current research has identified over 80 structural variants
or congeners of toxic microcystins with microcystin-LR
(Leucine-Arginine) being the most frequently detected
and extensively studied. The World Health Organization
(WHO) has set a provisional drinking water guideline
concentration of 1 microgram/liter (ug/L) and a
recreational water standard of 20 ug/L for microcystin-LR
(free and cell-bound). In addition to microcystin-LR,
microcystin-LA (Leucine-Alanine) is highly toxic, and
microcystin-RR (Arginine-Arginine) is highly prevalent but
less toxic.
Under the Safe Drinking Water Act (SDWA), EPA is
responsible for publishing a list of contaminants, called
the Contaminant Candidate List (CCL), that are not
subject to any current regulation but may require future
regulation under the SDWA. In 2005, EPA published a
second CCL (CCL2), including cyanobacteria and their
toxins. In June 2011, EPA published a draft CCL3 which
included three cyanotoxins: Anatoxin-a, Microcystin-LR,
and Cylindrospermopsin. The final CCL3 will be out for
public comment in mid-2012 and the final rule is expected
in August 2013.
A verified immunoassay technology.
The
The ETV Program operates largely as a public-private partnership through competitive cooperative agreements with non-profit research institutes.
program provides objective quality-assured data on the performance of commercial-ready technologies. Verification
does not imply product approval or effectiveness. ETV does not endorse the purchase or sale of any products and UfUfUf PUfi flfllf/Ptlf
services mentioned in this document. lW.U|HI.5|Ullf UIW
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Table 1. Description of Microcystin Test Kit Technologies
Vendors Technology
Abraxis, LLC
Microcystin ADDA Plate
Kit
Abraxis, LLC
Microcystin DM Plate Kit
Abraxis, LLC
Microcystin Strip Test Kit
Beacon Analytical
Systems
Microcystin Plate Kit
Beacon Analytical
Systems
Microcystin Tube Kit
Zeu-lmmunotec, S.L
MicroCystest Plate Kit
Type of Result
Quantitative
Quantitative
Semi-quantitative
Quantitative
Quantitative
Quantitative
Technology Description
Enzyme-linked immunosorbent assay (ELISA) utilizing polyclonal antibodies raised against the ADDA moiety of the mole-
cule, allowing recognition of microcystins, nodularins and their variants. Employs a 96-well assay plate that accommodates
a large number of samples.
ELISA utilizing monoclonal antibodies raised against the ADDA moiety of the molecule, allowing recognition of microcystins,
nodularins and their variants. Employs a 96-well assay plate that accommodates a large number of samples.
Rapid immunochromatographic test utilizing rapid cell lysis step (QuikLyse™) prior to testing. Strip kit allows single sample
analysis.
Immunological laboratory test utilizing polyclonal antibodies that bind microcystins and microcystin-enzyme conjugates that
compete for a limited number of antibody binding sites. Employs a 96-well assay plate that accommodates a large number
of samples.
Utilizes the same principles as the plate kit. Tube kit allows single sample analysis.
Utilizes protein phosphatase inhibition assay (PPIA) to detect toxicity of microcystins associated with inhibition of protein
phosphatases (PP) 1 and 2A in liver cells. Employs a 96-well assay plate that accommodates a large number of samples.
Selected Potential Outcomes of
Verified Microcystin Immunoassay
Technologies
Immunoassay technologies are faster
and less costly than traditional methods
of microcystin detection. The cost of
analyzing a sample for one analyte us-
ing immunoassay techniques can range
from $17 to $25, depending on the
number of replicates run, while GC-
MS analysis can cost between $500 to
$900 per sample. Therefore, test kits
can provide a cost-effective method to
screen for microcystins while GC-MS
and LC-MS methods provide true
quantitative confirmation. Also, the
analysis time for immunoassay tech-
niques is typically less than one day, as
compared to GC-MS, which can be two
to three weeks. Since all verified test
kits were reported by the operators as
easy to use, these kits can likely be
used by non-technical operators.
References:
Butler, N., et al., Microcystins: A brief overview of their toxicity and effects, with special reference to fish, wildlife, and livestock. January,
2009, Office of Environmental Health Hazard Assessment.
Guidelines for Safe Recreational Water Environments, Volume 1, Coastal and Fresh Waters. 2003: World Health Organization.
Hollrah, M., Standard Operating Procedure (SOP) Determination ofalgaltoxin residues in water extracts by liquid chromatography (LC)-
atmosphericpressure electrospray ionization tandem mass spectrometry (MS/MS). December, 2005, Water Sciences Laboratory, Univer-
sity of Nebraska.
Table 2. Selected Performance Results
(Microcystin-LR equivalents)
Technology
Microcystin
ADDA Plate
Microcystin DM
Plate
Microcystin Strip4
Microcystin Plate
Microcystin Tube
MicroCystest
Plate
Accuracy1
(%D)
-45% to 58%
24% to 94%
N/A
16% to 81%
-76% to 21%
20% to 280%
Precision2
(%RSD)
5% to 45%
2% to 11%
N/A
1%to15%
3% to 10%
1%to13%
Linearity (R2)
0.91
0.99
N/A
0.99
0.98
0.95
Method Detection
Limit3
(MDL, ppb)
0.14
0.11
N/A
0.15
0.18
0.24
1 . The accuracy %D range covers 0.1 , 0.5, 1 .0, 2.0, and 4.0 ppb of microcystin LR-equivalents.
2. Precision includes known Dl water spikes of the microcystin-LR variant and recreational water samples with
unknown variants.
3. MDL was calculated with seven replicate analyses with a toxin concentration of five times the vendor's esti-
mated detection limit.
4. Due to the semi-quantitative test design, parameters reported in this table cannot be calculated.
U.S. EPA Office of Water, http://water.epa.gov/scitech/drinkingwater/dws/ccl/
U.S. EPA ETVProgram, http://www.epa.gov/etv
EPA/600/S/12/511
April 2012
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