QuantiPlate™ Kit
for Paraquat
Catalog Number EP 023
Highlights:
•
•
Quantitative detection of
Paraquat pesticide residue
Results in 1½ hours—faster and
more cost effective than current
methods
Contents of Kit:
• 12 strips of 8 antibody-coated wells
•
•
•
•
•
•
•
•
•
•
each, in plate frame
1 vial of Assay Diluent
I vial Negative Control
1 vial of 0.02 ppb Paraquat
Calibrator (in deionized water)
1 vial of 0.04 ppb Paraquat
Calibrator (in deionized water)
1 vial of 0.2 ppb Paraquat
Calibrator (in deionized water)
1 vial of 0.4 ppb Paraquat
Calibrator (in deionized water)
1 vial of 1.0 ppb Paraquat
Calibrator (in deionized water)
1 bottle of Paraquat-enzyme
Conjugate
1 bottle of Substrate
1 bottle of Stop Solution
Intended Use
The EnviroLogix QuantiPlate Kit for Paraquat is designed for the quantitative
laboratory detection of Paraquat pesticide residues in ground and surface
water samples, with an assay quantitation range from 0.02 to 1.0 parts per
billion (ppb).
How the Test Works
The EnviroLogix Paraquat Plate Kit is a competitive Enzyme-Linked
ImmunoSorbent Assay (ELISA).
In the test, Paraquat pesticide residues in the sample compete with enzyme
(horseradish peroxidase)-labeled Paraquat for a limited number of antibody
binding sites on the inside surface of the test wells.
After a simple wash step, the outcome of the competition is visualized with a
color development step.
As with all competitive immunoassays, sample concentration is inversely
proportional to color development.
Darker color = Lower concentration
Lighter color = Higher concentration
Precision
Paraquat-fortified control solutions were repetitively analyzed both within a
single assay, and in different assays on different days. The data is expressed
as % CV for both the recovered concentration and for absorbance (OD).
Recovery
OD
(%CV)
(%CV)
Intra-Assay
n=7
0.1 ppb
7.8%
2.2%
0.75 ppb
6.3%
4.1%
Inter-Assay
n=11
0.1 ppb
7.5%
n/a
0.75 ppb
6.4%
n/a
Fortification and Recovery
Six ground and surface water samples were fortified with Paraquat to a concentration of 0.3 ppb. The average recovery was 103%, with a CV of 7.4%.
Cross-Reactivity
The EnviroLogix Paraquat Plate Kit does not distinguish between Paraquat
and certain other compounds, but detects their presence to differing degrees.
The following table shows the value for 50% B0 and the value for the 90.4%
B0 Limit of Detection for a list of compounds. Concentration is in ppb.
Rev. 10-25-04
QuantiPlate Kit for Paraquat
Page 2 of 6
Items Not Provided
•
•
marking pen (indelible)
•
tape or Parafilm®
•
timer (1 hour and 30 minutes)
•
cool tap or distilled water for
rinsing wells
•
microtiter plate reader or strip
reader
•
microtiter plate washer (optional)
•
multi-channel pipette that will
measure 25, 75 and 100 μL
(optional)
•
racked dilution tubes for loading
samples into the plate with a
multi-channel pipette (optional)
•
Compound
disposable tip adjustable airdisplacement pipette which will
measure 25, 75 and 100 μL
Paraquat
Diquat
50% B0
0.2
5000
LOD
90.4% B0
0.01
300
100% B0 equals the maximum amount of Paraquat-enzyme conjugate that is bound by the
antibody in the absence of any Paraquat in the sample (i.e. negative control). %B0 = (OD of
Sample or Calibrator/OD of Negative Control) x 100.
The following compounds were not detected at 10 ppm:
Aldrin, α-BHC, β-BHC, γ-BHC, δ-BHC, p,p’-DDT, Endrin, Heptachlor
p,p’DDD, p,p’-DDE, Dieldrin, Endosulfan I, Endosulfan II, Endosulfan
sulfate, Endrin aldehyde, Heptachlor epoxide (isomer B), Carbofuran,
Oxamyl, Methomyl, Aldicarb, Aldicarb sulfone, Aldicarb sulfoxide,
Hydroxycarbofuran, Methiocarb, Methylamine
Humic Acid did not interfere in the assay up to a concentration of 100 ppm.
Sodium thiosulfate did not interfere in the assay up to 0.0005N (the highest
concentration tested).
How to Run the Assay
•
Read all of these instructions before running the kit.
•
Allow all reagents to reach room temperature before beginning (at least
30 minutes with un-boxed strips and reagents at room temperature - do
not remove strips from bag with desiccant until they have warmed up).
•
Organize all samples, reagents and pipettes so that steps 1 and 2 can be
performed in 15 minutes or less.
•
If more than three strips are to be run at one time, the 15 minutes is likely
to be exceeded, and the use of a multi-channel pipette is recommended
(see “Note” below).
•
If three or fewer strips are to be run, use a disposable-tip air-displacement
pipette and a clean pipette tip to add each Calibrator and sample to the
wells. Conjugate, Substrate, and Stop Solution may be added in the same
manner; alternatively, use a repeating pipette with a disposable tip on the
end of the Combitip for the reagents.
•
If fewer than all twelve strips are used, reseal the unneeded strips and the
desiccant in the plastic bag provided.
•
Use the well identification markings on the plate frame to guide you
when adding the samples and reagents. Three strips may be used to run
the Negative Control (NC), five Calibrators (C1-C5) and six samples, in
duplicate. More samples require more strips. For an example plate
layout see Figure 1.
orbital plate shaker (optional)
Remove unneeded strips
1. Add 75 μL of Assay Diluent to each well. Immediately add 25 μL of
Negative Control (NC), 25 μL of each Calibrator (C1-C5) and 25 μL of
each sample (S1-S6) to their respective wells, as shown in Figure 1.
Follow this same order of addition for all reagents.
Add Calibrators and samples
NOTE: In order to minimize setup time it is recommended that a multichannel pipette be used in steps 1, 2, 6 and 8 when more than 3 strips are used.
2. Immediately add 100 μL of Paraquat-enzyme Conjugate to each well.
3. Thoroughly mix the contents of the wells by moving the strip holder in a
rapid circular motion on the benchtop for a full 20-30 seconds. Be careful
not to spill the contents!
Rev. 10-25-04
QuantiPlate Kit for Paraquat
Page 3 of 6
4. Cover the wells with tape or Parafilm to prevent evaporation and incubate
at ambient temperature for 1 hour. If an orbital plate shaker is available
shake plate at 200 rpm.
Mix plate
5. After incubation, carefully remove the covering and vigorously shake the
contents of the wells into a sink or other suitable container. Flood wells
completely with cool tap water, then shake to empty. Repeat this wash
step four times. Slap the plate on a paper towel to remove as much water
as possible. Alternatively, use a microtiter plate washer for the wash step.
6. Add 100 μL of Substrate to each well.
7. Thoroughly mix the contents of the wells, as in step 3. Cover the wells
with new tape or Parafilm and incubate for 30 minutes at ambient
temperature. Use orbital shaker if available.
Caution: Stop Solution is 1.0 N Hydrochloric acid. Handle carefully.
8. Add 100 μL of Stop Solution to each well and mix thoroughly. This will
turn the well contents yellow.
NOTE: Read the plate within 30 minutes of the addition of Stop Solution.
Bottle Wash method
How to Interpret the Results
Spectrophotometric Measurement and Interpretation
1. Set the wavelength of your microtiter plate reader to 450 nanometers (nm).
(If it has dual wavelength capability, use 600, 630 or 650 nm as the
reference wavelength.)
2. If the plate reader does not auto-zero on air, zero the instrument against
200 µL water in a blank well. Measure and record the optical density
(OD) of each well’s contents. Alternatively, measure and record the OD
in every well, then subtract the OD of the water blank from each of the
readings.
Complete protocol and add
Stop Solution
3. A semi-log or 4-parameter curve fit for the standard curve should be
used if the microtiter plate reader you are using has data reduction
capabilities. If not, calculate the results manually as described in the next
section.
How to Calculate the Results
1. After reading the wells, average the OD of each set of calibrators and
samples, and calculate the %B0 as follows:
%B0=average OD of Calibrator or sample x 100
average OD of Negative Control
Read plates in a Plate Reader
within 30 minutes of the
addition of Stop Solution.
The % B0 calculation is used to equalize different runs of an assay. While
the raw OD values of Negative Controls, Calibrators, and samples may
differ from run to run, the % B0 relationship of calibrators and samples to
the Negative Control should remain fairly constant.
The %B0 of each Calibrator should fall within these ranges:
Calibrator
%B0
0.02 ppb
80 – 90%
0.04 ppb
68 - 80%
0.2 ppb
38 - 50%
0.4 ppb
25 – 35%
1.0 ppb
12 - 20%
The CV for each pair of Calibrator and sample OD values should not
exceed 15%.
Rev. 10-25-04
QuantiPlate Kit for Paraquat
Page 4 of 6
Precautions and
Notes
•
•
Store all Plate Kit components at
4°C to 8°C (39°F to 46°F) when
not in use.
3. Determine the Paraquat concentration of each sample by finding its %B0
value and the corresponding concentration level on the graph.
4. Interpolation of sample concentration is only possible if the %B0 of the
sample falls within the range of %B0’s of the Calibrators.
Do not expose Plate Kit
components to temperatures
greater than 37°C (99°F) or less
than 2°C (36°F).
•
Allow all reagents to reach
ambient temperature (18°C to
27°C or 64°F to 81°F) before use.
•
Do not use kit components after
the expiration date.
•
Paraquat in water will adhere to
glass surfaces; samples and
standards should be handled with
plastics, preferably polypropylene
or HDPE. To remove dissolved
solids from surface water sample,
use of a disposable 0.2 µm nylon
filter in polypropylene or HDPE
housing is recommended.
•
If water samples have been
treated with acid as a
preservative, bring pH to 6 – 7.5
prior to analysis.
•
Do not use reagents or test well
strips from one Plate Kit with
reagents or test well strips from a
different Plate Kit.
•
2. Graph the %B0 of each Calibrator against its Paraquat concentration on a
semi-log scale (see Figure 3).
Do not expose Substrate to
sunlight during pipetting or while
incubating in the test wells.
•
Do not dilute or adulterate test
reagents or use samples not
called for in the test procedure.
•
It is recommended that positive
results be confirmed by an
alternate method (such as liquid
or gas chromatography).
•
Observe any applicable
regulations when disposing of
samples and kit reagents.
Rev. 10-25-04
If the %B0 of a sample is higher than that of the lowest Calibrator, the
sample must be reported as less than 0.04 ppb.
If the %B0 of a sample is lower than that of the highest Calibrator, the
sample must be reported as greater than 1.2 ppb. If a concentration must
be determined for these high level samples, dilute the sample 1:25 in
distilled water. Run this dilution in a repeat of the immunoassay. If the
result now falls within the range of the %B0’s of the Calibrators, you
must then multiply the concentration measured in the diluted sample by a
factor of 25.
Figure 1. Example of a typical plate setup.
A
B
C
D
E
F
G
H
1
2
3
NC
NC
C1
C1
C2
C2
C3
C3
C4
C4
C5
C5
S1
S1
S2
S2
S3
S3
S4
S4
S5
S5
S6
S6
4
5
6
7
8
9
10 11 12
Figure 2. Illustrative calculations
Well
contents
Negative
Control
0.02 ppb
Calibrator
0.04 ppb
Calibrator
0.2 ppb
Calibrator
0.4 ppb
Calibrator
1.0 ppb
Calibrator
Sample
OD
Average OD
± sd
%CV %B0 Paraquat
Concentration
(ppb)
100
NA
1.2
1.703 ± 0.021
1.688
1.718
1.412 1.434 ± 0.031 2.2
84.2 NA
1.456
1.250 1.237 ± 0.019 1.5
72.6 NA
1.223
0.710 0.689 ± 0.030 4.4
40.5 NA
0.667
0.452 0.469 ± 0.023 5.0
27.5 NA
0.485
0.256 0.261 ± 0.006 2.4
15.3 NA
0.265
1.017 1.034 ± 0.023 2.3
60.7 0.07 ppb
1.050
Actual values may vary; this data is for demonstration purposes only.
QuantiPlate Kit for Paraquat
Page 5 of 6
Figure 3. Illustrative standard curve
100
90
80
70
60
% Bo 50
40
30
20
10
0
0.01
0.1
Paraquat Concentration (ppb)
Rev. 10-25-04
1
QuantiPlate Kit for Paraquat
Page 6 of 6
LIMITED WARRANTY
EnviroLogix Inc. (“EnviroLogix”) warrants the products sold hereunder (“the
Products”) against defects in materials and workmanship when used in
accordance with the applicable instructions for a period not to extend beyond a
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option, any product or part thereof that proves defective in materials or
workmanship within the warranty period.
For Technical Support
Contact Us At:
EnviroLogix
500 Riverside Industrial
Parkway
Portland, ME 04103-1486
USA
Tel: (207) 797-0300
Toll Free: 866-408-4597
Fax: (207) 797-7533
e-mail:
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website:
www.envirologix.com
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Rev. 10-25-04