ELISA/assay

Microcystin DM ELISA kit

DEIABL454

96T

Microcystin DM ELISA kit

Microcystin

Microcystin DM ELISA kit

ELISA

Microcystin

Water

Quantitative and Qualitative

Water Samples

96T

The ELISA kit should be stored in the refrigerator (2-8°C). The solutions must be allowed to reach room temperature (20-25°C) before use. Reagents may be used until the last day of the month as indicated by the expiration date on the box. Consult state, local, and federal regulations for proper disposal of all reagents.

The assay exhibits very good cross-reactivity with all cyanobacterial cyclic peptide toxin congeners tested to date (see Specificity table below).

Water

Microcystin; nucleocapsid ; IMI; DM I virus

The Microcystins (ADDA)-DM (direct monoclonal) ELISA is an immunoassay for the quantitative and sensitive detection of Microcystins and Nodularins in water samples. This test is suitable for the quantitative and/or qualitative detection of Microcystins and Nodularins in water samples [please refer to the appropriate technical bulletins for sample collection, handling, and treatment of drinking (treated and untreated) and recreational water samples]. If necessary, positive samples can be confirmed by HPLC, protein phosphatase assay, or other conventional methods.

The test is a direct competitive ELISA for the detection of Microcystins and Nodularins. It is based on the recognition of Microcystins, Nodularins, and their congeners by a monoclonal antibody. Toxin, when present in a sample, and a Microcystins-HRP analogue compete for the binding sites of anti-Microcystins antibodies in solution. The anti-Microcystins antibodies are then bound by a second antibody (goat anti-mouse) immobilized on the wells of the microtiter plate. After a washing step and addition of the substrate solution, a color signal is generated. The intensity of the blue color is inversely proportional to the concentration of Microcystins present in the sample. The color reaction is stopped after a specified time and the color is evaluated using an ELISA reader. The concentrations of the samples are determined by interpolation using the standard curve constructed with each run.

1. Microtiter plate (12 × 8 strips) coated with a second antibody (goat anti-mouse) 2. Standards (6): 0, 0.15, 0.40, 1.0, 2.0, 5.0 ppb, 1.5 mL each 3. Control: 0.75 ± 0.185 ppb, 1.5 mL, prepared from a secondary source, for use as a Quality Control Standard (QCS) 4. Sample Diluent, 25 mL, for use as a Laboratory Reagent Blank (LRB) and for dilution of samples above the range of the standard curve 5. Microcystins-HRP Conjugate Solution, 6 mL 6. Microcystins-DM Antibody Solution (monoclonal anti-Microcystins), 6 mL 7. Wash Buffer (5×) Concentrate, 100 mL, must be diluted prior to use, see Reagent Preparation 8. Substrate (Color) Solution (TMB), 16 mL 9. Stop Solution, 12 mL

1. Micro-pipettes with disposable plastic tips (20-200 μL) 2. Multi-channel pipette (50-300 μL), stepper pipette (50-300 μL), or electronic repeating pipette with disposable plastic tips 3. Deionized or distilled water 4. Container with 500 mL capacity (for diluted 1× Wash Buffer, see Reagent Preparation) 5. Paper towels or equivalent absorbent material 6. Timer 7. Tape or parafilm 8. Microtiter plate reader (wavelength 450 nm) 9. Microtiter plate washer (optional)

Collect water samples in glass or PETG containers and test within 24 hours. Use of other types of plastic containers may result in adsorptive loss of Microcystins, producing inaccurate (falsely low) results. Drinking water samples should be treated with sodium thiosulfate immediately after collection (refer to appropriate technical bulletin). If samples must be held for longer periods (up to 5 days), samples should be stored refrigerated. For storage periods greater than 5 days, samples should be stored frozen. If total Microcystins concentration (free and cell bound) is required, an appropriate cell lysing procedure must be performed prior to analysis. Note: The use of sonication in cell lysing can negatively affect toxin concentrations, producing falsely low sample results. Please see the appropriate sample preparation technical bulletin for additional information on cell lysis. Samples may be filtered prior to analysis using glass fiber filters. The use of alternate filter types (non-glass fiber filters) may produce falsely low sample results, as Microcystins may bind to the filter material, removing it from the sample. Also, please note that some glass fiber filters are manufactured using a process which may cause interference which would cause inaccurate (falsely high) results. To avoid this potential bias in sample results, a total volume of at least 10 mL should be passed through the glass fiber filter, with the first 5 mL of filtered sample being discarded and the second 5 mL collected for testing (please see the Microcystins sample filtration technical bulletin for additional information on sample filtration). If determining total Microcystins concentration, samples should be lysed prior to filtration to prevent the removal of cell-bound Microcystins, which would cause inaccurate (falsely low) results.

Notes and Precautions 1. Micro-pipetting equipment and pipette tips for pipetting the standards and samples are necessary. 2. The use of a multi-channel pipette, stepping pipette, or electronic repeating pipette is recommended for the addition of the antibody, enzyme conjugate, substrate, and stop solutions in order to equalize the incubation periods on the entire microtiter plate. 3. To avoid drift and obtain accurate results, the addition of the antibody, color, and stop solutions should be performed in less than 2 minutes for each reagent. If additions to the entire microtiter plate cannot be completed in less than 2 minutes, run size should be decreased to the number of rows which can be pipetted in less than 2 minutes. 4. Please only use the reagents and standards from one kit lot in one test, as they have been adjusted in combination. Test Preparation 1. Allow the reagents and samples to reach ambient temperature before use. 2. Remove the number of microtiter plate strips required from the resealable pouch. The remaining strips are stored in the pouch with the desiccant (tightly closed). 3. The standards, control, sample diluent (LRB), enzyme conjugate, antibody, substrate, and stop solutions are ready to use and do not require any further dilutions. 4. Dilute the Wash Buffer (5×) Concentrate at a ratio of 1:5 with deionized or distilled water. If using the entire bottle (100 mL), add to 400 mL of deionized or distilled water and mix thoroughly.

1. Add 100 μL of the standard solutions, control, or samples into the wells of the test strips according to the working scheme given. Analysis in duplicate or triplicate is recommended. 2. Add 50 μL of the enzyme conjugate solution to the individual wells successively using a multi-channel pipette or a stepping pipette. 3. Add 50 μL of the antibody solution to the individual wells successively using a multi-channel pipette or a stepping pipette. Cover the wells with parafilm or tape and mix the contents by moving the strip holder in a circular motion on the benchtop for 30 seconds. Be careful not to spill the contents. Incubate the strips for 90 minutes at room temperature. 4. Remove the covering, decant the contents of the wells into a sink, and blot the inverted plate on a stack of paper towels. Wash the strips three times using the diluted wash buffer. Please use at least a volume of 250 μL of 1× wash buffer for each well and each washing step. Blot the inverted plate after each wash step on a stack of paper towels. After the last wash/blot, check the wells for any remaining buffer in the wells, and if necessary, remove by additional blotting. 5. Add 150 μL of substrate (color) solution to the individual wells successively using a multi-channel pipette or a stepping pipette. Cover the wells with parafilm or tape and mix the contents by moving the strip holder in a circular motion on the benchtop for 30 seconds. Be careful not to spill the contents. Incubate the strips for 20-30 minutes at room temperature. Protect the strips from sunlight. 6. Add 100 μL of stop solution to the wells in the same sequence as for the substrate (color) solution using a multi-channel pipette or a stepping pipette. 7. Read the absorbance at 450 nm using a microplate ELISA photometer within 15 minutes after the addition of the stopping solution.

The evaluation of the ELISA can be performed using commercial ELISA evaluation programs such as 4- Parameter (preferred) or Logit/Log. For a manual evaluation, calculate the mean absorbance value for each of the standards. Calculate the %B/B 0 for each standard by dividing the mean absorbance value of each standard by the Zero Standard (Standard 0) mean absorbance. Construct a standard curve by plotting the %B/B 0 for each standard on a vertical linear (y) axis versus the corresponding Microcystins concentration on the horizontal logarithmic (x) axis on graph paper. %B/B 0 for the control and samples will then yield levels in ppb of Microcystins by interpolation using the standard curve. The concentrations of the samples are determined using the standard curve run with each test. Samples showing a lower concentration of Microcystins than standard 1 (0.15 ppb) should be reported as containing Semi-quantitative results can be derived by simple comparison of the sample absorbances to the absorbances of the standards. Samples with lower absorbances than a standard will have concentrations of Microcystins greater than that standard. Samples which have higher absorbances than a standard will have concentrations of Microcystins less than that standard.

Coefficients of variation (CVs) for standards:

The detection limit for this assay, based on MC-LR, is 0.10 ppb (μg/L).

Four (4) groundwater samples were spiked with various levels of Microcystin-LR and assayed using the Microcystins (ADDA)-DM Assay:

1. Numerous organic and inorganic compounds commonly found in water samples have been tested and found not to interfere with this test. However, due to the high variability of compounds that might be found in water samples, test interferences caused by matrix effects cannot be completely excluded. 2. The presence of the following substances were found to have no significant effect on the Microcystins (ADDA)- DM assay results: calcium sulfate, magnesium sulfate, sodium chloride, magnesium chloride, sodium nitrate, potassium phosphate, calcium chloride, manganese sulfate, and aluminum oxide up to 10,000 ppm; copper chloride, sodium fluoride, sodium thiosulfate, ferric sulfate, and zinc sulfate up to 1,000 ppm; humic acid up to 10 ppm; Lugol's solution up to 0.01%. 3. Samples containing methanol must be diluted to a concentration ≤ 20% methanol to avoid matrix effects. 4. Seawater samples must also be diluted to a concentration ≤ 20% to avoid matrix effects. Alternately, if a lower detection limit is required, interfering compounds can be removed from seawater or brackish water samples prior to analysis. 5. No matrix effects have been observed with samples which have been treated with sodium thiosulfate at concentrations ≤ 1 mg/mL. 6. Mistakes in handling the test can cause errors. Possible sources for such errors can include: inadequate storage conditions of the test kit, incorrect pipetting sequence or inaccurate volumes of the reagents, too long or too short incubation times during the immune and/or substrate reaction, extreme temperatures during the test performance (lower than 10°C or higher than 30°C). The assay procedure should be performed away from direct sunlight. 7. As with any analytical technique (GC, HPLC, etc.), positive results requiring regulatory action should be confirmed by an alternative method.

The Microcystins (ADDA)-DM (direct monoclonal) ELISA is an immunoassay for the quantitative and sensitive detection of Microcystins and Nodularins in water samples. This test is suitable for the quantitative and/or qualitative detection of Microcystins and Nodularins in water samples [please refer to the appropriate technical bulletins for sample collection, handling, and treatment of drinking (treated and untreated) and recreational water samples]. If necessary, positive samples can be confirmed by HPLC, protein phosphatase assay, or other conventional methods.

1. Microtiter plate (12 × 8 strips) coated with a second antibody (goat anti-mouse) 2. Standards (6): 0, 0.15, 0.40, 1.0, 2.0, 5.0 ppb, 1.5 mL each 3. Control: 0.75 ± 0.185 ppb, 1.5 mL, prepared from a secondary source, for use as a Quality Control Standard (QCS) 4. Sample Diluent, 25 mL, for use as a Laboratory Reagent Blank (LRB) and for dilution of samples above the range of the standard curve 5. Microcystins-HRP Conjugate Solution, 6 mL 6. Microcystins-DM Antibody Solution (monoclonal anti-Microcystins), 6 mL 7. Wash Buffer (5×) Concentrate, 100 mL, must be diluted prior to use, see Reagent Preparation 8. Substrate (Color) Solution (TMB), 16 mL 9. Stop Solution, 12 mL

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet