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Rat IgM Quantitative EIA Kit Instructions
principle
This experiment used a double antibody sandwich ELISA method. The anti-rat IgM monoclonal antibody is coated on the microtiter plate, the IgM in the standard and the sample is combined with the monoclonal antibody, and the enzyme-labeled antibody is added to form an immune complex, which is attached to the plate, and the enzyme substrate TMB is added to appear blue. Add stop solution sulfuric acid, the color turns yellow, measure the OD value at 450nm, the IgM concentration is directly proportional to the OD value, and the IgM concentration in the sample can be obtained by drawing a standard curve.
Kit composition ( 2-8 ° C preservation)
Coated Wells | 96 holes | Enzyme Conjugate | 12ml |
10× specimen dilution (Sample Buffer) | 12ml | 20×Wash Buffer | 50ml |
Standards: 10ug/bottle | 2 bottles | Substrate working fluid (TMB Solution) | 12ml |
Cloth paper | One | Stop Solution | 12ml |
Prepare reagents and collect blood samples
1. Collection of specimens: serum, plasma (EDTA, heparin anticoagulation), cell culture supernatant, urine, etc., as early as possible, stored at 2-8 ° C for 48 hours; longer time must be frozen (-20 ° C or -70 ° C) Save to avoid repeated freezing and thawing. With urine specimen measured before dilution is 1:10 dilution (take 20ul, sample plus diluent 180ul, diluted 10 times). Serum or plasma is diluted at least 1:500,000 times.
2. Standard solution preparation: Add 1ml of distilled water before use and mix well to form a 10000ng/ml solution. Set the standard tube 8 tube, the first tube plus the standard dilution 900ul, the second to the eighth tube to add the sample dilution 500ul. Add 100 ul of the 10000 ng/ml standard solution to the first tube, and then aspirate 500 ul with the sampler and transfer to the second tube. Repeat the dilution as described above, and remove 500 ul from the seventh tube and discard it. The eighth tube is a blank control.
3. The 10× specimen dilution was diluted 1:10 with distilled water (example: 1 ml concentrated dilution + 9 ml distilled water).
4. Washing solution: diluted 1:20 with distilled water (example: 1 ml concentrated washing solution added to 19 ml of distilled water)
Test procedure
1. Loading: Add 100 ul of standard or sample to be tested in each well. Mix the reaction plate thoroughly and let it stand at 37 °C for 120 minutes.
2. Wash the plate: Wash the plate thoroughly with washing solution 4-6 times, and dry it on the filter paper. .
3. Add 100 ul of enzyme-labeled antibody working solution per well. The reaction plate was placed at 37 ° C for 60 minutes.
4. Wash the board: the same as before.
5. Add 100 ul of substrate working solution to each well and let it react at 37 ° C for 15 minutes in the dark.
6. Add 100 ul of stop solution to each well and mix.
7. Measure the absorbance at 450 nm using a microplate reader within 30 minutes.
Result calculation and judgment
1. All OD values ​​should be subtracted from the blank value before calculation.
2. Take the standard products 1000, 500, 250, 125, 62, 31, 15.6, 0 ng/ml as the abscissa and OD as the ordinate. Draw on the coordinate paper and draw the standard curve.
3. According to the OD value of the sample, find the corresponding IgM content on the graph, and then multiply the dilution factor.
Kit performance
1. Sensitivity: The minimum IgM detection concentration is less than 8 ng/ml.
2. Specificity: Recombinant or natural rat IgM can be detected simultaneously. Does not cross-react with other cytokines in rats.
3. Repeatability: The coefficient of variation in both the plate and the plate is less than 10%.
Precautions
1. It is recommended to make double holes for the above standard holes and samples to be tested. The standard curve should be made at the same time for each measurement.
2. The washing process is critical. Insufficient washing will result in an accuracy error and an erroneous rise in the OD value.
3. After the slats are opened, the remaining slats should be sealed again to keep the slats dry .
4. This kit should be stored in a 4oC refrigerator.
5. This kit is for scientific research only and cannot be used for clinical diagnosis!
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