Rat Immunoglobulin A (IgA) ELISA Kit Instructions for Use
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Rat Immunoglobulin A (IgA) ELISA Kit Instructions for Use
 ( for serum, plasma, cell culture supernatants and biological fluids )
principle
This experiment used double antibody sandwich ABC-ELISA. The anti-rat IgA monoclonal antibody was coated on the microtiter plate, the IgA in the standard and the sample was combined with the monoclonal antibody, and biotinylated anti-rat IgA was added to form an immune complex attached to the plate, horseradish peroxidation. Enzyme-labeled Streptavidin is combined with biotin, the substrate working solution is blue, and finally the stop solution sulfuric acid is added. The OD value is measured at 450 nm. The IgA concentration is directly proportional to the OD value. IgA can be obtained by drawing a standard curve. concentration.
Kit composition ( 2-8 ° C preservation)
Coated Wells
96 holes
Enzyme Conjugate
12ml
10× specimen dilution (Sample Buffer)
12ml
20×Wash Buffer
50ml
Standards: 500ng/bottle
2 bottles
Substrate working fluid (TMB Solution)
12ml
Primary antibody working solution (Biotinylated Antibody)
12ml
Stop Solution
12ml
Prepare reagents and collect blood samples
1. Collection of specimens: serum, plasma (EDTA, citrate, heparin anticoagulation), cell culture supernatant, tissue homogenate, etc., as early as possible, stored at 2-8 ° C for 48 hours; longer time must be frozen (-20 ° C Or -70 °C) to avoid repeated freezing and thawing. Before the determination of all specimens, dilute at least 1 : 20 with the specimen dilution (take 10 ul, add 190 ul of the dilution, and dilute 20 times).
2. Standard solution preparation: Add 1ml of distilled water before use and mix well to form a 500ng/ml solution. Set 8 tubes of standard tubes, and add 300 ul of standard dilution solution to each tube. Add 500 ul of the standard solution of 500 ng/ml to the first tube, mix and aspirate 300 ul with the sampler, and transfer to the second tube. Repeat the dilution in this way, and remove 300 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 the first antibody working solution to each well. The reaction plate was thoroughly mixed and placed at 37 ° C for 60 minutes.
4. Wash the board: the same as before.
5. Add 100 ul of enzyme-labeled antibody working solution per well. The reaction plate was placed at 37 ° C for 30 minutes.
6. Wash the board: same as before.
7. Add 100 ul of substrate working solution to each well and let it react at 37 ° C for 15 minutes in the dark.
8. Add 100 ul of stop solution to each well and mix.
9. 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 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 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 IgA content on the graph, and then multiply the dilution factor.
Kit performance
1. Sensitivity: The minimum IgA detection concentration is less than 2 ng/ml.
2. Specificity: Recombinant or natural rat IgA can be detected simultaneously. Does not cross-react with other cytokines in rats.
3. Repeatability: The coefficient of variation in the plate and plate is less than 10.6%.
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!