The enzyme-linked immunosorbent assay is a method for detecting unknown antigens or antibodies with enzyme-labeled antibodies (or SPA). This method has high sensitivity and strong specificity. Commonly used are ELISA method, indirect method, double antibody method and antigen method. This test introduces the enzyme-linked staphylococcal protein A immunoassay. The principle of ELISA method is as follows, see Figure 9
1. Add antigen to make it adsorb to the solid carrier
2. Wash and test serum
3. Wash with enzyme labeled SpA
4. Wash the substrate with enzyme added. Enzymatic catalysis produces colored products.
material:
1. Polyethylene plastic reaction plate (absorb protein Ag at PH9.6)
2. Antigen: boiled or sonicated antigen of typhoid bacillus O901
3. Serum to be tested
4. Freeze-dried enzyme-linked staphylococcus protein A (HRD-proteinA) pure product
5. O-phenylenediamine (OPD)
6. Coating solution: 0.05M sodium carbonate-sodium bicarbonate solution PH9.6
7. Diluent: 10% immune serum PBS-Tween 20, to prevent non-specific adsorption
8. Washing solution: 0.02MTrisTWeen20, Ph7.4 to prevent non-specific adsorption
9. Substrate solution: 0.02M disodium hydrogen phosphate 25.7ml
0.1M citric acid 24.3ml distilled water 50ml freshly prepared before use, dissolve 40mg o-phenylenediamine in the above buffer, then add 0.15ml 30% hydrogen peroxide, the substrate is sensitive to light, need to avoid light and use immediately.
10. Stop solution; 2M sulfuric acid method:
1. Coat the antigen with a clean polystyrene micro-reaction plate, add 0.1 ml of typhoid antigen to each well (diluted with coating buffer, protein content 10 μg / ml), set at 37 ℃ for 1 hour, discard the antigen solution and wash with Solution 3 times for 3 minutes each time.
2. Add test serum to each well and add different dilutions (such as 1: 5, 1:10, 1:20 ... 1:80) of test serum, PBS blank control, negative control serum, positive control serum 0.1 ml each . Incubate at 37 ° C for 30 minutes and wash 3 times.
3. Add enzyme-linked protein A to each well. Add 0.1 ml of enzyme-linked protein A to each well, set at 37 ° C for 20 minutes, and wash three times.
4. Add 0.1 ml each of the temporarily prepared substrate solution and place in the dark for 15 minutes. Add 1 drop of 2M carbonic acid to stop the reaction.
5. Judgment of results: (judgment by naked eyes)
If the color is the same as the negative control, it is negative, and if it is darker than the negative control, it is positive, which is indicated by () according to the color depth.
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