Enzyme-linked immunosorbent assay (ELISA) for the determination of antibodies against S. typhi "O"

Summary

Enzyme-linked immunosorbent assay (ELISA) is a method of detecting unknown antigens or antibodies with enzyme-labeled antibodies (or SPA). This method is highly sensitive and specific. Commonly used are ELISA method, indirect method, double antibody method and antigen method.

Operation method

Determination of antibodies to S. typhi "O"

Materials and Instruments

Typhoid bacillus
O-phenylenediamine Sodium carbonate Sodium bicarbonate Sodium bicarbonate PBS Disodium hydrogen phosphate Citric acid
Polyethylene plastic reaction plate Centrifuge Shaking table Incubator

Move

1. Antigen encapsulationTake clean polystyrene micro reaction plate, add typhoid antigen 0.1 ml (diluted with encapsulation buffer, protein content of 10 μg/ml) in each well, put it at 37℃ for 1 hour, discard the antigenic solution, use the washing solution 3 times for 3 minutes each time.
2. Add serum to be testedAdd 0.1 ml each of different dilutions (e.g. 1:5, 1:10, 1:20, 1:80) of the serum to be tested, PBS blank control, negative control serum and positive control serum into each well. Place at 37°C for 30 minutes and wash 3 times.
3. Enzyme-linked A proteinAdd 0.1 ml of each enzyme-linked A protein to each well, place at 37°C for 20 minutes, and wash three times.
4. Add 0.1 ml of each of the temporarily prepared substrate solutions and place in the dark for 15 minutes. Add 1 drop of 2M carbonic acid to terminate the reaction.
5. Judgment of results: (visual judgment)If the color is the same as the negative control, it is negative; if it is darker than the negative control, it is positive, and the color is indicated by (+) according to the depth of the color.

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