What Is an ELISA Assay?

In 1971, Swedish scholars Engvall and Perlmann, Dutch scholars Van Weerman and Schurs reported that the development of immune technology into solid-phase immunoassay methods for detecting trace substances in body fluids, that is, enzyme-linked immunosorbent assay (Enzyme-Linked ImmunoSorbent Assay, ELISA). ELISA has now become a frontier subject in the field of analytical chemistry. It is a special reagent analysis method, which is a new type of immunoassay technology developed on the basis of immunoenzymatic techniques.

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Make the antigen or antibody bind to the surface of a certain solid carrier and keep it

In the formal test, the positive control and the

ELISA uses serum

The enzyme used for labeling antibodies or anti-antibodies must have the following characteristics: high activity and sensitivity; stable at room temperature; reaction products are easy to visualize; can be produced commercially. At present, horseradish peroxidase (HRP), alkaline phosphatase, glucose oxidase, etc. are widely used, among which HRP is the most widely used.

Enzyme and antibody cross-linking, commonly used glutaraldehyde method and periodate oxidation method. The modified sodium periodate method of HRP-labeled antibody established by Guo Chunxiang is simple and easy, and the labeling effect is good, which is especially suitable for small batch preparation in the laboratory. The labeling procedure is as follows: 5 g HRP is dissolved in 0.5 ml of distilled water, 0.5 ml of a freshly prepared 0.06 mol / L sodium periodate (NaIO4) aqueous solution is added, and the mixture is placed in a refrigerator at 4 ° C for 30 minutes. 0.5ml of ethylene glycol aqueous solution, leave it at room temperature for 30 minutes, add 1ml of aqueous solution containing 5g of purified antibody, mix and put in a dialysis bag, and use 0.05mol / L carbonate buffer at pH9.5 in a refrigerator at 4 ° C The mixture was slowly dialyzed for 6 hours (or overnight) to combine to combine, and then aspirated. Add sodium borohydride (NaBH4) solution (5 (g / ml) 0.2ml), put in a refrigerator at 4 ° C for 2 hours, and add the above mixture solution An equal volume of saturated ammonium sulfate solution was placed in a refrigerator at 4 ° C for 30 minutes and centrifuged. The obtained precipitate was dissolved in a little 0.02mol / L, pH 7.4PBS, and dialyzed against it overnight (4 ° C). The insolubles were removed by centrifugation the next day. That is, an enzyme-labeled antibody was obtained, diluted to 5 ml with 0.02 mol / L, pH 7.4 PBS, and then measured, and then freeze-dried or stored at low temperature.

Determination of the effect of enzyme-labeled antibody labeling: The measurement includes enzyme and antibody activity, enzyme content and IgG content in the conjugate, molar ratio of enzyme to IgG, and binding rate.