Introduction of ELISA kit sandwich method for the detection of interleukin-8 - Database & Sql Blog Articles

Il-8

It is a polypeptide with a molecular weight of 8-10 kD. It has chemotactic effects on neutrophils, T lymphocytes and eosinophils, and activates neutrophils. It is an important inflammatory mediator. High levels of IL-8 can be detected in serum of severely infected patients and in local exudates of inflammation. In addition, recent studies have shown that IL-8 may be involved in the development of various tissue ischemia-reperfusion injury. We used two self-made monoclonal antibodies to establish a sandwich ELISA for the detection of IL-8 with a sensitivity of 156 Pg/ml. The method is simple and reproducible, and can be used for basic and clinical research of IL-8.

1. Principle: Two strains of anti-IL-8 monoclonal antibodies recognizing different epitopes were used, one of which (4D7) was used as a coating antibody to recognize and bind IL-8 in the specimen to be tested, and the other strain was used as an enzyme label. The antibody binds to another epitope that binds to the antibody-coated IL-8 and catalyzes the development of the substrate.

Second, reagent equipment

1.

Kit

Coated antibodies, enzyme-labeled antibodies, standards, substrate (ABTS), 96-well elisa plates are provided.

2. The coating buffer, PBS, and substrate buffer were prepared in the same manner as the conventional ELISA method.

Third, the operation steps:

1. Coating: pH 9.5 carbonate slow solution diluted 4D7 monoclonal antibody crude gamma globulin to 1 μg / ml, added to a 96-well plate, 100 μl / well, placed at 4 ° C, 36 hours.

2. Blocking: 0.1% Tween PBS (Buffer A) was rinsed three times with 96-well plates, 3% BSA Buffer A (Buffer B) 200 μl/well, and placed at 37 ° C for 1 hour.

3. Add the sample to be tested: Buffer A. Rinse the 96-well plate three times, add 100 μl/well of the sample to be tested and Buffer B diluted, and put it at 37 ° C for 3 hours.

4. Add enzyme labeling antibody: Buffer A was rinsed five times in 96-well plate, 3% PEG Buffer A diluted enzyme-labeled antibody to 1:800, added to 96-well plate, 100 μl/well, and placed at 37 ° C for 1 hour.

5. Color development: Buffer A rinse 96-well plate five times, pH5.4 citrate buffer solution substrate (ABTS), 0.2mg/ml, add 3%H2O2, 2μl/ml, add 96-well plate, 100μ/well , develop color at room temperature or 37 ° C.

Fourth, note:

1. If the specimen to be tested is serum, a disposable container should be used, and serum should be separated as soon as possible after blood collection (within 6 hours).

2. The blocking solution or antibody dilution should be freshly prepared or frozen.