Il-8 is a polypeptide with a molecular weight of 8-10 kD, which has chemotactic effects on neutrophils, T lymphocytes, and alkaliphilic granulocytes, and activates neutrophils, and is an important inflammatory mediator. High levels of IL-8 can be detected in the serum of patients with severe infections and in the exudate of inflammatory localizations.(Source: Laboratory guide to basic immunology in medicine, edited by Jin Boquan and Li Enshan, 1st edition, Beijing: World Book Publishing House, 1990)

Operation method

Sandwich ELISA

Principle

Two strains of anti-IL-8 monoclonal antibodies recognizing different epitopes were used, one (4D7) was used as a coated antibody to recognize and bind IL-8 in the specimen to be examined, and the other was used as an enzyme-labeled antibody that binds to another epitope of IL-8 bound to the coated antibody and catalyzes the substrate color development.

Materials and Instruments

Antibodies Standards ABTS
Buffer PBS
ELISA plates

Move

1. Envelope

Dilute 4D7 monoclonal antibody crude γ-globulin to 1 μg/ml in pH 9.5 carbonate buffer, add to 96-well plate, 100 μl/well, leave at 4 ℃ for 36 hours. Add 100 μl/well into 96-well plate and put at 4 ℃ for 36 hours.
2. Closure

Rinse 96-well plate three times with 0.1% Tween PBS (Buffer A), add 3% BSA Buffer A (Buffer B), 200 μl/well, put at 37 ℃ for 1 hour. Add 200 μl/well, put at 37 ℃ for 1 hour. 3.
3. Add samples to be examined

Buffer A rinse 96-well plate three times, add samples to be examined and Buffer B diluted standard 100 μl/well, put at 37 ℃, 3 hours. Add 100 μl/well of the sample to be examined and the standard diluted in Buffer B. Put the plate at 37 ℃ for 3 hours.
4. Add enzyme antibody

Rinse the 96-well plate five times with Buffer A. Dilute the enzyme antibody with 3% PEG Buffer A to 1:800 and add it into the 96-well plate, 100 μl/well. Dilute the enzyme antibody in 3% PEG Buffer A to 1:800 and add it into the 96-well plate, 100 μl/well, and put it at 37 ℃ for 1 hour.
5. Color development

Rinse the 96-well plate five times with Buffer A, dissolve the substrate (ABTS) in citrate buffer pH 5.4, 0.2 mg/ml, add 3% H2O2, 2 μl/ml, add to the 96-well plate, 100 μ/ml. ml, add 3% _H2O2, 2 μl/ml, add to 96-well plate, 100 μ/well, and develop color at room temperature or 37 ℃.

Caveat

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

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

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