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 Move 1. Envelope 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. For more product details, please visit Aladdin Scientific website.
Buffer PBS
ELISA plates
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 ℃.