The basis of ELISA is the immobilization of antigen or antibody and the enzymatic labeling of antigen or antibody. The antigen or antibody bound to the surface of the solid phase carrier still retains its immunological activity, and the enzyme-labeled antigen or antibody retains both its immunological activity and the enzymatic activity. The test sample reacts with the antigen or antibody on the surface of the solid support.
The antigen-antibody complex formed on the solid phase carrier is separated from other substances in the liquid by washing. The enzyme-labeled antigen or antibody is added, and it is also bound to the solid-phase carrier through the reaction. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme to become a colored product, and the amount of the product is directly related to the amount of the tested substance in the sample, and qualitative or quantitative analysis is carried out according to the color depth. The catalytic efficiency of the enzyme is very high, indirectly amplifying the results of the immune reaction, making the assay method reach a high sensitivity.
Certain secreted proteins can be detected by ELISA after interference with siRNA.
3. Reagents and consumables
(1) Coating buffer (pH 9.6 0.05 M carbonate buffer):
(2) Washing buffer (pH 7.4, 0.15 M PBS):
(4) Stop solution (2 M H2SO4):
(5) Substrate buffer (pH 5.0):
(6) Tetramethylbenzidine (TMB) solution:
(7) 2,2′-azino-bis-3-ethyl-phenprothiazoline sulfonamide (ABTS) using solution:
(8) Antigen, antibody and enzyme-labeled antibody: handle according to the instructions.
(9) Standard: Dilute with diluent to form gradient concentration solution.
(10) 96-well polystyrene plastic plate (ELISA plate).
4. Experimental steps (double antibody sandwich method):
(1) Coating: Dilute the antibody with coating buffer to a protein content of 1-10 μg/mL. Add 0.1 mL to the reaction well of the microtiter plate and keep it at 4°C overnight. The next day, the solution in the well was discarded, and the plate was washed 3 times with washing buffer, 3 min each time.
(2) Sample addition: Add 0.1 mL of the sample to be tested diluted at a certain concentration (at the same time as blank control, negative control well and positive control) to the above coated reaction wells, put in a humid box, 37 ℃, 1 hr. Wash the plate 3 times with wash buffer for 3 min each.
(3) Add enzyme-labeled antibody: Add 0.1ml of freshly diluted enzyme-labeled antibody (dilution after titration) to each reaction well. 37°C, 0.5 – 1 hr, wash the plate 3 times with washing buffer, 3 min each time.
(4) Add substrate solution for color development: add 0.1 mL of the ready-made TMB substrate solution to each reaction well, 37 ℃, 10-30min.
(5) Stop the reaction: add 0.05 mL of stop solution to each reaction well.
(6) Judgment of results: put the microplate on the microplate reader, read at 450 nm (if ABTS develops color, read at 410 nm), read and output to Excel.
(7) Take the standard concentration as the abscissa and the absorbance as the ordinate, generate a standard curve and a linear regression equation, calculate the concentration of the unknown sample according to the formula, and record it.