Principle of ELISA kit

ELISA (Shanghai Lianshuo Biotechnology Co., Ltd., one of the major suppliers of domestic immunology products)

ELISA is the abbreviation for Enzyme-Linked Immunosorbnent Assay. It is an immunoenzyme technology developed after immunofluorescence and radioimmunoassay. This technology has developed very rapidly since its introduction in the early 1970s, and it has been widely used in many fields of biology and medical sciences.

(1) Principle

ELISA is based on immunological reaction, a highly sensitive test technique that combines the specific reactions of antigen and antibody with the efficient catalytic effect of enzyme on substrate. Since the reaction of antigen and antibody is carried out in the well of a solid phase carrier-polystyrene microtiter plate, after each reagent is added to the incubation, excess free reactants can be removed by washing to ensure the specificity of the test results With stability. In practical applications, through different designs, there are many specific method steps. That is: the indirect method for detecting antibodies (Figure a), the double antibody sandwich method for detecting antigen (Figure b), and the antigen competition method for detecting small molecule antigens or haptens, etc. More commonly used are ELISA double antibody sandwich method and ELISA indirect method.

(2) Operation steps

Method 1: Double antibody sandwich method for detecting unknown antigens:

1. Coating: Dilute the antibody with 0.05M PH9. Carbonate coating buffer to a protein content of 1 ~ 10μg / ml. Add 0.1ml to the reaction well of each polystyrene plate, overnight at 4 ℃. The next day, the solution in the well was discarded and washed 3 times with washing buffer for 3 minutes each time. (Referred to as washing, the same below).

2. Add sample: add 0.1ml of the diluted test sample to the above-mentioned coated wells and incubate at 37 ℃ for 1 hour. Then wash. (Make blank wells, negative control wells and positive control wells at the same time).

3. Add enzyme-labeled antibody: add 0.1ml of freshly diluted enzyme-labeled antibody (dilution after titration) to each reaction well. Incubate at 37 ° C for 0.5 to 1 hour and wash.

4. Add substrate to develop color: add 0.1ml of temporarily prepared TMB substrate solution to each reaction well at 37 ℃ for 10 to 30 minutes.

5. Terminate the reaction: Add 0.05ml of 2M sulfuric acid to each reaction well.

6. Judgment of results: The results can be directly observed with the naked eye on a white background: the darker the color in the reaction well, the stronger the positive degree, and the negative reaction is colorless or extremely light. "-" Indicates. OD value can also be measured: on ELISA detector, at 450nm (410nm if ABTS color is developed), the OD value of each well is measured after zero adjustment with a blank control well, if it is greater than 2.1 times the OD value of the specified negative control , That is positive.

Method 2: Indirect method for detecting unknown antibodies:

Dilute the known antigen to 1 ～ 10μg / ml with coating buffer, add 0.1ml per well, and overnight at 4 ℃. Wash 3 times the next day.

Add 0.1ml of the diluted test sample (unknown antibody) to the coated wells, incubate at 37 ℃ for 1 hour, and wash. (Do blank, negative and positive well control at the same time) ↓

Add 0.1ml of freshly diluted enzyme-labeled secondary antibody (anti-antibody) to the reaction well, incubate at 37 ° C for 30-60 minutes, wash, and finally wash with DDW. ↓

The remaining steps are the same as 4, 5 and 6 of the "double antibody sandwich method".

(3) Reagent equipment

Reagent

(1) Coating buffer (PH9.6 0.05M carbonate buffer):

NaHCO3 1.59g

NaHCO3 2.93g

Add distilled water to 1000ml

(2) Washing buffer (PH7.4 PBS): 0.15M

KH2PO4 0.2g

Na2HPO4 · 12H2O 2.9g

NaCl 8.0g

KCl 0.2g

Tween-20 0.05% 0.5ml

Add distilled water to 1000ml

(3) Diluent:

Bovine serum albumin (BSA) 0.1 g

Add wash buffer to 100ml

Or use 5-10% of sheep serum, rabbit serum and other serums and washing solution.

(4) Stop solution (2M H2SO4):