The concept, principle and operation steps of ELISA

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 a highly sensitive test technique based on immunological reaction, which combines the specific reactions of antigens and tethers with the efficient catalytic action of enzymes on substrates. 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.

(B) Operating 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. Sample addition: 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 liquid to develop color: add 0.1ml of temporarily prepared TMB substrate solution to each reaction well at 37 ℃ for 10-30 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. O · D value can also be measured: On the ELISA detector, at 450nm (if ABTS color development, then 410nm), the blank control well is zeroed and the O · D value of each well is measured, if it is greater than the specified negative control OD 2.1 times the value 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. (Make blank, negative and positive well control at the same time) Add 0.1ml of fresh diluted enzyme-labeled secondary antibody (anti-antibody) to the reaction well, incubate at 37 ℃ for 30-60 minutes, wash, and wash with DDW one last time. 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):
NaCO3