ELISA technology’s technical details

ELISA: The full name is enzyme-linked immunosorbent test. Its principle is: during the measurement, the test specimen reacts with the antigen or antibody on the surface of the solid-phase carrier, and the antigen-antibody complex formed on the solid-phase carrier is washed into the solution. Separate the other substances, add enzyme-labeled antigen or antibody, and bind to the solid-phase carrier by reaction. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme into a colored product. The amount of the target substance is directly related, so it can be qualitatively or quantitatively analyzed according to the shade of the color.

Reagents and instruments: including 3 necessary reagents and other reagents and instruments

3 necessary reagents:

(1) Antigen or antibody in solid phase

The commonly used solid-phase carrier is polystyrene, which has strong protein adsorption performance, and the antibody or protein antigen retains its original immunological activity after being adsorbed on it. The process of immobilizing antigen or antibody on polystyrene is “coating”, and the two are combined by physical adsorption.

(2) Enzyme-labeled antigen or antibody

The lgG with higher purity is used when preparing the conjugate, so as to avoid the interference of other contaminated proteins when it is connected with the enzyme.

(3) Substrate for enzyme reaction

Other reagents and instruments:

Operation steps: Double antibody sandwich method for detecting unknown antigens (eg: ELISA double antibody sandwich method to determine the level of IFN-γ in the culture supernatant)

1) Coating: Take a blank 96-well plate, dilute the capture antibody with PBS according to COA, 100μl capture antibody working solution per well, overnight at room temperature;

 2) Wash the plate: 350μl washing solution per well, wash the plate 3 times;

3) Blocking: add 200 μL/well of blocking solution to block the ELISA plate, and incubate at room temperature for 1 hour;

4) Wash the plate: shake off the sealing liquid in the plate, add 350μL/well of the washing solution, and wash the plate 3 times;

6) Adding samples: Take out the coated and sealed 96-well plate, add 100μl standard or diluted sample respectively, make a duplicate well with the standard, and incubate for 2h at room temperature;

7) Wash the plate: 350μl washing solution per well, wash the plate 3 times;

8) Add enzyme-labeled antibody: Dilute detection antibody with diluent according to COA, 100μl detection antibody working solution per well, incubate at room temperature for 2h;

9) Wash the plate: 350μl washing solution per well, wash the plate 3 times;

10) Add substrate: 100μl color developing solution per well, incubate in the dark at room temperature for 20 minutes, and observe the color change continuously;

11) Add stop solution: 50μl stop solution per well, mix gently;

12) Reading the plate: detection According to the operation method of the enzyme-labeled instrument, the OD value is detected on the computer, and the concentration of the measured substance is obtained by calculation.

Precautions:

1. Sample collection and storage: hemolysis and lipemia samples should be avoided, which are prone to non-specific color interference;

2. The distilled or deionized water used in ELISA, including those used for washing, should be fresh and high-quality;

3. When adding samples, add the added substance to the bottom of the well of the ELISA plate, avoid it on the upper part of the well wall, and be careful not to splash or generate bubbles;

4. It is best to use a microplate shaker for washing.