ELISA, or enzyme-linked immunosorbent assay, refers to a qualitative and quantitative detection method that combines soluble antigens or antibodies with solid phase carriers such as polystyrene, and uses the specific binding of antigens and antibodies to perform immune reactions. ELISA is a comprehensive technology that organically combines the immune reactions of antigens and antibodies with the efficient catalytic reactions of enzymes. As one of the immunolabeling technologies (including immunofluorescence technology, immunoradiation technology, immunoenzyme technology, and immunocolloidal gold technology), it has been widely used in scientific research and clinical experiments, and has the characteristics of rapid, qualitative or quantitative, and even positioning.
1. Experimental principle
ELISA is mainly based on two characteristics of antigens or antibodies: 1) the antigens or antibodies fixed on the surface of the carrier will not be inactivated and still retain their immunoreactivity; 2) the enzyme-labeled antibodies or antigens have both immunological activity and enzyme catalytic activity. The basic principle of ELISA is to fix a certain concentration of antigens or antibodies on the surface of a polystyrene microplate by physical adsorption, add the specimen to be tested, and indirectly reflect the presence or absence or amount of the antigen or antibody to be tested by the depth of color development of the enzyme label. In practical applications, according to different designs, there are many specific methods and steps, including indirect methods for detecting antibodies, double antibody sandwich methods for detecting antigens, and antigen competition methods for detecting small molecule antigens or haptens. The more commonly used are double antibody sandwich methods and indirect methods.
The double antibody sandwich method is to fix a certain amount of coated antibodies on the surface of a polystyrene microplate by physical adsorption, add an irrelevant protein carrier to block the unbound sites, and then add the sample to be tested containing the antigen. After adding enzyme-labeled specific antibodies, the TMB substrate is used for color development. The depth of the color in the microplate is positively correlated with the concentration of the test substance. This method is suitable for the determination of divalent or more macromolecular antigens, but not for the determination of haptens and small molecule monovalent antigens, because they cannot form a two-site sandwich.
The indirect method is to coat a certain amount of antigen on a polystyrene microplate, add an irrelevant protein carrier to block the unbound sites, add the sample to be tested, and then add an enzyme-labeled secondary antibody. After incubation and washing, the substrate is added for color development. This method can detect various antibodies corresponding to the antigen with an enzyme-labeled antibody by simply replacing different solid-phase antigens. It is mainly used for the detection of pathogens and the diagnosis of infectious diseases.
2. Experimental flow chart
3. Experimental steps
3.1 Antigen detection by double antibody sandwich method
3.1.1 Coating: Dilute the antibody to a protein content of 1-10 μg/mL with 0.05 M PH9.6 carbonate coating buffer. Add 0.1 mL to each reaction well of the polystyrene plate and incubate at 4°C overnight. The next day, discard the solution in the well and wash 3 times with washing buffer, 3 min each time.
3.1.2 Sample addition: Add 0.1 mL of a certain dilution of the sample to be tested to the above-mentioned coated reaction wells, incubate at 37°C for 1 h and then wash. Blank wells, negative control wells and positive control wells must be set at the same time.
3.1.3 Add enzyme-labeled antibody: Add 0.1 mL of freshly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 0.5~1 h and then wash.
3.1.4 Add substrate solution for color development: Add 0.1 mL of freshly prepared TMB substrate solution to each reaction well, and color develop at 37°C in the dark for 10-30 min.
3.1.5 Stop reaction: Add 0.05 mL of 2 M sulfuric acid to each reaction well.
3.1.6 Determination: On the ELISA detector, adjust the blank well to zero, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The determination should be performed within 15 min after adding the stop solution.
3.2 Indirect method for antibody determination
3.2.1 Dilute the known antigen to 1-10 μg/mL with coating buffer, add 0.1 mL to each well, and incubate at 4°C overnight. Wash 3 times the next day.
3.2.2 Add 0.1 mL of the sample to be tested at a certain dilution to the coated reaction wells, incubate at 37 °C for 1 h, wash, and set up blank wells, negative control wells, and positive control wells.
3.2.3 Add 0.1 mL of freshly diluted enzyme-labeled second antibody to the reaction wells and incubate at 37 °C for 30-60 min. Finally, wash with DDW.
3.2.4 Add substrate solution for color development: Add 0.1 mL of freshly prepared TMB substrate solution to each reaction well, and color develop at 37 °C in the dark for 10-30 min.
3.2.5 Stop the reaction: Add 0.05 mL of 2 M sulfuric acid to each reaction well.
3.2.6 Determination: On the ELISA detector, adjust the blank well to zero, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The determination should be performed within 15 min after adding the stop solution.
4. Result Example
As shown in the figure, after the standard is diluted in multiples, the standard curve is drawn by measuring the OD value. By measuring the OD value of the sample, the concentration of the substance to be tested in the sample is obtained from the standard curve.
References
[1]. Lequin RM. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem. 2005 Dec;51(12):2415-8.
[2]. Aydin S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides. 2015 Oct;72:4-15. [3]. Hornbeck P, Winston SE, Fuller SA. Enzyme-linked immunosorbent assays (ELISA). Curr Protoc Mol Biol. 2001 May;Chapter 11:Unit11.2.
[4]. Liao JJ, Lewis JW. Qualifying ELISA data: combining information. J Biopharm Stat. 2000 Nov;10(4):545-58.