The principle of (ELISA) enzyme-linked immunosorbent assay) is as follows:
Immunoassay is a method that uses the principle of highly selective and specific recognition and binding of a specific antibody to an antigen or semi-antigen to analyze and determine the antibody or antigen to be measured.
In ELISA, immune recognition is performed by coating a 96-well plate with antibodies made of polystyrene and then using the antibodies to recognize the antigen to be measured (usually a protein biomarker of disease, virus, bacteria, etc.) and adsorbing the antigen from the complex solution to be measured onto the surface of the 96-well plate. Next, antibodies with Horseradish Peroxidase (HRP), fluorescent or radiolabeled antibodies are used to output the recognition signal either directly or indirectly. Finally, the concentration of the target antigen in the sample to be measured is calculated using information such as signal intensity and concentration gradient of the standard sample.
The antibody is encapsulated on a 96-well plate. Since in ELISA, the plate is made of polystyrene, which contains a benzene ring with amino acid residues of the antibody with a gravitational force similar to π-π stacking action, combined with electrostatic and hydrophobic effects, the antibody can be adsorbed on its surface. The unadsorbed antibodies are then washed with buffer and a blocking solution containing gelatin or Bovine Serum Albumin (BSA) is added to seal the unbound antibody portion of the enzyme plate. The purpose of adding the closure solution is to prevent other proteins from adsorbing on the 96-well plate due to electrostatic or hydrophobic effects, causing false positive signals and interfering with subsequent experiments.
Immunorecognition After coating the 96-well plate with the antibody, the sample to be tested is added and incubated at 37°C for some time, usually 1-2 hours. At this time, the antibody on the enzyme-labeled plate binds to the antigen to be tested for specific recognition. The quality of the antibody is critical here, as a good antibody will bind the antigen specifically and efficiently without being affected by other components of the sample to be tested, such as biomolecules, proteins, and inorganic salts.
Wash the plate which is in ELISA. Wash off the unbound antigen, add the recognition antibody corresponding to that antigen and continue to incubate at 37°C for 1-2 hours, followed by washing off the antibodies that are not attached to the antigen.
Enzyme-labeled signal output Enzyme-labeled secondary antibody with Horseradish Peroxidase (HRP) labeling is added for binding to the standard antibody. After 30 minutes of incubation, the unconjugated secondary antibody is washed off and a color developer is added to develop the color. The concentration of the antigen is determined based on the result of color development. It is generally believed that the concentration of antigen is positively correlated with the intensity of luminescence after color development.
In the case of antigen-competing antibody, for example, immediately after the addition of the antigen to be tested in the second step of the above immunosandwiching operation, the enzyme-labeled antigen is added so that it competes with the antigen to be tested to recognize the antibody on the enzyme-labeled plate. When the concentration of the antigen to be tested is higher, fewer enzyme-labeled antibodies can be attached to the antibodies on the enzyme plate, and less signal will be output. In this way, the concentration of the sample to be tested is inversely correlated with the enzyme chromogenic signal.