ELISA, the enzyme-linked immunosorbent assay, its basic principle is to immobilize a certain concentration of antigen or antibody on the surface of a polystyrene microplate by physical adsorption, add the sample to be tested, and indirectly reflect the color of the enzyme marker. The presence or absence or amount of the tested antigen or antibody. ELISA technology, as one of immunolabeling technologies (including immunofluorescence technology, immunoradiation technology, immunoenzyme technology and immunocolloidal gold technology), has been widely used in scientific research and clinical experiments, and has the characteristics of rapid, qualitative or quantitative and even localization. .
Among the ELISA experimental methods, the more common methods include double antibody sandwich method, competition method, indirect method, double antigen sandwich method and capture method, which will be described in detail below.
1. Double antibody sandwich method
The basic principle of the double-antibody sandwich method is to immobilize a certain amount of coated antibody 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. Antibody, enzyme-labeled secondary antibody is used for color development with TMB substrate, and the color depth in the microplate is positively correlated with the concentration of the analyte.
The applicable conditions of this method are: macromolecular substances containing multiple antigenic epitopes. Because this detection method requires two antibodies, the problem of antibody pairing is involved. Generally speaking, there are several commonly used antibody pairing methods: the coating antibody is monoclonal antibody, and the detection antibody is polyclonal antibody; the coating antibody is monoclonal antibody, and the detection antibody is monoclonal antibody, but the two monoclonal antibodies target antigenic The position is different; the coating antibody is polyclonal antibody, and the detection antibody is polyclonal antibody, and these two polyclonal antibodies are generally derived from different hosts.
When applying the enzyme-labeled secondary antibody in this detection method, one principle that should be noted is that the secondary antibody must only be directed against the detection antibody, but not against the coating antibody. In view of the limitations of enzyme-labeled secondary antibodies, most manufacturers now introduce biotin avidin amplification systems, which improve the sensitivity of the reaction and are more convenient to apply. We only need to label the detection antibodies with biotin, not It is necessary to enzymatically label each secondary antibody from different sources, and only HRP labeling of avidin can save a lot of tedious work.
2. Competition Law
Competition law is generally divided into direct competition law and indirect competition law. Here we take direct competition law as an example to explain the principle. The basic principle of the direct competition method is to coat the microplate with a suitable concentration of coated antibody, add an irrelevant protein carrier to block the unbound sites, and add a standard (sample) and a biotin-labeled antigen material for competitive binding. After incubation at a suitable temperature and a certain period of time, after washing, HRP-labeled streptavidin was added for reaction, TMB developed color, and the color depth in the microplate was negatively correlated with the concentration of the analyte.
This method is generally used to detect small molecules with fewer epitopes, of course, this method can also be used to detect macromolecular antigens and even antibodies. When detecting macromolecular antigenic substances, due to the influence of steric hindrance, the detection method is not as sensitive as the double antibody sandwich method for detecting macromolecular antigenic substances. When this method detects antibodies, the two competing antibodies may have different sources, resulting in low convergence of the two antibodies, which makes the detection results less reliable.
3. Indirect method
Indirect methods are generally used to detect antibodies. The basic principle is: 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, Add substrate to develop color. Generally speaking, in the indirect method for antibody detection, due to the limitations of enzyme-labeled secondary antibodies, the generally detected antibody is total IgG.
4. Double antigen sandwich method
The double-antigen sandwich method is similar to the double-antibody sandwich method. The basic principle is to immobilize a known concentration of antigen on the surface of a polystyrene microplate, block the unbound sites with an irrelevant protein carrier, add the sample to be tested, and then Biotin-labeled antigen and HRP-labeled streptavidin are added, and the reaction is developed and terminated by TMB, and the concentration of the analyte can be calculated after reading by the microplate reader. Both the double-antigen sandwich method and the indirect method can detect antibodies, but the former detects all types of antibodies against the antigen, including IgG, IgM, IgA, IgD, and IgE, which cannot be done by the indirect method.
5. Capture method
Capture methods are generally used to measure IgM antibodies. The basic principle is: coat the anti-IgM antibody on a solid-phase microplate, block the unbound sites with an irrelevant protein carrier, add the sample to be tested, then add the antigenic substance, and then add the biotinylated antibody against the antigen. The concentration of the labeled specific antibody and HRP-labeled streptavidin can be calculated after color development with TMB substrate and termination of the reaction.
Since we detect the IgM content in the sample, it also contains a higher concentration of IgG antibodies. When using the general indirect method, the IgG antibodies and IgM antibodies will compete with the solid phase antigen for binding. The absolute advantage will inevitably lead to a relatively limited amount of IgM antibodies that can bind, and the final result is a false negative.