Coating method of ELISA kit:
Fixing the antigen or antibody in a process is called coating. In Other words, coating is the process of binding an antigen or antibody to the surface of a solid support. The solid phase carrier of protein and polystyrene is combined by physical adsorption, relying on the interaction between the hydrophobic groups on the molecular structure of the protein and the hydrophobic groups on the surface of the solid phase carrier. This physical adsorption is non-specific and is affected by the molecular weight, isoelectric point, concentration, etc. of the protein. The adsorption capacity of the carrier to different proteins is different. Larger proteins and smaller molecules usually contain more hydrophobic groups, so they are more easily adsorbed on the surface of the solid phase carrier. IgG has a strong adsorption force on solid phases such as polystyrene, and its coupling occurs mostly on the Fc segment, and the antibody binding point is exposed to the outside. Therefore, the direct coating method is generally used for antibody coating. Most protein antigens can also be coated by methods similar to antibodies. When the antigenic determinant is in or adjacent to the hydrophobic region, the direct adsorption of the antigen and the solid phase carrier can make the antigenic determinant not fully exposed. In this case, the direct coating effect is not good, and indirect capture coating can be used The method is to pre-coat a specific antibody against the antigen, and then solidify the antigen through the antigen-antibody reaction. The antigen indirectly bound to the solid phase is far away from the surface of the carrier, and its antigenic determinants are also fully exposed. The indirect coated antigen is subjected to affinity chromatography with solid-phase antibodies, and the purity of the antigen coated on the solid phase is greatly improved. Therefore, the antigen with more impurities can also be captured by the coating method. The specificity and sensitivity of the test Both have been improved and repeatability is improved. Another advantage of indirect coating is that the amount of antigen is small, only 1/10 or even / 100 of direct coating. Non-protein antigens that are not easily adsorbed on polystyrene carriers can be coated in a special way. For example, when detecting anti-DNA antibodies, DNA is used as the coating antigen, and the general solid-phase carrier generally cannot directly bind to nucleic acids. The polystyrene board can be irradiated with ultraviolet rays (for example, 30W ultraviolet lamp, 75cm irradiation for 12 hours) to increase its adsorption performance. The solid-phase carrier is first pre-coated with alkaline proteins, such as polylysine, protamine, etc., which can also improve the binding capacity of nucleic acids. The avidin biotin system can also be used for indirect coating, that is, the carrier is coated with avidin first, and then biotinylated DNA is added. This coating method is uniform and firm, and has been widely applied to the quantification of various antigenic substances. Determination.
The lipid substance cannot be combined with the solid phase carrier. It can be dissolved in an organic solvent (such as ethanol) and added to the well of the ELISA plate. The lid is opened and placed in the refrigerator overnight or dried with cold air. After the alcohol volatilizes, let the lipid dry naturally On the solid surface. Anti-cardiolipin antibody ELISA reagents generally adopt this coating method.
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