Antigens are used to coat solid-phase carriers. Antigens can be divided into natural antigens, recombinant antigens and synthetic polypeptide antigens according to their sources. Natural antigens can be taken from animal tissues, microbial cultures, etc., and must be extracted and purified before being used for coating. For example, HBsAg can be extracted from the carrier's serum, general bacteria and viruses can be extracted from its culture, protein component antigens can be extracted from materials rich in this antigen, etc. (for example, AFP is extracted from cord blood or fetal liver) . Recombinant antigens are protein antigens expressed by antigen genes in plasmids, and E. coli or yeast are mostly used as plasmids. The advantage of recombinant antigens is that apart from the components of engineering bacteria, other impurities are few and non-infectious, but the purification technology is more difficult. Recombinant antigens using E. coli as a plasmid can not sufficiently remove E. coli components and used in ELISA. False positives may appear in the reaction. Anti-E. Coli antibodies are present in the serum because many subjects are infected with E. coli. Another characteristic of recombinant antigens is that they can use genetic engineering to prepare certain antigenic substances that cannot be separated from natural materials. For example, hepatitis C virus (HCV) has not been successfully cultured, and the content of HCV antigen in the serum of patients with hepatitis C is minimal. At present, most of the coating antigens used in the detection of anti-HCV ELISA are recombinant antigens prepared based on the HCV gene clone expression. In the diagnosis of infectious diseases, many recombinant antigens such as HBsAg, HBeAg and HIV antigens have been used in ELISA. Synthetic polypeptide antigen is a polypeptide fragment artificially synthesized based on the amino acid sequence of an antigenic determinant of a protein antigen molecule. Peptide antigens generally contain only one antigenic determinant, which has high purity and high specificity. However, because the molecular weight is too small, it is often difficult to directly adsorb on the solid phase. The coating of polypeptide antigen generally needs to be coupled with unrelated proteins such as bovine serum albumin (BSA), etc., and indirectly bound to the surface of the solid phase carrier by means of adsorption of the conjugate and the solid phase carrier. Another point of attention when using peptide antigens is that he can only detect the corresponding antibodies. A protein antigen often contains many different determinants that can cause antibody production, so other antibodies in the tested serum cannot react with the polypeptide antigen. In addition, some microorganisms often undergo antigen structure changes when they are mutated. In this case, coating with individual polypeptide antigens can cause missed detection of other antibodies.
The antibody coated with the solid phase carrier should have high affinity and high specificity, and can be obtained from antisera or ascites or culture fluid containing monoclonal antibodies. If the antigen used for immunization contains impurities (even a trace amount), hybrid antibodies will appear in the antiserum and must be removed (available by absorption method) before it can be used in ELISA to ensure the specificity of the test. Antiserum cannot be directly used for coating, IgG should be extracted first, usually using ammonium sulfate salting out and Sephadex gel filtration method. Generally, the crude IgG extracted by ammonium sulfate salting out can be used for coating, and the highly purified IgG is unstable. If high-affinity antibody coating is needed to improve the sensitivity of the test, affinity chromatography can be used to remove the non-specific IgG that contains more of the ELISA kit in the antiserum. The concentration of monoclonal antibody in ascites is higher and the specificity is stronger. Therefore, absorption and affinity chromatography treatments are not necessary. Generally, the ascites can be directly diluted after appropriate coating, and purified IgG can also be used if necessary. When applying monoclonal antibody coating, it should be noted that one monoclonal antibody is only directed against one epitope. In some cases, a mixed coating with multiple monoclonal antibodies can achieve better results.
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