Other methods related to ELISA technology

Other methods related to ELISA technology

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Two-site one-step method

Shanghai Yueyan Biotechnology Co., Ltd.

In the determination of antigen by the double antibody sandwich method, if monoclonal antibodies against two different antigenic determinants on the antigen molecule are used as solid-phase antibodies and enzyme-labeled antibodies, respectively, the addition of specimens and enzyme-labeled antibodies can be performed Two steps and one step. This dual site not only simplifies the operation and shortens the reaction time, such as the use of high-affinity monoclonal antibodies, the sensitivity and specificity of the assay are also significantly improved. The application of monoclonal antibodies has brought the ELISA for antigen determination to a new level.

In the one-step measurement, attention should be paid to the hook effect, which is similar to the phenomenon of post-banding of excess antigen in the precipitation reaction. When the concentration of the antigen to be tested in the specimen is quite high, the excess antigen will bind to the solid-phase antibody and the enzyme-labeled antibody, respectively, instead of forming a sandwich complex, and the result will be lower than the actual content. False negative results can even occur when the hook effect is severe.

Competition assay

When the interfering substances in the antigen material are not easy to remove, or it is not easy to obtain enough purified antigen, this method can be used to detect specific antibodies. The principle is that the antibody in the specimen competes with a certain amount of enzyme-labeled antibody for binding to the solid-phase antigen. The more antibody in the specimen, the less enzyme-labeled antibody bound to the solid phase, so the positive reaction is lighter than the negative reaction. If the antigen is of high purity, it can be directly coated with the solid phase. If there are interfering substances in the antigen, direct coating is not easy to succeed, you can use the capture coating method, that is, first coat the antibody corresponding to the solid phase antigen, and then add the antigen to form the solid phase antigen. Wash to remove impurities in the antigen, and then add specimens and enzyme-labeled antibodies for competitive binding reaction. There are multiple modes for the detection of antibodies by the competition method. The specimen and the enzyme-labeled antibody can be competitively combined with the solid-phase antigen. This method is generally used for anti-HBc ELISA. Another mode is to add the specimen and the antigen to the solid-phase antibody together for competitive binding. After washing, the enzyme-labeled antibody is added to react with the antigen bound to the solid phase. Anti-HBe detection generally uses this method.

Its methods and characteristics are:
â‘ Enzyme-labeled antigen (antibody) has the same ability to bind to solid-phase antibody (antigen) as the unlabeled antigen or antibody in the sample or standard body; â‘¡Solid-phase antibody (antigen) and enzyme-labeled antigen (antibody) in the reaction system ) Is a fixed limit, and the binding site of the former is less than the molecular weight of the enzyme-labeled and non-labeled antigen (antibody);
â‘¢ After the immune reaction, the amount of enzyme-labeled antigen (antibody) measured in the complex on the solid-phase carrier (enzyme activity) is inversely proportional to the concentration of the unlabeled antigen (antibody) in the sample or standard.

ABS-ELISA method
ABS is an abbreviation for avidin and biotin system. Avidin is a glycoprotein with a molecular weight of 60,000, and each molecule is composed of 4 subunits that can bind to biotin. Biotin is a small molecule compound with a molecular weight of 244. The derivative-hydroxysuccinimide ester prepared by chemical methods can form biotin-labeled products with various types of large and small molecules such as proteins and sugars. The labeling method is quite simple. The combination of biotin and avidin has a strong specificity, its affinity is much greater than that of antigen and antibody, and the two are extremely stable once they are combined. Since one avidin can be combined with 4 biotin molecules, the ABS and ELISA methods can be divided into enzyme-labeled avidin-biotin (LAB) method and bridged avidin-biotin (ABC) method. Kind. Both use biotin-labeled antibodies (or antigens) instead of enzyme-labeled antibodies (antigens) in the original ELISA system. In LAB, solid-phase biotin first reacts with unlabeled avidin, and then enzyme-labeled biotin is added to further increase the sensitivity. In the early days, avidin was extracted from egg whites. This egg avidin is a basic glycoprotein and has strong adsorption to polystyrene carriers. It can increase the background when used in ELISA. Streptavidin extracted from Streptomyces has no such shortcomings, and it tends to replace the former in ELISA applications. Because ABS-ELISA uses two more reagents than ordinary ELISA, it increases the operation steps, and ABS-ELISA is not widely used in clinical testing.