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Double antibody sandwich method:
The double antibody sandwich method is the most commonly used method for antigen detection. The sandwich method uses two primary antibodies to capture and immobilize the target antigen, which greatly increases the specificity of the reaction while ensuring sensitivity. The detection sensitivity of the method can be increased to the level of pg. The known antibody is coated on the surface of the solid phase, and the sample to be examined is added. If the sample contains the corresponding antigen, that is, the antibody is bound to the surface of the solid phase, the unbound component is washed and removed, and the antigen-specific enzyme-labeled antibody is added to remove the unbound antibody. The enzyme labels the antibody and develops color after adding the substrate. If there is no corresponding antigen in the specimen, there is no antigen binding on the surface of the solid phase, and the added enzyme-labeled antibody cannot be bound to the solid phase and is washed and removed. When a colorless substrate is added, no coloration occurs due to no enzyme catalysis. The double-antibody sandwich method is suitable for the determination of macromolecule antigens with a valence of 2 or more, but is not suitable for the determination of haptens and monomolecular monovalent antigens because it cannot form two-point sandwiches.
Indirect method:
The indirect method is the most commonly used method for detecting antibodies. The principle is to use an enzyme-labeled anti-antibody to detect a test antibody that has bound to a solid phase, and it is called an indirect method. The protein to be tested is first coated in the well plate, and then primary antibody, enzyme-labeled secondary antibody and substrate are sequentially added to develop color, and the antigen is quantitatively detected by an instrument (eg, microplate reader). This method is simple in operation but has a poor specificity due to its high background and has been gradually replaced by the sandwich method. The advantage of the indirect method is that a method of detecting the corresponding antibody can be established using the same enzyme-labeled secondary antibody as long as the coated antigen is changed. The key to the success of indirect methods lies in the purity of the antigen. Another interference factor in the indirect method is the high concentration of non-specific antibodies contained in normal serum.
Competition Law:
Competition assays can be used to measure antigens and can also be used to determine antibodies. When the interfering substance in the antigen material is not easily removed, or if it is not easy to obtain enough purified antigen, specific antibodies can be detected by this method. The principle is that the antibody in the specimen competes with a certain amount of enzyme-labeled antibody to bind with the solid-phase antigen. The more body size in the specimen, the less the enzyme-labeled antibody bound to the solid phase, and therefore the positive reaction coloration is lighter than the negative reaction. If the antigen is of high purity, it can be directly coated on the solid phase. If there are interfering substances in the antigen, direct coating is not easy to succeed. The capture coating method can be used, ie, the antibody corresponding to the solid phase antigen is first coated, and then the antigen is added to form the solid phase antigen. The impurities in the antigen are washed, and then the sample and the enzyme-labeled antibody are added to perform the competitive binding reaction. Small molecule antigens or semi-antibodies lack two or more sites that can be used in the sandwich method. Therefore, they cannot be measured by the double antibody sandwich method, and the competition mode can be used. The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete with the solid-phase antibody. The greater the amount of antigen present in the specimen, the less the enzyme-labeled antigen bound to the solid phase and the lighter the final color.
The ELISA commonly used methods include double antibody sandwich method, indirect method, competition method.