Negative color development and negative background in ELISA
The negative color development and negative background in ELISA are based on the commonly used indirect method to measure antibody and sandwich method to measure antigen as an example. The positive specimen contains specific antibodies or antigens in the detection system. It acts as a bridge between the immunosorbent and the enzyme conjugate, so that the enzyme conjugate is adsorbed on the solid phase. Negative color development, the depth of color development is proportional to the amount of antibody or antigen to be tested in the positive specimen. The negative specimen does not contain the antibody or antigen to be tested, and the enzyme conjugate cannot be bound to the solid phase, so it does not develop color. However, ELISA is a complex, multi-step reaction process. In each step of the reaction, the enzyme conjugate may be non-specifically adsorbed on the solid phase carrier and react with the substrate to exhibit color, which forms a negative background. The non-specific adsorption of enzyme conjugates on the solid phase usually has the following ways. The enzyme conjugate is directly adsorbed on the solid support. The enzyme conjugate is a protein and can be directly adsorbed on the surface of the solid support. However, in this step of ELISA, conditions that are not conducive to such adsorption are selected. Generally speaking, as long as the working concentration of the enzyme conjugate is appropriate and the reaction is thoroughly washed after the reaction, such adsorption can be avoided. The enzyme conjugate reacts with the protein component coated on the solid phase. When the coating antigen is not pure, the impurity protein will also be adsorbed on the solid phase carrier, and some impurities may be combined with the enzyme-labeled antibody due to "misrecognition". In the indirect method, hybrid antigens may also specifically or non-specifically bind to immunoglobulins in serum samples. In addition, non-specific immunoglobulin can also be adsorbed on the coated specific antigen, especially when this antigen is a basic protein, then the enzyme-labeled secondary antibody reacts with the adsorbed immunoglobulin to form a negative background. . In the indirect method, the content of non-specific immunoglobulin in the specimen far exceeds the specific antibody, so it can be directly adsorbed on the solid phase, which is the main reason for the formation of negative background. Polystyrene has good adsorption properties for immunoglobulins, the adsorption force of IgM is greater than IgG, and the adsorption force of polymerized IgG is greater than that of monomer IgG. The enzyme-labeled anti-Ig (secondary antibody) has the same affinity for the Ig adsorbed on the carrier and the specific Ig bound to the solid phase antigen. Therefore, in the reaction of adding the sample, although the conditions that are not conducive to adsorption are adopted, it is assumed Serum samples are not diluted appropriately, and some of the high-concentration Ig can still be adsorbed on the solid phase, resulting in a higher background. In the double antibody sandwich method, if the specimen contains rheumatoid factor, the negative serum can also be developed. Rheumatoid factor is an autoantibody that acts on the Fc segment of various animal IgGs, mostly IgM, and is a multivalent antibody. Serves as an antigen component in the sandwich method detection, and at the same time, the solid-phase antibody and the enzyme-labeled antibody react to bind the enzyme-labeled antibody to the carrier. The introduction of biotin-avidin system in ELISA can improve the sensitivity of detection, but if used improperly, it will deepen the background, but it will outweigh the gains. If the ratio of biotin-labeled protein is not appropriate, it is easy to form a high negative background. The avidin extracted from egg white is a basic glycoprotein, which can be adsorbed on polystyrene by electrostatic action, which is also one of the factors that cause BA-ELISA high background. Avidin extracted from streptococci improved this non-specific adsorption phenomenon.
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