In the ELISA kit, the selection considerations for each of the main constituent reagents include:
(1) Selection of solid phase support: Many substances can be used as solid phase carriers such as polyvinyl chloride, polystyrene, polyacrylamide and cellulose. The form may be a flat plate, a test tube, a bead or the like. Currently used is a 40-well polystyrene pit plate. Regardless of the carrier, it can be screened before use: it is coated with an equal amount of antigen, and the reaction is carried out under the same experimental conditions to observe whether the color reaction is uniform, and whether the adsorption performance is good.
(2) Selection of coated antibody: When the antibody is adsorbed on the surface of the solid phase carrier, the purity is required to be good, and the pH is generally required to be between 9.0 and 9.6. The adsorption temperature, time and the amount of protein also have a certain influence, and generally use 4 ° C for 18 to 24 hours. The optimum concentration of protein coating is titrated: after coating with different protein concentrations (0.1, 1.0, and 10 μg/ml, etc.), the OD value of the positive specimen is observed when the other test conditions are the same. The concentration with the highest OD value and the least amount of protein is selected. It is usually 1 to 10 μg/ml for most proteins.
(3) Selection of working concentration of enzyme-labeled antibody: First, titration of preliminary titer is carried out by direct ELISA (see enzyme-labeled antibody fraction). Then, other conditions are fixed or the "square matrix method" (the coating, the reference sample of the sample to be tested, and the enzyme-labeled antibody are respectively different dilutions) are accurately titrated in the formal experimental system.
(4) Enzyme substrate and hydrogen donor selection: The choice of hydrogen donor is cheap, safe, and has a distinct color reaction, but is itself colorless. Some hydrogen donors (such as OPD) have potential carcinogenic effects and should be protected. Those who are qualified should use hydrogen donors that are not carcinogenic and sensitive. For example, TMB and ABTS are currently satisfactory hydrogen donors. After the substrate has been applied for a period of time, a strong acid or a strong base should be added to terminate the reaction. Usually the substrate action time is preferably 10-30 minutes. The substrate used must be freshly prepared, especially H2O2 before use.
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