1. During the formal test, the test conditions should be controlled by a positive control and a negative control, respectively, and the samples to be tested should be made in duplicate to ensure the accuracy of the experimental results. Sometimes the background is high, indicating a non-specific reaction, which can be blocked with sheep serum, rabbit serum or BSA.
2. In ELISA, it is very important to choose various experimental conditions, including:
(1) Selection of solid-phase carrier: many substances can be used as solid-phase carrier, such as polyvinyl chloride, polystyrene, polyacrylamide and cellulose. The form can be concave hole plate, test tube, bead, etc. At present, the commonly used 40-hole polystyrene concave orifice plate. No matter what kind of carrier, it can be screened before use: it is coated with the same amount of antigen, and the reaction is carried out under the same experimental conditions. Observe whether the color reaction is uniform and determine whether its adsorption performance is good.
(2) The choice of coating antibody (or antigen): when adsorbing the antibody (or antigen) 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 during adsorption. Adsorption temperature, time and protein content also have a certain influence, generally use 4 ℃ for 18-24 hours. The optimal concentration of protein coating needs to be titrated: that is, after coating with different protein concentrations (0.1, 1.0, and 10 μg / ml, etc.), when the other test conditions are the same, observe the OD value of the positive specimen. Choose the concentration with the largest OD and the least protein. For most proteins, it is usually 1-10 μg / ml.
(3) Selection of working concentration of enzyme-labeled antibody: firstly perform direct titer titration by direct ELISA method (see enzyme-labeled antibody section). Then fix other conditions or adopt the "square matrix method" (coatings, reference samples of the samples to be tested and enzyme-labeled antibodies are at different dilutions) to accurately titrate its working concentration in the formal experimental system.
(4) Selection of enzyme substrates and hydrogen donors: The selection requirements for hydrogen donors are cheap, safe, and have a significant color reaction, while they are colorless. Some hydrogen donors (such as OPD, etc.) have potential carcinogenic effects, so attention should be paid to protection. Those with conditions should use non-carcinogenic, highly sensitive hydrogen donors, such as TMB and ABTS, which are currently more satisfactory hydrogen donors. After the substrate acts for a period of time, a strong acid or base should be added to stop the reaction. Usually the substrate action time is 10-30 minutes. Substrate use solution must be prepared fresh, especially
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