ELISA kit in the study of the matter - Database & Sql Blog Articles

Note on the elisa kit in the experiment The ELISA kit is divided into two key points in the ELISA experiment:
1. When conducting a formal experiment, the experimental conditions should be controlled by the positive control and the negative control. The samples to be tested should be made in duplicate to ensure the accuracy of the experimental results. Sometimes the background is high, clarifying that there is a non-specific response, you can choose to use sheep serum, rabbit serum or BSA to shut down.
Second, the selection of experimental conditions In the ELISA, it is important to select the various experimental conditions, including:
(1) Selection of solid phase carriers: Many substances can be used as solid phase carriers such as polyvinyl chloride, polystyrene, polyacrylamide and cellulose. The method can be such as a recessed flat plate, a test tube, a bead or the like. A 40-well polystyrene well plate is now commonly used. Regardless of the carrier, screening can be carried out before application: it is coated with the same amount of antigen and reacted under the same experimental conditions to investigate whether the color reaction is uniform or not, and it is determined whether the adsorption function is outstanding.
(2) Selection of coated antibody (or antigen): When the antibody (or antigen) 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 during adsorption. Adsorption temperature, time and its protein content also have a certain impact, generally choose 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 values ​​of the positive samples are investigated together with other experimental conditions. The concentration with the highest OD value and at least the minimum amount of protein is selected. Generally speaking, it is 1 to 10 μg/ml for most proteins.
(3) Selection of enzyme symbol antibody working concentration: First, the titration of the starting titer is carried out by direct ELISA (see the antibody part of the enzyme symbol). Then, other conditions are fixed or the "square matrix method" (the coating, the reference product of the sample to be tested, and the enzyme symbol antibody are different), and the working concentration is accurately titrated in the formal experimental system.
(4) Selection of enzyme substrate and hydrogen donor: The selection request for hydrogen donor is cheap, safe, and obviously resounding, but it is colorless. Some hydrogen donors (such as OPD) have potential carcinogenic effects and should be guarded against. Those who are qualified should use hydrogen donors that are not carcinogenic and sensitive, such as TMB and ABTS, which are now more satisfying hydrogen donors. After a period of substrate effects, you should participate in strong acid or alkali to stop the reaction. Generally, the effect of the substrate is 10-30 minutes. The substrate application solution must be freshly prepared, especially if H2O2 is used before use.