Hengyuan to share the choice of four ELISA experimental conditions - Database & Sql Blog Articles

EL-C1600N100013-B

Heng Jiaxing AO3401

The outer diameter of the probe domestically produced P125-Q2 four-pronged head nickel-plated needle is 2.02 needle

In the ELISA experiment, it is very important to select the various experimental conditions. Today, Shanghai Hengyuan will share the selection methods of the four Elisa experimental conditions, including the selection of the solid phase carrier, the selection of the coated antibody (or antigen), The selection of the working concentration of the enzyme-labeled antibody, the substrate of the enzyme and the choice of the hydrogen donor, let's take a look at it one by one:
First, the choice of solid phase carriers 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 recessed plate. Regardless of the carrier, screening can be carried out before use: the reaction is carried out under the same experimental conditions by coating with an equal amount of antigen, and whether the coloration reaction is uniform or not, and whether the adsorption performance is good.
Second, the choice of coating 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. 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, the titration of the initial titer is carried out by direct ELISA (see the enzyme-labeled antibody portion). 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.
Fourth, the substrate of the enzyme and the choice of hydrogen donors The choice of hydrogen donors is cheap, safe, and has a significant 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 while, 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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