Do a good contrast in the formal test, the test conditions of positive and negative control should be taken, and the samples to be tested should be made in two copies to ensure the accuracy of the test results.
Sometimes the background is higher, indicating a nonspecific reaction, can be closed by sheep serum, rabbit serum or BSA.
Selection of experimental conditions
In ELISA, it is important to select various experimental conditions, including: (1) Choice of solid-phase carriers: Many substances can be used as solid-phase carriers, such as PVC, polystyrene, polyacrylamide and cellulose. It can take the form of concave plate, test tube, bead, etc. At present, it is commonly used to have 40-well polystyrene concave plates.
No matter what kind of carrier, before use can be screened: with the same amount of antigen package is, under the same experimental conditions to react, to observe whether its color rendering reaction is homogeneity, according to which it is good adsorption performance. (2) Package by antibody (or antigen) selection: antibody (or antigen) adsorbed on the surface of the solid-phase carrier, the requirement severity is better, adsorption generally requires PH between 9.0 to 9.6. Adsorption temperature, time and protein amount also have a certain impact, generally more than 4 degrees C 18 to 24 hours. The optimum concentration of protein packs needs to be titrated: after being packaged with different protein concentrations (0.1, 1.0 and 10 ?g/ml, etc.), the OD values of the positive specimens are observed when other test conditions are the same. Select the concentration with the largest OD value and the lowest protein amount.
For most proteins, it is usually 1 to 10 ?g/ml. (3) Selection of enzyme-labeled antibody working concentration: first, the initial effect of titration using the direct ELISA method (see enzyme-labeled antibody part).
Then fix other conditions or take the "square array method" (pack edgy, reference for the sample to be examined, and enzyme-labeled antibodies are different dilutions) in the formal experimental system accurately titrates its working concentration. (4) The choice of the base of the enzyme and the hydrogen donor: the choice of the hydrogen donor is inexpensive, safe, with a clear color-rendering reaction, and itself is colorless. Some hydrogen donors (e.g. OPD, etc.) have potential carcinogenic effects, care should be taken to protect. Conditional people should use non-carcinogenic, high sensitivity of hydrogen donors, such as TMB and ABTS is currently more satisfactory hydrogen donors. After a period of action, strong acids or alkalis should be added to stop the reaction. Usually the substrate action time, to 10-30 minutes is appropriate. The substrate use fluid must be prepared freshly, especially if H2O2 is added before use.