Why do ELISA test serum samples need to be diluted? - Database & Sql Blog Articles

Each box of elisa kit has a corresponding manual, and each sample will have a dilution ratio of the sample and the diluent. Why do the samples need to be diluted? In today's article, Shanghai Renjie will analyze it for you. : Why do ELISA test serum samples need to be diluted?

Why is the serum in the ELISA kit diluted? There are two reasons:
1) Competition inhibition is obvious, and the competitor should be diluted;
2) To reduce the non-specific reaction, the specific antigen-antibody reaction is fully reflected.
Serum dilution can be done with ordinary PBS, 1% BSA can be added, can effectively reduce the ELISA background, of course, if you are too troublesome, I think it is not too big to replace PBS with saline. Regardless of whether you use direct competition or indirect competition, the dilution factor of the serum must be determined in advance, but it is not necessary to use a little bit. You can do a preliminary test and choose the dilution factor of OD at 1.0, which is the negative hole in your competition ELISA. The OD is at 1.0, and the resulting curve is more sensitive.

Steps for ELISA kit to dilute serum:
First dilute the protein by a certain multiple, for example, 10 times, 20 times dilution (so that one parameter can be roughly determined), and the antigen is subjected to a series of dilutions to coat the microplate, and then a certain dilution of the positive reference serum and the negative reference serum. After the incubation, the mixture is washed, and then the enzyme conjugate is added to carry out the reaction. After the incubation and washing, the substrate solution is colored, and the OD value is determined after the reaction is terminated. The dilution of the antigen with the OD value ≥ 1.0 is selected as the optimum coating concentration. It is generally preferred to select which OD value is slightly greater than 1.0, rather than selecting an antigen concentration less than 1.0. Negative reference serum OD values ​​are required to be <0.1-0.2. That is, there is a significant difference in the OD values ​​of the positive reference serum and the negative reference serum.