Basic types, uses and operating procedures of ELISA methods
1. Direct ELISA kit
This method is primarily used to detect antibodies. Taking the ELISA of duck viral hepatitis (DVH) antibody as an example, the procedure of direct ELISA is as follows:
(1) Information
1 coating liquid, washing liquid, heat preservation liquid, substrate liquid, stopping liquid;
2 DVH coated antigen, enzyme-labeled anti-antibody, negative and positive DVH refer to serum; duck serum to be tested;
3 ELISA detector, sampler, polystyrene microplate.
(2) Method process
1 Add antigen coating → 4 ° C overnight, wash three times, throw dry
2 add serum to be tested → 37 ° C for 2 hours, wash three times, throw dry
3 Add enzyme labeled antibody → 37 ° C for 2 hours, wash three times, throw dry
4 Add substrate solution → 37 ° C for 30 minutes, add stop solution
5 Determine the OD value using an ELISA tester and calculate the P/N ratio.
(3) Results Determination The ratio of known positive serum to known negative serum (P/N) ≥ 2.1, and the positive LD value of known positive serum is ≥ 0.4; in the case where the above conditions are established, if the serum to be tested is known The ratio of negative serum (P/N) ≥ 2.1, and the OD value of the serum to be tested is ≥ 0.4, then it is judged as positive, otherwise it is judged as negative.
2, double antibody sandwich ELISA kit
This method is primarily used to detect macromolecular antigens. The procedure of this method is described by taking the double sandwich antibody ELISA for detecting infectious bursal disease virus (IBDV) as an example.
1 add antibody coating → 4 ° C overnight, wash three times, throw dry
2 Add the antigen to be tested → 37 ° C for 30 minutes, wash three times, throw dry
3 Add enzyme-labeled antibody → 37 ° C for 30 minutes, wash three times, throw dry
4 Add substrate solution → 37 ° C for 15 minutes, add stop solution
5 Determine the OD value using an ELISA tester.
3, double sandwich ELISA kit
The primary difference between this method and the double antibody sandwich ELISA is that it uses an enzyme-labeled anti-antibody to view a variety of macromolecular antigens. It not only does not need to symbolize each antibody, but also improves the sensitivity of the assay.
The fundamental procedure of this law is:
1 Add antibody (Ab-1) coating → 4 ° C overnight, wash three times, throw dry
2 Add the antigen to be tested (Ag) → 37 ° C for 60 minutes, wash three times, throw dry
3 Add specific antibody (Ab-2) produced by non-same animals → 37 ° C for 60 minutes, wash three times, throw dry
4 Add enzyme-labeled anti-Ab-2 antibody (AB-3) → 37 ° C for 60 minutes, wash three times, throw dry
5 Add substrate solution → 37 ° C for 20 minutes, add stop solution
6 Determine the OD value using an ELISA tester.
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