Elisa detection kit and detection method for iga antibody against porcine rotavirus group a

An ELISA detection method for IgA antibody against Porcine rotavirus group A, comprising the following steps of:

• coating:
• diluting a VP6 fusion recombinant protein to 3.5 μg/mL using a 0.05 mol/L Na2CO3—NaHCO3 buffer solution with a pH of 9.6 to obtained a diluted protein solution, and adding 100 μL of the diluted protein solution to each well such that the VP6 fusion recombinant protein is adsorbed at 4° C. for 14 h;
• after the coating, discarding the diluted protein solution in the well, followed by adding 250 L of PBS wash solution containing 0.05% Tween-20 to each of the well, and washing the well for three times, with the final wash being tapped dry;
• blocking:
• adding 200 μL of PBS blocking solution containing 5% BSA and 0.05% Tween-20 to each of the well and blocked at 37° C. for 1 h;
• after the blocking, discarding the PBS blocking solution in the well, followed by adding 250 μL of the PBS wash solution to each of the well, and washing the well for three times, with the final wash being tapped dry;
• primary antibody incubation:
• diluting a milk sample to be tested with a sample diluent at a ratio of 1:100 or diluting a serum sample to be tested with the sample diluent at a ratio of 1:200 to obtain a primary antibody solution, adding 100 μL of the primary antibody solution to each of the well and incubated at 37° C. for 60 min;
• discarding the primary antibody solution in the well, followed by adding 250 μL of the PBS wash solution to each of the well, and washing the well for five times, with the final wash being tapped dry;
• secondary antibody incubation:
• diluting an enzyme-labeled secondary antibody with an antibody diluent at a ratio of 1:10000 to obtain a secondary antibody solution, followed by adding 100 μL of the secondary antibody solution to each of the well and incubated at 37° C. for 60 min;
• discarding the secondary antibody solution in the well, followed by adding 250 μL of the PBS wash solution to each of the well, and washing the well for five times, with the final wash being tapped dry;
• chromogenic time and reading:
• adding 100 μL of a single-component TMB substrate chromogenic solution to each of the well for a chromogenic reaction, and allowing the chromogenic reaction to proceed in dark at 37° C. for 15 minutes, subsequently, adding 100 μL of a stop solution to each of the well, and reading an OD value at 450 nm using an enzyme-labeled instrument;
• wherein the method is used for non-disease diagnostic purposes.