Preparation of Polyclonal Antibody

Polyclonal antiserum against GPV were prepared according to a previously published report with slight modifications (Ma et al., 2017). It was generated in rabbits by immunizing the animals with recombinant VP3 protein and used as a coating reagent fixed on NC membrane (test line). Specific steps are as follows: VP3 protein was blended and emulsified with the same volume of FCA or FIA, then two healthy rabbits were injected into the back by multiple sites subcutaneous injection with the 200 μg VP3 protein. The entire immunization procedure comprised four injections, wherein the first immunization was with FCA, later with FIA. The first two injections were carried out at an interval of 2 weeks, and the subsequent two injections were carried out at intervals of 1 week. The polyclonal antibody was purified from the serum by sequential precipitation with caprylic acid and ammonium sulfate. Indirect ELISA was performed to measure the antibody titers of negative serum, unpurified serum and purified serum. The concentration of the antibodies was determined with Bradford method.

The pAb was diluted with PBS (0.01 M, pH 7.4) into gradient concentration (0.2 mg/ml, 0.4 mg/ml, 0.8 mg/ml, 1.6 mg/ml, 3.2 mg/ml), then fixed to the NC membrane with ZX1000 Dispense Platform (BioDot Inc., United States), and goat anti-rabbit antibody (Control line) was fixed simultaneously. The blank control and positive samples containing GPV were tested to determine the optimal concentrations of the coating antibody.