Establishment of an indirect ELISA method for detecting porcine rotavirus antibodies by targeting the VP6 protein

Abstract:

[Background] Porcine rotavirus (PoRV) is one of the major pathogens causing porcine diarrhea, showcasing an increase in the infection rate recently. It is necessary to strengthen the clinical surveillance of PoRV. [Objective] To facilitate rapid detection and antibody screening in clinical practice, an indirect ELISA method targeting the VP6 protein of the prevalent PoRV strain was established. [Methods] The VP6 gene of the PoRV genotype G9P[23]I5 was cloned and expressed in a prokaryotic expression system, and the recombinant protein was purified. With the recombinant VP6 protein as the capture antigen, the checkerboard titration test was performed for PoRV-positive serum samples of pigs. The parameters of the indirect ELISA method were optimized, and the specificity, sensitivity, and repeatability of the method were examined. [Results] The parameters of the indirect ELISA method were optimized as follows: the VP6 antigen coating amount of 0.1 μg/well and coating overnight at 4 ℃; blocking with 5% skim milk at 37 ℃ for 2 h; positive serum dilution at 1:1 000 and incubation at 37 ℃ for 1 h; Goat pAb to Pig IgG H & L (HRP) at 1:15 000 and incubation at 37 ℃ for 1 h; color development with 3, 3', 5-5'-tetramethylbenidine (TMB) for 15 min. The established method showed the OD₄₅₀ cutoff value of 0.42 and the sensitivity of 1:8 000. It demonstrated great specificity, with the intra-batch and inter-batch coefficients of variation both less than 10%. A total of 130 clinical serum samples were detected by the established method and commercially available PoRV ELISA assay kit, which showed the result coincidence rate of 93.85%. The sensitivity of the indirect ELISA method established in this assay was higher than that of the commercially available PoRV ELISA assay kit. [Conclusion] The indirect ELISA method established in this study enables the rapid detection and large-scale screening of PoRV antibodies.