Development and application of a blocking ELISA method based on Cap protein for detecting antibodies against porcine circovirus 2

ABSTRACT Porcine circovirus type 2 (PCV2) serves as the key pathogen linked to porcine circovirus-associated disease, representing a considerable risk to the worldwide swine industry. A blocking enzyme-linked immunosorbent assay (ELISA) was established to identify antibodies specifically targeting PCV2, employing recombinant Cap protein as the antigen and a monoclonal antibody against PCV2 Cap protein as the detector antibody. Utilizing receiver operating characteristic curve analysis, a cutoff value of 33.8% was determined to distinguish between positive and negative serum samples. The sensitivity and specificity of this blocking ELISA method were reported at 94.7% and 96.1%, respectively. Notably, this approach exclusively identified antibodies for PCV2, showing no cross-reactivity with antibodies related to African swine fever virus (ASFV), Porcine epidemic diarrhea virus (PEDV), Porcine pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus. Both intra-assay and inter-assay coefficients of variation were less than 10%. In a comparison involving 402 porcine serum samples, the agreement rate with a commercial indirect ELISA kit reached 98.76%, with a kappa value of 0.888, reflecting high concordance between the two testing methods. This study showcases the blocking ELISA method as an efficient and standardized approach for serological monitoring of PCV2 in swine populations and for assessing seroconversion in vaccinated pigs.IMPORTANCEPorcine circovirus type 2 (PCV2) has become recognized as a pathogen of significant economic concern within the swine industry. PCV2 mainly affects the immune systems of pigs, leading to a reduction in lymphocytes and resulting in immune suppression in the affected animals. Co-infection with other porcine pathogens can enhance PCV2 infection and exacerbate porcine circovirus disease. Currently, the kits available for detecting PCV2 antibodies primarily employ indirect enzyme-linked immunosorbent assay (ELISA); however, this method is prone to false positives. In contrast, the blocking ELISA method offers enhanced specificity and provides a more straightforward interpretation of results. Previous studies utilizing blocking ELISA for PCV2 antibody detection have depended on plates coated with purified PCV2 virus, a process that is both technically challenging and time-consuming. Consequently, there is a pressing need to develop a new blocking ELISA method that is more efficient to detect antibodies against PCV2.