Abstract: To establish a blocking ELISA method for the detection of porcine circovirus type 3 (PCV3) antibodies, in this study, a hybridoma cell line 2E6, which secretes a good blocking antibody, was obtained by immunization of BALB/c mice with the prokaryotic expression of PCV3 Cap recombinant protein. The recombinant Cap protein was used as the coating antigen, and the horseradish peroxidase (HRP)-labeled 2E6 monoclonal antibody was used as the detection antibody, and the conditions were continuously optimized to finally establish a blocking ELISA method for detecting PCV3 antibody. The established blocking ELISA method was used to test 50 clinical negative sera, and the threshold value of blocking rate (PI) was calculated to determine the cut-off of the method. When PI ≤ 28.30%, the result was determined as negative; when PI ≥ 35.05%, the result was determined as positive; when 28.30%<35.05%, the result was determined as suspicious. The suspicious results should be tested again and the still suspicious results will be judged as positive. Specificity tests showed no cross-reactivity with positive sera for porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) and swine fever virus (CSFV). The sensitivity test showed that the detection potency could reach 1:128. The repetition test showed that the intra- and inter-batch coefficients of variation were less than 10%. The conformity test showed that the method was highly consistent with the gold standard immunoperoxidase monolayer test (IPMA) for PCV detection with a Kappa value of 0.9. In conclusion, the blocking ELISA method established in this study has good specificity and high compliance rate, which can be used for later detection of PCV3 antibodies and provides technical support for epidemiological investigation and clinical diagnosis of PCV3.

Key words: porcine circovirus type 3, Cap recombinant protein, monoclonal antibody, blocking ELISA