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Preparation of Monoclonal Antibodies Against Porcine Circovirus Type 2 Capsid Protein and Development of a Blocking ELISA for Detection of the Antibody Against the Virus
by
Haifeng Sun
1, 
Qingqing Liu
1,
Shuyan Zhai
1,
Biyue Wu
1,
Zicheng Ma
1,
Yangyang Sun
1,
Kaiyuan Ye
1,
Haoyuan Wang
1,
Yanni Gao
1,
Xianwei Wang
1,2,
Juan Bai
1,2,* and
Ping Jiang
1,2,* 1
Key Laboratory of Animal Disease Diagnostics and Immunology, Ministry of Agriculture, MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
2
Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China
*
Authors to whom correspondence should be addressed.
Vet. Sci. 2026, 13(7), 617; https://doi.org/10.3390/vetsci13070617 (registering DOI)
Submission received: 1 June 2026
/
Revised: 23 June 2026
/
Accepted: 24 June 2026
/
Published: 25 June 2026
Simple Summary
Porcine circovirus type 2 (PCV2) severely damages the global pig breeding industry. In this study, 10 monoclonal antibodies (mAbs) targeting the PCV2 Cap protein were prepared and characterized, among which mAb 4C4 with excellent reactivity, neutralizing and blocking activity was selected for HRP labeling. Using the recombinant Cap protein as the coating antigen, a blocking ELISA (bELISA) was optimized and established. The cutoff value was set at 40% via ROC analysis, with 98.66% sensitivity and 100% specificity. No cross-reaction with other common swine pathogens was found, and the assay repeatability was favorable. The detection of 312 clinical sera showed a high coincidence with a commercial kit, and the inhibition rate closely correlated with serum neutralizing antibody titers. This bELISA is reliable for PCV2 antibody detection and epidemiological surveillance.
Abstract
Porcine circovirus type 2 (PCV2) is the primary causative agent of a spectrum of porcine circovirus-associated diseases (PCVDs) and remains a major threat to the global swine industry. In this study, ten monoclonal antibodies (mAbs) targeting the Cap protein of PCV2 were generated and characterized. One mAb, designated 4C4, which exhibited high reactivity, strong neutralizing activity, and superior blocking efficacy, was selected for horseradish peroxidase (HRP) labeling. After optimizing the reaction parameters, a blocking ELISA was developed for the detection of the anti-PCV2 antibody. Using receiver operating characteristic (ROC) curve analysis, a cutoff value of 40% was established to distinguish positive from negative serum samples. The sensitivity and specificity of this blocking ELISA method were 98.66% and 100%, respectively. No cross-reactivity was observed with serum antibodies against classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine reproductive and respiratory syndrome virus (PRRSV), or pseudorabies virus (PRV). Intra-assay and inter-assay repeatability tests yielded coefficients of variation (CVs) all below 10%, confirming the assay's excellent reproducibility. Simultaneous testing of 312 clinical porcine serum samples using the developed bELISA and a commercial indirect ELISA kit revealed an overall coincidence rate of 99.04%. In addition, the percentage inhibition (PI) in the bELISA was strongly correlated with serum anti-PCV2 neutralizing antibody titers. In conclusion, the blocking ELISA developed herein demonstrates high sensitivity, strong specificity, and good reproducibility, serving as a potentially effective tool for the detection of the anti-PCV2 antibody and epidemiological investigation.
