A Nanobody-horseradish Peroxidase Fusion Protein-based Competitive ELISA for Rapid Detection of Antibodies Against Porcine Circovirus Type 2

Abstract Background: The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)‑horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum.Results: Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15‑HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti‑PCV2 antibodies. The cut‑off value of the cELISA was 20.72%, 360 porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68% and 95.92%, respectively. The coincidence rate of the two methods was 99.17%. When detecting 620 clinical porcine serum samples, a good consistent (kappa value=0.954) was found between the result of the cELISA and that of commercial kits.Conclusions: In brief, the newly developed cELISA based PCV2-Nb15‑HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccines efficacy and indirect diagnosis of PCV2 infection.