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 is 20.72 %. Three hundreds and sixty 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 results of the cELISA and those 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 vaccine efficacy and the indirect diagnosis of PCV2 infection.

Easily Established and Multifunctional Synthetic Nanobody Libraries as Research Tools

“…Anti-GFP and anti-YFP nanobodies immobilized to Sepharose resin have been used to facilitate the purification of lowly expressed GFP and YFP fusions from mammalian cell lysate [48]. Moreover, antigen-specific nanobodies can be fused to alkaline phosphatase or horseradish peroxidase used in ELISAs, and they have been successfully used in virus detection and toxic material detection [49][50][51]. The small size and high specificity of nanobodies allow for the very limited adherence to non-specific proteins.…”

Development and validation of a PCR-free nucleic acid testing method for RNA viruses based on linear molecular beacon probes

“…Immunodiagnostic methods, such as ELISA [ 6 ], utilize a series of classic virus detection techniques involving viral proteins and specific antibodies. However, even without considering the long cycle or high cost of antibody preparation, sensitivity is still a challenge for many immunology diagnostic technology platforms.…”

Development of a Nanobody-Based Competitive Enzyme-Linked Immunosorbent Assay for Efficiently and Specifically Detecting Antibodies against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses

“…Compared with traditional antibodies, nanobodies exhibit more attractive features for diagnostic application, such as small volume (15 kDa), easy genetic manipulation, and high stability ( 17 , 18 ). Recently, nanobodies have been fused with horseradish peroxidase (HRP) for the development of competitive ELISA (cELISA) to detect antibodies against some animal disease viruses ( 19 , 20 ). However, the production of the nanobody-HRP fusion protein needs to transfect the HEK293T cell with the plasmid each time, which impedes mass production of the diagnostic kit using the nanobody-HRP fusion protein as reagents.…”