[PMC free article] [PubMed] [CrossRef] [Google Scholar] 31

[PMC free article] [PubMed] [CrossRef] [Google Scholar] 31. for detecting antibodies against PRRSV in pig sera, exhibiting a cut-off value of 23.19% and good sensitivity, specificity, and reproducibility. Importantly, the cELISA specifically detect anti-genotype 2 PRRSV antibodies. The cELISA showed more sensitive than the commercial IDEXX ELISA kit by detecting the sequential sera from the challenged pigs. The compliance rate of cELISA with the commercial IDEXX ELISA kit was 96.4%. In addition, the commercial IDEXX ELISA kit can be combined with the developed cELISA for the differential detection of antibodies against genotype 1 and 2 PRRSV in pig sera. Collectively, the developed nanobody-based cELISA showed advantages of simple operation and low production cost and can be as an assay for epidemiological investigation of genotype 2 PRRSV infection in pigs and evaluation after vaccination. (3). It contains at least 10 open reading frames (ORFs), including ORF1a, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5, ORF5a, ORF6, and ORF7 (4, 5). PRRSV is divided into two types based on genetic distances: genotype 1 (European) Ziprasidone hydrochloride monohydrate and genotype 2 (North American) (6, 7). Particularly, the genotype 2 PRRSV strains are the predominant pathogens that cause clinical outbreaks of PRRS in North America and China (8). However, the genotype 1 PRRSV has attracted increasing attention, and multiple strains have been recently isolated and identified in China (8, 9). These two genotypes share only approximately 60% nucleotide identities and do not produce cross-protection (10). The capsid protein of PRRSV (PRRSV-N protein) encoded by ORF7 gene is relatively conserved and accounts for 20%C40% of the total amount of viral particle. It has good antigenicity and immunogenicity, and anti-PRRSV-N protein antibodies can be detected at 7?days postinfection (11, 12). Therefore, the PRRSV-N protein is an ideal target for the development of a diagnostic kit for detecting anti-PRRSV antibodies (11). To date, the main commercial ELISA kits for detecting anti-PRRSV antibodies in pig sera are developed with indirect ELISA (iELISA) using PRRSV-N protein as a coated antigen (13) and goat anti-pig IgG as the second antibody. The assays were universally used to be diagnosis of PRRSV infection and evaluation after vaccination. However, this method requires a higher purity antigen and an enzyme-labeled secondary antibody, resulting in a large production cost of the commercial kit. Nowadays, conventional polyclonal and monoclonal antibodies are widely used as indispensable reagents for the development of disease diagnostic kits (14). Nevertheless, the traditional antibodies have some shortcomings that limit their application in related fields. For example, polyclonal antibodies suffer from batch-to-batch variability, while monoclonal antibodies have high costs and difficult genetic manipulation for production. Thus, there is an urgent need to develop strategies aimed at the production of alternative scaffolds (15). In recent years, single-domain antibodies (sdAbs), also known as nanobodies, are derived from the heavy chain antibody variable region (VHH) in camelids and have attracted much attention in disease diagnosis and treatment (16). 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. In the present Ziprasidone hydrochloride monohydrate study, the specific nanobodies against PRRSV-N protein were screened Ziprasidone hydrochloride monohydrate and isolated. Based on the nanobodies, Ziprasidone hydrochloride monohydrate a simple and fast platform for synthesizing nanobody-HRP fusion proteins was developed (Fig. 1A). Then, using the nanobody-HRP fusion proteins as a reagent, a cELISA was developed for detecting anti-genotype 2 PRRSV antibodies in pig sera (Fig. 1B). The developed nanobody-based cELISA showed advantages of simple operation and low-cost production of nanobody-HRP fusion proteins and good sensitivity, specificity, Ziprasidone hydrochloride monohydrate and reproducibility. In addition, the cELISA showed high agreement with the commercial IDEXX ELISA kit and more sensitivity than the kit by detecting the sequential sera from the challenged pigs. Thus, we think that the developed nanobody-based cELISA for detecting anti-genotype 2 FAZF PRRSV antibodies was an ideal.

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