Abstract
BackgroundPorcine epidemic diarrhea virus (PEDV), which is characterized by severe watery diarrhea, vomiting, dehydration and a high mortality rate in piglets, leads to enormous economic losses to the pork industry and remains a large challenge worldwide. Thus, a rapid and reliable method is required for epidemiological investigations and to evaluate the effect of immunization. However, the current diagnostic methods for PEDV are time-consuming and very expensive and rarely meet the requirements for clinical application. Nanobodies have been used in the clinic to overcome these problems because of the advantages of their easy expression and high level of stability. In the present work, a novel biotinylated nanobody-based blocking ELISA (bELISA) was developed to detect anti-PEDV antibodies in clinical pig serum.ResultsUsing phage display technology and periplasmic extraction ELISA (PE-ELISA), anti-PEDV N protein nanobodies from three strains of PEDV were successfully isolated after three consecutive rounds of bio-panning from a high quality phage display VHH library. Then, purified Nb2-Avi-tag fusion protein was biotinylated in vitro. A novel bELISA was subsequently developed for the first time with biotinylated Nb2. The cutoff value for bELISA was 29.27%. One hundred and fifty clinical serum samples were tested by both newly developed bELISA and commercial kits. The sensitivity and specificity of bELISA were 100% and 93.18%, respectively, and the coincidence rate between the two methods was 94%.ConclusionsIn brief, bELISA is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PEDV vaccines efficacy and indirect diagnosis of PEDV infection.
Highlights
Porcine epidemic diarrhea virus (PEDV), which is characterized by severe watery diarrhea, vomiting, dehydration and a high mortality rate in piglets, leads to enormous economic losses to the pork industry and remains a large challenge worldwide
Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea (PED), a highly contagious enteric disease characterized by severe watery diarrhea, vomiting and dehydration leading to enormous economic losses in the swine industry worldwide [1, 2]
Total RNA was extracted from peripheral blood lymphocytes (PBLs), and reverse transcribed to cDNA to construct the variable domain of heavy-chain only antibody (VHH) library. cDNA was used as a template to amplify specific VHH fragments using nesting PCR
Summary
Porcine epidemic diarrhea virus (PEDV), which is characterized by severe watery diarrhea, vomiting, dehydration and a high mortality rate in piglets, leads to enormous economic losses to the pork industry and remains a large challenge worldwide. Conventional PEDV diagnostic methods are based on laboratory tests and include virus isolation, conventional reverse transcription-polymerase chain reaction (RT-PCR) [13, 14], real-time RT-PCR [15,16,17], indirect fluorescent antibody (IFA) assay [18] and enzyme-linked immunosorbent assay (ELISA) [19]. These conventional methods are time-consuming, and expensive, exhibit low specificity and sensitivity, and require welltrained technicians and special instruments. Different types of ELISAs, including indirect [19, 21], competitive and blocking ELISA, have been widely applied to detect PEDV in large-scale blood or feces samples, but these assays are based on the use of PEDV-specific monoclonal or polyclonal antibodies that require more support cost and exhibit low expression yields and high levels of instability [22]
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