Establishment And Application Of A Capture ELISA For Detecting Antibodies Against PRRSV

Porcine reproductive and respiratory syndrome (PRRS) has become one of the major infectious diseases in the swine industry since1980s. PRRS causes reproductive failure of pregnant sows and respiratory disease of all ages, especially the suckling piglets. An outbreak of PRRS took place in China for the first time in1995, and the pathogen was isolated in the next year which confirmed the existence of PRRSV in China.Enzyme-linked immunosorbent assay (ELISA) exhibits the advantages of simple operation, easy standardization, and convenient promotion. In this study, the anti-GST fusion protein monoclonal antibody was adopted to establish the capture ELISA method for the detection of negative and positive PRRSV sera. Monoclonal antibody possesses the merits of high specificity and the consistent activity before and after the coating process. The capture ELISA method is expected to improve the sensitivity, specificity and reproducibility of the PRRSV antibody detection. Meanwhile, the capture ELISA method can be used as a generic detection means for the detection of other pathogenic serum samples.1. Prokaryotic expression of the fusion protein GSTAfter the designation of the specific primers, the GST tag sequence was amplified by PCR using the pGEX-KG empty vector as template, and was cloned into the prokaryotic expression vector of pET-28a to obtain the recombinant plasmid. The plasmid was transformed into E. coli cells subsequently, and was induced by IPTG. The SDS-PAGE test confirmed that the GST protein expressed with His tag existed mostly in the form of inclusion bodies, and the expression level reached the summit in5hr. The molecular weight of the fusion protein was about32kDa.2. Preparation and validation of the anti-GST monoclonal antibodyAfter its expression and purification, the purified fusion protein was vaccinated to the female BALB/C mouse. The serum antibody titer was measured after the second immunity and the cell fusion was processed when the immune titer was more than1:10000.8strains of cell lines, which could stably produce the monoclonal antibody responsible for a single epitope, were screened through indirect ELISA method and cloned by means of limiting dilution. The Western Blotting test showed that the monoclonal antibody could well react with the expressed GST protein and the PRRSV N (GST-N) protein combined with GST. Monoclonal antibody cell lines were amplified and vaccinated to the female BABL/C mouse to induce ascites. The titer of the ascites were all above1:500*25, which indicated the application of the followed testing method.3. Establishment of the capture ELISA method for PRRSV antibodyThe obtained anti-GST monoclonal antibody was purified and coated onto the elisa plate, and then, the purified fusion protein GST-N was added to create the ELISA method to detect the positive and negative PRRSV serum samples. The experiment confirmed that the G2E1mono-strain responsible for anti-GST was suitable to be adopted as the coating antibody due to its final specificity and sensitivity. The best package concentration was optimized at1μg/mL by cross method, and the best combining concentration of GST-N protein was2μg/mL. The optimum serum dilution was1:80, and the best reaction time was30min. The best act concentration of secondary antibody was1:3000, and the best reaction time was30min. The best reaction time of the substrate was10min. The cut-off value was identified as0.31after testing100samples of PRRSV antibody-negative serum samples.4. Application of the capture ELISA method for PRRSV antibodyThe optimized detection method was used for detecting727clinical samples. Comparing with the IDEXX HerdChek PRRS ELISA kit, the positive samples of capture ELISA method and IDEXX ELISA kit were458and484, respectively. The negative samples were269and243, respectively. The positive and negative rates were accordance with the IDEXX HerdChek PRRS ELISA kit in a ratio of82.23%and75.3%, respectively, and the overall coincident rate was79.91%. The result reveals that this detection method possesses well specificity and sensitivity, and is suitable in the clinical detection.