Preparation And Application Of Liquid Blocking ELISA Kit And Immunocolloidal Gold Strip For PRRS

Porcine reproductive and respiratory syndrome (PRRS)， also known as blue-eardisease，is a highly contagious infectious diseases characterized by reproductivefailure in sows, and respiratory disease in pigs. The causative agent of this infection isporcine reproductive and respiratory syndrome virus（PRRSV）, a member of familyof Arterivirus. Since its first report in1987in North America, PRRS has caused ahuge economical loss of swine industry. PRRS was first identified in China in1995., Since2001, a severe form of PRRS with a high fever was identified, which ispredominant type currently in China and demonstrated to be caused by a high virulentstrain of PRRSV (highly virulent porcine reproductive and respiratory syndrome virus,HP-PRRSV), and the infection was named highly pathogenic porcine reproductiveand respiratory syndrome (HP-PRRS). Sequence comparison revealed the HP-PRRSVin China has a30aa (481and533–561) deletion in its Nsp2coding region in relationto the North American isolate (VR-2332).Although lots of methods for diagnosing PRRSV infection have been reported bydetecting either PRRSV antigens or antibody, those method are laborious andtime-consuming, and not suitable for distinshing the PRRSV and HP-PRRSVinfections in particular. Therefore, to prepare a simple, specific and rapid diagnosticmethod for those is necessary. In China, the main used vaccine for PRRSV wasinactivated vaccines and attenuated vaccines, but in order to avoid virulence return,virus transmission，shed or vertical transmission, we only use the inactivated vaccinesin breeding pig farms. For these reasons, it is imperative to prepare a diagnosticmethod of antibody property for rapid on-site application, which can distinguishbetween the wild virus infection and vaccination, and provide technical method forcomprehensive prevention and control HP-PRRS of clinical，swine purification andmolecular epidemiology. In this study, the monoclonal antibody1E9against the synthetic30aa polypeptiddeleted in HP-PRRSV-encoded Nsp2was prepared and employed to establish theblocking ELISA kit and immunocolloidal gold strip for the detection of antibodyaginast PRRS detection liquid by using1E9.The Nsp2-protein sequence of VR-2332and several other HP-PRRSV were separated from our country, and the conserved30aa deletion was selected as a template to synthesize polypeptide. The monoclonalantibody1E9,4D1were prepared to against PRRSV Nsp2-deletion using the30aapolypeptide.1E9has the good activity and a subclass of IgG2a which confirmed byDot-ELISA, indirect immunofluorescence, etc. The liquid blocking ELISA andimmunocolloidal gold strip were prepared through the monoclonal antibody1E9. Andthen220local samples were examined by liquid blocking ELISA detection, and100local samples were done by immunocolloidal gold strip detection. The detectionshown that blocking ELISA detection rate is53.2%, while the commercial kitdetection rate of69.5%. There had59positive samples in the detection of theimmunocolloidal gold strip and65positive samples in the detection of commercial kit.The liquid blocking ELISA and the immunocolloidal gold strip detection has areliability which had compared with commercial kits and RT-PCR results.These methods can be applied to epidemiological investigation in the pigindustry，prevent enlarge of epidemic focus, remove infected pigs and provide thebasis for the purification of pig farms.