A ELISA Detection Method For PRRSV Virus Based On Monoclonal Antibody With Broad Viral Recognition And Characterization Of Its Coding Sequence

Porcine reproductive and respiratory syndrome(PRRS,commonly known as blue-ear disease)is a disease which cause serious loss to the pig farming industry all over the world.Over the years,PRRSV has been endemic in China and caused extremely economic losses.The key to control PRRSV requires accurate diagnosis methods for monitoring the epidemic situation.However,it is very difficult to make accurate diagnosis according to the clinical symptoms and epidemiological characteristics of PRRS.Therefore,there is urgent need to establish a rapid,simple,accurate diagnostic technique.The purpose of this study was to establish an ELISA detection method by using PRRSV M protein monoclonal antibody to capture PRRSV virion,which could be used for the diagnosis of PRRSV.Meanwhile,the monoclonal antibody was purified and the coding sequence was determined,so as to develop an efficient PRRSV antigen diagnostic kit.The results mainly include the following aspects:1.After purification,the monoclonal antibody was identified as Ig G2 b,and then purified Ig G with Protein G Resin column.After purification,the strips were clear and no redundant strips in the electrophoresis diagram of PRRSV anti-m-mab,indicating that the purified anti-m-mab was of higher purity and less heteroprotein.By RNA isolation and cloning of c DNA sequence coding for Mabs from hybridoma cells,sequencing data showed that this monoclonal antibody was encoded by germ line VH and VL genes.Sequence comparison showed that the homology of the whole coding sequence and the germ line genes was up to 99%,and there was no somatic mutation and affinity maturation process.2.SD16,GD-hd,JXA1,VR2332,VR2385,CH1 a,and NADC30L-Lik were inoculated with susceptible cell Mac145 respectively,and the above strains were detected by the IFA on hybridization.The results showed that the above 7 strains could be detected by anti-M-Mab,so it could be concluded that anti-M-Mab had the characteristics of broad-spectrum recognition of hetergenous PRRSV strain.Based on the above conclusions,the i ELISA of SD16 and anti-m-mab was conducted to further verify the conclusion.3.Due to the Double antibody sandwich ELISA can catch the virus directly and high sensitivity,thus we investigate to establish a sandwich ELISA detection method of PRRSV viron.The ELISA protocol was optimized.Based on the results,the experiment was carried out with SD16,GD-hd,JXA1,VR2332,VR2385 and CH1 a strain,and the results showed that all of them could be specifically combined with anti-M-Mab.Subsequently,the virus ratio was diluted,and the specificity was significant at the level of 10-6,so it could be applied to the laboratory PRRSV test diagnosis.