Preparation And Applying Of PRRS Monoclonal Antibody

Porcine Reproductive and Respiratory Syndrome (PRRS) is the new infection diseasecaused by Porcine Reproductive and Respiratory Syndrome virus (PRRSV) has raised greatconcerns. Clinical symptom of PRRS are characterized by reproductive failure is sows andbirth of weak piglets that often die soon after birth of respiratory disease and secondaryinfection. This sickness for was exploded the first time in 1987, at present has proliferated allover the world, has created the huge economic loss for the various countries pig-breedingindustry in many countries.Monoclonal antibodies was invented in the 1970's is a high-tech of biology. Monoclonalantibodies are produced by cell fusion technology. Compared whit polyclonal antibodies,monoclonal antibodies have following virtues: identical structures, uniform component, highspecificity, animate bio-activity and fewer crossing reactivity, has the obvious superiority.How to creatively realize the application value of the specified monoclonal antibody in thedifferent domain is investigative focal point at present.Balb/c mouse was selected for the experiment and immunized with PRRSV that waspurified by SephadexG200 laminar analysis and was mixed with Freund's adjuvant thoroughly.The Balb/c mouse was boosted once before fusing. The spleen cell from immunized Balb/emouse was fused with SP2/0 and then incubated in cell culture plates to screen hybridoma cellline. The fusion cells were got from 144 holes of the cell culture plate and the rate ofcellular fusion was 52.17％. Indirect ELISA method was used to screen the fusion cells, theresults showed that 11 positive holes were got and the rate of fusion was7.64％.The cells werecloned by limiting dilution assay and three hybridoma cell strains secreting monoclonalantibodies for PRRSV were got and named 2B7, 5F9 and 3G2, respectively. Meanwhile thevalence of purified abdominal dropsy was 1: 1.28×106,1:6.4×105,1:3.2×105, respectively,and they were belong to IgG1 subgroup. The detected results showed that the specificity andstability of the three monoclonal antibodies were better.Sensitive and effective ELISA testing method was constructed for detecting PRRS andthe method has the vital significance and application value for the PRRS research ondiagnostics and immunology. Rabbits were immunized by PRRSV to get rabbit-anti-PRRVpolyclonal antibodies that were purified by ammonium sulfate precipitation method. Thepolyclonal antibody was used as coating antibody and the McAb2B7 marked with HRP wereused as enzyme labeled antibody to construct double sandwich enzyme labeledimmunosorbent assay (ELISA) for the detection of PRRSV. The best coating concentration of the coating antibodies and the best working concentration of the enzyme labeled antibodywere tested and the results showed that the best concentration was7.08μg /ml (1:800diluted) and 2.96μg/ml(1:1600 diluted), respectively. This method is special, sensitive,reliable, conveniently and needn't specific installation and technology to promotion andapplication.