Development Of Latex Agglutination Detection Of Antibody Against Infectious Bovine Rhinotracheitis Virus

Infectious Bovine Rhinotracheitis (IBR) is a disease of cattle and bison, caused by bovine herpesvirus type1. IBR is characterized by respiratory and/or genital manifestation, The clinical symptoms include infectious rhinotracheitis, conjunctivitis, infectious purulent vaginitis, orchitis and abortion.It is widespread in the world, serological testing and removal of infected animals are best methods to control the disease. At present, Austria, Sweden, Italy (province of Bolzano), Switzerland, Denmark, Norway Finland and parts of Germany (the ’Oberfranken’ and ’Oberpfalz’ districts of Bavaria have sucessfully eliminated IBR. However, with respect to the developing countries, eradication would be difficult, perhaps impossible, and very expensive.For the diagnostic of the disease, the methods recommended by OIE in international trade, including the separation of the virus in the semen, real-time PCR, virus neutralization test, enzyme-linked immunosorbent assay. But these methods are either relatively time-consuming, or require specific equipment, also requires the detection of certain experimental skills. This study was designed to establish a rapid and easy detection method for field usage.In this study, sucrose gradient centrifugation, separation and purification processing complete BHV-1virus particles, Mice immunized with purified virus particles, and preparation of monoclonal antibodies, and then sensitized latex, to determine the optimal sensitization and blocking conditions. Sensitivity, specificity, repeatability of LAT was compared with serum neutralization assay. Finally,512clinical samples collected from Xinjiang, Inner Mongolia, Jiangsu were detected by LAT. The contents of studies include the following:1. Preparation of monoclonal antibodies against BHV-1(1)BHV-1was proliferation with MDBK, using ultra-high-speed sucrose gradient centrifugation method to purify and concentrate virus particles, and gained the virus particles which has been processed completely.(2) Using inactivated purified virus particles to immune Balb/c mice, and fusion, screening, monoclonal obtain three stable hybridoma cell lines which are secrete against BHV-1monoclonal antibody. Expand after the intraperitoneal injection of Balb/c preparation of ascites.2. Establishment of the LAT and the primary application(1) Suitable concentration of BHV-1antigen was used to sensitize and appropriate blocking conditions are determined. And then specificity test and repeatability test results show that the method has good specificity and reproducibility.(2) The developed LAT was compared with SNT screened antibodies to BHV-1by simultaneously detecting252bovine serum samples that are from Xinjiang and Inner Mongolia, the total coincidence rate was96.4%, the two methods has a high match rate, the difference was not significant (P>0.05), and therefore, the LAT of detecting antibodies to BHV-1was established which is rapid and accurate.(3) The established method to Detected512clinical sample are randomly collected from Inner Mongolia, Xinjiang, Jiangsu regions. A total of177positive sera, the positive rate was34.6%, this is basically the same with other laboratory test results.Take together, this study successfully prepared against BHV-1monoclonal antibody, and developed the latex agglutination test as rapid diagnostic tools for disease prevention and control.