Development And Preliminary Application Of ELISA Kit For Detection Of PORcine Pseudorabies Virus GB Blocking Antibody

Pseudorabies(PR),also known as Aujeszky’s disease(AD),is a highly infectious disease caused by pseudorabies virus(PRV).Pseudorabies is not specific to the host,and has strong infectivity to ruminants,rodents,mammals and carnivores.Recent studies have suggested that humans may also be potential hosts of PRV.PRV infection in pigs can cause severe clinical symptoms and acute death,and the main clinical symptoms are diarrhea,vomiting,nervous system disorder,dyspnea,slow growth,breeding disorder and so on.Although scientists have spent the past few years working on diagnostic methods and vaccine development,PRV remains an important infectious disease widely prevalent in China’s pig industry.Currently,the pig pseudo rabies for the global pig industry,in the event of the disease will be may cause enormous economic losses,should build practical and reliable can rapid testing method of the pig pseudo rabies virus,in order to realize the timely diagnosis of the disease,so that they can respond quickly,blocked its route of transmission,thereby lowering its brings the economic loss.Current studies have found that gB gene is very important in PRV as the main protective antigen gene with high conserved and immunogenicity.Therefore,this paper developed a PORcine PSEUDorabies virus gB blocking ELISA kit and applied it preliminarily.This study mainly through the establishment of the method,the determination of critical value,raw materials screening,validation test,clinical serum detection and coincidence rate detection and other experimental methods to establish and optimize its conditions.According to the results,the establishment method was as follows:the purified pseudorabies virus was diluted to 5 μg/mL and added to the imported enzyme-plate,then added to the blocking solution at 2-8℃ for 14 h,and then dried at 37℃ for 2 h.During the detection,the detected serum was twice diluted and added to the coated plate,cultured at 37℃ for 30 min,then added the enzyme-labeled antibody,cultured at 37℃ for 30 min,washed,added the chromogenic solution and placed at 37℃,added the termination solution after 10 min,and read at 450 nm of the enzyme-labeled reader.According to the test,the critical value of negative and positive is 0.6;The coefficient of variation between batches was within 15%.The coefficient of variation within the batch was less than 10%.The serum to be tested can be diluted up to 2048 times and the test result is still not positive;No cross-reaction with other six swine viruses;The coincidence rate reached 95.37%.The institute built pig pseudo rabies virus gB blocking antibody ELISA detection method,shows that the sensitivity and specificity were relatively high,has the advantages of operation simple and convenient and fast,can quickly realize the pig pseudo rabies virus detection,is able to make farming pseudo rabies prevention and diagnosis of pigs and relevant epidemiological studies offer certain help.According to the preliminary application of the kit,detect the level of antibodies generated after pig pseudorabies immunization,and vaccinate the pigs with reduced antibody level again to prevent pseudorabies.