Establishment And Application Of IPMA And IFA Clinical Antibody Detection Methods For Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome is a kind of respiratory infectious disease caused by porcine reproductive and respiratory syndrome virus,which has a high transmission speed and mortality,and seriously endangers the development of pig industry.The purpose of this study is to establish a stable clinical serum detection method of PRRSV immunoperoxidase monolayer cell test(IPMA)and indirect immunofluorescence method(IFA).The established IFA method is used to detect the antibody titer and neutralizing antibody titer of clinical PRRSV serum,to study the relationship between antibody and neutralizing antibody titer,to evaluate and analyze antibody immunity,so as to prevent disease And provide some technical support for vaccine evaluation and immune evaluation.In this experiment,the filtered PRRS virus solution was inoculated into Marc145 cells,fixed with pre-cooled absolute ethanol after 48 h,added PRRSV standard positive serum,incubated at 37 ℃ for 1h,and then added 1: 700 diluted goat anti-pig Ig G-HRP(IFA add 1: 200 diluted Ig G-FITC fluorescent antibody),incubate at 37 ℃for 1h,and finally DAB color development for 5min,observe the result under the microscope(IFA can be directly observed under the fluorescence microscope),that is,establish the IPMA and IFA clinical of PRRSV Serum antibody detection method.The detection method of IPMA and IFA neutralizing antibody is based on the above method,incubating an equal volume of PRRSV and clinical serum at 37 ℃ for 1h,and then inoculating Marc145 cells,other steps are the same as the above method.In this study,PRRSV’s IPMA and IFA clinical serum antibody and neutralizing antibody detection methods were successfully established.After optimizing the IFA reaction conditions,it was finally determined that 100 TCID50 viruses were inoculated into Marc145 cells.After 48 hours of culture,they were pre-chilled at-20 ℃ Fix with absolute ethanol for 30 min to prepare an IFA cell reaction plate.The serum to be tested starts at 1:50.Based on the strength of the antibody,the antibody can be tested by serial dilution based on this ratio.Sheep anti-pig Ig G-FITC worksThe concentration is 1: 200.Compared with the detection results of the IDEXX kit and the IFA antibody detection method,the total coincidence rate reached 96.5%.And this method only reacts with PRRSV standard positive sera specifically for antigen and antibody,and has no cross reaction with other common swine virus standard positive sera.Antibodies can still be detected when the serum dilution factor is 1: 400,indicating the specificity and sensitivity of this method Sexuality is higher.Using the established IFA detection method,the antibody titer and neutralizing antibody titer of the two farm sera were detected,and the antibody titer was found to be 6.25,12.5,25,50,100 times the neutralizing antibody titer,and the antibody titer was determined.It has a positive correlation with the neutralizing antibody titer.The ratio of neutralizing antibody to antibody is about 1:10 to 1: 100,of which 25 times is the most,accounting for 48%.The neutralizing antibody can be roughly predicted based on the antibody titer The titer provides certain technical support for PRRS prevention,vaccine evaluation,and immunization evaluation.The IPMA and IFA antibody detection methods have the characteristics of strong specificity,high sensitivity and simple operation.They can not only detect antibodies,but also be used for the detection of antigens such as viruses.