Development Of A Duplex Fluorescent Microbead-based Immunoassay For The Detection Of Swine Pseudorabies Virus GE And GB IgG Antibodies

Pseudorabies?PR?,also known as Aujezsky's disease,is an acute viral infection caused by the pseudorabies virus?PRV?.As a natural host of PR,swines are also very important storage hosts and detoxifiers,which bring huge economic losses to the swines industry.PRV is a member of Herpesviridae,swines infected with PRV at different ages show different symptoms.Piglets are generally deadly encephalitis.Pregnant sows will cause miscarriage,stillbirth,and mummification.Fattening pigs disordered breathing occurs,adult pigs were infected with latent infection,long-term carriers of the virus detox.In our PR eradication plan,although attenuated gE vaccines are now widely used,simply using the vaccine does not solve the problem fundamentally.Establishing a double efficient differential diagnosis technique that can effectively distinguish between wild-type infected swines and vaccine-vaccinated swines,and at the same time capable of monitoring vaccine-immune swine IgG is the key to eventually eradicate pseudorabies.For the current status of PR,the purpose of this study is to establish a rapid,highly specific,and highly sensitive immunoassay for single-fluorescent microbead-based immunoassay.On this basis,we developed an efficient dual detection method,which can rapid identification of porcine IgG for wild-type PRV infection and vaccine immunization.In this study,pMAL-c5x prokaryotic expression system was used to construct PRV gE and gB recombinant plasmids,and PRV gE and gB fusion MBP and His-tagged recombinant proteins were obtained.SDS-PAGE and WB were analysed afer Ni²⁺affinity chromatography.Recombinant proteins with good immunogenicity and antigenicity were obtained,laying the foundation for the further establishment of indirect ELISA and FMIA methods.In present study,PRV gE and gB recombinant proteins were used as coating antigens.After optimization of various factors affecting ELISA,the indirect ELISA assay was established.The ROC curve analysis showed that when the critical value of gE was 0.399,the maximum AUC was 0.970,the sensitivity was 95.83%,and the specificity was 91.37%;when the critical value of gB was 0.324,the AUC maximum was 0.928 and the sensitivity was 91.67%,and the specificity was 88.89%,indicating that the established ELISA method has diagnostic value for the detection of PRV.In this study,PRV gE and gB recombinant proteins were used as antigens to couple with microbead 12 and 28 respectively.Using gE-12 and gB-28 coupled complexes as capture carriers,FMIA was established separately.The specificity of the two methods is good,and there is no cross-reaction between the conjugating antigen and other common swine virus positive serum.The results of the analysis by ROC curve showed that when the critical value of gE was 5991.5,the maximum AUC was 0.981,the sensitivity was 92.3%,and the specificity was 99.26%.When the gB critical value was 2862,the AUC maximum was 0.989 and the sensitivity was 96.3%,and the specificity is 95.74%.The ACU of both FMIAs was greater than that of ELISA?gE 0.981>0.970;gB 0.989>0.928?,indicating that FMIA is superior to the ELSA method established in this study.Based on single FMIA,this study placed gE and gB in the same reaction system and established and validated a dual FMIA that can be used to simultaneously detect PRV gE and gB IgG antibody,which is linearly related to the detection results of the single detection method and can reflect the same index.In conclusion,the present study uses the prokaryotic expression vector pMAL-c5x for the soluble expression of gE and gB recombinant protein.The prepared recombinant protein was used as an antigen to couple with the microspheres,and the pig serum was used as a sample to establish a single FMIA detection method for gE and gB IgG antibody,respectively.Based on this,a duplex FMIA detection method for gE and gB IgG antibody was established.The establishment of the method can lay the foundation for the further establishment of multiple detection methods for porcine viral diseases and provide ideas for the establishment of new serological diagnostic techniques for other animal disease antibodies.