Development Of Monoclonal Antibodies Against Transmissible Gastroenteritis Virus And Establishment Of Detection Methods

Transmissible gastroenteritis(TGE)is a highly contagious gastrointestinal infectious disease caused by transmissible gastroenteritis virus(TGEV).It occurs mostly in winter and spring.The main clinical symptoms are diarrhea and vomiting.The mortality of piglets after infection is extremely high and caused serious economic losses.Therefore,a rapid and accurate diagnosis of TGE at the beginning of the infection is critical.By studying the TGE antigen and antibody detection methods,it is possible to better prevent and control the epidemic.In this experiment,TGEV was purified by ultracentrifugation and sucrose density gradient centrifugation,BALB/c mice were immunized,cells were fused after three immunizations,positive hybridoma cells were screened by indirect ELISA,and positive cells were cloned by limiting dilution method.After sub-cloning,hybridoma cell lines stably secreting antibodies were obtained,which were 3G11,1D6,2E5 and 5D1,respectively.Four monoclonal antibodies were found to be specific for TGEV-N protein by western blot;among the four cells,the 3G11,2E5 and 5D1 subclasses were all Ig G1,and the subclass of 1D6 was Ig M;indirect immunofluorescence assay identified four monoclonal antibodies that bind to cells infected with TGEV;in the specific reaction experiment,four monoclonal antibodies did not react with Porcine Epidemic Diarrhea virus(PEDV),Porcine Rotavirus(PRV),and Porcine Circovirus 2(PCV2).In this experiment,ascites of 3G11 cells was prepared from BALB/c mice,and the ascites was purified by a purification column,and the titer was determined to be 10~5.A double antibody sandwich ELISA assay was established using laboratory-preserved TGEV-N monoclonal antibody E10F10D5 and 3G11 monoclonal antibody.Compared with the traditional Das-ELISA,the detection of antibody 3G11 was coupled with HRP,which eliminated the incubation of the enzyme-labeled secondary antibody,reducing the operation steps and time.Through the exploration and optimization of the reaction conditions,it was determined that when the concentration of E10F10D5 monoclonal antibody was 4 ?g/m L and the concentration of HRP-3G11 monoclonal antibody was 1?800,the detection effect was the best.Moreover,when processing the stool sample,it is not necessary to add the virus lysate,and only need to mix and freeze.Through further analysis,it was confirmed that the method has good specificity,sensitivity and reproducibility,and the coincidence rate of clinical sample detection reached 92.6%,which can be used for clinical detection.In this experiment,an immunoenzymatic histochemical method for detecting viruses in tissues was also established using HRP-labeled 3G11 monoclonal antibody.To verify whether HRP-labeled 3G11 m Ab can be used to detect TGEV infection in tissue lesions,the response of the virus to HRP-3G11 was first detected by cell culture TGEV.As a result,it was found that HRP-3G11 m Ab can specifically bind to TGEV.By injecting TGEV into one-week-old piglets,the duodenum,jejunum and ileum were taken respectively,and HRP-labeled 3G11 antibody was used as the primary antibody to establish an immunohistochemical method for detecting tissue virus.There was no significant difference compared with the results of unlabeled antibodies,indicating that it can be used for the detection of TGEV in small intestine tissue.To establish an indirect ELISA method for detecting TGEV antibodies,the laboratory-preserved TGEV-N protein-expressing strain p Pro Ex HTb-TGEV-N/BL21 was activated to induce expression and purification of TGEV-N protein,an indirect ELISA method for detecting serum antibodies was established using the purified protein as a coating antigen.After the conditions were optimized,the coating concentration was 0.5 ?g/m L,the optimal blocking time was 37? 1 h,the optimal dilution of the serum sample was 1?160,and the action time was 37?1 h.The optimal duration of secondary anti-HRP-labeled rabbit anti-porcine Ig G was 37°C for 45 min.The method can specifically detect TGEV positive serum,and has good sensitivity and reproducibility,and can be used for clinical detection of TGEV antibodies.In summary,this experiment used HRP-labeled 3G11 monoclonal antibody to establish a double antibody sandwich ELISA method for detecting TGEV fecal material and an immunohistochemical method for detecting tissue materials.Using purified TGEV-N protein,a TGEV was established.An indirect ELISA method for antibodies.The establishment of these three detection methods laid the foundation for the effective prevention and control of clinical TGE.