| Porcine transmissible gastroenteritis(TGE)is an acute,highly contagious enteric infectious disease,which is caused by porcine transmissible gastroenteritis virus(TGEV),characterized by severe vomiting,diarrhea and high mortality in piglets within 2 weeks old.It is difficult to clinically differentially diagnose TGE with other enteric infectious diseases with similar clinical signs caused by porcine epidemic diarrhea virus(PEDV),porcine delta coronavirus and porcine rotavirus.Colloidal gold immunochromatographic strip has some characteristics of high specificity and simple operation,and it is suitable for veterinarians to rapidly diagnose diseases clinically.TGEV N protein is known to be an important target protein for TGEV antigen and antibody detection method study.Therefore,this study used N protein as the target protein to prepare monoclonal antibodies,and developed colloidal gold immunochromatographic strip for rapid detection of TGEV antigen,aiming to provide important technical support for the rapid diagnosis and prevention of TGE.In this study,three female BALB/c mice were immunized with recombinant TGEV N protein emulsified with Freund's adjuvant,and immunized 3 times at intervals of 2 weeks.On day 10 after the third immunization,the serum specific antibody of 3 mice were detected by ELISA and the antibody titers between 1:10240 and 1:20480.The mice with antibody titer of 1:20480 was selected for subsequent cell fusion and booster immunization via intraperitoneal injection.On the third day after the booster immunization,the mouse spleen cells were mixed with SP20 cells,and the positive wells secreting TGEV N protein-specific antibody were screened by ELISA.After 3 times of subcloning of the positive wells,four hybridoma cell strains secreting TGEV N protein monoclonal antibodies(McAb)were obtained,which were named 2F5,1G11,1F12,and 1B10,respectively.Western blot analysis showed that all four McAbs could specifically bind to TGEV N protein.Immunoperoxidase monolayer assay showed that 4 monoclonal antibodies were able to specifically bind to TGEV in infected PK15 cells.The antibodies secreted by the four hybridoma cells were all IgG1 subclasses and Kappa chains(IgG1?).Furthermore,4 hybridoma cells were intraperitoneally injected into 10-week-old female BALB/c mice,and ascites of 4 monoclonal antibodies were prepared.The titer of ascites antibody by ELISA was 1:655360,1:655360,1:1310720,1:655360.Meanwhile,anti-N protein polyclonal antibodies were prepared by immunizing two healthy New Zealand white rabbits with the N protein emulsified by Freund's adjuvant.After three immunizations,the ELISA antibody titer of N-protein specific antibodies were>1:10~5.The rabbit anti-N antibody could specifically bind with TGEV N protein and TGEV identified by Western blot.Furthermore,the 2F5 monoclonal antibody purified by the mouse monoclonal antibody purification kit was used as a detection line(3 mg/mL),the purified rabbit anti-TGEV N antibody(150?g/mL,pH 8.0)was used as a gold-labelled antibody,and goat anti-rabbit IgG(1 mg/mL)was used as a quality control line to assemble a test strip.The test strip can specifically react with the TGEV N protein,and the lowest concentration of TGEV N protein that can be detected was 3.125?g/mL.The test strip did not crossly react with PEDV,pseudorabies virus,porcine circovirus,swine fever virus,porcine reproductive and respiratory viruses.Finally,15 of small intestines and contents from natural sick piglets with diarrhea signs were detected using the assembled test strips and RT-PCR,the positive detection rates of test strips and RT-PCR were 20.0%(3/15)and26.7%(4/15),respectively,and the coincidence rate of the two methods is 93.33%.The results indicated that the colloidal gold immunochromatographic antigen test strip based on TGEV N protein monoclonal antibody preliminarily prepared could rapidly and specifically detect TGEV in clinical samples,and can be used for on-site diagnosis of TGE. |
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