Preparation Of Monoclonal Antibodies Against Recombinant Nucleoprotein Of TGEV And Preliminary Development Of A Double Antibody Sandwich ELISA

Transmissible gastroenteritis virus（TGEV）cause severe diarrhea in pigs and death in piglets.In particular,the mortality rate of piglets within 2 weeks of age is up to 100%.It is easy to have a mixed infection with other enterovirus,and it is very similar to the clinical symptoms of porcine epidemic diarrhea virus（PEDV）.All of these factors make the detection of the pathogen a problem.In this experiment,a double antibody sandwich ELISA for detection of antigen was established by using monoclonal antibody against TGEV recombinant N protein and polyclonal antibody.The well-tried method can be used as a reliable and simple method for the diagnosis of pathogens.This study is carried out from the following aspects:1.Preparation of monoclonal antibodies against TGEV recombinant N protein.Recombinant N protein was expressed by Escherichia coli prokaryotic expression system.After purification and identification,BALB/c mice were immunized.After cell fusion,2 hybridoma cell lines,2G7 and 2C2,capable of stabilizing antibody against recombinant N protein,were obtained.The isotypes of the two monoclonal antibodies were found to be both IgG1.And ascites titer of two monoclonal antibodies was 1:10⁸.The result of indirect ELISA showed that the antibody titers of supernatant of the 20^th passage of cells were 1:12800,which indicates stable and strong antibody secreting capacity of the two hybridoma.2.Preparation of polyclonal antibodies against TGEV and recombinant N protein.The purified virus and recombinant N proteins were used to immunize rabbits.Two kinds of polyclonal antibody with high purity were obtained by purification of serum.The titers of indirect ELISA were 1:204800 and 1:25600.The determination concentration was51.64mg/ml and 37.28mg/ml.3.Establishment of a double antibody sandwich ELISA detection method.Experimental results showed the best detection when 2C2 monoclonal antibody used as the capture antibody and N polyclonal antibody used as testing antibody.The optimal coating for 2C2McAb was diluted 1000 times at 37℃for 2 hours.The testing antibody N protein PcAb was diluted 1000 times with 1 hour of incubation and antigen reaction for 2 hours at 37℃.The enzyme labeled antibody was diluted 2000 times at 37℃for 30 minutes.The substrate is coloured at room temperature for 15 minutes.Through the detection of specificity,sensitivity and reproducibility,it showed that the method could capture TGEV in a special way and have no cross reaction with PEDV and PRV.The lowest detection concentration of the virus was 13.37μg/ml.The coefficient of variation within the batch was less than 5%,the intraassay coefficient of variation was less than 10%,and the reproducibility was good.