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 13.37?g/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.