Preparation And Identification Of Monoclonal Antibodies Against S Protein Of TGEV

In this research, recombinant spike protein of transmissible gastroenteritis virus(TGEV) whichwas expressed with baculovirus expression system and a recombinant plasmid named PCI-Sa withprimary epitopes of TGEV were used to immune BALB/c mouses respectively. Cell fusion wasperformed according to the traditional method. Indirect ELISA assay was carried out to screenhybridoma cell lines using supernatant of TGEV propagation as for the first kind of antigen, andpurified recombinant spike protein expressed in Lactococcus lactics for the second kind of antigen.By limiting dilution and 3 serials of clones, 2 hybridoma cell lines (designated C7C8B2 andB5G8G7) producing antibodies against TGEV were obtained, and 1 hybridoma cell line(designated E6D9A12) secreting antibodies recognizing S protein was also gained. Theantibodies of 3 hybridoma cell lines were respective belong to IgM,IgM,IgG2a subgroup. Afterextended culture and several freeze thaw cycles, monoclonal antibodies could still be stablysecreted. The average number of chromosomes in 3 hybridomas was 88±10 couples. Investigatedthrough indirect ELISA, the titers of 3 cell-cultured antibodies were measured to be 1:200, 1:100,1:500 and titers of antibodies produced in ascities were respectively 1:2×10~5, 1:1×10~4, 1:6×10~5.Using the supernatants of TGEV, PEDV, PRV and PrV propagation as antigens, 3 antibodiesall exposed specific recognition characters. And they could not react with PEDV, PRV or PrV. Itwas confirmed that these specific antibodies could be used for TGEV diagnosis.Immunofluorescence assays were performed with ST cell monolayers infected with TGEVprobed by the antibodies or supernatant of SP2/0 cell culture. After staining with FITC labeledsecondary antibodies, strong yellow-green fluorescence could be observed in plasma and onmembrane of cells. However, no fluorescence was detected with the addition of supernatant ofSP2/0 cell culture. With recombinant S protein as immunogen, antibodies were found tospecifically bind to pathogens with the confirmation of indirect immunofluorescence assays.Using purified TGEV as antigen, dot-ELISA was performed to determine the correlationbetween the monoclonal antibodies and TGEV. It was revealed that the supernatants of 3hybridomas but not supernatant of SP2/0 cell culture could recognize TGEV. The specificity ofantibodies to virus was further ensured.All the results indicate that with recombinant S protein as immunogen, the screenedmonoclonal antibodies own rather excellent specificity to TGEV. And this could provide foundationfor TGEV pathogenesis research and rapid diagnosis.