Development Of Monoclonal Antibody ELISA For Detection Type Asia Ⅰ FMDV Antigen And Molecular Identification Of Vaccine Strains

Taking sucrose desity gradient centrifugation method to pure 146S antigen of type Asia I (xinjiang vaccine strains) inactivated foot-and-mouth disease virus, then immunized mouse(blab/c) and fuse the SP2/0 myeloma cells with the spleen cells, after screening by IS-ELISA, four hybridoma cell line secreting stably monoclonal antibody against type Asia I FMDV were prepared. By identifving, the IS-ELISA antibody titers of culture supernatant and ascities to 1^#G₃C₂, 2^#G₆B₉ and 10^#C₂G₁ were not less than 1:20000, 1:10⁶ respectively, the IS-ELISA antibody titers of culture supernatant and ascities to 8^#E₃C₁₀ were 1:5120, 1:81920 respectively. The immunoglobulin subclass of the McAb to 1^#G₃C₂, 8^#E₃C₁₀,10^#C₂G₁,2^#G₆B₉ were IgG₁, IgG₁, IgG₁, IgM respectively By SDS-PAGE and immunihistochemistry staining, we identified that the McAbs of 1^#G₃C₂,2^#G₆B₉,10^#C₂G₁ recognized discontinuous epitopes, and the McAbs of 8^#E₃C₁₀ recognized continuous epitope. All are the neutralizing McAbs, but the McAb of 8^#E₃C₁₀ have low PD₅₀. All have highly specific.A IS-ELISA based on the mixtue of three different monoclonal antibodys(1^#G₃C₂,2^#G₆B₉,10^#C₂G₁) has been developed and it was applied to the testing of the type Asia I FMDV inactivated antigens, type O FMDV inactivated antigens and BHK₂₁ cell surpernatant. The experiment result demonstrated that the array have highly specific and there is a better correlation between our determined OD value and type Asia I antigen diluted multiple. We had studied totally 11 batch the antigen yields of the formulation of FMD vaccines series, including serotype Asia I, O and bivalent (O-Asia I), the data indicated that the whole virions of commercial vaccines is different in differ corporation production and differ batch. Then it is expected to apply in developing the testing of effective antigen yields in type Asia I FMD commercial vaccines and it will become a alternative proposal for assessment of vaccine potency.In order to identified the identity of viral seeds in manufacture plant, we amplified and secquenced the VP1 gene of two FMD vaccine strains(lc vaccine strain and xinjiang vaccine strain) by using RT-PCR. The result indicated that all corporation have taken FMD vaccine with standard procedures. At the same time, we compared the VP1 nucleotide acid sequence and deduced amino acid sequence of two vaccine strains with jiangsu strain, which is the main prevalence strain of type Asia I in China. The homology analysis showed that xinjiang strain to jiangsu strain's nucleotide acid sequence majority is 84.4%, amino acid majority is 89.6%; LC vaccine strain to jiangsu strain's nucleotide acid sequence majority is 84.4%, amino acid majority is 90.0%; xinjiang strain to LC vaccine strain's nucleotide acid sequence majority is 83.4%, amino acid majority is 87.2%. Through the analysis of VP1 gene and deduced amino acid sequence, we found that two vaccine strains and jiangsu strain are not belong to one genotype(VP1 nucleotide acid sequence majority＞85% classified to the same genotype). There are 9 and 7 mutation respectively in VP1 G-H loop amino acid 140 to 160 and carboxyl extremity amino acid 203 to 211 for xinjiang vaccine strain and LC vaccine strain, but both arginine-glycine-aspartic acid (RGD) sequence didn't change, and they didn't lose the ability for using integrin approach to recognize epitopes of host cell. So we didn't know the vaccine come fi-om FMDV mxinjiang strian and LC strain whether could protect host from attacking of prevail strain (jiangsu strain) or not. It need to research further.