Study On Humoral Immunity Of Echovirus 11 Candidate Vaccine And Establishment Of ELISA Antigen Quantitative Assay

Objectives:An Echovirus 11（Echo-11）strain was isolated in Vero cells and polyclonal antibodies against the structural proteins VP1,VP2,VP3 and VP4 of Echo-11 were prepared using recombinant proteins expressed in E.coli cells.As vaccine candidates,the empty and full particles（EP and FP）of the Echo-11 strain were used to study the immunogenicity and immune persistence in Balb/c mice.The purified Echo-11 viral particles were characterized by the polyclonal antibodies and the monoclonal antibodies and polyclonal antibodies against Echo-11 viral particles were prepared.Using two kinds of antibodies,a double antibody sandwich ELISA method was established to quantitatively detect the antigen of Echo-11.Methods:The plasmids with inserted genes of Echo-11 VP1-VP4 were constructed.The prokaryotic expression vectors p GEX-6p-1-VP1 to-VP4 was used to express the VP1-VP4 fragments which were purified and used to raise antibodies in rabbits.The harvest of Echovirus-infected cells was obtained from the 10-layer Vero cell factory.After purification EP and FP,their components were characterized by rabbit polyclonal antibodies.The purified viral particles were as the immunogens to generate mouse anti-monoclonal antibody and rabbit anti-virion-polyclonal antibody.A double antibody sandwich ELISA method was established.The poly-and monoclonal antibodies were conjugated with HRP as detection antibodies and the same un-conjugated antibodies were used as capturing antibodies,respectively.The sensitivity of the two ELISA methods to detect and quantitate Echo-11 antigen was compared,and one of the methods with higher sensitivity was chosen.After the optimal working concentrations were determined by chessboard titration,the established method was verified for the linear range,accuracy,precision,specificity and stability of the method.Results:Rabbit polyclonal antibodies and mouse monoclonal antibodies with ELISA titers of 1:106 and 1:108 were generated.Both antibodies could specifically recognize Echo-11 antigen without cross reactions with other Enteroviruses.The heavy chain subclass of monoclonal antibody is Ig G2 b,the light chain subclass is Ig K,and the recognition protein of monoclonal antibody is VP1.The vaccine candidate strain was isolated in Vero cells.The components and purity of the two particles were analyzed by SDS-PAGE and WB.The protein components of EP and FP particles were similar to other Enteroviruses.EP was composed of VP0,VP1 and VP3 with a purity of78%;FP is composed of VP1,VP2,VP3 and VP4 with a purity of 98%.The immunogenicity of virus particles was studied in a mouse model.The data showed that the differences in immunogenicity between EP,FP and EP+FP were not statistically significant.A quantitative ELISA for antigen detection was established.The detection range was 4.0-16.0 μg/ m L,with a R2 value greater than 0.99.Samples with high,medium and low concentrations were used to verify the ELISA kit.The accuracy validation results showed that the recovery rates were between 96.9% and107.5%.Both the CVs in intra-and inter-assays on precision were not higher than12%.No cross reactions with the antigens other than Echo-11 were observed.After incubation of ELISA kit at 37℃for 6 d,the CVs of results in stability test were lower than 3%.The developed method might be used for quantitative determination of samples at various stages of preparation of Echo-11 antigen.The established method can be used to detect the antigen content of samples in the antigen preparation processes and finished vaccine candidates.Conclusions:The immunogenicity of Echo-11-49/XY/CHN/2016 strain isolated in Vero cells showed that it’s EP and FP could be used as inactivated vaccine candidates,which provided theoretical basis and guiding experience for the subsequent research and development of Echo-11 vaccine.The established antigen quantitative detection method can be applied to the antigen quantification of candidate vaccines,and has guiding significance for the antigen detection of samples in different stages of antigen preparation.