The Development And Application Of A Double Antibody Sandwich ELISA For Quantitative Detection Of MERS-CoV Antigen

Middle East respiratory sydrome?MERS?is a newly emerging infectious respiratory disease with high mortality caused by the infection of MERS coronavirus?MERS-CoV?.MERS-CoV has a certain human-to-human transmission ability and still has no sign of disappearance,which has raised concerns about the global spread of MERS-CoV.However,there are no effective vaccines and therapeutic agents available for MERS-CoV.To prevent and control of MERS,it is very necessary to build fast and simple detection methods of MERS-CoV for the quantification of vaccines and early diagnosis of the disease.The spike?S?glycoprotein of MERS-CoV consists of N-terminal S1 subunit and C-terminal S2 subunit.The receptor-binding domain?RBD?located on S1 subunit can mediate the entry of virus by binding DPP4 receptor on the surface of host cells.RBD is the main region to induce neutralizing antibody and becomes the target of developing various candidate vaccines and therapeutic agents.In this study,the four mouse monoclonal antibodies?mAbs?against MERS-CoV S-RBD protein were successfully prepared.The MERS-CoV S1 protein with high purity was purified by using the immunoaffinity chromatography column coupled with the selected monoclonal antibody and anion exchange chromatography column.A double antibody sandwich ELISA method was established for quantitative detection of MERS-CoV antigen.Chapter one:Preparation of mouse?mAbs?against MERS-CoV S-RBD protein and immunoaffinity chromatography column.Objective To prepare mouse?mAbs?against MERS-CoV S-RBD protein and immunoaffinity chromatography column.Methods Mice were intraperitoneally injected with four recoveried hybridoma cell linesagainst MERS-CoV S-RBD protein.The four mAbs?mAb-1,mAb-3,mAb-5 and mAb-13?were purified from ascites by binding saturated ammonium sulfate precipitation method and immunoaffinity chromatography with protein A column.The purified mAbs were identified by Western-blot and indirect IFA.The mAb with the highest affinity was selected by indirect ELISA and coupled to CNBr-activated Bestarose 4B gel to prepare specific immunoaffinity chromatography column.Results The four purified?mAbs?were proved to bind specifically with RBD protein and S protein by Western-blot and indirect IFA,respectively.The mAb-13 was identified as the mAb with the highest affinity by indirect ELISA.The coupling efficiency of mAb-13 was 99.80%.Conclusion The specific mAbs and the immunoaffinity chromatography column were successfully prepared.It laid a foundation for the rapid diagnosis of MERS-CoV and the purification of candidate vaccines.Chaper two:Expression,purification and identification of MERS-CoV S1 protein.Objective To express,purify and identify the MERS-CoV S1 protein with natural conformations.Methods The amplified S1 gene was ligated into the pFastBac1 plasmid to construct the recombinant plasmid pFB1-S1.The recombinant bacmid BpFB1-S1 was constructed by transforming the pFB1-S1 plasmid into DH10Bac competent cells and then transfected into sf9 cells to rescue the recombinant baculovirus rpFB1-S1.The suspension culture supernatants of sf9 cells infected with the rpFB1-S1 baculovirus were purified by immunoaffinity chromatography method and anion exchange chromatography method.The expression,molecular weight,purity and reactivity of the S1 protein were detected by indirect IFA,SDS-PAGE,thin slice scanning and Western blot,respectively.Results The size of PCR product was 2212bp.The pFB1-S1 plasmid and the BpFB1-S1 bacmid were proved to be constructed correctly.The rpFB1-S1 infected sf9 cells showed the specific green fluorescence while there was no fluorescence in rpFB1 infected sf9 cells.A protein band of interest appeared at a molecular weight of about 100 kDa on SDS-PAGE gel.The purity of the purified protein was above 95%.The purified protein was identified to be bound specifically by mAb-13 by Western blot.Conclusion The MERS-CoV S1 protein was successfully expressed,purified and identified,which laid the foundation for the establishment of MERS-CoV detection methods and the development of candidate vaccines and therapeutic agents.Chaper three:Development and application of a double antibody sandwich ELISA for quantitative detection of MERS-CoV antigen.Objective To establish a double antibody sandwich ELISA method for quantitative detection of MERS-CoV antigen and to apply it to the quantification of vaccines.Methods mAb-13,horse anti-MERS-VLPs specific IgG and MERS-CoV S1 protein were correspondingly used as the capture antibody,the detection antibody and the standard substance to establish a double antibody sandwich ELISA.The sensitivity,specificity and reproducibility of the method were also detected.The method was applied to quantify S1 protein in MERS virus-like particles?MERS-VLPs?,MERS pseudovirus and recombinant rabies virus expressing MERS-CoV S1 protein(rSRV9-MERS_S1).Results The linear range of the method was 31.2¹000.0 ng/ml.The limit of quantification and detection of the method were 31.2 ng/ml and 7.8 ng/ml,respectively.The method had no specific reaction with SARS-CoV S1 protein.The coefficient of variation of intra-assay and inter-assay were less than 10%.The concentrations of S1 protein in different supernatants containing MERS-VLPs,MERS pseudovirus(400 TCID₅₀/ml)and rSRV9-MERS_S1(2×10⁸ TCID₅₀/ml)were 186.4 ng/ml,211.9 ng/ml and 490.3 ng/ml,respectively.Conclusion The double antibody sandwich ELISA described in this sudy had good sensitivity,specificity and reproducibility.It could be used for the quantitative detection of MERS-CoV S1 protein antigen.