Preparation Of Monoclonal Antibody Against Chikungunya Virus E2 Protein And Establishment Of ELISA

Background:Chikungunya fever（CHIKF）is a disease caused by Chikungunya virus（CHIKV）infection transmitted by vectors Aedes aegypti and Aedes albopictus mosquitoes,with fever and joint pain as the main symptoms.Since 2000,CHIKF has caused several large outbreaks worldwide,resulting in huge economic losses.However,CHIKV infection is often misdiagnosed as dengue virus infection because the clinical symptoms caused by CHIKV infection are very similar to those of dengue virus infection and the two viruses are often co-infected.Therefore,establishment of specific detection methods for CHIKV infection is of great importance for prevention and control of CHIKF.Objective:To prepare monoclonal antibodies with high affinity against CHIKV and cross-reactivity with wide spectrums of CHIKV.To establish an enzyme-linked immunosorbent assay（ELISA）on basis of screened monoclonal antibodies in order to provide a tool for rapid and accurate diagnosis of CHIKV infection.Methods:（1）Immunize mice with the recombinant envelope protein E2（CHIKV-E2）of Chikungunya virus（CHIKV）to produce anti-CHIKV-E2 monoclonal antibodies using the standard hybridoma technique.（2）Express CHIKV-E2 proteins of three genotypes using a Eukaryotic expression system,and validate bindings of monoclonal antibodies to different genotypes of CHIKV-E2 via Western blotting.（3）Use CHIKV-E2 recombinant protein as an antigen to evaluate the binding affinity of monoclonal antibodies to CHIKV-E2 via indirect ELISA or Bio-Layer Interferometry（BLI）.（4）Screen pairwise combination of monoclonal antibodies via a double-antibody sandwich ELISA to obtain the optimal combination of antibodies.（5）Evaluate the sensitivity of optimal recombination of antibodies in sandwich ELISA capturing serially-diluted CHIKV-E2 recombinant protein,and capturing other inactivated arboviruses or their E proteins for the specificity of ELISA.（6）Finally,the specific epitopes that monoclonal antibodies were against were analyzed via Western blotting and ELISA using deletion mutants of CHIKV-E2 protein structural domains and CHIKV-E2 peptide library as antigens.The conserved nature of antigenic epitopes was analyzed by bioinformatic methods.Results:In this study,25 monoclonal antibodies that reacted with CHIKV-E2 antigen were obtained by the conventional hybridoma technique,among which 6 monoclonal antibodies showed above 10 S/N values in ELISA.Western blot analysis showed that these six monoclonal antibodies were able to bind to different spectrums of CHIKV-E2 protein.Indirect ELISA analysis of the antibodies showed that the EC50（ng/m L）of five antibodies（1F1:18.62;2F10:6.724;3B9:5.512;3F4:19.43;4G10:3.02）was higher than that of the commercially available antibody CHK265（EC50=33.94 ng/m L）,and the antibody 1G10 had the lowest EC50（ng/m L）（61.13）.Further BLI analysis revealed that the KD（n M）values of the antibodies ranged from1.17×10^-8 to 9.43×10^-10,all of which were lower than 10^-7.These results indicated these six antibodies had high affinity with CHIKV-E2 protein.In sandwich ELISA,the combination of 2F10 as the capture antibody and 1G10 as the detection antibody,showed the highest S/N values.The lowest concentration of capturing CHIKV-E2recombinant protein was 771.7 pg/m L.The optimal combination of antibodies did not capture other arboviruses in sandwich ELISA.Finally,we revealed via WB and ELISA that the antibodies 1G10 and 2F10 recognize the residues 281aa-294aa in the domain C,and 190aa-203aa in the domain B of the CHIKV-E2 protein,respectively.Conclusion:In this study,a pair of monoclonal antibodies with high-affinity against conserved epitopes of CHIKV-E2 protein was obtained,and the ELISA method established with this pair of antibodies could be used to specifically detect CHIKV-E2protein of different CHIKV spectrums.Therefore,this study lays a foundation for further development of ELISA with high specificity and sensitivity for CHIKV.