Screening Of Neutralizing Antibodies Against Foot-and-Mouth Disease Virus Serotype A From Bovine Single B Cells And Development Of Competitive ELISA Method

Foot-and-mouth disease virus(FMDV)is the causative agent of foot-and-mouth disease(FMD)of cloven-hoofed animals like cattle,pig,goat,etc.FMDV is a single-stranded positive-sense RNA virus with seven serotypes:O,A,C,ASIA1 and SAT1?3.Among the seven serotypes of FMDV,serotype A has the greatest antigenic variability.In China,the frequency of FMDV serotype A decreased significantly since 2018,however,it still poses a serious threat to the health of cloven-hoofed animals due to the source of the epidemic has not been eliminated completely.Inactivated vaccine is an important strategy for prevention of FMD,how to evaluate the quality and immune effect of inactivated vaccine is an important topic in the research of prevention of FMD.Monoclonal antibody is an important tool for analyzing structural of viral antigens and developing new diagnostic and detection techniques.In recent years,it has become a new technology to develop monoclonal antibodies by isolating antigen-specific B cells from susceptible animals to develop single B cell antibodies.The purpose of this study was to develop FMDV-specific single B cell neutralizing antibodies from immunized cattle by using single B cell antibodies technology,and to develop ELISA method based on these neutralizing antibodies that can be used to evaluate the immune effect of FMDV serotype A vaccine.In this study,single B cells specific to 146S were sorted by flow cytometry,biotin-labeled FMDV A/AF72 146S was used as bait antigen,and PE-labeled 12S was used as negative screening antigen.The Ig G variable region gene of heavy chain and light chain of sorted single B cell was amplified by nest PCR and inserted into vector to construct expression plasmids.The plasmids of heavy chain and light chain were co-transfected into CHO-S cell for antibody expression.Flow analysis,indirect ELISA and indirect immunofluorescence assay(IFA)were used to verify the reaction characteristics of antibodies,and virus neutralization test(VNT)was used to verify the neutralization activity of antibodies.The results showed that a total of 72 strains antibodies were screened,including 29 strains of neutralizing antibodies,and 6 strains of neutralizing antibodies that could neutralized the three lineages of FMDV(Serial number:W2,W21,W125,W145,W151,W153).By comparing the reactivity and affinity of the6 strains of neutralizing antibodies,the neutralizing antibody W145 and single B cell antibody E32produced previously in our lab with broad reactivity with FMDV serotypes A,O and ASIA1 were selected to develop a competitive ELISA method for detecting neutralizing antibodies(NAC-ELISA).The specificity and sensitivity of NAC-ELISA were estimated to be 98.1%and 100%by using serum samples from na?ve animals and virus infected animals,respectively,with a cutoff value of 1.4(log₁₀).The results of the NAC-ELISA showed a statistically significant correlation with those of VNT(P<0.01,R²=0.876)and there was no cross-reaction with FMDV serotypes O and ASIA1.In conclusion,29 strains of neutralizing antibodies against FMDV serotype A were screened in this study,which provided rich antibody tools for antigen structure analysis of FMDV serotype A.A competitive ELISA method for detecting neutralizing antibodies of FMDV serotype A was developed,which provided a candidate method for evaluating the immune effect of vaccine.