牛分枝杆菌融合蛋白ESAT-6/CFP-10/Rv3872抗体间接ELISA检测方法的建立与应用OA

To develop an indirect enzyme-linked immunosorbent assay(iELISA)for detecting anti-bodies against Mycobacterium bovis(Mb),the Mb genes ESAT6,CFP10,and Rv3872 were selected and connected via a flexible(GGGS)4 linker.The fused sequence was cloned into the pET-28a(+)vector and expressed as a recombinant protein ESAT-6/CFP-10/Rv3872(E-C-R).This recombinant protein was employed as the coating antigen for the establishment and performance evaluation of the iELISA.Optimization experi-ments identified the following conditions as optimal:antigen coating concentration of 100 ng/mL,blocking with 10 g/L betaine solution at 4℃ for 12 h,serum dilution at 1∶100 and incubation at 37℃for 1 h,HRP-conjugated sheep anti-bovine IgG at 1∶10 000 dilution with incubation at 37℃ for 60 min,and substrate color development for 15 min.Under these conditions,the cut-off value(D450nm)was deter-mined to be 0.39.Comparative validation demonstrated that the developed iELISA exhibited higher sen-sitivity than a commercial indirect ELISA kit,and no cross-reactivity with sera positive for Salmonel-la,Mycobacterium avium subsp.paratuberculosis,Mycoplasma bovis,Brucella,or Escherichia coli.Both intra-and inter-assay coefficients of variation were below 10％.The developed iELISA demonstrated good sensitivity,specificity,and reproducibility.Moreover,the antigen-coated plates and reagents maintained stability for over 12 months under storage at 2 to 8℃.When applied to 100 clinical serum samples,the overall agreement rates with the tuberculin skin test,a commercial antibody strip,and a commercial iELISA kit were 87％(87/100),86％(86/100),and 93％(93/100),respectively.In conclusion,this study successfully established a highly sensitive,specific,reproducible,and stable iELISA for detecting Mb antibodies,providing a novel tool for the serological diagnosis of bovine tuberculosis.