基于LppA蛋白的丝状支原体山羊亚种抗体间接ELISA检测方法的建立与应用OA

An indirect enzyme-linked immunosorbent assay(ELISA)based on recombinant LppA was de-veloped for the detection of antibodies against Mycoplasma mycoides subsp.capri(Mme).The LppA gene was cloned and expressed in Escherichia coli,and the recombinant protein was used as the coating an-tigen.The recombinant LppA protein had an apparent molecular mass of approximately 49 ku and was spe-cifically recognized by Mmc-positive goat serum in Western blot analysis.The optimal antigen coating con-centration was 4 μg/mL,and the optimal dilutions of the serum and HRP-conjugated rabbit anti-goat IgG were 1:100 and 1:2 500,respectively.The cutoff OD450 value was determined to be 0.378.Both the intra-assay and inter-assay coefficients of variation were below 10％.No cross-reactivity was observed with sera positive for Chlamydia abortus,Coxiella burnetii,or caprine astrovirus.Mmc-positive serum remained positive at a dilution of 1:1 024.Among 288 clinical goat serum samples tested using the developed assay,126 were positive,corresponding to a seropositivity rate of 43.75％.The indirect ELISA developed in this study for detecting antibodies against Mmc provides a useful technical tool for epidemiological investiga-tions of Mmc.