应用B.melitensis 16M单克隆抗体建立羊布病的胶体金免疫层析检测体系

Establishment a Colloidal gold-Immunochromatographic Assay of B.melitensis by Monoclonal Antibodies Against B.melitensis 16M

[目的]用B.melitensis16M提纯的LPS和O链抗原作为检测抗原,研制出针对B.melitensis16M的单克隆抗体(McAb),将其标记胶体金,建立一种诊断布病的胶体金免疫层析方法。[方法]B.melitensis16M全菌免疫Balb/C小鼠,采用杂交瘤细胞技术制备McAb,测定免疫球蛋白亚类及亲和力。用HiTrapProteinGHP亲和层析纯化抗体并用胶体金标记,建立胶体金免疫层析方法。选择最佳反应模式组装成试纸条,对其特异性、敏感性、稳定性、阳性病料试验。[结果]筛选出能稳定分泌抗B.melitensis16M的种特异性单克隆抗体杂交瘤细胞株2D10、3C6、1E10,单抗亚类分别为IgG2a、IgG1,亲和常数(k)介于1×10-8￣2.6×10-10。根据配对实验,以3C6为最佳包被抗体,2D10为最佳金标抗体。抗体IgG标记浓度为30μg/ml时,检测效果最佳。同时,与B.melitensis16M多抗IgG作为包被抗体、2D10为金标抗体的检测体系对比,两种包被方法组装的试纸条最低检出量为5.0×103CFU/ml,且不与其它菌发生交叉反应,保持期≥100d。以荧光定量PCR为参照,试纸条检测80份阳性病料,检检出率为100%。[结论]建立的胶体金免疫层析方法检测B.melitensis16M快速、简便、可靠,有望开发成为布病快速诊断试剂盒。

[Objective] To prepare a monoclonal antibodies （mAbs） against LPS and O-chain of B. melitensis 16M and establish colloidal gold-immunochromatographic assay （GICA） for diagnosis of Brucellosis. [Methods] B.melitensis 16M was used to immunize Balb/C mice and the mAbs were prepared using hybridoma technique followed by identification of IgG isotype and its affinity. HiTrap Protein G HP affinity chromatography was employed to purify the antibodies, which were labeled with colloidal gold for establishment of GICA .The best optimization reaction pattern was obtained and installed dipstick, which of specificity, sensitivity, stability and positive samples were tested. [Results] The hybridoma ceils of 2D10, 3C6, 1El0 were obtained and identified as IgG2a and IgG1 of IgG isotypes with affinity constants （Kaff） ranging from 1 xl0-s to 2.6×10^-9. 3C6 and 2D10 were each optimized coating and labeling antibodies. When labeling IgG were 30μg/ml,the detection result were best, meanwhile,to contrast the polyclonal antibody IgG of B.melitensis 16M and 2D10 were coating antibody and labeling antibodies, two different dipsticks＇ limit of detection were 5.0×10^3CFU/ml,and no cross-reacting.The keep time were ≥100d.To contrast fluorescent quantitation PCR,the detection rate was 100% from 80 postive samples. [Conclusion ] The established GICA is rapid and accurate for B.melitensis 16M detection with such potential utility as for instant diagnosis of Brucellosis.