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| 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 | |
| 引用本文： | 唐景峰,李晓艳,梅建军,刘 锴,王兴龙.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[J].中国农学通报,2006,22(11):28-28. |
| 作者姓名： | 唐景峰 李晓艳 梅建军 刘 锴 王兴龙 |
| 作者单位： | 1. 吉林大学,长春,130062;军事医学科学院十一所,长春,130062 2. 军事医学科学院十一所,长春,130062 3. 吉林大学,长春,130062 |
| 基金项目： | 吉林省科技厅计划项目“布氏杆菌病快速检测技术的研究”（20050549）. |
| 摘 要： | 目的 用B.melitensis 16M提纯的LPS和O链抗原作为检测抗原，研制出针对B.melitensis 16M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.melitensis 16M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HiTrap Protein G HP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.melitensis 16M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.melitensis 16M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.melitensis 16M快速、简便、可靠，有望开发成为布病快速诊断试剂盒。 |
| 关 键 词： | B.melitensis 16M 单克隆抗体 胶体金免疫层析方法 |
| 收稿时间： | 2006-08-13 |
| 修稿时间： | 2006-08-132006-08-22 |
Establishment a Colloidal gold-Immunochromatographic Assay of B.melitensis by Monoclonal Antibodies Against B.melitensis 16M |
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| Tang Jingfeng,Li Xiaoyan,Mei Jianjun,Liu Kai,Wang Xinglong.Establishment a Colloidal gold-Immunochromatographic Assay of B.melitensis by Monoclonal Antibodies Against B.melitensis 16M[J].Chinese Agricultural Science Bulletin,2006,22(11):28-28. | |
| Authors: | Tang Jingfeng Li Xiaoyan Mei Jianjun Liu Kai Wang Xinglong |
| Institution: | (1Jilin University, Changchun 130062; 2Eleven Institue, Academy of Military Medical Sciences, Changchun 130062) |
| Abstract: | 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 cells of 2D10、3C6、1E10 were obtained and identified as IgG2a and IgG1 of IgG isotypes with affinity constants (Kaff) ranging from 1×10-8 to 2.6×10-9. 3C6and 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 5.0×103CFU/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. |
| Keywords: | B melitensis 16M Monoclonal antibody Colloidal gold-immunochromatographic assay |
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