布病胶体金免疫层析检测方法的建立Development of Colloidal Gold-Immunochromatographic Assay for Brucellosis
唐景峰,李晓艳,王兴龙,梅建军
TANG Jing-feng~△,LI Xiao-yan,WANG Xing-long,et al (()~△Faculty of Agriculture
摘要(Abstract)：
目的建立诊断布病的胶体金免疫层析方法。方法以B.abortus 544A全菌免疫BALB/c小鼠,以杂交瘤细胞技术制备单抗,纯化后以胶体金标记,选择最佳反应模式组装试纸条,并对其进行鉴定。结果已筛选4B8、1G9、1C9和1E24株稳定分泌单抗的细胞株,以其单抗互相配对组合的试纸条均能检出B.abortus 544A菌。其中以1G9为包被抗体、4B8为金标抗体的试纸检测效果最佳,与以B.abortus 544A菌多抗为包被抗体、4B8单抗为金标抗体的试纸对比,检测结果一致,且不与其他菌发生交叉反应。对PCR检测为阳性的70份牛血清样品,两种试纸条检测结果的符合率均为100%。结论所建立的诊断布病的胶体金免疫层析法快速、简便、准确,有望用于布病的诊断。
Objective To develop a colloidal gold-immunochromatographic assay(GICA) for brucellosis.Methods Immunize BALB/c mice with B.abortus 544A for preparation of McAb by hybridoma cell technique.Purify the McAb and label with colloidal gold for formulation of GICA indicator paper under an optimal condition.Results Four cell strains stably secreting McAbs,i.e.4B8,1G9,1C9 and 1E2,were screened.The GICA indicator papers formulated with McAbs secreted by any two of the four strains were suitable for the detection of B.abortus,of which the paper using McAb 1G9 as coating antibody and McAb 4B8 as colloidal gold-label antibody showed a satisfactory detection result consistent with that using PcAb against B.abortus 544A as coating antibody and McAb 4B8 as colloidal gold-labeled antibody.The two papers were further used for the detection of 70 bovine serum specimens proved as B.abortus positive by PCR,and the coincidence rate of detection results was 100%.No cross reaction with other bacteria was observed.Conclusion The developed GICA was rapid,simple and accurate,and might be used for the diagnosis of brucellosis.
关键词(KeyWords)：
布鲁菌病;单克隆抗体;胶体金免疫层析方法
Brucellosis;Monoclonal antibody;Colloidal gold-immunochromatographic assay
基金项目(Foundation): 吉林省科技厅计划项目(20050549)
作者(Author):
唐景峰,李晓艳,王兴龙,梅建军
TANG Jing-feng~△,LI Xiao-yan,WANG Xing-long,et al (()~△Faculty of Agriculture
DOI: 10.13200/j.cjb.2007.02.52.tangjf.015
参考文献(References)：
- [1]Sohn AH,Probert WS,Glaser C,et al.Human neurobrucellosiswith intracerebral granuloma caused by a marine mammal brucellaspp.Emerging Infect Dis,2003,9(4):485-488.
- [2]Fu K,Zhao YY,Tang WX,et al.Preparation and identification ofmonoclonal antibodies against daintain.Hybridoma(Larchmt),2006,25(2):95-97.
- [3]Swinnen K,Krul A,Van Goidsenhoven I,et al.Performance compari-son of protein Aaffinity resins for the purification of monoclonal anti-bodies.Chromatogr B Analyt Technol Biomed Life Sci,2006,8(9):20-24.
- [4]Jin Y,Jang JW,Han CH,et al.Development of immunoassays forthe detection of kanamycin in veterinary fields.J Veterinary Sci-ence,2006,7(2):111-117.
- [5]Shyu RH,Shyu HF,Liu HW,et al.Colloidal gold based immuno-chromatographic assay for detection of ricin.Toxicon,2002,40(3):255-258.
- [6]Baskar LK,Srikanth TR,Suba S,et al.Development and evaluationof a rapid flowthrough immuno filtration test using recombinant filar-ial antigen for diagnosis of brugian and bancroftian filariasis.Micro-biol Immunol,2004,48(7):519-525.