目的:制备抗结核分支杆菌的特异单克隆抗体,经纯化标记荧光后,建立直接荧光抗体染色方法用于痰标本的检测.方法:采用常规方法制备单克隆抗体.分泌单克隆抗体阳性杂交瘤细胞株的筛选采用ELISA法,确认采用免疫印迹法.单克隆抗体的纯化采用饱和硫酸铵粗提,Sephadex G-50层析纯化.单抗纯化后采用透析法标记异硫氰酸荧光素,初步应用于临床痰涂片的检测. 结果:经筛选获得4株单克隆抗体杂交瘤细胞株.ELISA检测小鼠腹水单克隆抗体效价达到1:6 400～1:12 800.免疫印迹实验表明4株单抗均为抗结核杆菌 38 KD 蛋白单抗,其中两株产生较强的免疫反应条带.单抗纯化后标记异硫氰酸荧光素,建立直接荧光抗体染色方法,对41份结核病患者的痰标本进行检测,阳性率为 90.24%,与抗酸染色法比较,直接荧光抗体染色方法敏感性明显高于抗酸染色法(P<0.05).结论:制备抗结核杆菌 38 KD 蛋白的单克隆抗体,并建立直接荧光抗体染色方法,初步应用于临床痰涂片检测中,对于辅助诊断结核病具有一定价值.

Objective To observe the immune effect of recombinant outer surface protein(rOspA) of Chinese Borrelia garinii in animals and provide a basis for the development of rOspA vaccine.Methods Immunize New Zealand rabbits,sheep and dogs with purified rOspA at different dosages.Determine the serum specific IgG by IFA and perform neutralization test in vitro.Results The serum IgG titers of the animals increased significantly compared with those before immunization and could still be detected 6 months after immunization.Neutralization test in vitro proved that each milliliter of rabbit anti-rOspA serum could kill 10~5 Lyme disease spirochetes,however,the killing capacities of anti-rOspA sera of sheep and dog immunized with various dosages of rOspA were also different.Each milliliter of sheep or dog anti-rOspA serum could kill 10~6 Lyme disease spirochetes at most.Conclusion rOspA showed good immune protection and might be an effective component of Lyme disease rOspA subunit vaccine in China.