重组人类肥胖抑素抗血清的研究  

Preparation and application of antiserum against recombinant human leptin

作　　者：许婉宁[1] 丁宗一[1] 唐建国[2] 

机构地区：[1]北京儿科研究所营养研究室,100045 [2]北京大学生命科学学院

出　　处：《中华儿科杂志》2001年第2期88-91,共4页Chinese Journal of Pediatrics

基　　金：北京市自然科学基金资助项目!(7972 0 12 )

摘　　要：目的　研究重组人类肥胖抑素抗血清的制备及应用。方法　以高度纯化的体外重组肥胖抑素免疫兔子 ,获得兔抗人类肥胖抑素抗血清 ,应用双向免疫扩散电泳、免疫沉淀法、蛋白质印迹法及放射免疫法鉴定其滴度并研究应用其检测重组肥胖抑素的特异性和灵敏度 ,建立标准曲线并应用免疫沉淀法和蛋白质印迹法进行临床样本的初步检测。结果　双向免疫扩散电泳测定抗血清滴度为1∶16。应用放射免疫法确定抗血清工作稀释度为 1∶2 0 0 0 ,并建立检测范围在 0 78～ 10 0 μg/L的标准曲线。应用免疫沉淀法结合蛋白质印迹法进行检测 ,可见抗血清与重组人类肥胖抑素特异性结合 ,检测灵敏度可达 1μg/L ;抗血清与儿童血浆中肥胖抑素特异性结合 ,吸光度扫描定量为 10 μg/L。同时以具有生物活性的重组人类肥胖抑素作为免疫原所获得的抗体与变性及非变性重组蛋白均可特异性结合。结论　获得特异性兔抗人类肥胖抑素抗血清 ,并可用于临床检测生物样品中的肥胖抑素水平。Objective To explore the preparation and application of antiserum againgst recombinant human leptin(r leptin). Methods Antihuman leptin antiserum was raised in rabbits by immunizing with highly purified recombinant human leptin. The concentration of r leptin in the first immune reaction was 2 μg/L,then reduced to 1 μg/L. After 20 weeks,the serum was obtained from rabbit ear vein. The authors determined its titer by radioimmunoassay(RIA), then constructed the reference curve. The serum containing no leptin was obtained from children's serum through Sep Pak C18 Cartridge. The natural leptin in children's serum was extracted by immunoprecipitation. The r leptin was diluted with children's serum containing no leptin,then the r leptin and natural leptin were detected by Western blotting. The quantity was determined by densitometric scanning. Results The titer of antihuman leptin by counter immunoelectrophoresis was 1∶16. The working titer of antiserum by RIA was 1∶2 000. Using this titer, the authors constructed the RIA reference curve whose limits were 0.78 100 μg/L. Recombinant human leptin and children plasma leptin could be detected by antihuman leptin antiserum specifically with Immunoprecipitation and Western blotting. The lowest amount detectable was one microgram. The antiserum raised against recombinant human leptin with biological activity was able to detect both denatured and undenatured protein molecules. Conclusion The specificity and sensitivity of the radioimmunoassay which utilized the antiserum were high and it was possible to do research in clinical samples by Western blotting and radioimmunoassay with the antiserum.

关 键 词：肥胖症 抗体 放射免疫测定 蛋白质类 血清 肥胖抑素 

分 类 号：R341[医药卫生—基础医学]