摘要
建立高灵敏度和特异性的白介素 8(IL - 8)ELISA法。用人工重组的IL - 8多次免疫兔 ,获取高效价抗体。纯化抗体经生物素标记 ,同辣根过氧化酶 (HRP)标记的亲和素可特异结合。采用纯化的抗体包被 96孔板 ,形成固相抗体。加入不同浓度标准品后 ,反应 1.5h ,洗净后再加入生物素标记抗体 ,以酶标记亲和素作为探针 ,经酶底物显色反应可获得良好的ELISA标准曲线。该方法测定范围为 0 .2 - 10 .8ng/mL ,最低测出值为0 .0 2ng/mL ,批内和批间CV小于 8%和 10 %。用该法测得 18名正常人血浆中IL - 8水平为 0 .15± 0 .0 6ng/mL。 18份高低不同浓度IL - 8水平的血清样品经本法和RIA测定其均值为 0 .5 4± 0 .35ng/mL和 0 .6 3±0 .5 9ng/mL ,相关性强 (r =0 .819,t=5 .89)。在U93 7细胞系体外培养液中 ,LPS刺激 2 4h和 4 8h可测出IL - 8水平明显上升 ;而经TNF -α刺激 2 4h和 4 8h后同对照没有明显变化。该方法操作简便 ,具有较强灵敏度和特异性 ,可满足人血清和细胞培养液中IL - 8水平的检测。
To develop a high sensitive and high specific ELISA for IL-8, the high effect antibody was obtained by immunizing rabbits with recombinant IL-8 more times.The purified anti-IL-8 IgG was labeled with biotin and then specially linked to avidin which was labeled with HRP.The purified antibody was used to coat 96 hole board as capture antibody.After adding different concentration of IL-8 standards,reaction was kept for 1.5 hours.Then biotin-IgG, avidin-HRP were added in turn to the reaction system.IL-8 levels were determined by the color, and standard curve was obtained.The IL-8 determine range of this ELISA was 0.2-10.8ng/mL.CVs within batch and between batch were less than 8% and 10%,respectively.The plasma IL-8 tested in normal persons(n=18) was 0.15±0.06ng/mL.But in their serum, IL-8 level was tested to be 0.54±0.35ng/mL by RIA and 0.63±0.59ng/mL by ELISA.The two methods have obvious correlation in IL-8 values(r=0.819,t=5.89).It was discovered that in cell culture U 937 cell stimulated by LPS for 24 or 48 hours produced more IL-8, but U 937 cell stimulated by TNF-α for 24 or 48 hours had not produced more IL-8 compared to control.This is one of simple, sensitive and specific method for measurement of IL-8, it can be used to measure IL-8 level in human plasma or serum and the supernatant of cell culture.
出处
《标记免疫分析与临床》
CAS
2004年第3期151-154,共4页
Labeled Immunoassays and Clinical Medicine
基金
国家自然科学基金资助项目 (NO :39970 717)
