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利用细菌荧光素酶建立酶联免疫吸附试验检测抗-HBs的研究

Establish enzyme linked immunosorbent assay with bacterial fluorescein enzyme to detect Anti - HBs
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摘要 目的利用细菌荧光素酶作为示踪物,建立一种灵敏度高、特异性强、性能稳定、操作简便、无环境污染的生物发光酶酶联免疫检测技术(Bioluminescent enzyme linked immunosorbent assay BELISA)。方法用戊二醛作为交联剂,将细菌荧光素酶标记到乙型肝炎表面抗原上,使酶标记物具有酶和抗原的双重活性,采用双抗原夹心法检测。结果最适的标记条件为:细菌荧光素酶与乙型肝炎表面抗原的比例2:1,pH值7.0,反应温度室温(22℃),反应时间2小时,0.1%戊二醛用量50μl/ml标记物。所建立的生物发光酶酶联免疫试验检测抗-HBs灵敏度2.5mIU/ml,变异系数(CV)<15%。与抗-HAV·IgM阳性血清、抗-HCV阳性血清无交叉反应,阻断试验成立。对200份抗-HBs阴性血清(RIA-、ELISA-)进行BELISA检测,确定抗-HBs阴性血清光量子数为50MV/20s。对98份抗-HBs阳性血清(RIA+)进行BELISA和ELISA检测,BELISA检出98份阳性,与RIA符合率100%,而ELISA与RIA的符合率为89.9%,可见BELISA灵敏度与RIA相同。结论用细菌荧光素酶为示踪物建立的生物发光检测技术灵敏度高,特异性强,可取代放射免疫分析和以辣根过氧化物酶为示踪物建立的酶联免疫检测技术。 Objective Establish a enzyme linked Immunosorbent assay technique with bacterial fluorescein enzyme (BELISA) .The technique has high sensitivity . high specificity and steady nature.Is simple and convenient to operate.Doesn' t have environmental pollution.Methods HBsAg was labeled with bacterial fluorescein enzyme by glutaric dialdehyde.Made enzyme labeled compound have dual activity of enzyme and antigen. Used dual antigen sandwith to detect. Results Best labeling condition as follows: Proportion of enzyme and antigen is 2:1, pH 7.0, reactive temperature is indoor temperature (22℃) . Reactive time is 2 hours. Quantity of utilization of 0.1 % glutaric dialdehyde is 50μl/ml labeled commpound. The BELISA technique to detect Anti - HBs sensitivity is 2. 5mIU/ml. Coefficient of variation is smaller than 15 % . Hasn' t cross reaction with positive serum of Anti - HAV·IgM and Anti - HCV.Obstructive experiment succeed.The value of photoquantum of negative serum of Anti - HBs (RIA- . ELISA - ) through detecting in BELISA to 200 portions is 50MV/20s.Detecting 98 portions positive serum (RIA + ) of Anti - HBs in BELISA and ELISA.BELISA discovered 98 portions positive.The detectable coincidence rate is 100% with RIA.The detectable coincidence rate of ELISA is 89.9% with RIA.Sensitivity of BELISA is identical with RIA.Conclusion The BELISA established with bacterial fluorescein enzyme has high sensitivity and specificity.The method can replace RIA and ELISA established with horse radish peroxidase.
作者 王斌 唐心利
出处 《医学研究通讯》 2003年第1期9-12,共4页 Bulletin of Medical Research
关键词 细菌荧光素酶 酶联免疫吸附试验 抗-HBS Bacterial fluorescein enzyme Enzyme linked immunosonbent assay Anti - HBs
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