【摘要】 将PCR技术的极高灵敏度与ELISA技术检测肿瘤坏死因子-α(TNF-α)抗原的特异性相结合,利用生物素标记的腺病毒六邻体基因重组质粒(AdAT)作为PCR的模板DNA,以链亲和素(strep-tavidin)作连接分子,与生物素标记的兔抗鼠IgG抗体相连接,后者再与TNF-α的单克隆抗体结合。设计一对扩增六邻体基因的引物进行PCR,通过对DNA扩增片段浓度的定量分析,替代ELISA方法中的酶-底物显色反应而建立TNF-α的免疫PCR检测方法。结果表明,该技术灵敏度比ELISA方法灵敏10~3倍,可检出100fg/ml的TNF-α。借助此方法可检出脑脊液中极微量的TNF-α。更多还原

【Abstract】 The very high sensitivity of polymerase chain reaction (PCR) was combined with the specifici-ty of ELISA method for the determination of TNF-α. By using streptavidin as linker molecules,a Bio-dUTP labeled recombinant plasmid (AdAT) containing adenovirus hexon gene that was a template DNA for PCR was linked to the biotin labeled rabbit antibody against mouse IgG.and then the latter connected with the spe-cific monoclonal antibody against TNF-α. After a pair of primers for hexon gene were designed,the PCR was carried out and the immuno-PCR assay technique was therefore established through the quantitative analysis of the amount of amplified DNA fragments instead of the enzyme-substrate reaction of ELISA. The result in-dicated that the sensitivity of immuno-PCR assay for TNF-α was 103 times as sensitive as that of ELISA,and 100 fg/ml of TNF-α could be detected by this method. This technique can be employed to detect very low-leveled TNF-α present in cerebrospinal fluid.更多还原