应用核酸适配子检测细胞因子的新方法-ELONA法  

Novel Methods to Detect Cytokines by Enzyme-linked Oligonucleotide Assay

作　　者：严馨蕊[1] 高绪文[1] 姚立红[1] 张智清[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所基因室,北京100052

出　　处：《生物工程学报》2004年第5期679-682,共4页Chinese Journal of Biotechnology

基　　金：国家高技术 863计划项目基金资助 (No .2 0 0 1AA2 15 2 5 1)~~

摘　　要：以人肿瘤坏死因子 (Humantumornecrosisfactor,hTNF α)特异性的核酸适配子为检测分子建立了酶联寡聚核苷酸吸附试验 (Enzyme linkedOligonucleotideassay ,ELONA)方法 ,用于hTNF α的检测。通过SELEX(SystematicEvolutionofLigandsbyExponentialEnrichment)方法从随机RNA库中筛选到与hTNF α特异结合的RNA适配子。根据其序列 ,用体外转录方法合成生物素标记的RNA适配子 ,并对其进行了氨基修饰以增加其稳定性。以hTNF α的单克隆抗体为捕获分子 ,生物素标记的hTNF α特异性RNA适配子为检测分子建立了ELONA方法 ,并对这种检测方法的灵敏度、精密度和准确度等进行了分析。同时用ELONA和ELISA方法检测了正常人血清中的hTNF α水平 ,并对检测结果进行比较。结果显示 ,ELONA方法的灵敏度为 10 0pg mL ,具有较好的精密度和准确度。ELONA法的检测结果与ELISA法检测结果基本一致。该方法适用于血清。The development of the systematic evolution of ligands by exponential enrichment (SELEX) process has made it possible to isolate oligonucleotide sequences with the capacity of recognizing virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as “aptamers', are useful as a class of molecules that rival antibodies in diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. To meet the shortcomings of antibodies, aptamers have the following advantages. Aptamer does not depend on animals, cells, or even in vivo conditions and produced by chemical synthesis with extreme accuracy and reproducibility. Once denatured, functional aptamers could be regenerated easily within minutes. They are stable to long term storage and can be transported at ambient temperature. We describe here an enzyme linked oligonucleotide assay that use a SELEX derived RNA aptamer to detect hTNFα. In order to protect from nuclease attack, the RNA aptamer was modified by replacement of 2′ NH2 for 2′ OH at all ribo purines. In a sandwich micro plate assay, hTNFα monoclonal antibody was coated on the surface of the plate, biotin labeled RNA aptamer was used as a detect molecle. HTNFα was diluted by pooled human serum as standard, and streptavidin horseradish peroxidase substrate system was added for detection. Accuracy, precision, sensitivity, specificity of ELONA method were analyzed. The levels of hTNF α in normal human serum samples were assayed by the ELONA and the ELISA processes. The resultes demonstrate that a sandwich assay using a SELEX derived RNA aptamer has parameters for accuracy, precision, sensitivity, specificity well within the limits expected of a typical enzyme linked assay. There is no significant difference between the results of ELONA and ELISA. The minimum detection level was 100 pg/mL. This method will be useful for detection of almost all the cytokin

关 键 词：人肿瘤坏死因子 核酸适配子 细胞因子 酶联寡聚核苷酸吸附试验 生物素标记 

分 类 号：R346[医药卫生—基础医学]