应用核酸适配子检测细胞因子的新方法—ELONA法

以人肿瘤坏死因子(Human tumor necrosis factor,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—linked Oligonucleotide assay,ELONA)方法,用于hTNF—α的检测。通过SELEX(Systematic Evolution of Ligands by Exponential Enrichment)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100pg／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、细胞培养上清等多种生物标本中各种细胞因子及其它蛋白的检测。

Novel Methods to Detect Cytokines by Enzyme-linked Oligonucleotide Assay

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 hTNFalpha. 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, hTNFalpha monoclonal antibody was coated on the surface of the plate, biotin-labeled RNA aptamer was used as a detect molecle. HTNFalpha 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-alpha 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 cytokines and other protein molecules.