Cytokine aptamers as therapeutic and diagnostic agents / 生物工程学报

Aptamers are oligonucleotides derived from an in vitro evolution process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Aptamers specially binding to targets could recognize and inhibit the function of targets. Using this method, many powerful antagonists of cytokines have been found. In order for these antagonists to work in animal models of disease and in humans, it is necessary to modify the aptamers. First of all, 2'-F, 2'-NH2 and 2'-CH3O modifications of nucleoside triphosphates could prolong half-lives in blood. Aptamers can be kept in the circulation from hours to days by conjugating them to higher molecular weight vehicles. After modified, conjugated aptamers are injected into animals, they inhibit physiological functions known to be associated with their target cytokines. Exhibiting binding characteristics comparable to or even better than monoclonal antibodies, these ligands can be used as detection probes, highly efficient inhibitors of protein function or specific competitors in high-throughput screening (HTS) assays. Recently several aptamers of cytokines have been characterized. Some of them have been used as diagnostic agent for the detection of target cytokines. The first aptamer that has proceeded to phase II clinical studies is NX-1838, an injectable angiogenesis inhibitor that can be potentially used to treat macular degeneration-induced blindness. Aptamers will be versatile tools that can greatly enhance the efficiency of modern diagnose and therapy development.

Novel methods to detect cytokines by enzyme-linked oligonucleotide assay / 生物工程学报

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.