Aptamer sandwich assays

A method for identifying a pair of aptamers that binds to separate non-overlapping sites on a target molecule, the method comprising:

• (a) preparing a plurality of candidate aptamers having a binding affinity to the target molecule by synthesizing an oligonucleotide library comprising a plurality of oligonucleotides that consist of random nucleotide sequences having lengths of 35-45 nucleotides flanked by two fixed nucleotide sequences having lengths of 15-25 nucleotides, one of the two fixed nucleotide sequences being located at a 4′-end and the other one of the two fixed nucleotide sequences being located at a 3′-end, and by selecting the plurality of candidate aptamers from the oligonucleotide library using a SELEX (systematic evolution of ligands by exponential enrichment) process;
• (b) selecting a first aptamer having a dissociation constant (Kd) of 10 nM or less from the plurality of candidate aptamers by measuring the dissociation constants of the plurality of candidate aptamers binding to the target molecule under conditions of 35-40° C. and pH 7.0-8.0 and determining candidate aptamers having a Kd of 10 nM or less as the first aptamer;
• (c) selecting a second aptamer from the plurality of candidate aptamers, wherein the second aptamer has a dissociation constant of 100 nM or less and is capable of binding to a non-overlapping site of the target molecule, and wherein the second aptamer is determined by labeling the first aptamer with a detectable label, forming a complex of the labeled first aptamer and the target molecule;
• (d) contacting the complex with the plurality of candidate aptamers, and detecting candidate aptamers having non-competitive binding affinity to the complex.