Processing
Background
Insulin-like growth factor-I (IGF-I; aka somatomedin C) can be used as a biomarker for detection of athletes doping with recombinant human growth hormone (rhGH). This research group describes the development of a simple, aptamer-magnetic bead (MB)-based assay for serum human IGF-I. The IGF-I aptamer-MB assay could offer a significant improvement over immunoassays, which currently require purification of IGF-I from other binding proteins and deliver inconsistent performance results. Use of DNA aptamers in place of antibodies provides a binding reagent of a defined composition that can be manufactured with high fidelity. Aptamers also display superior target affinity, when appropriately selected. In addition, they can be produced more quickly and cost-effectively, as they require no animal host.
Experiment
The researchers identified 72-base aptamer candidates using 9 rounds of MB-SELEX DNA aptamer development. 36 aptamer candidates, supplied by IDT as 5′-biotinylated DNA oligonucleotides, were tested for affinity against microplate-bound, recombinant human IGF-I (rhIGF-I). The top 4 aptamer candidates were tested in both capture aptamer-MB and reporter aptamer roles, and were compared in an enzyme-linked aptamer-MB sandwich format (ELASA, enyzme-linked aptamer sorbent assay) in undiluted human serum.
Results
The assay was reproducibly sensitive with a probable limit of detection <16 ng/mL in human serum and did not require the separation if rhIGF-I from IGF binding proteins (IGFBPs). Based on these results, the authors suggest that the aptamer with superior binding, binds to an exposed epitope in rhIGF-I:IGFBP complexes.
In ongoing experiments, the authors will assay actual isolated rhIGF-I:IGFBP complexes in buffer but here conclude that the assay works to some extent in human serum, especially when excess rhIGF-I is present.
Products from IDT
All of the DNA templates, primers, and the library of biotinylated candidate aptamers used in these experiments were synthesized by IDT. The 72-base DNA aptamers were designed with 18 fixed-sequence bases at each end to serve as primer binding regions for PCR amplification. The intervening 36 bases were randomized in the initial SELEX DNA template library.
