An aptamer was previously selected against the anaphylactic allergen β-conglutin (β-CBA I), which was subsequently truncated to an 11-mer and the affinity improved by two orders of magnitude. The work reported here details the selection and characterisation of a second aptamer (β-CBA II) selected against a second aptatope on the β-conglutin target. The affinity of this second aptamer was similar to that of the 11-mer, and its affinity was confirmed by three different techniques at three independent laboratories. This β-CBA II aptamer in combination with the previously selected β-CBA I was then exploited to a dual-aptamer approach. The specific and simultaneous binding of the dual aptamer (β-CBA I and β-CBA II) to different sites of β-conglutin was confirmed using both microscale thermophoresis and surface plasmon resonance where β-CBA II serves as the primary capturing aptamer and β-CBA I or the truncated β-CBA I (11-mer) as the secondary signalling aptamer, which can be further exploited in enzyme-linked aptamer assays and aptasensors.
Keywords: Dual aptamer; SELEX; Sandwich assay; Truncation studies; β-Conglutin.