Purpose: To evaluate the binding affinity and specificity of ssDNA aptamers previously selected against human norovirus GI virus-like particles (VLPs).
Methods: Six aptamer candidates (AP1-AP6) obtained after four rounds of a novel selection process were screened. Aptamer binding affinity was evaluated using an Enzyme-Linked Aptamer Sorbent Assay (ELASA) performed on a panel of human norovirus VLPs. Briefly, VLPs were bound to microtiter wells, blocked, and incubated with biotinylated aptamer. Plates were developed by the sequential addition of streptavidin peroxidase followed by a TMB 2-component microwell peroxidase substrate system. Absorbance was recorded at 450 nm and binding affinity was expressed as ratio of the absorbance of test sample to that of the negative control. Similar assays were done using 20% stool suspensions obtained from infected individuals.
Results: Of the six aptamers tested, AP4 displayed the greatest binding affinities for both GI and GII human norovirus VLPs. Average ratios for GI VLPS were 6.4±0.4 (GI.1), 5.2±0.6 (GI.6), 5.5±0.4 (GI.7), 5.0±0.4 (GI.8); and for GII VLPs were 4.0±0.4 (GII.1), 7.8±0.7 (GII.2 Snow Mountain), 6.0±0.3 (GII.4 Grimsby), 6.1±0.5 (GII.4 Houston), 6.6±0.7 (GII.12), 5.3±0.2 (GII.17). Difference in binding affinity were statistically significant by VLP type (P<0.05). Aptamers affinity for virus in diluted human fecal samples was dampened [GI.I (3.8±0.1), GI.6 (1.5±0.1), and GII.4 Sydney (1.1±0.03)], most likely due to matrix-associated inhibition.
Significance: The aptamers screened had broad reactivity and high binding affinity for a variety of human norovirus VLPs and are promising ligands for use in future capture and detection assays.