Fig. 2: Isolation and validation of patient M-Ig aptamers.
a Patient M-IgG, one from a patient with stable disease (SD) and one from a patient who had achieved complete remission (CR), were purified from serum by Melon-resin columns and then immobilized on protein G columns for SELEX. b–c Nucleotide sequence and 2D structure prediction at 25 °C on PBS, 2 mM MgCl2 for aptS that was isolated after 7 SELEX rounds for the M-Ig of patient SD (ΔG = − 7.21 kcal mol−1) (b), and for aptC that was isolated after 6 SELEX rounds for the M-Ig of patient CR (ΔG=−8.94 kcal mol−1) (c). d Specificity of aptS and aptC. Polyclonal immunoglobulins (poly-Igs), daratumumab, M-Ig from patient SD (SD), M-Ig from patient CR (CR), and M-Igs from unrelated patients (unrel. M-Igs) were immobilized on protein G-coated plates and incubated for 30 min with no aptamer (-), biotin-labeled scrambled aptamers (scr), biotin-labeled aptD (D), biotin-labeled aptS (S), or biotin-labeled aptC (C). Binding was detected with streptavidin-HRP and visualized with TMB (370 nm). AptS, aptC, and aptD were specific and did not cross-react. e–f Binding affinity by surface plasmon resonance (SPR). Biotin-labeled aptS or aptC were immobilized on SwitchAvidin sensors, exposed to increasing concentrations (6.25–200 nM) of target M-Ig, or control polyclonal human IgG (dotted line, representative 100 nM). Results show specific binding that was obtained after subtracting control (biotin-scrambled aptamer) that was analyzed in parallel. Sensograms show dissociation constant (KD) of 8.75 nM for aptS (e) and 1.85 nM for aptC (f). No significant signal was observed for polyclonal IgG control (dotted line, 100 nM). All data represent means of at least n = 3 biologically independent samples ± SD, two-tailed Student t- test. **p < 0.01; ***p < 0.001; ****p < 0.0001.
