Fig. 4: Specificity and sensitivity of aptC in patient serum.

a Serum protein electrophoresis (SPEP, top) and immunofixation (IFE, bottom), composite of chronological data. Control (Ctrl) and sequential M-Ig serum samples (0, 6, 12, 14, 17, 22, and 25 months) analyzed by SPEP show M-Ig in the cathodal gamma fraction (arrow) at 0 and 25 months at concentrations of 2.9 g dl⁻¹ (0 months, Pre-ASCT) and 0.5 g dl⁻¹ (25 months, relapse). M-Ig was not detected (ND) in months 6–22. IFE identified the M-Ig as an IgG Kappa (asterisks) at 0 months (pre-ASCT) and at 25 months. M-Ig was not detected by IFE in months 6–22. Alb (albumin), SP = serum protein (no immunofixation), G = SP fixed with anti-IgG antibody (Ab), A = SP fixed with anti-IgA Ab, M = SP fixed with anti-IgM Ab, Κ = SP fixed with anti-Kappa light chain Ab, λ = SP fixed with anti-Lambda light chain Ab. b–c Specificity and sensitivity in serum by ELONA. Patient CR serum obtained at 0 months and controls (MM, no MM) were diluted 1:125–1:40,000 (b). Patient CR sera obtained at months 6–25 and controls were diluted 1:4–1:1,000 (c). b–c 0 months, 6 months, 12 months, 14 months, 17 months, 22 months, 25 months and control sera (Ctrl MM, Ctrl no MM) were incubated for 30 min with biotin-labeled aptC immobilized on streptavidin-coated plates. Biotin-scrambled aptamer (scr) and no aptamer (no apt) controls were also included for: 0 months, control no MM, control MM, 6 months, and 22 months. Binding was detected with goat anti-human IgG-HRP and visualized with TMB (370 nm). Binding was observed in all conditions where aptC was incubated with patient CR serum. No signal was obtained when aptC was incubated with control sera, or when controls (aptC scrambled (scr), no apt) were incubated with patient or control sera. d Quantitation of residual disease in serum obtained during remission. M-Ig in remission sera c were quantified using samples with known concentrations (0 months, 25 months) as standards. Concentrations were 0.37 × 10⁻³ g dl⁻¹ (6 months), 0.45 × 10⁻³ g dl⁻¹ (12 months), 0.48 × 10⁻² g dl⁻¹ (14 months), 0.13 × 10⁻¹ g dl⁻¹ (17 months), 0.79 × 10⁻¹ g dl⁻¹ (22 months) to 4.8 × 10⁻¹ g dl⁻¹ at 25 months demonstrating exponential increase after 12 months. e Specificity and sensitivity in serum by HTRF. FRET occurs when aptC [biotin-aptC-SA-Tb] was incubated with CR serum, but not with controls (no MM, MM, buffer), nor with scrambled aptamer (scr) or no aptamer (no apt) in any condition. f Pull-down of M-Ig from CR serum. Serum from CR (lanes 1–4) and MM control (Ctrl MM: lanes 5–8) were incubated with biotin-aptC (aptC) or biotin-aptC scrambled (scr) on streptavidin (SA) beads. Flow-through was analyzed by SPEP and protein fractions were quantified by densitometry. CR and Ctrl MM sera were incubated with buffer (1 and 5), SA-beads (2 and 6), aptC + SA beads (3 and 7) or scr + SA beads (4 and 8). AptC reduced M-Ig in CR serum from 0.6 g dl⁻¹ (lane 1) to 0.3 g dl⁻¹ (lane 3). No effect was observed with scr control (lane 4). AptC had no effect on M-Ig in control serum (lanes 5–8). g Effect of aptC on serum protein fractions (based on f). Serum protein fractions albumin (Alb), alpha 1 (α1), alpha 2 (α2), beta 1 and beta 2 (β), and polyclonal gammaglobulins (γ) were quantified after pull down and graphed as percentage of control (lanes 2 and 6 in f). CR serum (top): AptC ‘pulled out’ CR M-Ig from serum but did not affect other CR serum protein fractions. Scrambled aptC (scr) had no effect. Control MM serum (bottom): AptC and scr control did not affect the concentrations of an unrelated M-Ig or serum protein fractions in control serum (Ctrl MM). 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.