Fig. 3: Specificity and sensitivity of aptS in patient serum.

a Serum protein electrophoresis (SPEP, top) and immunofixation (IFE, bottom), composite of chronological data. Commercial serum assay control (Ctrl) and sequential serum samples (0, 4, 18 months) obtained from patient SD were analyzed by SPEP. The M-Ig migrates in the cathodal gamma fraction (arrow). Concentrations were 2.3 g dl⁻¹ (0 months), 0.7 g dl⁻¹ (4 months), and 2.1 g dl⁻¹ (18 months). IFE identified the M-Ig as an IgG Kappa in each sample (asterisks). 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 Specificity and sensitivity in serum by ELONA. Biotin-aptS and controls (biotin-scrambled (scr), no aptamer (no apt)) were immobilized on streptavidin-coated plates and incubated for 30 min with increasing dilutions (1:1000–1:40,000) of patient SD sera that had been analyzed by SPEP. Serum from 0, 4, and 18 months or controls from 10 patients with or without MM (Ctrl MM, Ctrl no MM) were incubated with biotin aptS. Biotinylated scrambled aptamer (scr) and no aptamer (no apt) were included (0; 4; 18 months; control no MM and control MM). Binding was detected with goat anti-human IgG-HRP and visualized with TMB (370 nm). AptS was specific for SD serum and sensitive in all dilutions (tested up to 1:40,000). c Specificity and sensitivity in serum by HTRF. FRET occurs when aptS [biotin-aptS-SA-Tb] was incubated with SD serum, but not with control (no MM, MM, buffer), nor with no apt or scrambled controls (biotin-scr-SA-Tb) in any condition. d Pull-down of M-Ig from SD serum. Serum from SD (lanes 1–4) and MM control (lanes 5–8) were incubated with biotin-aptS (aptS) or biotin-aptS scrambled (scr) on streptavidin (SA) beads. Flow-through was analyzed by SPEP and protein fractions were quantified by densitometry. SD and Ctrl MM sera were incubated with buffer (1 and 5), SA-beads (2 and 6), aptS + SA beads (3 and 7) and scr + SA beads (4 and 8). Incubation with aptS reduced M-Ig from 0.8 g dl⁻¹ (lane 2) to 0.5 g dl⁻¹ (lane 3). No effect was observed with scr control (lane 4). AptS had no effect on M-Ig in control serum (lanes 5-8). e Effect of aptS on serum protein fractions (based on d). 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 on d). SD serum (top): AptS ‘pulled out’ SD M-Ig but did not affect other SD serum protein fractions. Scrambled aptS (scr) had no effect. Control MM serum (bottom): AptS 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.