Journal: Communications Biology

Article Title: Personalized immunoglobulin aptamers for detection of multiple myeloma minimal residual disease in serum

doi: 10.1038/s42003-020-01515-x

Figure Lengend Snippet: a Left: Target and counter-targets antibodies were immobilized on protein G columns to optimize exposure of Fab to ssDNA. Right: SELEX approach. During selection (1) immobilized target (e.g., daratumumab) was incubated with a randomized 40- mer ssDNA oligonucleotide SELEX library. Non-binding ssDNA were washed off while binding sequences were retained and eluted for incubation with counter-targets (e.g., polyclonal antibodies, beads) (2, ‘Counter selection’) Flow-through was collected, PCR amplified (3), strand-separated (4, ‘ssDNA recovery’) and incubated with target in the next SELEX round. b Left: Nucleotide sequence and 2D structure prediction for aptD at 25 °C, PBS, 2 mM MgCl 2 : Δ G = − 7.62 kcal mol −1 . Right: Specificity. Daratumumab and control IgGs rituximab, M-Ig from three unrelated MM patients and polyclonal IgG were immobilized on protein G coated plates and incubated for 30 min with biotin-aptD or controls (biotin-scrambled aptamer (scr), no aptamer (no apt)). Binding was detected with streptavidin-HRP and visualized with TMB chromogenic substrate (370 nm). c Limit of detection. Biotin-aptD, immobilized on streptavidin coated plates, was incubated with serum containing decreasing concentrations of daratumumab. Six independent measurements performed in triplicates determined a limit of detection of 4.5 × 10 −6 g dl −1 . d Binding affinity ( K D ) by surface plasmon resonance (SPR). Biotin-aptD was immobilized on SwitchAvidin sensors, exposed to increasing concentrations (6.25–200 nM) of daratumumab or control polyclonal human IgG (dotted line, representative 100 nM) and graphed after subtracting results obtained with control (biotin-scrambled aptD) analyzed in a parallel channel. Sensograms show aptD specific binding that determined a dissociation constant ( K D ) of 2.67 nM. e Specificity and sensitivity for daratumumab in serum by ELONA. Biotin-aptD, immobilized on streptavidin coated plates was incubated with serum from three patients (D1-D3) obtained before (pre-treatment) and after daratumumab treatment (treatment) at different dilutions (dil x1000). Biotin-scrambled aptD (scr) was used as a control with pre-treatment and treatment samples. No aptamer (no apt) control was included for treatment samples only. Sera were diluted up to 1:40,000 with buffer. No significant signal was detected in pre-treatment samples or with scrambled control (scr) or no aptamer (no apt). AptD detected daratumumab in all sera obtained after treatment at all dilutions. f Specificity and sensitivity in serum by homogenous time-resolved fluorescence (HTRF). Insert: HTRF measures fluorescence resonance energy transfer (FRET) that occurs when aptD labeled with terbium (streptavidin-terbium (SA-Tb), ex. 340/em. 620 nm) is in proximity of acceptor fluorophore d2 labeled anti-human IgG (anti IgG-d2; ex. 620/em. 665 nm). Graph: FRET occurs when aptD [biotin-aptD-SA-Tb] was incubated with treatment serum (patient D1), but not when incubated with pre-treatment serum (Pre-Tx) or control sera (MM, no-MM), nor when controls (scrambled aptD, no apt) were incubated with treatment serum. 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, n.s. not significant.

Article Snippet: 620/em 665 nm) conjugated to goat polyclonal anti-human IgG-Fc (Cisbio cat # 61HFCDAF) was used as acceptor.

Techniques: Selection, Incubation, Binding Assay, Amplification, Sequencing, SPR Assay, Fluorescence, Förster Resonance Energy Transfer, Labeling, Two Tailed Test

Journal: Communications Biology

Article Title: Personalized immunoglobulin aptamers for detection of multiple myeloma minimal residual disease in serum

doi: 10.1038/s42003-020-01515-x

Figure Lengend Snippet: 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 MgCl 2 for aptS that was isolated after 7 SELEX rounds for the M-Ig of patient SD (Δ G = − 7.21 kcal mol −1 ) ( b ), and for aptC that was isolated after 6 SELEX rounds for the M-Ig of patient CR (Δ G=− 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 ( K D ) 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.

Article Snippet: 620/em 665 nm) conjugated to goat polyclonal anti-human IgG-Fc (Cisbio cat # 61HFCDAF) was used as acceptor.

Techniques: Purification, Sequencing, Isolation, Incubation, Labeling, Binding Assay, SPR Assay, Two Tailed Test

Journal: Communications Biology

Article Title: Personalized immunoglobulin aptamers for detection of multiple myeloma minimal residual disease in serum

doi: 10.1038/s42003-020-01515-x

Figure Lengend Snippet: 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.

Article Snippet: 620/em 665 nm) conjugated to goat polyclonal anti-human IgG-Fc (Cisbio cat # 61HFCDAF) was used as acceptor.

Techniques: Protein Electrophoresis, Serum Assay, Incubation, Binding Assay, Two Tailed Test

Journal: Communications Biology

Article Title: Personalized immunoglobulin aptamers for detection of multiple myeloma minimal residual disease in serum

doi: 10.1038/s42003-020-01515-x

Figure Lengend Snippet: 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.

Article Snippet: 620/em 665 nm) conjugated to goat polyclonal anti-human IgG-Fc (Cisbio cat # 61HFCDAF) was used as acceptor.

Techniques: Protein Electrophoresis, Incubation, Labeling, Binding Assay, Quantitation Assay, Two Tailed Test

Journal: Communications Biology

Article Title: Personalized immunoglobulin aptamers for detection of multiple myeloma minimal residual disease in serum

doi: 10.1038/s42003-020-01515-x

Figure Lengend Snippet: Clinical characteristics of patient CR.

Article Snippet: 620/em 665 nm) conjugated to goat polyclonal anti-human IgG-Fc (Cisbio cat # 61HFCDAF) was used as acceptor.

Techniques: