Figure 6.
Serum treatment with DTT or IdeS and protein G spin columns identify IgM as the predominant immunoglobulin species leading to complement activation. (A) Nonreducing, no heat SDS-PAGE gel of IgG and IgM with increasing concentrations of DTT. IgG (3 μg), IgM (3 μg), or both were incubated with DTT (0 mM, 0.5 mM, 1 mM, and 3 mM) in phosphate-buffered saline (PBS) (pH 7.4) in a final reaction volume of 10 μL at room temperature for 30 minutes. Some experimental details removed throughout legend. IgM′ represents a partially reduced form of IgM. (B) Sera from patient with hemolysis, elevated liver enzymes, low platelets (HELLP) with or without the addition of additional C5 inhibitor (eculizumab), sutimlimab, DTT, or FDi on PIGAKO cells. (C) CM12 sera was processed over a protein G spin column to purify IgG (protein G eluate [GE]) or isolate flow-through (protein G flow-through [GF]). GE and GF underwent centrifugal concentration (as required), desalting, and buffer exchange into PBS. Final preparation was added to healthy control sera in the bioluminescent mHam on CD46KO cells with or without IdeS treatment. (D) CM04 sera prepared as per panel C. After preparation, GF samples were treated with IdeS, DTT (3-mM pretreatment; 0.6-mM final), or sutimlimab (30 μg/mL). Final preparation per well was added to HC sera in the bioluminescent mHam on CD46KO cells. (E) Example tracing of CM04 in bioluminescent mHam on CD46KO cells with or without HI, temperature control (untreated serum heated to 37°C for 30 minutes), eculizumab, sutimlimab, IdeS pretreatment, or DTT before treatment. (F) Summary of relative luminescence at 1 hour for CM-TMA samples treated with DTT (n = 5) or IdeS (n = 5) compared with acute, remission, or eculizumab spiked samples. (G) Healthy control sera spiked with polyclonal IgM (extra 5, 10, or 20 μg per well) and run in bioluminescent mHam on CD46KO cells. (H) Healthy control sera spiked with myeloma IgM (10 μg per well), polyclonal IgM (10 μg per well), myeloma IgG1 (10 μg per well), polyclonal IgG (10 μg or 60 μg per well) and run in bioluminescent mHam on PIGAKO cells. (I) Healthy control sera spiked with polyclonal IgM (5 or 10 μg per well), polyclonal IgG (20 or 60 μg per well), myeloma IgG1 (20 μg per well), or myeloma IgM (10 μg per well) and run in bioluminescent mHam on DKO cells. (B-E,G-I) Example traces plotted as mean ± SD for each triplicate. (F) P values were calculated using 1-way ANOVA for Dunnett multiple-comparisons test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. HCh, heavy chain; LC, light chain; ns, not significant; RLU, relative luminescence units.

Serum treatment with DTT or IdeS and protein G spin columns identify IgM as the predominant immunoglobulin species leading to complement activation. (A) Nonreducing, no heat SDS-PAGE gel of IgG and IgM with increasing concentrations of DTT. IgG (3 μg), IgM (3 μg), or both were incubated with DTT (0 mM, 0.5 mM, 1 mM, and 3 mM) in phosphate-buffered saline (PBS) (pH 7.4) in a final reaction volume of 10 μL at room temperature for 30 minutes. Some experimental details removed throughout legend. IgM′ represents a partially reduced form of IgM. (B) Sera from patient with hemolysis, elevated liver enzymes, low platelets (HELLP) with or without the addition of additional C5 inhibitor (eculizumab), sutimlimab, DTT, or FDi on PIGAKO cells. (C) CM12 sera was processed over a protein G spin column to purify IgG (protein G eluate [GE]) or isolate flow-through (protein G flow-through [GF]). GE and GF underwent centrifugal concentration (as required), desalting, and buffer exchange into PBS. Final preparation was added to healthy control sera in the bioluminescent mHam on CD46KO cells with or without IdeS treatment. (D) CM04 sera prepared as per panel C. After preparation, GF samples were treated with IdeS, DTT (3-mM pretreatment; 0.6-mM final), or sutimlimab (30 μg/mL). Final preparation per well was added to HC sera in the bioluminescent mHam on CD46KO cells. (E) Example tracing of CM04 in bioluminescent mHam on CD46KO cells with or without HI, temperature control (untreated serum heated to 37°C for 30 minutes), eculizumab, sutimlimab, IdeS pretreatment, or DTT before treatment. (F) Summary of relative luminescence at 1 hour for CM-TMA samples treated with DTT (n = 5) or IdeS (n = 5) compared with acute, remission, or eculizumab spiked samples. (G) Healthy control sera spiked with polyclonal IgM (extra 5, 10, or 20 μg per well) and run in bioluminescent mHam on CD46KO cells. (H) Healthy control sera spiked with myeloma IgM (10 μg per well), polyclonal IgM (10 μg per well), myeloma IgG1 (10 μg per well), polyclonal IgG (10 μg or 60 μg per well) and run in bioluminescent mHam on PIGAKO cells. (I) Healthy control sera spiked with polyclonal IgM (5 or 10 μg per well), polyclonal IgG (20 or 60 μg per well), myeloma IgG1 (20 μg per well), or myeloma IgM (10 μg per well) and run in bioluminescent mHam on DKO cells. (B-E,G-I) Example traces plotted as mean ± SD for each triplicate. (F) P values were calculated using 1-way ANOVA for Dunnett multiple-comparisons test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. HCh, heavy chain; LC, light chain; ns, not significant; RLU, relative luminescence units.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal