Human igm

CP ELISA assays were carried out as previously described (26 ). Briefly, human IgM (3 μg/ml) (MP Biomedical) in coating buffer (100 mM Na₂CO₃/NaHCO₃ [pH 9.6]) was immobilized on a high-binding ELISA plate (Greiner Bio-One). Dose-dependent ElpQ or ElpB inhibition was determined using a 12-point, two-fold dilution: ElpQ_181-343 (4.4–0.002 μM), ElpQ_Y240A (10–0.005 μM), ElpQ_A4 (4.4–0.002 μM), ElpQ_Δ290-296 (22–0.011 μM), ElpQ_181-340 (2.2–0.001 μM), ElpQ_182-378 (4.4–0.002 μM), or ElpB_Y266A (4.0–0.002 μM). Normal human serum (2%, Innovative Research) was added, along with either ElpQ or ElpB inhibitor in CP buffer (10 mM Hepes [pH 7.3], 0.1% [w/v] gelatin, 140 mM NaCl, 2 mM CaCl₂, 0.5 mM MgCl₂). All assays were performed in triplicate and normalized to positive (100%, no inhibitor) and negative (0%, no Normal human serum) controls. Inhibition curves were fitted using nonlinear regression to a normalized variable slope model in GraphPad Prism (v 10.0.2, GraphPad Software, Inc) (www.graphpad.com).