Concentration of free and IgG-bound GM-CSF in human serum. (A) Total IgG was isolated individually from the sera of healthy subjects (HC) or patients with PAP (PAP) using protein G and evaluated by Western blotting to detect GM-CSF (top panels) or IgGκ (κ) and -λ (λ) light chains (as a loading control, bottom blots). Each lane represents one subject. (B) Detection of free GM-CSF and autoantibody-bound GM-CSF in serum. A set of “standard” samples composed of recombinant human GM-CSF (Leukine) at various concentrations ranging from 0 to 30 ng/mL were prepared in mouse serum in the absence (○) or presence (▽) of purified human GM-CSF autoantibodies (30 μg/mL). Standard samples were diluted 1/30 with 1% BSA in PBS and then GM-CSF was measured using a commercial human ELISA kit (R&D Systems) as directed by the manufacturer. (C) Use of a novel ELISA (SDS-HD ELISA, see “Methods”) to quantify GM-CSF in PBS in the absence or presence of GM-CSF autoantibody (1 μg/mL) and in the absence or presence of a pretreatment with SDS and heat denaturation. Each bar represents the mean of duplicate determinations for 1 of 4 separate experiments with similar results. (D) GM-CSF level evaluated using a novel human GM-CSF ELISA as described in “Methods.” Symbols represent the same samples and conditions as described in the legend to panel B above. GM-CSF was detectable in the absence of GM-CSF autoantibody (○), undetectable in the presence of GM-CSF autoantibody in the absence of SDS-HD pretreatment (▽), and detection was restored in the presence of GM-CSF autoantibody by SDS-HD pretreatment (△). (E) Free GM-CSF (▨) or total GM-CSF (free and autoantibody-bound; ■) were measured in sera of healthy subjects (HC) or patients with PAP (PAP) using a commercially available ELISA or the SDS-HD ELISA, respectively, as described in “Methods.” Total serum GM-CSF levels in HC and PAP were not different (3048 ± 484, n = 11; PAP 2360 ± 668, n = 5; respectively, P = .43).