Binding affinity, capacity, and specificity of purified anti-GM-CSF autoantibodies in iPAP patients. (A) Saturation binding plot of [¹²⁵I]-GM-CSF binding to purified anti-GM-CSF autoantibody. Vertical axis represents the molar fractional binding of GM-CSF to autoantibody. Binding capacity (maximal binding) was determined from the asymptote of the curve, and binding affinity (K_AV) was determined from the concentration of GM-CSF at 50% of maximum binding. (B) Binding of autoantibodies to various forms of human GM-CSF. Reactivity of autoantibodies was measured by antigen capture and compared with binding to recombinant human GM-CSF produced in E coli, which was taken as 100%. Various forms were tested, including recombinant human GM-CSF produced in CHO cells; the large trypsin fragment that contained residues 31-58, 86-107, and 112-127, connected by disulfide bonds; and carboxymethylated GM-CSF, which had an intact primary amino acid sequence but lost the second and tertiary structure because of the loss of the 2 disulfide bonds. NS indicates not significant. (C) Xenospecificity of autoantibody binding. Reactivity of autoantibodies to murine and bovine GM-CSF was measured as above. (D) Specificity of autoantibody binding with respect to other human cytokines. Reactivity of autoantibodies to various human cytokines was measured as above. Error bars indicate SD.