Analysis of the structural integrity of the hIL-6 mutants using conformation-dependent anti–hIL-6 MoAbs. (A) Differential recognition of dot blots of native (N) and denatured (D) WT hIL-6 by anti–hIL-6 MoAbs. Dot blot analysis was performed at two concentrations of untreated (native) or boiled hIL-6. Boiling was performed in the presence of 0.1% SDS and 0.167% (vol/vol) β-mercaptoethanol for 7 minutes (denatured). Results of one of two independent experiments are shown. Both experiments gave very similar results. (B) Recognition of hIL-6 mutants by three different conformation-dependent anti–hIL-6 MoAbs as determined by sandwich ELISA. The concentrations of equal amounts of each hIL-6 variant were determined using microtiter plates coated with either anti–hIL-6 MoAbs AH-65 or MoAb 16 as phase and biotinylated anti–hIL-6 MoAb B-E4 as tracer. Results are presented as the percentage of the values obtained for WT hIL-6. Values are the mean ± SD of at least two independent experiments performed in duplicate. ND, not determined.