Analysis of the structural integrity of the shIL-6R mutants using conformation-dependent anti–hIL-6R MoAbs. (A and B) Differential recognition of native (N) and denatured (D) WT shIL-6R by anti–hIL-6R murine MoAbs 17.6, 22.1, 32.3, 34.4, 38.8, M-91, and M-182 and Pc-Rb, a rabbit anti–hIL-6R polyclonal antiserum. (A) Dot blot analysis of two concentrations of untreated (native) or denatured shIL-6R as described in Fig 6A. (B) Immunoprecipitation of equal amounts of S35-labeled shIL-6R by anti–shIL-6R Abs in the absence (untreated) or presence (SDS) of 0.1% SDS or after 10 minutes of boiling (boiled). The amounts of immunoprecipitated shIL-6R were measured and are presented as the percentage of the values obtained with untreated shIL-6R. The effect of boiling was tested once, whereas that of SDS was tested twice.
Analysis of the structural integrity of the shIL-6R mutants using conformation-dependent anti–hIL-6R MoAbs. (C) Recognition of shIL-6R mutants by five different conformation-dependent anti–hIL-6R MoAbs as determined by sandwich ELISA. The concentrations of equal amounts of each shIL-6R variant were determined using microtiter plates coated with anti–hIL-6R MoAbs 22.1, M-182, 34.4, or 17.6 as phase and alkaline-phosphatase conjugated anti–hIL-6R MoAb M-91 as tracer. Results are presented as the percentage of the values obtained for WT shIL-6R. Values are the mean ± SD of at least two independent experiments performed in duplicate. ND, not determined.
