Fig. 3.

Immunoprecipitation analyses of murine mabs specific for IDR-A and -B with single amino acid mutants of TPO using ³⁵S-methionine labelled translated proteins. The results are expressed as percentage values of wild type TPO ectodomain precipitated in the same experiment. The TPO mutants used were: (a) K713A and (b) E716A, selected as controls within the epitope of mab 47 (c) E461T (d) D553N (e) D624S and (f) N642D selected to probe the vicinity of IDR-B. Background binding was assessed using an irrelevant IgG mab. Generally, the background binding obtained in the assay with different mutants ranged from 361cpm to 580cpm. Specific binding to wild type TPO ectodomain with the variety of anti-TPO mabs used in the study ranged from 8997cpm to 1287cpm. The binding of mab 47 was abrogated for the K713A (a) and E716A (b) mutants respectively), whilst the binding of mab 15, specific for IDR-B, was significantly reduced (p = 0·006) in the N642D mutant (f). Data represents the results of at least two experiments.