EPITOPE ANALYSIS OF HUMAN IL-6 RECEPTOR GP80 MOLECULE WITH MONOCLONAL-ANTIBODIES

Gp80 human IL-6R was studied using 7 murine mAb (M37, M91, M113, M139, M164, M182 and M195) obtained after fusion of splenocytes of Balb/c m ice immunised with a mixture of recombinant IL-6 receptor (rIL-6R) and cells from 2 cell lines expressing IL-6R. These were U266, which is I L-6 independent and XG-1 which is IL-6-dependent. In ELISA the 7 mAb r eacted against the rIL-6R and against the natural soluble form found i n plasma (nIL-6R), which both lack transmembrane and cytoplasmic domai ns. However, M195 reacted less with the natural than with the recombin ant soluble IL-6R. Using FACS analysis, the 7 mAb were shown to bind t o U266 cells but not to the Namalva cell line which is deprived of IL- 6R. This showed that they all recognised the membrane form of the IL-6 R. Three of the anti-IL-6R mAb reacted with rIL-6R by Western blotting . Four different epitopes of the molecule were identified, either by c ross-blocking experiments of mAb binding to IL6R in ELISA or by the bi osensor Biacore technology. A group of 4 mAb (M37, M113, M139 and M164 ) and another mAb (M195) identified 2 different epitopes involved in I L-6 binding. These antibodies were able to inhibit the binding of IL-6 to IL-6R and the proliferation of the IL-6-dependent XG-1 cell line. M91 and M182 recognized 2 other epitopes that were not involved in IL- 6 binding. As expected, M91 did not inhibit XG-1 proliferation; in con trast, M182 interfered with the proliferative response of the XG-1 cel l line. This suggests that the epitope recognized by M182 is involved in the interaction between IL-6-gp80 complex and gp130 necessary for s ignal transmission. Using 2 mAb recognizing 2 different epitopes, we d esigned a sandwich ELISA to measure soluble IL-6R in biological fluids .