Human GM-CSF interaction with the alpha-chain of its receptor studied using surface plasmon resonance

A surface plasmon resonance (SPR) based biosensor has been used for studyin g the interaction of recombinant human granulocyte-macrophage colony-stimul ating factor (GM-CSF) with genetically engineered alpha-chain subunits of i ts specific receptor (GM-R alpha). Western blot analysis of GM-R alpha conf irmed the correct size (80 kDa) and reactivity of these proteins against an ti-GM-R alpha polyclonal or monoclonal antibodies. GM-CSF was immobilized, using standard amine coupling methods, to the dextran-modified gold biosens or surface in order to capture GM-R alpha subsequently injected over the se nsing layer. GM-R alpha were shown to specifically form complexes with the immobilized ligand. Pre-incubation of constant amounts of GM-R alpha with d ilutions of soluble GM-CSF before injection of the mixture over the GM-CSF matrix, prevented ligand binding in a dose dependent manner. In contrast, u nrelated soluble cytokines or serum proteins (e.g. G-CSF, albumin, etc.) we re found to exert no inhibition. Complexes formation blockage by pre-incuba tion of constant amounts of GM-R alpha with dilutions of neutralizing anti- GM-R alpha antibodies was concentration dependent, further assessing the sp ecificity of the interaction. To investigate the possibility of relating th e effect on binding affinity of critical conformational changes at the cont act site, experiments of multisite binding were performed, flowing a set of neutralizing monoclonal antibodies reacting to different epitopes on GM-CS F over the GM-CSF matrix, before injecting GM-R alpha. The results indicate d that antibody interaction with helix D and helix A of GM-CSF markedly inh ibited GM-CSF binding to GM-R alpha. Comparable results were obtained using the biosensor technology and enzyme-linked immunoassays, in representative experiments performed with the same reagents. These experiments demonstrat e that SPR can be successfully used for studying complementary interactions between GM-CSF and its receptor alpha-chains in solution without using lab els or secondary tracers and, compared with conventional immunoanalysis met hods, significantly saving time. (C) 1999 Elsevier Science S.A. All rights reserved.