
<ArticleTitle>Human <prot>GM-CSF</prot> interaction with the alpha-chain of its receptor studied using surface plasmon resonance.</ArticleTitle>
<AbstractText>A surface plasmon resonance (SPR) based biosensor has been used for studying the interaction of recombinant human <prot>granulocyte-macrophage colony-stimulating factor</prot> (<prot>GM-CSF</prot>) with genetically engineered alpha-chain subunits of its specific receptor (<prot>GM-Ralpha</prot>). Western blot analysis of <prot>GM-Ralpha</prot> confirmed the correct size (80 kDa) and reactivity of these proteins against anti-<prot>GM-Ralpha</prot> polyclonal or monoclonal antibodies. <prot>GM-CSF</prot> was immobilized, using standard amine coupling methods, to the dextran-modified gold biosensor surface in order to capture <prot>GM-Ralpha</prot> subsequently injected over the sensing layer. <prot>GM-Ralpha</prot> were shown to specifically form complexes with the immobilized ligand. Pre-incubation of constant amounts of <prot>GM-Ralpha</prot> with dilutions of soluble <prot>GM-CSF</prot> before injection of the mixture over the <prot>GM-CSF</prot> matrix, prevented ligand binding in a dose dependent manner. In contrast, unrelated soluble cytokines or serum proteins (e.g. <prot>G-CSF</prot>, <prot>albumin</prot>, etc.) were found to exert no inhibition. Complexes formation blockage by pre-incubation of constant amounts of <prot>GM-Ralpha</prot> with dilutions of neutralizing anti-<prot>GM-Ralpha</prot> antibodies was concentration dependent, further assessing the specificity of the interaction. To investigate the possibility of relating the effect on binding affinity of critical conformational changes at the contact site, experiments of multisite binding were performed, flowing a set of neutralizing monoclonal antibodies reacting to different epitopes on <prot>GM-CSF</prot> over the <prot>GM-CSF</prot> matrix, before injecting <prot>GM-Ralpha</prot>. The results indicated that antibody interaction with helix D and helix A of <prot>GM-CSF</prot> markedly inhibited <prot>GM-CSF</prot> binding to <prot>GM-Ralpha</prot>. Comparable results were obtained using the biosensor technology and enzyme-linked immunoassays, in representative experiments performed with the same reagents. These experiments demonstrate that SPR can be successfully used for studying complementary interactions between <prot>GM-CSF</prot> and its receptor alpha-chains in solution without using labels or secondary tracers and, compared with conventional immunoanalysis methods, significantly saving time.</AbstractText>
