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 studying the interaction of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) with genetically engineered alpha-chain subunits of its specific receptor (GM-Ralpha). Western blot analysis of GM-Ralpha confirmed the correct size (80 kDa) and reactivity of these proteins against anti-GM-Ralpha polyclonal or monoclonal antibodies. GM-CSF was immobilized, using standard amine coupling methods, to the dextran-modified gold biosensor surface in order to capture GM-Ralpha subsequently injected over the sensing layer. GM-Ralpha were shown to specifically form complexes with the immobilized ligand. Pre-incubation of constant amounts of GM-Ralpha with dilutions of soluble GM-CSF before injection of the mixture over the GM-CSF matrix, prevented ligand binding in a dose dependent manner. In contrast, unrelated soluble cytokines or serum proteins (e.g. G-CSF, albumin, etc.) were found to exert no inhibition. Complexes formation blockage by pre-incubation of constant amounts of GM-Ralpha with dilutions of neutralizing anti-GM-Ralpha 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 GM-CSF over the GM-CSF matrix, before injecting GM-Ralpha. The results indicated that antibody interaction with helix D and helix A of GM-CSF markedly inhibited GM-CSF binding to GM-Ralpha. 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 GM-CSF and its receptor alpha-chains in solution without using labels or secondary tracers and, compared with conventional immunoanalysis methods, significantly saving time.
Revoltella
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RP
Institute of Mutagenesis and Differentiation, CNR, Pisa, Italy. r.revoltella@imd.pi.cnr.it
Laricchia Robbio
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L
Vikinge
T
T
Pardi
E
E
Levantini
E
E
Beffy
P
P
D000818:Animals; D000906:Antibodies; D000941:Antigens; D001667:Binding, Competitive; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D009500:Neutralization Tests; D021122:Protein Subunits; D011817:Rabbits; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D000906:Antibodies; D000941:Antigens; D021122:Protein Subunits; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor