Binding of model soluble immune complexes to modified C-reactive protein.
We have identified a binding interaction between a modified form of C-reactive protein (mCRP) and model immune complexes. We have characterized these interactions in terms of their relative affinity, specificity, pH dependence, and recognition site on mCRP. First, binding isotherms were generated for the reaction of immobilized mCRP with heat-aggregated IgG (HAG) which gave an estimate for the value of the dissociation constant, Kd, of 1.6 nM. Second, competitive binding assays were performed using immobilized HAG to determine the relative affinity (IC50) for the interaction between HAG or monomeric IgG and mCRP in the fluid phase. While the binding of HAG to mCRP occurred with high affinity (IC50=0.72 nM), the binding of monomeric IgG to mCRP occurred with >2000-fold lower affinity (IC50>1.66 muM). Third, immune complex binding to immobilized mCRP was studied using defined ratios of horseradish peroxidase and rabbit anti-peroxidase Ab. The binding of these complexes to mCRP was strongly pH dependent, with a maximum at pH 5.5. At this optimal pH, preformed peroxidase-antiperoxidase immune complexes (approximately 2:3 Ab:Ag molar ratio) were shown to bind immobilized mCRP with a Kd of 111 nM. Fourth, using mCRP-specific mAbs, HAG and peroxidase- antiperoxidase binding sites were localized to CRP sequences to 130 to 138 and 200 to 206. Since inflammatory processes often cause microenvironments to become acidic, and since mCRP selectively binds immune complexes at acidic pH, we propose that mCRP mediates the specific binding of immune complexes in vivo and potentiates effector reactions for immune complex removal.
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Immtech International, Inc., Evanston, IL 60201, USA.
Brockmeier
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Potempa
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D000818:Animals; D019138:Antibodies, Blocking / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000936:Antigen-Antibody Complex / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002097:C-Reactive Protein / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D006735:Horseradish Peroxidase / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008854:Microscopy, Electron; D008954:Models, Biological; D011485:Protein Binding; D011817:Rabbits; D012995:Solubility
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D002097:C-Reactive Protein; D006735:Horseradish Peroxidase