title: Recombinant soluble human Fcgamma receptor I with picomolar affinity for immunoglobulin G.
abstract: The ectodomain of human FcgammaRI (rsCD64) was expressed in HEK 293T cells and purified by immobilized-metal affinity chromatography. Binding activity to human IgG was verified by ELISA and the isotype-specificity determined by a surface plasmon resonance inhibition assay was found to be the same as for native CD64. The active concentration of the rsCD64 preparation was derived using a solution competition assay and was used for the subsequent kinetic analysis. Binding curves were well described by a simple monovalent interaction model confirming the known stoichiometry of the interaction. Mass-transport limitation was prevented by using sufficiently low surface capacities. For binding to the recombinant mouse/human chimeric antibody cPIPP (IgG1/kappa) a high association rate of k(ass)=1.7 x 10(6) (M s)(-1) and a low dissociation rate of k(diss)=1.8 x 10(-4) s(-1) were observed. The derived dissociation equilibrium constant of K(D)=110 pM was significantly lower than that reported for binding to native FcgammaRI.
authors.affiliation: Aachen University, Department of Molecular Biotechnology, 52074 Aachen, Germany.
authors.forename: Antje
authors.initials: A
authors.lastname: Paetz
authors.forename: Markus
authors.initials: M
authors.lastname: Sack
authors.forename: Theo
authors.initials: T
authors.lastname: Thepen
authors.forename: Mehmet K
authors.initials: MK
authors.lastname: Tur
authors.forename: Daniela
authors.initials: D
authors.lastname: Bruell
authors.forename: Ricarda
authors.initials: R
authors.lastname: Finnern
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
mesh_terms: D000818:Animals; D000937:Antigen-Antibody Reactions; D002460:Cell Line; D002678:Chimera / Q000276:immunology; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology* / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D017452:Receptors, IgG / Q000276:immunology* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D007074:Immunoglobulin G; D017452:Receptors, IgG; D011994:Recombinant Proteins