Recombinant soluble human Fcγ receptor I with picomolar affinity for immunoglobulin G

The ectodomain of human FcγRI (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/κ) a high association rate of kass = 1.7 × 106 (M s)−1 and a low dissociation rate of kdiss = 1.8 × 10−4 s−1 were observed. The derived dissociation equilibrium constant of KD = 110 pM was significantly lower than that reported for binding to native FcγRI.