AI Article Synopsis
- The ectodomain of human FcgammaRI (rsCD64) was successfully expressed and purified from HEK 293T cells, showing binding activity to human IgG via ELISA.
- The binding specificity matched that of native CD64, and kinetic analysis revealed a simple monovalent interaction model.
- The association and dissociation rates for binding to a chimeric antibody were measured, showing a strong interaction with a low dissociation equilibrium constant compared to native FcgammaRI.
Article 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.
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| http://dx.doi.org/10.1016/j.bbrc.2005.10.162 | DOI Listing |
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