A rapid and quantitative technique for assessing IgG monomeric purity, calibrated with the NISTmAb reference material.

The fraction of intact monomer in a sample (moles/moles), the monomeric purity, is measured as a quality control in therapeutic monoclonal antibodies but is often unknown in research samples and remains a major source of variation in quantitative antibody-based techniques such as immunoassay development. Here, we describe a novel multiplex technique for estimating the monomeric purity and antigen affinity of research grade antibody samples. Light scattering was used to simultaneously observe the mass of antibody binding to biosensor surfaces functionalised with antigen (revealing Fab binding kinetics) or protein A/G (PAG).

Kinetic Analysis of the Multivalent Ligand Binding Interaction between Protein A/G and IgG: A Standard System Setting.

Recombinant protein A/G (PAG) has a sequence coding for eight IgG binding sites and has enhanced interspecies affinity. High-frequency sampling of a PAG titration with IgG produces concentration profiles that are sensitive to the kinetic availability of the binding sites. The full kinetic model developed here for IgG binding sequentially to PAG shows only two distinct kinetic processes, describing an initial rapid association of two antibodies to PAG with a rate constant k-fast = (1.