Antibody immobilization to high-performance liquid chromatography supports. Characterization of maximum loading...

This study examined various factors that affect the maximum amount of intact immunoglobulin G (IgG) or Fab fragments that can be covalently immobilized to silica and other HPLC-grade supports for use in immunoaffinity chromatography or immunoextractions. Factors that were considered included the amount of surface area available for immobilization, the pore size of the support, the type of immobilization method and the nature of the support matrix. The main factor in determining the extent of immobilization was found to be the relationship between the support's surface area and the ability of the IgG or Fab fragments to reach this surface. Access to the support surface was a function of the size of the protein being immobilized and the support porosity, with maximum immobilization being obtained with supports having pore sizes of approximately 300 A for intact IgG and 100 A for Fab fragments. Some differences in the maximum level of immobilization were noted between different coupling methods. Supports like Poros and Emphaze gave similar results to those seen with HPLC-grade silica when a comparison was made between materials with comparable pore sizes. Many of the trends observed in this work for IgG and Fab fragments should apply to other proteins that are to be immobilized to HPLC supports.