Covalent immobilization of antibodies for the preparation of immunoaffinity chromatographic supports

Immunosorbents in immunoaffinity chromatography (IAC) are prepared by immobilizing expensive antibodies without guidelines for ensuring the best coupling efficiencies, and avoiding low binding capacities. Covalent immobilization of antibodies on N-hydroxysuccinimide (NHS)-activated Sepharose 4 Fast Flow resin was optimized using human IgG via full factorial design with incubation times (4, 9, 14, 19 and 24 h), temperatures (4 degrees C and 20 degrees C) and coupling reaction buffers (sodium bicarbonate and triethanolamine). The best coupling efficiency (CE) (83.4 +/- 8.7%) was reached with triethanolamine buffer, 14 h and 4 degrees C. Comparison of antibody isotypes (IgG or IgM) by a nested factorial analysis suggested that antibodies in the IgG isotype presents the best coupling efficiency.