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°C and 20°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°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.