ALBUMIN-BINDING SURFACES - SYNTHESIS AND CHARACTERIZATION

The nature of the proteinaceous film deposited on a biomaterial surfac e following implantation is a key determinant of the subsequent biolog ical response. To achieve selectivity in the formation of this film, m onoclonal antibodies have been coupled to a range of solid substrates using avidin-biotin technology. Antibody clones varied in their antige n-binding activity following insertion of biotin groups into lysine re sidues. Biotinylated antibodies coupled to solid substrates via an imm obilized avidin bridge retained their biological activity. During immo bilization of avidin a significant proportion of the protein molecules were passively adsorbed rather than covalently attached to the surfac e. This loosely bound material could be removed by stringent elution p rocedures which resulted in a surface density of 5.4 pmol avidin cm(-2 ). Although these conditions would be harsh enough to denature monoclo nal antibodies, they did not destroy the biotin-binding activity of th e residual surface-coupled avidin, enabling the subsequent immobilizat ion of biotinylated antibodies. The two-step immobilization technique allowed the use of gentle protein modification procedures, reduced the risk of surface-induced denaturation and removed loosely bound materi al from the surface. The versatility of the technique encourages its a pplication to a wide range of immobilization systems where retention o f biological activity is a key requirement.