Photolinker-polymer-mediated immobilization of monoclonal antibodies, F(ab')2 and F(ab') fragments

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Gao, Hui
Kislig, Elisabeth
Norbert, Oranth
Sigrist, Hans
Photolinker-polymer-mediated covalent immobilization of antibodies, F(ab') and F(ab')2 fragments has been achieved by light-dependent coupling procedures. Anti-alpha-foetoprotein (anti-AFP) monoclonal antibodies were covalently linked to microplates by layer-coating procedures, which entail antibody photoimmobilization to a photolinker-polymer-precoated surface. In this and the co-coating procedure described, diazirine-functionalized BSA (T-BSA) served as the multifunctional light-activatable linking agent (photolinker polymer). Prior to photo-activation, F(ab')2 or F(ab') fragments derived from anti-(prostate-specific antigen) monoclonal antibodies were mixed and co-coated with the photolinker polymer on to polystyrene microplates. The immunoreagents remained immunologically active after 350 nm irradiation (irradiance 0.7 for 20 min). Immuno-responses of photoimmobilized monoclonal anti-AFP antibodies were equivalent to signal intensities obtained with physically adsorbed antibodies. Photoimmobilization of anti-PSA F(ab') fragments in the presence of T-BSA revealed exponential binding characteristics indicating stabilizing molecular co-operativity of the BSA constituent. Co-coating procedures yielded 62 and 65% binding of applied 14C-labelled F(ab')2 and F(ab') fragments respectively. Covalency of antibody binding was inferred from: (i) the strict dependence of photoreagent availability; (ii) the light-dependence of the immobilization process; and (iii) the reversibility of immunocomplexation after acid treatment.
Publication Reference
Biotechnol Appl Biochem . 1994 Oct;20(2):251-63.