Photolinker-polymer-mediated immobilization of monoclonal antibodies, F(ab')2 and F(ab') fragments.
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 mW.cm-2 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.
Gao
H
H
Institute of Biochemistry, University of Bern, Switzerland.
Kislig
E
E
Oranth
N
N
Sigrist
H
H
D000327:Adsorption; D000911:Antibodies, Monoclonal; D001389:Azirines / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments; D010777:Photochemistry; D011108:Polymers; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D019809:Streptavidin; D013462:Sulfur Radioisotopes
C090804:3-(trifluoromethyl)-3-(3-isothiocyanophenyl)diazirine; D000911:Antibodies, Monoclonal; D001389:Azirines; D001426:Bacterial Proteins; D007140:Immunoglobulin Fab Fragments; D011108:Polymers; D013462:Sulfur Radioisotopes; D012710:Serum Albumin, Bovine; D019809:Streptavidin