Conjugation of antibodies with bifunctional chelating agents: isothiocyanate and bromoacetamide reagents, methods of analysis, and subsequent addition of metal ions.
Preparation of the chelating agent (S)-4-[2,3-bis[bis(carboxymethyl)am ino]propyl]phenyl isothiocyanate is reported. Procedures for conjugation of this and (S)-N-4-[2,3-bis[bis-(carboxymethyl)amino] propyl]phenyl bromoacetamide to monoclonal antibodies and other proteins are described. The conjugates may be purified quickly by centrifugation through Sephadex G-50. The number of protein-bound chelating groups may be measured by titration with standard 57Co2+, using thin-layer chromatography to monitor binding. The labeled products retain their immunoreactivity, as illustrated by experiments in vivo with chelate-conjugated antibody to mouse I-AK antigen.
D000818:Animals; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D001402:B-Lymphocytes / Q000276:immunology; D002614:Chelating Agents / Q000138:chemical synthesis; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003037:Cobalt Radioisotopes; D003432:Cross-Linking Reagents / Q000138:chemical synthesis; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000138:chemical synthesis; D007204:Indium; D008198:Lymph Nodes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008670:Metals; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding; D011868:Radioisotopes; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D003037:Cobalt Radioisotopes; D003432:Cross-Linking Reagents; D008670:Metals; D011868:Radioisotopes; D007204:Indium; C042007:1-(4-bromoacetamidobenzyl)EDTA; C043981:4-(2,3-bis(bis(carboxymethylamino))propyl)phenyl isothiocyanate; D004492:Edetic Acid