title: N-terminal modification of immunoglobulin polypeptide chains tagged with isothiocyanato chelates.
abstract: Conjugates of monoclonal antibodies with drugs, toxins, radionuclides, and other agents are in widespread use in therapeutic trials and as clinical research tools. The characterization of these immunoconjugates generally does not include determining the individual sites at which such agents are attached. We have begun to explore the attachment of the bifunctional chelating agent isothiocyanatobenzyl-EDTA (CITC1) to the N-termini of the light chains of the Lym-1 monoclonal antibody. The similarity between this bifunctional chelating agent and Edman's reagent, phenyl isothiocyanate, led us to develop methods to distinguish between chelate-conjugated alpha-amino groups and epsilon-amino groups by Edman degradation. Practically all the N-terminal Asp alpha-NH2 groups of Lym-1 can be modified at neutral pH, while attachment at lysine side chains predominates at pH 9. Comparison of the immunoreactivities of Lym-1-CITC conjugates with and without N-terminal conjugation shows that both are almost fully active. This implies that modification of light-chain N-termini has little or no effect on immunoreactivity, despite the fact that these residues lie near the antigen-binding sites.
authors: Chemistry Department, University of California, Davis 95616.
authors: T M
authors: TM
authors: Rana
authors: C F
authors: CF
authors: Meares
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D001224:Aspartic Acid / Q000737:chemistry; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004140:Dinitrophenols; D004492:Edetic Acid / Q000737:chemistry* / Q000031:analogs & derivatives; D005998:Glycine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D000922:Immunotoxins / Q000737:chemistry* / Q000276:immunology; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D013861:Thiocyanates / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D004140:Dinitrophenols; D007147:Immunoglobulin Light Chains; D000922:Immunotoxins; D013861:Thiocyanates; D001224:Aspartic Acid; C043981:4-(2,3-bis(bis(carboxymethylamino))propyl)phenyl isothiocyanate; D004492:Edetic Acid; D008239:Lysine; D005998:Glycine