title: Conjugation of a reactive thiol at the nucleotide binding site for site-specific antibody functionalization.
abstract: Described here is a UV photo-cross-linking method that utilizes the NBS (nucleotide binding site) for site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBS(Thiol)), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBS(Thiol) method by successfully functionalizing a prostate specific antigen antibody (IgG(PSA)) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5-1.5 J/cm(2) was determined to yield the most efficient photo-cross-linking and resulted in 1-1.5 conjugations per antibody while preserving antibody/antigen binding activity and Fc recognition. Utilizing the IBA-Thiol ligand allows for an efficient means of site-specifically conjugating UV sensitive functionalities to antibody NBS that would otherwise not have been amenable by the previously described UV-NBS photo-cross-linking method. The UV-NBS(Thiol) conjugation strategy can be utilized in various diagnostic and therapeutic applications with nearly limitless potential for the preparation of site-specific covalent conjugation of affinity tags, fluorescent molecules, peptides, and chemotherapeutics to antibodies.
authors.affiliation: Department of Chemical and Biomolecular Engineering, §Advanced Diagnostics and Therapeutics, and ⊥Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Alves
authors.forename: Nur
authors.initials: N
authors.lastname: Mustafaoglu
authors.forename: Basar
authors.initials: B
authors.lastname: Bilgicer
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D000941:Antigens / Q000378:metabolism*; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D007211:Indoles / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: D000906:Antibodies; D000941:Antigens; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D007211:Indoles; D009711:Nucleotides; D013438:Sulfhydryl Compounds; C014612:indolebutyric acid