title: Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.
abstract: Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab')2fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab')2fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications.
authors.affiliation: Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0575, United States. Electronic address: terry.kirley@uc.edu.
authors.forename: Terence L
authors.initials: TL
authors.lastname: Kirley
authors.affiliation: Department of Cancer Biology, Proteomics Laboratory, College of Medicine, University of Cincinnati, 3125 Eden Avenue, Cincinnati, OH 45267-0521, United States.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Greis
authors.affiliation: Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0575, United States.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Norman
mesh_terms: D000906:Antibodies / Q000032:analysis* / Q000737:chemistry*; D004220:Disulfides / Q000032:analysis* / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods*; D007140:Immunoglobulin Fab Fragments / Q000032:analysis* / Q000737:chemistry*; D010084:Oxidation-Reduction; D013194:Staining and Labeling
chemical_list: D000906:Antibodies; D004220:Disulfides; D007140:Immunoglobulin Fab Fragments
keywords: Antibody conjugation; Fab fragments; Monoclonal antibody; Selective disulfide reduction; Sulfhydryl modification; TCEP agarose