Labeling of antibodies by in situ modification of thiol groups generated from selenol-catalyzed reduction of native disulfide bonds.
A new method for labeling antibodies which involves selenol-catalyzed reduction of native disulfide bonds in antibodies to generate thiol groups, which then are labeled using thiol-reactive reagents, is described. The reduction and labeling steps of this rapid procedure are carried out in one vessel, without requiring any separation step to remove the reductant before labeling. It results in a quantitative and homogenous incorporation of about seven labeled groups per antibody molecule in less than 5 min. All reagents used are commercially available-selenocystamine (catalyst precursor), dithiothreitol or tris(2-carboxyethyl)phosphine (reductant), and thiol-reactive labeling reagents such as biotin-poly(ethylene oxide)-maleimide. This method is broadly applicable for labeling proteins such as immunoglobulins with reducible disulfide bonds, whose reduction and labeling does not result in a significant loss of activity. Biotinylated murine antibodies (anti-phosphotyrosine and anti-EGF receptor) prepared by this reduced-disulfide labeling method perform comparably or better than amino-group biotinylated antibodies in applications such as enzyme-linked immunosorbent assay, immunohistochemistry, and immunoprecipitation. This reduced-disulfide labeling method is superior to amino-group labeling methods because it is not inhibited by the presence of amines in solution, as demonstrated by the biotinylation of an antibody in a hybridoma culture supernatant containing amino acids and serum proteins.
Singh
Rajeeva
R
ImmunoGen, Inc., 128 Sidney Street, Cambridge, MA 02139, USA. rajeeva.singh@immunogen.com
Maloney
Erin K
EK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001667:Binding, Competitive; D019921:Biotinylation / Q000379:methods; D004220:Disulfides / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007158:Immunologic Techniques; D007700:Kinetics; D051379:Mice; D010084:Oxidation-Reduction; D019000:Phosphotyrosine / Q000276:immunology; D051381:Rats; D018036:Selenium Compounds / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004220:Disulfides; D018036:Selenium Compounds; D013438:Sulfhydryl Compounds; C442270:selenol; D019000:Phosphotyrosine; D066246:ErbB Receptors