Site-directed biotinylation of antibodies for controlled immobilization on solid surfaces.
Site-directed biotinylation of antibodies at the hinge region was developed to immobilize antibodies in an oriented manner via biotin-streptavidin linkage. When intact antibody was biotinylated with maleimide-activated biotin after reduction, the reaction preferentially occurred at the sulfhydryl groups between the C(H1) and the C(L) domains and, provided that the reagent concentration exceeded a certain level, at those between the C(H2) and the C(H2) domains at the hinge. Based on this result, we devised an approach in which free maleimide was added to compete with the activated biotin for the preferential sites between the C(H1) and the C(L) domains. Since the smaller molecular size of free maleimide made it more accessible for the reaction than biotin, maleimide bound to the groups between the C(H1) and the C(L) domains first and thus conceded the groups between the C(H2) and the C(H2) domains to biotin under optimal conditions. In an alternative approach, selective biotinylation at the hinge was also achieved by reacting activated biotin with F(ab')(2) fragment prepared by enzymatic cleavage. This result indicated that, when free of Fc, the hinge structure, which contains the functional groups, of the fragment was open, allowing easy access to the biotin derivative from the aqueous medium. Both site-directed biotinylation preparations were tested as capture antibodies in sandwich-type immunoassays and compared to whole antibody randomly biotinylated at amino groups on the molecule. Preparations of both the intact antibody and the F(ab')(2) showed consistently enhanced detection capabilities that were 2.6 and 20 times that of the control, respectively.
Cho
Il-Hoon
IH
Program for Bio-Microsystem Technology, Korea University, 1, 5-ka, Anam-dong, Sungbuk-ku, Seoul 136-701, Korea.
Paek
Eui-Hwan
EH
Lee
Haiwon
H
Kang
Ji Yoon
JY
Kim
Tae Song
TS
Paek
Se-Hwan
SH
D000345:Affinity Labels / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D000941:Antigens / Q000276:immunology; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation / Q000379:methods*; D015153:Blotting, Western / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D008301:Maleimides / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry; D013499:Surface Properties
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D000941:Antigens; D007128:Immunoglobulin Fragments; D008301:Maleimides; C043592:maleimide; D001710:Biotin; D019809:Streptavidin