Synthesis of bifunctional antibodies for immunoassays.
The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.
DeSilva
B S
BS
Procter & Gamble Pharmaceuticals, Route 320, Woods Corners, Norwich, New York 13815, USA. Desilva.bs@pg.com
Wilson
G S
GS
D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D001331:Automation; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D003543:Cysteamine / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism; D006041:Goats; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D008301:Maleimides / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D015394:Molecular Structure; D010434:Pepsin A / Q000378:metabolism; D013997:Time Factors
D018033:Antibodies, Bispecific; D003432:Cross-Linking Reagents; D004220:Disulfides; D004800:Enzymes, Immobilized; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008301:Maleimides; D003543:Cysteamine; D010434:Pepsin A