Extended length heterobifunctional coupling agents for protein conjugations.
A series of extended length heterobifunctional coupling agents is described. The successive aminocaproic acid homologation of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, a known 9-atom long maleimide active ester linker, yielded 16-, 23-, and 30-atom long maleimide active ester homologues. The performance study of these coupling agents in automated microparticle enzyme immunoassays showed that, in the alpha fetoprotein assay, in which the linkers were employed in the construction of the alkaline phosphatase-antibody conjugates, the signal increased 64% when the length of the linker was incremented from 9 atoms to 23 atoms and 82% for the 30-atom long linker as compared with the 9-atom homologue. Similar improvements were observed in the performance of carbohydrate antigen, marker of ovarian cancer (CA-125), immunoassay where the linkers were used for conjugation of the capture antibody anti-CA-125 to the microparticle. Thus, a 300% signal improvement resulted when a 30-atom linker was used instead of the 9-atom homologue. The observed differences in the performance of the conjugates are interpreted as resulting from improved antibody binding and lowering of the steric hindrance of the complementarity-determined region of the antibody when longer coupling agents were used.
Bieniarz
C
C
Department of Immunochemistry, Abbott Laboratories, North Chicago, Illinois 60064, USA.
Husain
M
M
Barnes
G
G
King
C A
CA
Welch
C J
CJ
D000469:Alkaline Phosphatase; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D018394:CA-125 Antigen / Q000276:immunology; D002417:Cattle; D003432:Cross-Linking Reagents / Q000138:chemical synthesis* / Q000737:chemistry; D007118:Immunoassay; D007074:Immunoglobulin G; D008301:Maleimides / Q000138:chemical synthesis*; D011506:Proteins; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013329:Structure-Activity Relationship; D000509:alpha-Fetoproteins / Q000276:immunology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D018394:CA-125 Antigen; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D008301:Maleimides; D011506:Proteins; D000509:alpha-Fetoproteins; C025285:N-(4-carboxycyclohexylmethyl)maleimide N-hydroxysuccinimide ester; D000469:Alkaline Phosphatase