The use of N-(aminobenzoyloxy) succinimide as a two-level heterobifunctional agent for the preparation of hapten-protein conjugates. Daunomycin as a model hapten with an amino group.
Three geometric ortho-, meta-, and para-isomers of N-(aminobenzoyloxy)succinimide (ABS) were synthesized, and their usefulness as a two-level heterobifunctional cross-linking agent in the preparation of hapten-protein conjugates was evaluated. The conjugation was based on the principle that ABS reacts immediately with an amino group of a hapten, and an aminobenzoyl group incorporated into the hapten is then activated by diazotization to a functional diazobenzoyl group acting on tyrosine or histidine residues of the protein. Using the anti-tumor antibiotic daunomycin (DM) as a model hapten, the three isomers of ABS were compared for their ability to conjugate DM with bovine serum albumin (BSA); DM incorporation onto a BSA molecular was found to occur to the highest degree with m-ABS, followed by p-ABS. while o-ABS completely failed to conjugate under the same coupling conditions. Using m-ABS it was possible to introduce more than 10 molecules of DM per BSA molecule. One of the DM-BSA samples was used as the immunogen for the production of anti-DM serum in a rabbit. The antibody specificity was shown to be direct to DM but not to other anti-cancer drugs (bleomycin, mitomycin C, actinomycin D and 5-fluorouracil) by the double antibody enzyme immunoassay (DEIA) using DM-beta-galactosidase conjugate as a label. An enzyme-linked immunosorbent assay (ELISA) for anti-DM IgG was developed using a DM-human serum albumin (DM-HSA) conjugate similarly prepared with m-ABS and horseradish peroxidase-conjugated goat anti-rabbit IgG as the solid-phase antigen and the labelled second antibody, respectively. This ELISA permitted us to measure accurately as little as 50 ng of anti-DM IgG per ml using a standard anti-DM IgG which had been purified from the anti-DM serum using an affinity column of Sepharose 4B with DM-HSA as the ligand. Using this ELISA as well as a sandwich enzyme immunoassay (SEIA) for total IgG, serum levels of anti-DM IgG and total IgG levels were easily monitored in a rabbit following immunization with DM-BSA. These results indicate that the use of DBS provides a novel method for preparing hapten-protein conjugates which will be useful in biochemistry and immunochemistry.
Fujiwara
K
K
Faculty of Pharmaceutical Sciences, Nagasaki University, Japan.
Matsumoto
N
N
Kitagawa
T
T
Inouye
K
K
D000107:Acetylation; D000588:Amines; D000818:Animals; D000906:Antibodies / Q000032:analysis; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000138:chemical synthesis; D003630:Daunorubicin / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006241:Haptens / Q000276:immunology*; D007536:Isomerism; D011817:Rabbits; D012710:Serum Albumin, Bovine; D013388:Succinimides
D000588:Amines; D000906:Antibodies; D003432:Cross-Linking Reagents; D006241:Haptens; D013388:Succinimides; C066593:N-(3-aminobenzoyloxy)succinimide; C066595:N-(4-aminobenzoyloxy)succinimide; D012710:Serum Albumin, Bovine; C066592:N-(2-aminobenzoyloxy)succinimide; D003630:Daunorubicin