THE USE OF N-(AMINOBENZOYLOXY)SUCCINIMIDE AS A 2-LEVEL HETEROBIFUNCTIONAL AGENT FOR THE PREPARATION OF HAPTEN-PROTEIN CONJUGATES - DAUNOMYCIN AS A MODEL HAPTEN WITH AN AMINO GROUP

被引:9
作者
FUJIWARA, K [1 ]
MATSUMOTO, N [1 ]
KITAGAWA, T [1 ]
INOUYE, K [1 ]
机构
[1] TOKYO RES CTR,BIOTECHNOL RES LABS,AYASE,KANAGAWA 252,JAPAN
关键词
Daunomycin; ELISA; Hapten; Hapten-protein conjugate; N-(Aminobenzoyloxy)succinimide;
D O I
10.1016/0022-1759(90)90384-8
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 sample 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-ß-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 IgD 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. © 1990.
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页码:227 / 235
页数:9
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