Development and application of a sensitive enzyme immunoassay for 7-N-(p-hydroxyphenyl)mitomycin C

An antibody specific for 7-N-(p-hydroxyphenyl)mitomycin C (M-83) was developed and used in a simple and sensitive enzyme immunoassay for detection of this anticancer drug in serum. The imino group of M-83 was covalently coupled to mercaptosuccinyl groups introduced into bovine serum albumin with a cross-linker, N-maleoylaminobutyric acid. The resulting conjugate was then used for the production of a specific antibody to M-83 in rabbits. Enzyme labeling of M-83 was performed using beta-D-galactosidase (EC 3.2.1.23) via m-maleoylbenzoic acid. Antibody production was of sufficiently high titer in rabbits to allow the development of a highly sensitive enzyme immunoassay for the free drug which accurately measures 15 pg of M-83 per assay tube. This assay was monospecific to M-83 and showed almost no cross-reactivity with a variety of other mitomycin analogues (mitomycin A, B, and C; porfiromycin; and acetylmitomycin C). Using this assay, preliminary pharmacokinetic study was undertaken using serial serum samples obtained from a patient who received the drug i.v., revealing a biphasic fashion of the kinetics, with an alpha-serum half-life of 9.7 min and a beta-serum half life of 80 min. An i.v. injection of M-83 into rats and assay of serum concentration revealed a similar biphasic response with an alpha-serum half-life of 8.3 min and a beta-serum half-life of 62 min.