Enzyme Immunoassay for the Quantification of Mitomycin C Using β-Galactosidase as a Label¹

A mitomycin C (MMC) antibody was produced following immunization of rabbits with a MMC-bovine serum albumin conjugate, which was newly synthesized by coupling MMC to mercaptosuccinylated bovine serum albumin via a cross-linker, N-maleoyl aminobutyric acid. Enzyme labeling of MMC was performed using β-d-galactosidase (EC 3.2.1.23) via m-maleoyl benzoic acid. An enzyme immunoassay for MMC was developed utilizing these reagents by a double-antibody technique. The standard curve of the assay was linear on a logitlog plot, and the lower limit of detection was 12 nm (0.2 ng/tube) so the enzyme immunoassay was found to be approximately 25 times more sensitive than a microbiological assay. Further, the enzyme immunoassay is practically free from interference by any other anticancer drugs. No significant decrease in MMC immunoreactivity was observed following 24 hr of incubation of the drug in normal human serum or urine at 37°. Using this assay, serum or urine levels of MMC can be determined accurately after administration of the drug to rats at a single dose of 600 µg/kg. The sensitivity and specificity of the enzyme immunoassay for MMC should provide a valuable new tool for use in pharmacokinetic and toxicity studies of MMC.