Sensitive enzyme immunoassay for the quantification of bleomycin using beta-D-galactosidase as a label

A sensitive and specific enzyme immunoassay (EIA) for an anticancer drug, bleomycin (BLM), has been developed, which allows measurement of as little as 25 pg of the antibiotic/tube. An antibody to BLM was obtained by immunizing rabbits with an antigen prepared by conjugating BLM with mercaptosuccinylated bovine serum albumin via N-(gamma-maleimidobutyryloxy)succinimide as a coupling agent. Enzyme labeling of BLM was performed using beta-D-galactosidase (beta-Gal; EC 3.2.1.23) via N-(m-maleimidobenzoyloxy)succinimide. Free and antibody-bound BLM-beta-Gal conjugates were separated by precipitation of the antibody-bound fraction with a second antibody (anti-rabbit IgG). Displacement of BLM-beta-Gal by unlabeled BLM when plotted as a logit-log function was linear over a concentration range of 10 pg-1 ng. The antibody distinguished alterations in the terminal structure of BLM, showing 277% cross-reaction with BLM B2, 4.6% with A2, 0.42% with A2'-b, 0.13% with A5, and 0.07% with the second-generation analog peplomycin. The EIA is free from interference by other anticancer drugs. Using this assay, drug levels were easily determined in tissues of rats following sc administration at a dose of 500 micrograms/kg. The sensitivity and specificity of the EIA should provide a useful tool for developing pharmacokinetic and toxicity studies of BLM.