Enzyme immunoassay for the quantification of mithramycin using beta-D-galactosidase as a label

A sensitive enzyme immunoassay for mithramycin (MTM) has been developed by using antibody induced in rabbits, beta-D-galactosidase-labeled MTM, and a double-antibody separation technique, which allowed us to measure accurately as little as 100 pg of MTM per assay tube. MTM-antibody was produced against MTM-bovine serum albumin conjugate prepared by the use of diazotized p-aminobenzoic acid as a cross-linker. The beta-D-galactosidase-labeled MTM conjugate was similarly prepared by a geometric m-isomer of diazotized aminobenzoic acid. This enzyme immunoassay was specific to MTM and showed a very slight cross-reactivity with MTM analogues, chromomycin A3 (5.6%) and olivomycin (2.4%), but no cross-reactivity with drugs commonly used with MTM in combination chemotherapy for cancer treatment. The values of MTM concentrations detected by this assay were comparable to those detected by the high-pressure liquid chromatography method. However, the enzyme immunoassay method was 100 times more sensitive in detecting MTM in lower concentrations. Using this assay, drug levels were easily determined in the blood and urine of rats during 6 h after i.v. administration of MTM in a single dose of 2.0 mg/kg. Since MTM has long been used against a variety of human cancers, the enzyme immunoassay of the drug will be a valuable new tool in clinical pharmacological studies.