Fujiwara K, Isobe M, Saikusa H, Nakamura H, Kitagawa T, Takahashi S
Cancer Treat Rep. 1983 Apr;67(4):363-9.
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.
已开发出一种针对抗癌药物博来霉素（BLM）的灵敏且特异的酶免疫测定法（EIA），该方法能够检测低至25 pg/管的抗生素。通过用经N-（γ-马来酰亚胺丁酰氧基）琥珀酰亚胺作为偶联剂将BLM与巯基琥珀酰化牛血清白蛋白偶联制备的抗原免疫兔子，获得了针对BLM的抗体。使用β-D-半乳糖苷酶（β-Gal；EC 3.2.1.23）通过N-（间马来酰亚胺苯甲酰氧基）琥珀酰亚胺对BLM进行酶标记。游离的和与抗体结合的BLM-β-Gal缀合物通过用第二抗体（抗兔IgG）沉淀抗体结合部分来分离。当将未标记的BLM对BLM-β-Gal的置换绘制为对数几率-对数函数时，在10 pg至1 ng的浓度范围内呈线性。该抗体能够区分BLM末端结构的变化，与BLM B2的交叉反应率为277%，与A2的交叉反应率为4.6%，与A2'-b的交叉反应率为0.42%，与A5的交叉反应率为0.13%，与第二代类似物培洛霉素的交叉反应率为0.07%。该EIA不受其他抗癌药物的干扰。使用该测定法，在以500微克/千克的剂量皮下给药后，很容易测定大鼠组织中的药物水平。EIA的灵敏度和特异性应为开展BLM的药代动力学和毒性研究提供有用的工具。
