Gabor F, Pittner F, Spiegl P
Institute of Pharmaceutical Technology, University of Vienna, Austria.
Arch Pharm (Weinheim). 1995 Dec;328(11-12):775-80. doi: 10.1002/ardp.19953281109.
A radioimmunoassay has been developed for the quantitation of triamcinolone-acetonide (TAAc) at the picogram level. For use of TAAc as an antigenic epitope first the drug was hemisuccinoylated at C-21 as confirmed by 13C-NMR- and mass spectroscopy after derivatization. This hapten was conjugated to the carrier-protein bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) by different amide-bond generating methods (imidazolide-, carbodiimide-, carbodiimide/sulfo-N-hydroxysuccinimide-, mixed anhydride-method) yielding antigens of quite different conjugation number, solubility and usefulness. The mixed anhydride-method yielded most useful soluble conjugates bearing 0.3-31.5 mol TAAc per mol carrier-protein. Coupling by the carbodiimide-method yielded insoluble conjugates, inappropriate for antigen synthesis in hapten immunoassays because of formation of coupling agent modified residues and crosslinking of the carrier-protein. Specificity of the antisera obtained by immunization with TAAc-BSA and TAAc-KLH was assessed by isolation of the soluble hapten-antibody complex and a RIA protocol was developed providing a detection limit of 200 pg (0.46 pmol) TAAc/ml sample.
已开发出一种放射免疫分析法，用于皮克级曲安奈德-丙酮化物（TAAc）的定量分析。为了将TAAc用作抗原表位，首先通过衍生化后的13C-NMR和质谱确认，该药物在C-21位进行了半琥珀酰化。通过不同的酰胺键生成方法（咪唑化物法、碳二亚胺法、碳二亚胺/磺基-N-羟基琥珀酰亚胺法、混合酸酐法）将该半抗原与载体蛋白牛血清白蛋白（BSA）或钥孔血蓝蛋白（KLH）偶联，得到了共轭数、溶解度和实用性差异很大的抗原。混合酸酐法产生了最有用的可溶性偶联物，每摩尔载体蛋白含有0.3-31.5摩尔TAAc。碳二亚胺法偶联产生不溶性偶联物，由于偶联剂修饰残基的形成和载体蛋白的交联，不适用于半抗原免疫分析中的抗原合成。通过用TAAc-BSA和TAAc-KLH免疫获得的抗血清的特异性通过分离可溶性半抗原-抗体复合物进行评估，并开发了一种放射免疫分析方案，其检测限为200 pg（0.46 pmol）TAAc/ml样品。
