Chen P, Tai H H
Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082, USA.
Res Commun Mol Pathol Pharmacol. 1995 Jun;88(3):317-26.
A simple and sensitive enzyme immunoassay for cyclosporin A has been developed. Cyclosporin A (CsA) antibodies were generated in rabbits using a novel hapten (CsA-C6) derived from ozonolysis of a double bond at residue 1 followed by reaction with 6-amino hexanoic acid. The antibodies generated from CsA-C6 recognized the spacer arm between the hapten and the carrier protein. A heterologous bridge strategy for haptenenzyme conjugate was used to improve the sensitivity. A hemisuccinyl ester of cyclosporin A alcohol (CsA-C4) was synthesized, conjugated to horseradish peroxidase and used as the enzyme labeled hapten. A high sensitivity enzyme immunoassay for CsA was developed. The concentration that gave 50% inhibition of binding was 1 ng/ml (8.3 x 10(-10)M) of CsA. The specificity of the antibodies was examined against various metabolites. Metabolites with structural modifications at distal residues 4, 8 and 9 showed the least crossreactivity, whereas metabolites derived from residue 1 exhibited full crossreaction as expected.
已开发出一种用于环孢素A的简单且灵敏的酶免疫测定法。使用一种新型半抗原（CsA-C6）在兔体内产生环孢素A（CsA）抗体，该半抗原由残基1处双键的臭氧分解产物与6-氨基己酸反应得到。由CsA-C6产生的抗体识别半抗原与载体蛋白之间的间隔臂。采用半抗原-酶缀合物的异源桥策略来提高灵敏度。合成了环孢素A醇的半琥珀酸酯（CsA-C4），将其与辣根过氧化物酶偶联并用作酶标记半抗原。开发出一种用于CsA的高灵敏度酶免疫测定法。产生50%结合抑制的CsA浓度为1 ng/ml（8.3×10⁻¹⁰M）。针对各种代谢物检测了抗体的特异性。在远端残基4、8和9处有结构修饰的代谢物显示出最低的交叉反应性，而源自残基1的代谢物如预期表现出完全交叉反应。
