Steiner G, Küllinger B, Mayer G, Jie Y, Leibl H, Kovarik J, Woloszczuk W
Second Medizinische Universitätsklinik, University of Vienna, Austria.
Clin Chem. 1991 Mar;37(3):403-10.
A competitive protein-binding assay for cyclosporine based on use of the intracellular cyclosporine-binding protein cyclophilin (CYP) was used to measure cyclosporin A (CsA) and its bioactive metabolites in whole blood. CYP from cytoplasmic extracts or erythrocyte lysates was applied in the binding assay with use of [3H]CsA as tracer and charcoal adsorption for separating bound from free tracer. Binding affinities of various CsA analogs and metabolites correlated well with their reported in vitro immunosuppressive activities. The assay detected as little CsA as 50 micrograms/L (1 g = 0.832 mmol of CsA), analytical recovery was greater than 80%, and CVs were less than 8% for intra-assay and less than 11% for interassay precision in the range of 150-1000 micrograms/L. We used this assay to measure CsA concentrations in blood and compared the results with those measured by HPLC or by CsA-specific (monoclonal) and CsA-nonspecific (polyclonal) radioimmunoassays. Binding assay results were, in nearly all cases, less than those measured by the nonspecific RIA and frequently greater than 20% above the values determined by the CsA-specific assays. Individual patients had pronounced differences in the relative proportions of CsA, CYP-binding (bioactive) metabolites, and cross-reacting CsA metabolites. Because the presence of bioactive metabolites may considerably contribute to the immunosuppressive activity of CsA, we consider the binding assay clinically useful for measuring CsA in biological fluids.
一种基于细胞内环孢素结合蛋白亲环蛋白（CYP）的环孢素竞争性蛋白结合测定法，用于测量全血中环孢素A（CsA）及其生物活性代谢产物。细胞质提取物或红细胞裂解物中的CYP用于结合测定，使用[3H]CsA作为示踪剂，并通过活性炭吸附分离结合型和游离型示踪剂。各种CsA类似物和代谢产物的结合亲和力与其报道的体外免疫抑制活性密切相关。该测定法可检测低至50微克/升的CsA（1克 = 0.832毫摩尔CsA），分析回收率大于80%，在150 - 1000微克/升范围内，批内变异系数小于8%，批间精密度小于11%。我们使用该测定法测量血液中的CsA浓度，并将结果与通过高效液相色谱法（HPLC）或CsA特异性（单克隆）和CsA非特异性（多克隆）放射免疫测定法测得的结果进行比较。在几乎所有情况下，结合测定结果均低于非特异性放射免疫测定法测得的结果，且常常比CsA特异性测定法确定的值高出20%以上。个体患者在CsA、CYP结合（生物活性）代谢产物和交叉反应性CsA代谢产物的相对比例上存在显著差异。由于生物活性代谢产物的存在可能对CsA的免疫抑制活性有很大贡献，我们认为该结合测定法在临床上可用于测量生物体液中的CsA。
