MacFarlane G, Scheller D, Ersfeld D, Jensen T, Jevans A, Wong P Y, Kobayashi M
INCSTAR Corporation, Stillwater, Minnesota, USA.
Ther Drug Monit. 1996 Dec;18(6):698-705. doi: 10.1097/00007691-199612000-00012.
Tacrolimus (FK506) is a macrolide antibiotic with potent immunosuppressive properties. FK506 is 10- to 100-fold more potent than cyclosporin A in preventing organ rejection and in toxicity. Extreme inter- and intrapatient variability and lack of correlation between drug dosage and drug blood levels necessitate consistent and reliable therapeutic drug monitoring. Previous methods for monitoring drug levels have been lengthy and labor intensive and have required organic solvents for sample extraction. We have developed a manual enzyme-linked immunosorbent assay (ProTrac II ELISA) that employs standardized reagents and a nonorganic solvent extraction and yields good assay sensitivity and precision. The calculated mean sensitivity of the assay was 0.18 ng/ml. Interassay precision ranged from 6.3% to 13.1% (coefficient of variation) for FK506 in whole blood (concentration range, 1.0-25.0 ng/ml). Recovery of the drug from spiked EDTA whole blood was 91-98% over the same concentration range. Mean recovery of the drug from clinical samples spiked with kit standards was 99.5%. Dilutions of high-concentration spiked EDTA whole blood samples and high-concentration samples from patients exhibited linearity of observed versus expected values (y = 0.86x - 1.15; r = 0.99). Comparison of ProTrac II with the INCSTAR ProTrac FK506 ELISA by paired Student's t test showed no statistical difference between the methods (p = 0.46). Comparison of ProTrac II ELISA to the IMx microparticle enzyme immunoassay (MEIA) by linear regression resulted in a line with a slope of 1.22 (r = 0.91). Analysis by t test resulted in a p value < 0.001, indicating that the absolute values obtained in these assays are significantly different. The mean (+/-SD) difference in the absolute values was 4.2 +/- 2.6 ng/ml, with the MEIA values consistently higher. These results indicate that ProTrac II is a sensitive, precise ELISA for the determination of tacrolimus in whole blood; it can be performed in < 4 h.
他克莫司（FK506）是一种具有强效免疫抑制特性的大环内酯类抗生素。在预防器官排斥和毒性方面，FK506的效力比环孢素A强10至100倍。患者之间以及患者自身的药物差异极大，且药物剂量与血药浓度之间缺乏相关性，因此需要进行持续且可靠的治疗药物监测。以往监测药物水平的方法耗时且费力，还需要有机溶剂进行样品提取。我们开发了一种手动酶联免疫吸附测定法（ProTrac II ELISA），该方法采用标准化试剂和非有机溶剂提取，具有良好的测定灵敏度和精密度。该测定法计算得出的平均灵敏度为0.18 ng/ml。全血中FK506的批间精密度范围为6.3%至13.1%（变异系数）（浓度范围为1.0 - 25.0 ng/ml）。在相同浓度范围内，加标EDTA全血中药物的回收率为91% - 98%。用试剂盒标准品加标的临床样品中药物的平均回收率为99.5%。高浓度加标EDTA全血样品和患者高浓度样品的稀释液显示，观察值与预期值呈线性关系（y = 0.86x - 1.15；r = 0.99）。通过配对学生t检验比较ProTrac II与INCSTAR ProTrac FK506 ELISA，结果显示两种方法之间无统计学差异（p = 0.46）。通过线性回归比较ProTrac II ELISA与IMx微粒酶免疫测定法（MEIA），得到一条斜率为1.22的直线（r = 0.91）。t检验分析得出p值< 0.001，表明这些测定中获得的绝对值存在显著差异。绝对值的平均（±标准差）差异为4.2 ± 2.6 ng/ml，MEIA值始终较高。这些结果表明，ProTrac II是一种用于测定全血中他克莫司的灵敏、精确的ELISA方法；该方法可在4小时内完成。
