Modulation of nonspecific binding in ultrafiltration protein binding studies
Modulation of nonspecific binding in ultrafiltration protein binding studies
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DOI：
10.1023/a:1024406221962
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发表时间：
2003-07-01
影响因子：
3.7
通讯作者：
Lee, YH
中科院分区：
文献类型：
--
作者：
Lee, KJ;Mower, R;Lee, YH
目的.本研究的目的是减少或防止化合物与超滤（UF）蛋白结合（PB）测试单元的非特异性结合（NSB）。使用UF装置（再生纤维素，MWCO 10 K）进行PB和NSB测量，并在滤膜上使用或不使用5%吐温80（TW 80）或5%苯扎氯铵（巴克）进行预处理。在UF测试装置中孵育1小时后，将人血清和pH 7.4磷酸盐缓冲盐水中的给药溶液（10 μ M）在3，000 g和室温下离心。平行地，使用96孔平衡透析器在平衡透析（艾德）中在37 ℃下进行4小时的PB和NSB测量。采用LC/MS或LSC对UF和艾德样品进行分析。在UF中观察到依托泊苷、氢化可的松、普萘洛尔和长春碱的重度NSB。与此相反，TW 80或巴克预处理过滤膜上的NSB从87-95%降低到13-64%，而不会引起膜完整性的显着变化。当NSB低于50%作为预处理剂的结果时，标记化合物的PB数据与ED的PB数据相当。用TW 80或巴克预处理的膜对于具有高膜结合倾向的化合物显示出显著较少的NSB。一种改进的UF方法与预处理提高了UF的性能，并能够产生可比的PB结果ED。
Purpose. The aim of this study was to reduce or prevent nonspecific binding (NSB) of compounds to ultrafiltration (UF) protein binding (PB) testing units.Methods. UF units (regenerated cellulose, MWCO 10K) were used for PB and NSB measurements with or without pretreatment with 5% tween 80 (TW 80) or 5% benzalkonium chloride (BAK) on the filter membrane. Dosing solutions (10 muM) in human serum and pH 7.4 phosphate-buffered saline were centrifuged at 3,000 g and room temperature after 1-h incubation in UF testing units. In parallel, a 96-well equilibrium dialyzer was used for PB and NSB measurements in equilibrium dialysis (ED) at 37degreesC for 4 h. Samples of UF and ED were analyzed by LC/MS or LSC.Results. Severe NSB was observed for etoposide, hydrocortisone, propranolol, and vinblastine in UF. In contrast, TW 80 or BAK pretreatment on the filter membrane decreased the NSB from 87-95% to 13-64% without causing a significant change in membrane integrity. When NSB was below 50% as a result of pretreating agents, PB data of marker compounds were comparable to those of ED.Conclusions. The pretreated membrane with TW 80 or BAK showed significantly less NSB for compounds that had a tendency toward high membrane binding. A modified UF method with pretreatment improved the performance of UF and was able to produce comparable PB results to ED.