Department of Radiopharmacy, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
Nucl Med Biol. 2013 Oct;40(7):896-900. doi: 10.1016/j.nucmedbio.2013.06.010. Epub 2013 Jul 31.
Radioligand receptor binding assays are a common method to evaluate the affinity of newly synthesized benzodiazepine ligands for the receptor. [(3)H]-flumazenil is an antagonist of benzodiazepine receptors and is generally used as a radioligand. In this study, the binding parameters of [(3)H]-flumazenil to rat cortical membranes were evaluated using two separation methods: filtration with GF/C filters and centrifugation. Additionally, the effects of vacuum pressure, exposure time to the cocktail, and geometry on the filtration method were studied. The binding parameters of [(3)H]-flumazenil (Kd and Bmax) were determined through saturation studies using two methods. The results from this study showed that the filtration method is time consuming and requires more steps to be completed. Because filtration causes partial elution of bound [(3)H]-flumazenil into the liquid scintillation cocktail, the results are not reproducible, which result in inaccurate estimation of the binding parameters. The centrifugation method in contrast to filtration is straightforward and produces reproducible as well as reliable results, all of the steps are performed in a single polypropylene tube, which eliminates the loss of tissue and avoids other systematic errors associated with transfer and handling.
放射性配体受体结合分析是评估新合成苯二氮䓬类配体与受体亲和力的常用方法。[(3)H]-氟马西尼是苯二氮䓬受体的拮抗剂，通常用作放射性配体。在这项研究中，使用两种分离方法评估了[(3)H]-氟马西尼与大鼠皮质膜的结合参数：GF/C 过滤器过滤和离心。此外，还研究了真空压力、对鸡尾酒的暴露时间和几何形状对过滤方法的影响。通过两种方法的饱和研究确定了[(3)H]-氟马西尼（Kd 和 Bmax）的结合参数。本研究结果表明，过滤法耗时且需要更多步骤才能完成。由于过滤会导致部分结合的 [(3)H]-氟马西尼洗脱到液体闪烁鸡尾酒中，因此结果不可重复，从而导致结合参数的估计不准确。相比之下，离心法简单直接，产生可重复且可靠的结果，所有步骤都在单个聚丙烯管中进行，这消除了组织损失，并避免了与转移和处理相关的其他系统误差。
