人血清白蛋白（HSA）是人血浆中酸性药物的最重要载体之一，并且已表现出可逆地结合大量不同的化合物。已为HSA确定了几个不同的配体结合位点。其中，Site II被确定为主要的药物结合位点之一。Amplite™人血清白蛋白（HSA）Site II结合测定试剂盒是一种基于荧光的高通量测定法，用于确定小分子与HSA的结合。该测定法基于新型荧光探针HSA Blue™S2。它的特征是具有独特的光谱和结合特性，可结合至HSA的位点2。HSA Blue™S2在蛋白质结合状态和蛋白质未结合状态之间显示出较大的荧光强度差异。在HSA Blue™S2存在下，小分子竞争结合HSA导致荧光强度低。该测定法可用作高通量筛选工具，以确定在II位点与HSA的总结合。

样品分析方案

概述

向孔中添加HSA工作溶液（50 μL）和HSA Blue™S2工作溶液（50 μL）

向各个孔中添加各种浓度的测试药物（50 μL）

在室温下孵育15至45分钟

使用荧光酶标仪在Ex / Em = 365/480 nm（截止= 435 nm）下测量响应 

储备溶液配制

工作溶液配制

操作步骤

以下协议可用作指导，应根据需要进行优化。

1. 在孔中加入50 μL HSA Blue™S2工作溶液。
2. 在孔中加入50 μL HSA工作溶液。
3. 在各自的孔中加入50 μL药物工作溶液。（总体积= 150 μL /孔）。
4. 将样品在室温下孵育30分钟。
5. 使用荧光酶标仪在Ex / Em = 365/480 nm（截止= 435 nm）处检测荧光的增加。 

图示

图1.使用Amplite™人血清白蛋白（HSA）Site II结合测定试剂盒测量了华法林（Site-1药物）和布洛芬（Site-2药物）的反应。使用Spectra Max Gemini XS（分子设备）以Ex / Em = 365/480 nm和截止= 435 nm检测荧光。

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