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 I被确定为主要的药物结合位点之一。Amplite 人血清白蛋白（HSA）I位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™S1。它的特征是具有独特的光谱和结合特性，可与HSA的位点1结合。HSA Blue™S1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™S1存在下，小分子竞争HSA结合会导致荧光强度低。该测定法可用作高通量筛选工具，以确定在站点I处与HSA的总结合。

样品分析方案

概述

在微孔板孔中制备样品

从样品中取出液体

添加鬼笔环肽-iFluor 488标记溶液（100μL/孔）

在室温下染色细胞20至90分钟

清洗细胞在显微镜下观察样品

注意：将小瓶加热至室温并在打开前短暂离心。

储备溶液配制

将100 μL DMSO（组分D）加入HSA Blue™S1（组分A）中并充分混合。
注意：将未使用的HSA Blue™S1储备溶液分装成一小份保存在-20°C下，以避免冻融循环。

工作溶液配制

将50 μL HSA Blue™S1储备溶液加入5 mL HSA分析缓冲液（组分B）中，并充分混合。
注意：HSA Blue™S1工作溶液不应储存，应立即使用。
注意：5 mL HSA Blue™S1工作溶液足以用于一个96孔板。

将250 μL HSA溶液（组分C）加入5 mL HSA分析缓冲液（组分B）中并充分混合。
注意：5 mL HSA工作溶液足以用于一个96孔板。

使用HSA分析缓冲液（组分B）将药物原液在3X工作溶液中稀释至所需浓度。
注意：对于此处提到的方案，每孔的建议体积为50 μL。

操作步骤

以下方案可用作参考，实际应根据需要进行调整。

1. 在孔中加入50 μL HSA Blue™S1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）I位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Spectramax Gemini XS（分子设备）以Ex / Em = 365/480 nm和截止= 435 nm检测数据。

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