Amplite™人血清白蛋白（HSA）I位结合检测试剂盒

人血清白蛋白（HSA）是人血浆中酸性药物的重要载体之一，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。

 

参考文献

Anionic versus neutral Pt(II) complexes: The relevance of the charge for human serum albumin binding.
Authors: Ricciardi, Loredana and Guzzi, Rita and Rizzuti, Bruno and Ionescu, Andreea and Aiello, Iolinda and Ghedini, Mauro and La Deda, Massimo
Journal: Journal of inorganic biochemistry (2020): 111024

Human Serum Albumin Binding in a Vial: A Novel UV-pH Titration Method To Assist Drug Design.
Authors: Dargó, Gergő and Bajusz, Dávid and Simon, Kristóf and Müller, Judit and Balogh, György T
Journal: Journal of medicinal chemistry (2020): 1763-1774

An integrated quantitative structure and mechanism of action-activity relationship model of human serum albumin binding.
Authors: Serra, Angela and Önlü, Serli and Coretto, Pietro and Greco, Dario
Journal: Journal of cheminformatics (2019): 38

Anticancer activity, calf thymus DNA and human serum albumin binding properties of Farnesiferol C from Ferula pseudalliacea.
Authors: Tanzadehpanah, Hamid and Mahaki, Hanie and Samadi, Pouria and Karimi, Jamshid and Moghadam, Neda Hosseinpour and Salehzadeh, Sadegh and Dastan, Dara and Saidijam, Massoud
Journal: Journal of biomolecular structure & dynamics (2019): 2789-2800

Multi-Spectroscopic Characterization of Human Serum Albumin Binding with Cyclobenzaprine Hydrochloride: Insights from Biophysical and In Silico Approaches.
Authors: Baig, Mohammad Hassan and Rahman, Safikur and Rabbani, Gulam and Imran, Mohd and Ahmad, Khurshid and Choi, Inho
Journal: International journal of molecular sciences (2019)

Redox properties and human serum albumin binding of nitro-oleic acid.
Authors: Zatloukalova, Martina and Mojovic, Milos and Pavicevic, Aleksandra and Kabelac, Martin and Freeman, Bruce A and Pekarova, Michaela and Vacek, Jan
Journal: Redox biology (2019): 101213

The displacement study of 99m Tc-DTPA-Human serum albumin binding in presence of furosemide and metformin by using equilibrium dialysis and molecular docking.
Authors: Chemlal, Laila and Akachar, Jihane and Makram, Sanaa and Zoubir, Brahim and Cherrah, Yahia and Eljaoudi, Rachid and Ibrahimi, Azeddine and Faouzi, Mly A
Journal: IUBMB life (2019): 2003-2009

Alteration of human serum albumin binding properties induced by modifications: A review.
Authors: Maciążek-Jurczyk, Małgorzata and Szkudlarek, Agnieszka and Chudzik, Mariola and Pożycka, Jadwiga and Sułkowska, Anna
Journal: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy (2018): 675-683

Comparative studies on the human serum albumin binding of the clinically approved EGFR inhibitors gefitinib, erlotinib, afatinib, osimertinib and the investigational inhibitor KP2187.
Authors: Dömötör, Orsolya and Pelivan, Karla and Borics, Attila and Keppler, Bernhard K and Kowol, Christian R and Enyedy, Éva A
Journal: Journal of pharmaceutical and biomedical analysis (2018): 321-331

Human serum albumin binding of certain antimalarials.
Authors: Marković, Olivera S and Cvijetić, Ilija N and Zlatović, Mario V and Opsenica, Igor M and Konstantinović, Jelena M and Terzić Jovanović, Nataša V and Šolaja, Bogdan A and Verbić, Tatjana Ž
Journal: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy (2018): 128-139