应用荧光相关光谱技术定量研究抗原-抗体相互作用
发布时间：
2023-05-06
分类：
美国雅培制药公司的Sergey Y. Tetin团队综述了荧光相关光谱（FCS）及其衍生技术定量分析抗原-抗体结合反应的应用研究。抗体分子在基础研究和医疗检测等领域中有着广泛应用。因此，可在溶液样品中定量、均相分析抗原-抗体相互作用的分析技术具重大学术和商业价值。传统生物学技术（比如ELISA）较难在微量（数个微升）样品中快速、灵敏、准确地表征抗原-抗体结合反应，包括该反应的平衡常数、动力学常数、抗原特异性结合位点、抗原-抗体结合模型等。而荧光自相关（FACS）和交相关（FCCS）光谱技术可在微量溶液中，无需抗原-抗体结合物分离（均相），定量分析抗原-抗体结合反应的多种特性。FACS技术可应用于测定抗原-抗体结合反应的平衡解离常数，以及确定抗原分子上的关键结合位点；FCCS技术与夹心法结合可测定抗原-抗体结合结合反应的动力学解离常数；矩分析技术（Moment analysis）可用于抗体的结合活性分析；时间积分荧光累计分析法（TIFCA）可建立抗原-抗体结合模型。综上，将FCS及其衍生技术应用于抗原-抗体相互作用研究具有灵敏度高、样品用量少、快速与均相检测等优点，对抗体药物研发具有重要意义。
Abstract
Antibodies are excellent binding proteins that have found numerous applications in biological research, biotechnology, and medicine. Characterization of their ligand binding properties has long been, and continues to be, the focus of many researchers. Antibodies are also perfect test systems which can be used for the evaluation of newly introduced biophysical techniques. Working with many different antibodies, we continuously implement the growing arsenal of methods offered by fluorescence fluctuation spectroscopy (FFS) and apply them for antibody research. In this chapter, we will describe applications of FFS for antibody binding characterization and also provide examples how studying of antibodies helps to develop and enhance the tool set offered by FFS technology. In addition to traditional affinity evaluations, we will describe how resolving molecular populations enables determinations of the binding stoichiometry and provides further information about the system. Even though all our examples include antibodies, the same experimental procedures can also serve well for characterizing various proteins and other ligand binding systems.
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