Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.
GSK, Via Fiorentina 1, 53100 Siena, Italy.
Anal Chem. 2021 Aug 31;93(34):11669-11678. doi: 10.1021/acs.analchem.1c00696. Epub 2021 Jul 24.
Epitope mapping of antibodies (Abs) is crucial for understanding adaptive immunity, as well as studying the mode of action of therapeutic antibodies and vaccines. Especially insights into the binding of the entire polyclonal antibody population (pAb) raised upon vaccination would be of unique value to vaccine development. However, very few methods for epitope mapping can tolerate the complexity of a pAb sample. Here we show how hydrogen-deuterium exchange mass spectrometry (HDX-MS) can be used to map epitopes recognized by pAb samples. Our approach involves measuring the HDX of the antigen in absence or presence of varied amounts of pAbs, as well as dissociating additives. We apply the HDX-MS workflow to pAbs isolated from rabbit immunized with factor H-binding protein (fHbp), a vaccine antigen. We identify four immunogenic regions located on the N- and C-terminal region of fHbp and provide insights into the relative abundance and avidity of epitope binding Abs present in the sample. Overall, our results show that HDX-MS can provide a unique and relatively fast method for revealing the binding impact of the entire set of pAbs present in blood samples after vaccination. Such information provides a rare view into effective immunity and can guide the design of improved vaccines against viruses or bacteria.
抗体（Abs）的表位作图对于理解适应性免疫至关重要，同时也有助于研究治疗性抗体和疫苗的作用模式。特别是深入了解疫苗接种后产生的整个多克隆抗体群体（pAb）的结合情况，对于疫苗开发将具有独特的价值。然而，能够耐受 pAb 样本复杂性的表位作图方法却寥寥无几。本文展示了如何利用氢氘交换质谱（HDX-MS）来绘制 pAb 样本所识别的表位图谱。我们的方法包括测量抗原在不存在或存在不同浓度的 pAb 以及解离添加剂时的 HDX。我们将 HDX-MS 工作流程应用于从用因子 H 结合蛋白（fHbp）免疫的兔子中分离的 pAb 上，fHbp 是一种疫苗抗原。我们鉴定了位于 fHbp 的 N 端和 C 端区域的四个免疫原性区域，并深入了解了样品中存在的结合表位 Abs 的相对丰度和亲和力。总体而言，我们的结果表明，HDX-MS 可以提供一种独特且相对快速的方法，用于揭示疫苗接种后血液样本中存在的整套 pAb 的结合影响。此类信息提供了对有效免疫的罕见观察，并可指导针对病毒或细菌的改良疫苗的设计。
