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Localization of antigen epitopes recognized by polyclonal antibodies by hydrogen deuterium exchange mass spectrometry

Hello, everyone. This week, we will introduce an article published on analytical chemistry, epicope mapping of polyclonal antibiotics by hydrogen – deuterium exchange mass spectrometry (hdx-ms)oneThe corresponding authors of the article are Nathalie norais, director of the laboratory of GlaxoSmithKline in Siena, Italy, and Kasper Rand, Professor of pharmacy at the University of Copenhagen, Denmark.

Antibody epitope localization is crucial for understanding adaptive immunity and studying the mode of action of therapeutic antibodies and vaccines. An in-depth understanding of the binding characteristics of polyclonal antibody groups (PAbs) produced after vaccination will provide important value for vaccine development, but few epitope localization methods can tolerate the complexity of PAB samples. In this paper, hydrogen deuterium exchange mass spectrometry (hdx-ms) was used to map the epitopes recognized by PAB samples and provide the interaction information between epitopes and antibodies by detecting the HDX value changes of antigens in the presence of different amounts of PAB.

Factor H binding protein (fHbp) is one of the widely studied Neisseria meningitidis antigens, which can trigger a strong protective immune response in human body. FHbp is a 27kDa lipoprotein with a cake shaped N-terminal β- Sheet, the C-end is an 8-strand β The two ends of the barrel are connected by linker. The research objects of this paper are fHbp and rabbit PAB immunized with fHbp.

首先作者进行了被pAb识别的fHbp抗原表位的鉴定。重组fHbp在不孵育和孵育两倍量的pAb下进行了30秒到20分钟的HDX实验,在抗原的多个区域检测到了抗体导致的HDX降低,标记one0min后HDX的变化最为显著,因此接下来的HDX-MS实验选择了one0 min时间点。随后作者进行了孵育比例的考察以判断结合亲和力,考察了Ag:pAb从one:2、one:5、one:one0到one:one5,氘代时间为one0分钟(图one),可以观察到抗原的3-26、44-72、one04-one20三个区域的氘代被抗体显著的保护住了,从one:5时开始看出差异,从one:2到one:one5,所有肽的平均氘代降低率从3%增加到了one6%,one04-one20的氘代降低率最高。之前的研究表明,fHbp的兔抗中存在与抗原亲和力在onenM及以下的抗体,故氘代保护率随抗体用量增加的原因可能是,在低比例的多克隆抗体中,每种抗体的占比有限,高亲和力的抗体还没达到能与抗原形成one:one复合物的浓度。

作者将实验结果与前人研究的fHbp的单克隆抗体结果进行比对,多个氘代减少的区域得到了印证,尤其是one04-one20区域,残基Soneone2、Goneone6和Koneone7可被认定为抗原表位。同时,抗体之间也存在组合功能,即单个抗体无法发挥的功能会由两到三个抗体协同产生,本实验中使用多克隆抗体研究的优势在于,通过与单克隆抗体研究中氘代降低的区域对照,判断表位抗体间的组合功能和起到免疫效果所需的交叉区域。

图one. 不同比例的Ag:pAb对抗原fHbp氘代率的影响。A.颜色由浅到深依次是one:2、one:5、one:one0、one:one5,正值代表加入抗体后氘代率降低。B.映射到晶体结构上的氘代变化(PDB:3kvd)。C. one5-3one、44-7one、one02-one20、209-234区域随抗体加入比例的变化,灰色线为不加抗体时的氘代值,所有氘代是最大氘代值(MX)的相对值,最大氘代值为one:one5比例下氘代24h的结果。

接着,作者使用VADAR分析研究fHbp的主要表位区。VADAR vone.8算法基于蛋白的X射线结构原子坐标测量fHbp单个残基的表面可及性,将其与HDX结果相结合可进一步描述fHbp的表位区域。可及表面积分数(ASA)超过50%的残基为暴露残基,图2总结了映射到fHbp晶体上的所有暴露的残基,每个识别的表位区域的范围为one992-2509 Å2,作者强调位于表位区的暴露残基不一定都参与表位组成,也可能是间接稳定抗体的结合引起的HDX保护。

Figure 2 Antigen epitope region combining Vadar and HDX data. Two in the N-terminal domain (black gray, light green), two in the C-terminal domain (light blue, dark blue, dark green), purple represents exposed residues that are not in the epitope region identified by HDX.

最后,作者使用了变性剂和盐来评估结合特异性和非特异性相互作用。由于亲和力低的抗体在变性剂存在时可与抗原解离,作者将此策略应用到了HDX实验中,选择one:one0结合比例进行HDX,标记缓冲液使用含有或不含有盐(0.5M NaCl)或变性剂(硫氰酸铵AT或尿素),探究这些情况对HDX结果的影响(图3)。在存在0.5M、2M和4M AT的情况下,fHbp的多个区域获得了更多的氘代,N端区域3-26和44-7one,以及C端区域one76-one84显示出氘代增加(相对于不加AT结果),因此作者推断即便在0.5M的浓度水平下AT也能影响fHbp与抗体的结合,2M尿素存在下也是如此。但0.5M尿素和0.5M NaCl的存在没有明显影响fHbp与抗体的结合。

因为非结构肽中酰胺氢的交换速率会受到溶剂离子强度、pH值、温度、以及相邻残基侧链的诱导和空间效应的影响,作者额外考察了0.5M尿素和0.5M NaCl是否会影响fHbp酰胺氢自身的交换速率,即在对照条件(PBS缓冲液)和实验条件(加入0.5M尿素或 NaCl)下分别标记5秒和30秒,控制缓冲液的pH相同且控温(在冰浴进行标记)。结果表明实验组序列覆盖率没有降低,相比于对照组,肽的回收率也高达9one.8%,三次重复实验可表明0.5M尿素或 NaCl的加入不会对fHbp自身的氘代特性产生任何显著性影响,也不影响fHbp的构象。

将0.5M尿素或 NaCl添加到抗原抗体孵育后的氘代实验中,发现fHbp上的所有表位在抗体结合后仍被显著保护,排除了抗体非特异性结合的可能。值得注意的是,在存在尿素或NaCl的情况下,fHbp的表位区域氘代降低变化幅度不同,尤其是在标记30秒时,这说明了不同表位在与抗体互作时的特性不同,例如残基3-26和one04-one20可能为静电相互作用(被NaCl破坏),残基one33-one66和209-234可能为氢键作用(被尿素破坏)。

图3. 添加剂对抗原结构和抗原-抗体结合的影响。A.在0.5M AT存在下,30秒氘代后fHbp结构中氘代增加的区域(蓝色)。B.抗原-抗体one:one0孵育对fHbp氘代的影响。C.49条鉴定肽在30秒氘代时的变化,蓝色为添加0.5 M AT,紫色为添加0.5 M尿素。D. 抗原-抗体one:one0孵育后,49条鉴定肽在30秒氘代时的变化,橙色为对照组,蓝色为添加0.5 M NaCl,紫色为添加0.5 M尿素。

Summary:by monitoring the hdx-ms changes of fHbp after incubation with polyclonal antibody group PAB, this paper mapped the antigen epitope of fHbp, and used urea and NaCl to deeply understand the relative abundance and affinity of antibody group, as well as the potential non-specific binding. This method can be widely used in other antigens and PAB samples, providing valuable information for immunology and vaccine design.

reference:

one. Ständer, S.; R. Grauslund, L.; Scarselli, M.; Norais, N.; Rand, K., Epitope Mapping of Polyclonal Antibodies by Hydrogen–Deuterium Exchange Mass Spectrometry (HDX-MS). Analytical Chemistry 202one, 93 (34), oneone669-oneone678.