Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response
Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response
复制标题
DOI：
10.1073/pnas.1213737110
复制
发表时间：
2013-02-19
影响因子：
11.1
通讯作者：
Georgiou, George
中科院分区：
文献类型：
--
作者：
Wine, Yariv;Boutz, Daniel R.;Georgiou, George
我们已经开发并验证了一种方法学，用于确定免疫后多克隆血清反应的抗体组成。胃酶消化的血清免疫球蛋白经标准抗原亲和层析后，用自下而上的高效液相色谱-高分辨串联质谱仪分析洗脱、洗脱和流过组分。对单个单抗的鉴定需要建立一个由成熟B细胞的NextGen测序构建的免疫球蛋白可变区基因(V基因)序列数据库。抗体V基因序列具有高度多样性的短互补决定区(CDR)，与数千个IGG共享的骨架区域相邻，极大地增加了从蛋白质组观察到的多肽中识别抗原特异性IGG的难度。通过定位标记唯一的V-H CDRH3序列的多肽，我们鉴定了一组在亲和层析洗脱中大量富含的V-基因，构成了血清多克隆反应。在一只兔的加强免疫后，我们发现抗原特异性的血清免疫反应是寡克隆的，包括编码34个不同CDRH3的抗体，这些抗体分成30个不同的抗体V-H克隆型。在这34个CDRH3中，有12个占抗原特异性CDRH3多肽质量谱计数的60%。作为比较，18个不同CDRH3(12个克隆型)的抗体出现在一只未免疫的兔的抗原特异性IgG组分中，该兔碰巧显示出中等效价的BSA。蛋白质组学鉴定的抗体被合成并显示出亚纳摩尔亲和力。了解多克隆血清反应的能力对于分析疫苗接种后的抗体反应以及更全面地了解健康和疾病中的适应性免疫反应可能是至关重要的。
We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V-H CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigenspecific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V-H clonotypes. Of these 34 CDRH3s, 12 account for similar to 60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.