Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells.
Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells.
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DOI：
10.1016/j.bbagen.2015.03.009
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发表时间：
2015-07
期刊：
影响因子：
--
通讯作者：
González-Sapienza G
中科院分区：
文献类型：
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作者：
Rossotti M;Tabares S;Alfaya L;Leizagoyen C;Moron G;González-Sapienza G
重组骆驼单链抗体的可变区(纳米体)具有体积小、产量高、通用性强、稳定性好等优点，在研究、诊断和治疗方面具有重要的应用价值。虽然它们针对纯化抗原的制备是直接的，但针对多通道或复合膜细胞受体等困难靶点的纳米抗体的产生仍然具有挑战性。在这里，我们设计了一个基于生物素手柄的高通量识别纳米体细胞受体的平台。利用生物素受体多肽标签，优化了96孔培养块中纳米体的体内生物素化，允许用流式细胞仪和ELISA法并行分析，并直接用于下拉/MS靶标鉴定。从小鼠骨髓来源的树突状细胞免疫的骆驼的VHH文库中选择和鉴定了针对Mac-1(CD11b/CD18)、MHC II和小鼠Ly-5白细胞共同抗原(CD45)受体的纳米体板，证明了该策略的潜力。通过开启和关闭添加生物素，该方法还允许选择的NBS直接与细胞上的表位结合。这一策略将有效的纳米小体的选择简化为复杂的抗原，所选择的纳米小体构成了即用的生物素化试剂。这种方法将加速发现纳米体到细胞膜的受体，这些受体构成了最大的药物和分析靶点。
Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domain (nanobody) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct used for pull-down/MS target identification. The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. This strategy streamline the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.