Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays.
Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays.
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DOI：
10.1021/acs.analchem.5b03561
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发表时间：
2015-12-01
影响因子：
7.4
通讯作者：
González-Sapienza G
中科院分区：
文献类型：
--
作者：
Rossotti MA;Pirez M;Gonzalez-Techera A;Cui Y;Bever CS;Lee KS;Morisseau C;Leizagoyen C;Gee S;Hammock BD;González-Sapienza G
从骆驼抗体库中获得的单域重链结合物（纳米抗体）为免疫分析的发展带来了巨大的希望。然而，没有简单的方法可以从初步筛选后获得的阳性克隆群中选择最有价值的纳米抗体。在本文中，我们描述了一种基于纳米抗体的新型平台，该平台可以比较数百个克隆与标记抗原的反应性，并确定用于双位点免疫测定开发的最佳纳米抗体对。输出的克隆在 96 孔培养块中体内生物素化，然后用于使抗生物素蛋白包被的孔的生物素结合能力饱和。这标准化了捕获的抗体的量，允许通过对它们与标记抗原的反应性进行排序来对其进行分类。使用人可溶性环氧化物水解酶 (sEH) 作为模型抗原，我们能够对四个家族的 96 个克隆进行分类，并通过测序确认这种分类。这提供了选择一组由 5 种捕获抗体组成的限制性抗体的标准，并针对其余 96 个克隆来测试它们中的每一种。该方法构成了表位分箱的强大工具，在我们的案例中，可以开发用于 sEH 的夹心 ELISA，检测限为 63 pg/mL，动态范围为 4 个对数，在不同组织提取物中具有出色的回收率。该策略提供了一种测试纳米抗体成对组合的系统方法，并且对于开发高灵敏度夹心免疫测定具有广泛的用途。
Single domain heavy-chain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays.