Multiplexed fluid array screening of phage displayed anti-ricin single domain antibodies for rapid assessment of specificity
Multiplexed fluid array screening of phage displayed anti-ricin single domain antibodies for rapid assessment of specificity
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DOI：
10.2144/000112600
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发表时间：
2007-12-01
期刊：
影响因子：
2.7
通讯作者：
Goldman, Ellen R.
中科院分区：
文献类型：
--
作者：
Anderson, George P.;Matney, Russell;Goldman, Ellen R.
噬菌体展示是一种众所周知的技术，其有助于选择与所需靶标结合的肽或蛋白质。使用该工具，可以选择包含在来源动物的天然免疫库中或来自合成产生的集合的结合元件。美洲驼的免疫球蛋白的仅重链类别的未配对可变结构域代表了创建噬菌体展示文库的遗传物质的理想来源。半合成的美洲驼文库的初始淘选仅产生一种毒素蓖麻毒素的结合剂。为了增加选择的单克隆噬菌体结合剂的数量，除了酶联免疫吸附测定（ELISA）之外，还使用Luminex xMAP技术（Luminex，Austin，TX，USA）在三轮选择后筛选噬菌体的克隆群体。xMAP技术检测在平衡条件下固定在微球表面的与蓖麻毒素结合的噬菌体展示单域抗体（sdAb）。这种增强的能力直接导致了另外的感兴趣的单结构域抗体的鉴定。表达、纯化选择的sdAb，然后评价其特异性以及与常规免疫球蛋白G（IgG）相比增强的热稳定性。我们确定了平衡解离常数，并证明了它们在夹心免疫测定中作为有效捕获分子的用途。
Phage display is a well-known technique that facilitates the selection of peptides or proteins that bind to a desired target. Using this tool, binding elements contained in the natural immune repertoires of the source animal or from a synthetically generated collection may be selected. The unpaired variable domain of the llama's heavy-chain-only classes of immunoglobulins represents an ideal source of genetic material to create phage display libraries. Initial panning of a semi-synthetic llama library yielded only one binder to the toxin ricin. In an effort to increase the number of monoclonal phage binders selected, the Luminex xMAP technology (Luminex, Austin, TX, USA) was used in addition to the enzyme-linked immunosorbent assay (ELISA) to screen clonal populations of phage after three rounds of selection. The xMAP technology detected phage displayed single domain antibody (sdAb) bound to ricin immobilized on the surface of microspheres under equilibrium conditions. This enhanced capability led directly, to the identification of additional single domain antibodies of interest. The selected sdAbs were expressed, purified, and then evaluated for their specificity as well as enhanced thermal stability in comparison to conventional immunoglobulin G (IgG). We determined equilibrium dissociation constants and demonstrated their use as effective capture molecules in sandwich immunoassays.