An immuno-precipitation assay for determining specific interactions between antibodies and phage selected from random peptide expression libraries
An immuno-precipitation assay for determining specific interactions between antibodies and phage selected from random peptide expression libraries
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DOI：
10.1016/s0022-1759(02)00080-7
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发表时间：
2002-06-01
影响因子：
2.2
通讯作者：
Todd, I
中科院分区：
文献类型：
--
作者：
Al-bukhari, TAMA;Tighe, P;Todd, I
可以筛选噬菌体显示的随机肽库以选择表达特异性结合抗体的噬菌体表达肽，从而使噬菌体表达的肽序列基序有助于定义抗体的表位，通常需要筛选抗体抗体噬菌体噬菌体噬菌体噬菌体噬菌体噬菌体为了结合免疫测定中选择抗体的结合，以验证相互作用的特异性。酶联免疫吸附测定（ELISA）通常用于此目的。但是，对于许多抗体，测量特定高亲和力相互作用的最佳技术是免疫沉淀测定。因此，研究了免疫沉淀作为测量从随机肽显示库中选择的抗体和噬菌体克隆之间相互作用的一种手段。对谷氨酸脱羧酶特异的三种小鼠单克隆抗体被用于在T7噬菌体上选择肽作为9-mers，在M 13噬菌体的PIII上进行了12-mers，或在M13噬菌体的PVIII上约束15-MERS。在选定的噬菌体的克隆和测序之后，将抗体和噬菌体的混合物在溶液中孵育，并将免疫复合物与蛋白质G一起沉淀在与塞膦糖珠的结合。为了检测和定量形成免疫复合物并被沉淀的噬菌体，通过通过沉淀的噬菌体诱导大肠杆菌感染噬菌体的生物学特性。目的是通过确定产生的斑块数量来量化沉淀的噬菌体，因此与溶液中噬菌体和抗体之间的相互作用程度成正比，此处介绍的结果表明，这种测量单克隆抗体与噬菌体相互作用的方法选择用于通过单克隆抗体识别的肽的表达是高度特异性和敏感的。 （c）2002 Elsevier Science B.V.保留所有权利。
Libraries of random peptides displayed by bacteriophage can be screened to select phage expressing peptides that specifically bind antibodies, so that the peptide sequence motifs expressed by the phage can help to define the epitopes of the antibodies, It is often desirable to screen antibody-selected phage for binding of the selecting antibody in an immunoassay in order to verify the specificity of the interaction. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for this purpose. However, for many antibodies, the best techniques for measuring specific, high affinity interactions are immunoprecipitation assays. Immuno-precipitation was therefore investigated as a means of measuring interactions between antibodies and phage clones selected from random peptide display libraries. Three mouse monoclonal antibodies specific for glutamic acid decarboxylase were used to select peptides as 9-mers on T7 phage, linear 12-mers on pIII of M 13 phage, or constrained 15-mers on pVIII of M13 phage. Following the cloning and sequencing of selected phage, mixtures of antibody and phage were incubated in solution and the immune complexes were precipitated with Protein G bound to Sepharose beads. In order to detect and quantitate the phage that had formed immune complexes and been precipitated, advantage was taken of the biological properties of the phage by inducing infection of Escherichia coli by the precipitated phage. The aim was to quantitate the phage precipitated by determining the number of plaques produced, which would therefore be proportional to the degree of interaction between the phage and the antibody in solution, The results presented here indicate that this method of measuring monoclonal antibody interactions with phage selected for expression of peptides recognised by the monoclonal antibody is highly specific and sensitive. (C) 2002 Elsevier Science B.V. All rights reserved.