Methods for comprehensive identification of membrane proteins recognized by a large number of monoclonal antibodies
Methods for comprehensive identification of membrane proteins recognized by a large number of monoclonal antibodies
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DOI：
10.1016/j.jim.2009.09.003
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发表时间：
2009-12-31
影响因子：
2.2
通讯作者：
Kurosawa, Yoshikazu
中科院分区：
文献类型：
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作者：
Kurosawa, Gene;Sumitomo, Mariko;Kurosawa, Yoshikazu
为了分离与肿瘤相关抗原(AGS)结合的单抗，我们开发了以下策略。利用噬菌体展示单抗文库，我们分离到了大量与人肿瘤细胞表面结合的单抗。通过免疫染色对单抗进行筛选，筛选出对恶性细胞有较强染色的克隆。此后，单抗识别的AGS被鉴定出来。为了用MS鉴定AGS，候选分子必须通过免疫沉淀或亲和层析进行纯化。我们分离了数百株呈现癌症特异性染色模式的单抗。为了在短时间内鉴定出被众多单抗识别的AGS，我们开发了两种方法。用流式细胞术(FCM)[克隆分组]方法，通过比较FCM的染色模式，可以对多种抗体进行分组。事实证明，在许多情况下，每组成员都与相同的分子结合。在发现一个候选抗原后，制备与其胞外部分相对应的多肽，并用于在所有单抗中按位置鉴定与该抗原结合的克隆(通过三维ELISA同时鉴定克隆)。这两种方法均可普遍适用于各种膜蛋白及其单抗。(C)2009爱思唯尔B.V.保留所有权利。
In order to isolate monoclonal antibodies (mAbs) that bind to tumor-associated antigens (Ags) we developed the following strategy. Using the phage-display Ab library we isolated a large number of mAbs that bind to the surface of human tumor cells. The mAbs were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. Thereafter, the Ags recognized by the mAbs were identified. For identification of the Ags by MS candidate molecules had to be purified either by immunoprecipitation or by affinity chromatography. We isolated several hundred mAbs that showed cancer-specific staining patterns. In order to identify the Ags that were recognized by the numerous mAbs within a short time we developed two methods. Using the GFC [grouping of clones by flow cytometry (FCM)] method many Abs could be grouped by comparing the staining patterns of FCM. Members in each group turned out to bind to the same molecule in many cases. After a candidate Ag was revealed, the polypeptide corresponding to its extracellular portion was prepared and used for identification of clones that bound to the Ag among all the mAbs by SITE (simultaneous identification of clones through three dimensional ELISA) method. Both methods can be generally applicable to various kinds of membrane proteins and the mAbs against them. (C) 2009 Elsevier B.V. All rights reserved.