Rapid identification of monospecific monoclonal antibodies using a human proteome microarray.
Rapid identification of monospecific monoclonal antibodies using a human proteome microarray.
复制标题
DOI：
10.1074/mcp.o111.016253
复制
发表时间：
2012-06
期刊：
影响因子：
--
通讯作者：
Blackshaw S
中科院分区：
文献类型：
--
作者：
Jeong JS;Jiang L;Albino E;Marrero J;Rho HS;Hu J;Hu S;Vera C;Bayron-Poueymiroy D;Rivera-Pacheco ZA;Ramos L;Torres-Castro C;Qian J;Bonaventura J;Boeke JD;Yap WY;Pino I;Eichinger DJ;Zhu H;Blackshaw S
为了扩大可用于蛋白质组学研究的工具范围，我们建立了一个包含16,368个独特的全长人类ORF的文库，这些ORF可表达为N-末端GST-His6融合蛋白。在酵母中表达后，这些蛋白被单独纯化并用于构建人蛋白质组微阵列。为了证明这种试剂的有效性，我们开发了一种简化的策略来生产单特异性单抗，该策略使用活的人类细胞免疫和基于微阵列的抗体特异性分析作为其中心组件。我们证明了基于微阵列的抗体特异性分析可以使用二维池策略有效地执行。我们还证明，我们的免疫和选择策略产生了很大一部分免疫印迹和免疫沉淀级的单特异性单抗。我们的数据表明，该管道为产生具有特殊特异性的单抗提供了一个强大的平台。
To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His6 fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.