Optimal construction of non-immune scFv phage display libraries from mouse bone marrow and spleen established to select specific scFvs efficiently binding to antigen
Optimal construction of non-immune scFv phage display libraries from mouse bone marrow and spleen established to select specific scFvs efficiently binding to antigen
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DOI：
10.1016/j.bbrc.2004.08.131
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发表时间：
2004-10-15
影响因子：
3.1
通讯作者：
Tsutsumi, Y
中科院分区：
文献类型：
--
作者：
Okamoto, T;Mukai, Y;Tsutsumi, Y
单克隆抗体广泛应用于基础研究、医学和制药工业。近年来，随着基因工程和抗体工程的发展，单克隆抗体的大量生产成为可能，单克隆抗体的应用和代次研究越来越受到人们的关注。另一方面，近年来，已经开发了噬菌体展示系统用于高通量分离和产生与各种抗原具有高亲和力的新型单克隆抗体。该技术能够构建含有数十亿个展示各种抗体片段的噬菌体库，并从该噬菌体库中快速筛选出特异性单克隆抗体。此外，该技术具有很大的优势，即可以在不免疫动物的情况下产生MAb。然而，仍然有相对较少的报道证实，有用的单克隆抗体可以从非免疫抗体库中获得。后者，如目前的方法所采用的，似乎不能达到生产任何所需抗原的有用MAb所需的高质量，因为从非免疫供体克隆抗体基因是低效的。这一问题是由于它们的RT-PCR引物组、PCR条件和通过构建抗体基因文库进行亚克隆的效率不能涵盖所有抗体多样性的事实造成的。在试图克服这些早期的问题，在这里，我们描述了一种优化的方法，建立一个高质量的，非免疫库从小鼠骨髓和脾脏，并评估其多样性的内容，多种抗体的广泛的抗原库。作为该方法的应用的一个例子，我们描述了特异性单克隆抗体结合荧光素酶的选择，并确定至少18个不同的克隆。使用该非免疫小鼠抗体库，我们还获得了针对VEGF、VEGF受体2、TNF-α和假单胞菌外毒素的单克隆抗体。证实了文库的高质量及其对该应用的适用性。(C)2004年爱思唯尔公司All rights reserved.
Monoclonal antibodies (MAbs) are widely applied in basic research, medicine, and the pharmaceutical industry. Recently, applications and generations of MAbs have been increasingly attracting attention in many research areas since MAbs could be produced in large quantities with the development of genetic technology and antibody engineering. On the other hand, in recent years, phage display system has been developed for high-throughput isolation and generation of novel MAbs that have high affinity with various antigens. This technology is capable of constructing "Library" containing billions of phage repertoires displaying various antibody fragments, and rapid selection of a specific MAb from this phage library. Additionally, this technology has a great advantage that MAbs can be generated without immunization to animals. However, there are still relatively few reports confirming that useful MAbs can be derived from non-immune antibody libraries. The latter, as undertaken by current methods, seem unable to achieve the high quality required to produce useful MAbs for any desired antigen because cloning of antibody gene from non-immune donors is inefficient. This problem is caused by the fact that their RT-PCR primer sets, PCR conditions, and efficiency of subcloning through construction of antibody gene library cannot encompass all the antibody diversity. In an attempt to overcome some of these earlier problems, here we describe an optimized method to establish a high quality, non-immune library from mouse bone-marrow and spleen, and assess its diversity in terms of content of multiple antibodies for a wide antigenic repertoire. As an example of the application of the methodology, we describe the selection of specific MAbs binding to Luciferase and identify at least 18 different clones. Using this non-immune mouse antibody library, we also obtained MAbs for VEGF, VEGF receptor 2, TNF-alpha, and Pseudomonas Exotoxin. confirming the high quality of the library and its Suitability for this application. (C) 2004 Elsevier Inc. All rights reserved.