Development of an antigen microarray for high throughput monoclonal antibody selection.
Development of an antigen microarray for high throughput monoclonal antibody selection.
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DOI：
10.1016/j.bbrc.2013.12.033
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发表时间：
2014-03-21
影响因子：
3.1
通讯作者：
Wright, Gavin J.
中科院分区：
文献类型：
--
作者：
Staudt, Nicole;Mueller-Sienerth, Nicole;Wright, Gavin J.
用于高通量单克隆抗体筛选的抗原阵列的开发。在筛查的早期阶段识别交叉反应抗体。鉴定功能性重组抗体的简便筛选方法。单克隆抗体是有价值的实验室试剂，并且越来越多地被用作治疗一系列疾病的治疗剂。尽管有几种不同的技术可用，但选择经验证在特定应用中有效的新单克隆抗体可能是资源密集型的，结果不确定。为了解决这个问题，我们开发了一种方法，该方法能够早期筛选用多达五种不同抗原免疫的动物产生的杂交瘤上清液，然后将抗体克隆到单个表达质粒中。虽然这种方法缓解了细胞克隆瓶颈，并具有在克隆前筛选抗体功能的理想能力，但可用于筛选的小体积杂交瘤上清液限制了用于合并免疫的抗原数量。在这里，我们报告的抗原微阵列，显着减少功能筛选所需的上清液的体积的发展。这种方法允许从单个动物中进行平行单克隆抗体选择的抗原数量显著增加。最后，我们展示了一种方便的小规模转染方法的成功使用，以快速识别编码功能性克隆抗体的质粒，解决了这种方法中的另一个瓶颈。总之，我们表明，建立杂交瘤抗体技术与精细筛选和抗体克隆方法相结合的混合方法可用于选择所需的功能特性的单克隆抗体对许多不同的抗原从一个单一的免疫宿主。
Development of an antigen array for high throughput monoclonal antibody selection. Identification of cross-reactive antibodies at an early stage of screening. Convenient screening method to identify functional recombinant antibodies. Monoclonal antibodies are valuable laboratory reagents and are increasingly being exploited as therapeutics to treat a range of diseases. Selecting new monoclonal antibodies that are validated to work in particular applications, despite the availability of several different techniques, can be resource intensive with uncertain outcomes. To address this, we have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five different antigens followed by cloning of the antibody into a single expression plasmid. While this approach relieved the cellular cloning bottleneck and had the desirable ability to screen antibody function prior to cloning, the small volume of hybridoma supernatant available for screening limited the number of antigens for pooled immunisation. Here, we report the development of an antigen microarray that significantly reduces the volume of supernatant required for functional screening. This approach permits a significant increase in the number of antigens for parallel monoclonal antibody selection from a single animal. Finally, we show the successful use of a convenient small-scale transfection method to rapidly identify plasmids that encode functional cloned antibodies, addressing another bottleneck in this approach. In summary, we show that a hybrid approach of combining established hybridoma antibody technology with refined screening and antibody cloning methods can be used to select monoclonal antibodies of desired functional properties against many different antigens from a single immunised host.
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影响因子：
2.9
作者：
Sun Y;Gallagher-Jones M;Barker C;Wright GJ
通讯作者：
Wright GJ
影响因子：
3.5
作者：
Mizuno, T;Kawasaki, M;Yoshihara, Y
通讯作者：
Yoshihara, Y
影响因子：
64.8
作者：
Wrammert, Jens;Smith, Kenneth;Wilson, Patrick C.
通讯作者：
Wilson, Patrick C.
DOI：
10.3791/3881
发表时间：
2012-03-05
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Kerr JS;Wright GJ
通讯作者：
Wright GJ
影响因子：
46.9
作者：
Love, J. Christopher;Ronan, Jehnna L.;Ploegh, Hidde L.
通讯作者：
Ploegh, Hidde L.