Highly sensitive detection of antibody nonspecific interactions using flow cytometry.
Highly sensitive detection of antibody nonspecific interactions using flow cytometry.
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DOI：
10.1080/19420862.2021.1951426
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发表时间：
2021-01
期刊：
影响因子：
5.3
通讯作者：
Tessier PM
中科院分区：
文献类型：
--
作者：
Makowski EK;Wu L;Desai AA;Tessier PM
抗体药物开发的快速演变性质导致了在早期发现期间产生大量(数百至数千)领先抗体候选的技术。必须迅速削减这些候选药物，以确定最像药物的候选药物，以便深入分析它们的安全性和有效性，由于时间和资源要求，只能对有限数量的抗体进行这一分析。成功的抗体疗法的一个关键生物物理性质是高特异性，定义为低水平的非特异性结合或多特异性。虽然在开发抗体多特异性检测方法方面取得了一些进展，但这些检测方法大多受到灵敏度低或需要专有抗体表面展示方法的检测格式的限制，其中一些检测使用复杂且定义不明确的多特异性试剂。在这里，我们报告了多特异性颗粒(PSP)分析，这是一种灵敏的流式细胞术，用于评价抗体的非特异性相互作用，克服了以往的限制，可用于评价不同类型的IgG、多特异性抗体和Fc融合蛋白。我们的方法使用涂有蛋白A的微米大小的磁珠来捕获极稀浓度(0.02 mg/mL)的抗体。结合这些结合物的多特异性试剂的流式细胞术分析可以灵敏地检测临床期抗体的非特异性相互作用的差异。我们的PSP分析使用以前报道的多特异性试剂强烈区分具有不同水平多特异性的抗体，这些多特异性试剂要么是明确定义的蛋白质，要么是高度复杂的蛋白质混合物。此外，我们还发现，一种独特的试剂，即卵清蛋白，产生了最好的检测灵敏度和特异性。重要的是，我们的检测比ELISA等标准检测要敏感得多。我们期待我们的简单、灵敏和高通量的PSP检测将加速安全和有效的抗体疗法的发展。
The rapidly evolving nature of antibody drug development has resulted in technologies that generate vast numbers (hundreds to thousands) of lead antibody candidates during early discovery. These candidates must be rapidly pared down to identify the most drug-like candidates for in-depth analysis of their safety and efficacy, which can only be performed on a limited number of antibodies due to time and resource requirements. One key biophysical property of successful antibody therapeutics is high specificity, defined as low levels of nonspecific binding or polyspecificity. Although there has been some progress in developing assays for detecting antibody polyspecificity, most of these assays are limited by poor sensitivity or assay formats that require proprietary antibody surface display methods, and some of these assays use complex and poorly defined polyspecificity reagents. Here we report the PolySpecificity Particle (PSP) assay, a sensitive flow cytometry assay for evaluating antibody nonspecific interactions that overcomes previous limitations and can be used for evaluating diverse types of IgGs, multispecific antibodies and Fc-fusion proteins. Our approach uses micron-sized magnetic beads coated with Protein A to capture antibodies at extremely dilute concentrations (<0.02 mg/mL). Flow cytometry analysis of polyspecificity reagent binding to these conjugates results in sensitive detection of differences in nonspecific interactions for clinical-stage antibodies. Our PSP assay strongly discriminates between antibodies with different levels of polyspecificity using previously reported polyspecificity reagents that are either well-defined proteins or highly complex protein mixtures. Moreover, we also find that a unique reagent, namely ovalbumin, results in the best assay sensitivity and specificity. Importantly, our assay is much more sensitive than standard assays such as ELISAs. We expect that our simple, sensitive, and high-throughput PSP assay will accelerate the development of safe and effective antibody therapeutics.
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影响因子：
4.9
作者：
Shan, Lu;Mody, Neil;Esfandiary, Reza
通讯作者：
Esfandiary, Reza
影响因子：
--
作者：
Birtalan, Sara;Fisher, Robert D.;Sidhu, Sachdev S.
通讯作者：
Sidhu, Sachdev S.
影响因子：
5.3
作者：
Bailly, Marc;Mieczkowski, Carl;Fayadat-Dilman, Laurence
通讯作者：
Fayadat-Dilman, Laurence
影响因子：
5.6
作者：
Birtalan, Sara;Zhang, Yingnan;Sidhu, Sachdev S.
通讯作者：
Sidhu, Sachdev S.
DOI：
10.1007/978-1-4939-8648-4_8
发表时间：
2018-01-01
期刊：
ANTIBODY ENGINEERING, 3 EDITION
影响因子：
--
作者：
Krah, Simon;Grzeschik, Julius;Zielonka, Stefan
通讯作者：
Zielonka, Stefan