Developing high-quality mouse monoclonal antibodies for neuroscience research - approaches, perspectives and opportunities.
Developing high-quality mouse monoclonal antibodies for neuroscience research - approaches, perspectives and opportunities.
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开发用于神经科学研究的高质量小鼠单克隆抗体 - 方法，观点和机会。
DOI：
10.1016/j.nbt.2015.11.007
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发表时间：
2016-09-25
影响因子：
5.4
通讯作者：
Trimmer JS
中科院分区：
文献类型：
--
作者：
Gong B;Murray KD;Trimmer JS
高质量抗体（Abs）对神经科学研究至关重要，因为它们仍然是用于标记和捕获神经系统内源性表达蛋白靶点的主要亲和蛋白质组学试剂。正如在其他领域一样，神经科学家经常面临不准确和不可复制的基于抗体的结果和/或报告。加州大学戴维斯分校/美国国立卫生研究院神经单抗设施的创建，其使命是通过应用一种独特的方法来生成和验证针对哺乳动物大脑（即神经单抗）优化的小鼠单克隆抗体（mab），以解决神经科学研究中对高质量抗体的未满足需求。在这里，我们描述了我们的多步骤单抗筛选方法，重点是鉴定在标记哺乳动物脑样本中表现出功效和特异性的单抗。我们提供了来自NeuroMab筛选的示例，以及来自随后选定的NeuroMab的专门验证。我们强调了确定脑免疫标记特异性的特殊挑战和考虑因素。我们还描述了为什么我们强调通过免疫印迹和免疫组织化学对脑样本进行大量候选者的广泛验证对于确定那些在这些应用中表现出功效和特异性的候选药物成为神经单抗至关重要。我们描述了对不太常见的非igg1 IgG亚类的候选物的特别关注，这些候选物可以促进亚类特异性二抗同时多重标记。我们详细介绍了我们最近使用重组克隆神经单抗作为存档所有神经单抗的方法，在DNA序列水平上明确定义神经单抗，并将IgG1神经单抗重新设计为不太常见的IgG亚类，以促进其在多重标记中的使用。最后，我们提出了一些建议，以促进Ab的开发和使用，以及基于Ab的神经科学实验的设计、执行和解释。随着抗体验证的增强，已发表实验中使用的抗体的明确鉴定，以及最终用户对基于抗体的分析的熟练程度，神经科学研究的可重复性将得到改善。
High-quality antibodies (Abs) are critical to neuroscience research, as they remain the primary affinity proteomics reagent used to label and capture endogenously expressed protein targets in the nervous system. As in other fields, neuroscientists are frequently confronted with inaccurate and irreproducible Ab-based results and/or reporting. The UC Davis/NIH NeuroMab Facility was created with the mission of addressing the unmet need for high-quality Abs in neuroscience research by applying a unique approach to generate and validate mouse monoclonal antibodies (mAbs) optimized for use against mammalian brain (i.e., NeuroMabs). Here we describe our methodology of multi-step mAb screening focused on identifying mAbs exhibiting efficacy and specificity in labeling mammalian brain samples. We provide examples from NeuroMab screens, and from the subsequent specialized validation of those selected as NeuroMabs. We highlight the particular challenges and considerations of determining specificity for brain immunolabeling. We also describe why our emphasis on extensive validation of large numbers of candidates by immunoblotting and immunohistochemistry against brain samples is essential for identifying those that exhibit efficacy and specificity in those applications to become NeuroMabs. We describe the special attention given to candidates with less common non-IgG1 IgG subclasses that can facilitate simultaneous multiplex labeling with subclass-specific secondary antibodies. We detail our recent use of recombinant cloning of NeuroMabs as a method to archive all NeuroMabs, to unambiguously define NeuroMabs at the DNA sequence level, and to re-engineer IgG1 NeuroMabs to less common IgG subclasses to facilitate their use in multiplex labeling. Finally, we provide suggestions to facilitate Ab development and use, as to design, execution and interpretation of Ab-based neuroscience experiments. Reproducibility in neuroscience research will improve with enhanced Ab validation, unambiguous identification of Abs used in published experiments, and end user proficiency in Ab-based assays.