Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE.
Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE.
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DOI：
10.1016/j.bbrep.2018.10.004
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发表时间：
2018-12
影响因子：
2.7
通讯作者：
Norman AB
中科院分区：
文献类型：
--
作者：
Kirley TL;Greis KD;Norman AB
单克隆抗体和衍生片段在实验和治疗上广泛使用。对这些抗体的彻底表征是必要的，包括对其热稳定性和储存稳定性的评估。因此，评估抗体的潜在构象稳定性也很重要。我们最近记录了非还原性SDS-PAGE可用于评估SDS中的单克隆和多克隆IgG结构域热解折叠。利用该相同的h2 E2抗可卡因mAb，在该研究中，我们产生并分析了各种mAb抗体片段，以描绘在各种加热方案和通过非还原SDS-PAGE分析后导致观察到的离散条带的抗体的结构域。先前，基于不存在SDS时这些CH 2、CH 3和Fab结构域的相对热稳定性（通过差示扫描量热法测量），假设这些结构域解折叠转变和凝胶带源自已知的mAb结构域。在本研究中，我们产生并分析了F（ab '）2、Fab和Fc片段，以及仅由重链组成的mAb，并通过非还原性SDS-PAGE检查了这些片段中的每一个中的热诱导结构域解折叠。对结果进行解释和整合，以产生SDS中mAb IgG的热去折叠的改进模型。这些结果和提出的模型应普遍适用于许多单克隆和多克隆抗体，并允许一个给定的抗体的化学或遗传修饰的版本之间的构象稳定性的新的比较。这种修饰的抗体和抗体药物缀合物是常用的，并且对于实验和治疗应用是重要的。mAb F（ab '）2片段在非还原性SDS-PAGE中表现出多个未折叠状态。Fab和Fc mAb片段不表现出类似的多个未折叠状态条带。基于片段分析，修订了SDS中先前的mAb结构域解折叠途径。检测到仅重链mAb变体，并显示多个未折叠状态。这些结果可能与许多单克隆抗体和多克隆抗体的分析相关。
Monoclonal antibodies and derived fragments are used extensively both experimentally and therapeutically. Thorough characterization of such antibodies is necessary and includes assessment of their thermal and storage stabilities. Thus, assessment of the underlying conformational stabilities of the antibodies is also important. We recently documented that non-reducing SDS-PAGE can be used to assess both monoclonal and polyclonal IgG domain thermal unfolding in SDS. Utilizing this same h2E2 anti-cocaine mAb, in this study we generated and analyzed various mAb antibody fragments to delineate the structural domains of the antibody responsible for the observed discrete bands following various heating protocols and analysis by non-reducing SDS-PAGE. Previously, these domain unfolding transitions and gel bands were hypothesized to stem from known mAb structural domains based on the relative thermal stability of those CH2, CH3, and Fab domains in the absence of SDS, as measured by differential scanning calorimetry. In this study, we generated and analyzed F(ab’)2, Fab, and Fc fragments, as well as a mAb consisting of only heavy chains, and examined the thermally induced domain unfolding in each of these fragments by non-reducing SDS-PAGE. The results were interpreted and integrated to generate an improved model of thermal unfolding for the mAb IgG in SDS. These results and the model presented should be generally applicable to many monoclonal and polyclonal antibodies and allow novel comparisons of conformational stabilities between chemically or genetically modified versions of a given antibody. Such modified antibodies and antibody drug conjugates are commonly utilized and important for experimental and therapeutic applications. mAb F(ab’)2 fragments exhibit multiple unfolded states in non-reducing SDS-PAGE. Fab and Fc mAb fragments do not exhibit similar multiple unfolded state bands. Previous mAb domain unfolding pathway in SDS is revised based on fragment analyses. A heavy chain only mAb variant is detected and exhibits multiple unfolded states. These results are likely relevant to analyses of many monoclonal and polyclonal Abs.
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影响因子：
4.8
作者：
Kirley TL;Norman AB
通讯作者：
Norman AB
DOI：
10.1016/j.bbrc.2007.02.042
发表时间：
2007-04-13
影响因子：
3.1
作者：
Garber, Ellen;Demarest, Stephen J.
通讯作者：
Demarest, Stephen J.
影响因子：
5.6
作者：
Demarest, SJ;Rogers, J;Hansen, G
通讯作者：
Hansen, G
影响因子：
--
作者：
Martell, Bridget A.;Orson, Frank M.;Poling, James;Mitchell, Ellen;Rossen, Roger D.;Gardner, Tracie;Kosten, Thomas R.
通讯作者：
Kosten, Thomas R.
影响因子：
7.3
作者：
Paula, S;Tabet, MR;Ball, WJ
通讯作者：
Ball, WJ