Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.
Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.
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DOI：
10.1016/j.bbrc.2016.10.128
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发表时间：
2016-11-25
影响因子：
3.1
通讯作者：
Norman AB
中科院分区：
文献类型：
--
作者：
Kirley TL;Greis KD;Norman AB
已经开发了许多方法用于化学标记和增强抗体及其共同片段（包括Fab和F（ab '）2片段）的性质。之前已经实现了一些抗体二硫键的某种选择性还原，产生了可以在指定位点标记的抗体和抗体片段，增强了它们的实用性和性质。使用温和还原选择性还原F（ab '）2片段中存在的两个铰链二硫键是有用的。然而，这种还原通常不是定量的，并且导致多个二硫键的还原，因此随后的多个标记或缀合位点既不是同质的也不是化学计量的。这里，利用固定在琼脂糖珠上的三（2-羧乙基）膦（TCEP）完成连接组成Fab片段的部分重链和完整轻链的单个二硫键的简单且有效的选择性还原。用几种半胱氨酸选择性荧光试剂以及通过半胱氨酸定向PEG化标记所得还原的半胱氨酸残基。这两个半胱氨酸残基也可以通过双功能半胱氨酸交联剂二溴异丙亚胺重新连接，从而在远离抗原结合位点的该位点处恢复重链和轻链之间的共价连接，并且还引入荧光探针。这些选择性部分还原的Fab片段还有许多其他的研究和临床用途，包括生物素化、毒素和药物缀合以及放射性同位素的掺入，并且该技术能够简单地产生具有许多潜在应用的非常有用的Fab片段衍生物。
Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’)2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’)2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications.