Labeling of antibodies by in situ modification of thiol groups generated from selenol-catalyzed reduction of native disulfide bonds
Labeling of antibodies by in situ modification of thiol groups generated from selenol-catalyzed reduction of native disulfide bonds
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DOI：
10.1006/abio.2002.5624
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发表时间：
2002-05-15
影响因子：
2.9
通讯作者：
Maloney, EK
中科院分区：
文献类型：
--
作者：
Singh, R;Maloney, EK
描述了一种标记抗体的新方法，该方法涉及硒醇催化抗体中天然二硫键的还原以生成巯基，然后使用巯基反应试剂进行标记。这种快速程序的还原和标记步骤在一个容器中进行，在标记之前不需要任何分离步骤来去除还原剂。在不到5分钟的时间内，每个抗体分子可定量且均匀地结合约7个标记基团。所有使用的试剂都是市售的-硒半胺（催化剂前体），二硫代苏糖醇或三（2-羧基乙基）膦（还原剂）和巯基反应性标记试剂，如生物素-聚（环氧乙烷）-马来酰亚胺。该方法广泛适用于标记具有可还原二硫键的免疫球蛋白等蛋白质，其还原和标记不会导致活性的显着丧失。通过这种还原二硫标记方法制备的生物素化小鼠抗体（抗磷酸酪氨酸和抗egf受体）在酶联免疫吸附测定、免疫组织化学和免疫沉淀等应用中表现与氨基生物素化抗体相当或更好。这种还原二硫标记方法优于氨基标记方法，因为它不受溶液中胺的存在的抑制，正如杂交瘤培养上清中含有氨基酸和血清蛋白的抗体的生物素化所证明的那样。(C) 2002 Elsevier Science （USA）。
A new method for labeling antibodies which involves selenol-catalyzed reduction of native disulfide bonds in antibodies to generate thiol groups, which then are labeled using thiol-reactive reagents, is described. The reduction and labeling steps of this rapid procedure are carried out in one vessel, without requiring any separation step to remove the reductant before labeling. It results in a quantitative and homogenous incorporation of about seven labeled groups per antibody molecule in less than 5 min. All reagents used are commercially available-selenocystamine (catalyst precursor), dithiothreitol or tris(2-carboxyethyl)phosphine (reductant), and thiol-reactive labeling reagents such as biotin-poly(ethylene oxide)-maleimide. This method is broadly applicable for labeling proteins such as immunoglobulins with reducible disulfide bonds, whose reduction and labeling does not result in a significant loss of activity. Biotinylated murine antibodies (anti-phosphotyrosine and anti-EGF receptor) prepared by this reduced-disulfide labeling method perform comparably or better than amino-group biotinylated antibodies in applications such as enzyme-linked immunosorbent assay, immunohistochemistry, and immunoprecipitation. This reduced-disulfide labeling method is superior to amino-group labeling methods because it is not inhibited by the presence of amines in solution, as demonstrated by the biotinylation of an antibody in a hybridoma culture supernatant containing amino acids and serum proteins. (C) 2002 Elsevier Science (USA).