Sulfhydryl Site-Specific Cross-Linking and Labeling of Monoclonal Antibodies by a Fluorescent Equilibrium Transfer Alkylation Cross-Link Reagent
Sulfhydryl Site-Specific Cross-Linking and Labeling of Monoclonal Antibodies by a Fluorescent Equilibrium Transfer Alkylation Cross-Link Reagent
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DOI：
10.1021/bc00001a006
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发表时间：
1990-01-01
影响因子：
4.7
通讯作者：
Smith, Richard H.
中科院分区：
文献类型：
--
作者：
del Rosario, Renato B.;Wahl, Richard L.;Smith, Richard H.
描述了通过一类新的“平衡转移烷基化交联（ETAC）试剂”实现单克隆抗体巯基的位点特异性分子内交联。在用二硫苏糖醇（DTT）完全或部分还原链间二硫化物后，使两种鼠IgG 2a单克隆抗体225.28 S和5G 6.4与α，α-双[（对甲苯磺酰基）甲基]-间氨基苯乙酮（ETAC 1a）和荧光缀合衍生物磺酰罗丹明B m-（α，α-双（对甲苯磺酰基甲基）乙酰基）苯胺衍生物（ETAC 1B）反应。1b产物的还原SDS-聚丙烯酰胺凝胶电泳分析表明，形成了S-ETAC-S链间重链和轻链交联（通过摄像机光密度测定法，总产率为23-34%），其在100 ℃下不与DTT进行二硫键-硫醇交换。相比之下，在未还原或还原的抗体反应时没有观察到链间交联，其中硫醇已经预先用碘乙酰胺烷基化。这些结果表明链间巯基的位点特异性交联，并且将它们的距离置于3-4埃内。使用77个IP 3人卵巢癌靶细胞的ETAC 1b 5G6.4交联产物的流式细胞术显示阳性结合和免疫反应性保留。I-131标记的完整5G6.4和I-125标记的还原5G6.4 + ETAC Ia产物在大鼠中的体内生物分布在24小时内基本相同。本研究说明了可标记ETAC试剂作为硫醇特异性探针用于各种免疫学研究的潜在应用。
The site-specific intramolecular cross-linking of sulfhydryls of monoclonal antibodies via a new class of "equilibrium transfer alkylation cross-link (ETAC) reagents" is described. Following complete or partial reduction of interchain disulfides with dithiothreitol (DTT), two murine IgG2a monoclonal antibodies, 225.28S and 5G6.4, were reacted with alpha,alpha-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC 1a) and a fluorescent conjugated derivative, sulforhodamine B m-(alpha,alpha-bis(p-tolylsulfonylmethyl)acetyl)anilide derivative (ETAC 1b). Reducing SDS-polyacrylamide gel electrophoresis analysis of the products from 1b indicated the formation of S-ETAC-S interchain heavy and light chain cross-links (similar to 23-34% overall yield by video-camera densitometry) which do not undergo disulfide-thiol exchange with DTT at 100 degrees C. In contrast, no interchain cross-links were observed upon reaction of unreduced or reduced antibody wherein the thiols have been previously alkylated with iodoacetamide. These results indicated site-specific cross-linking of interchain sulfhydryls and places their distance within 3-4 angstrom. Flow cytometry of the ETAC 1b 5G6.4 cross-linked product using 77 IP3 human ovarian carcinoma target cells showed positive binding and retention of immunoreactivity. The in vivo biodistributions of I-131-labeled intact 5G6.4 and I-125-labeled reduced 5G6.4 + ETAC la product in rats were essentially identical over a period of 24 h. The present study illustrates the potential applications of labelable ETAC reagents as thiol-specific probes for a wide variety of immunological studies.