Chung Wen Yuan, Sack Markus, Carter Rachel, Spiegel Holger, Fischer Rainer, Hirst Timothy R, Williams Neil A, James Roger F L
Royal College of Surgeons, Department of Surgery, Connolly Hospital, Blanchardstown, D15, Ireland.
J Immunol Methods. 2008 Dec 31;339(2):115-23. doi: 10.1016/j.jim.2008.08.005. Epub 2008 Sep 9.
Previously we have described studies on in vitro pentamer assembly of Escherichia coli (E. coli) derived heat-labile enterotoxin B subunit (EtxB) using conventional monoclonal antibodies (Amin et al., JBC 1995: 270, 20143-50 and Chung et al., JBC 2006: 281, 39465-70). To extend these studies further we have used phage-display to select single-chain Fragment variable (scFv) antibodies against different forms of the B-subunit. Two clones exhibiting strong and differential binding were chosen for detailed characterization. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions and a nonsense mutation present in one of these (scFv-B1.3.9) was corrected. Binding analysis showed that scFv-B1.3.9 bound in ELISA to both heat-denatured monomeric B-subunits (EtxB1) and also displayed cross-reactivity towards pentameric EtxB (EtxB5), although there was no reactivity towards monoganglioside (GM1) captured EtxB5. Another antibody (scFv-B5.2.14) had a different reactivity profile and, in ELISA, bound only to EtxB5 but not to EtxB1 or to EtxB5 captured via GM1. Surprisingly, in competition experiments, the assembled pentameric B-subunit inhibited binding of scFv-B5.2.14 to immobilized EtxB5 only weakly, whereas reduced, but not oxidized, monomeric EtxB1 was an efficient competitor. These results clearly demonstrate that B1.3.9 and B5.2.14 have different specificities for cryptic epitopes not accessible in the fully assembled GM1 bound pentameric form of EtxB. Taken together our results show that we were able to successfully isolate and characterize recombinant scFvs that differentially recognize diverse denatured forms or assembly intermediates of the heat-labile enterotoxin B subunit of E. coli.
此前，我们已经描述了使用传统单克隆抗体对源自大肠杆菌（E. coli）的热不稳定肠毒素B亚基（EtxB）进行体外五聚体组装的研究（Amin等人，《生物化学杂志》1995年：270, 20143 - 50；Chung等人，《生物化学杂志》2006年：281, 39465 - 70）。为了进一步扩展这些研究，我们使用噬菌体展示技术筛选针对不同形式B亚基的单链可变片段（scFv）抗体。选择了两个表现出强结合和差异结合的克隆进行详细表征。进行了全面的序列分析以确定框架区和互补决定区，并校正了其中一个克隆（scFv - B1.3.9）中存在的无义突变。结合分析表明，scFv - B1.3.9在酶联免疫吸附测定（ELISA）中与热变性的单体B亚基（EtxB1）均有结合，并且对五聚体EtxB（EtxB5）也表现出交叉反应性，尽管对通过单唾液酸四己糖神经节苷脂（GM1）捕获的EtxB5没有反应性。另一种抗体（scFv - B5.2.14）具有不同的反应模式，在ELISA中仅与EtxB5结合，而不与EtxB1或通过GM1捕获的EtxB5结合。令人惊讶的是，在竞争实验中，组装好的五聚体B亚基仅微弱地抑制scFv - B5.2.14与固定化EtxB5的结合，而还原型但非氧化型的单体EtxB1是一种有效的竞争者。这些结果清楚地表明，B1.3.9和B5.2.14对在完全组装的GM1结合的EtxB五聚体形式中无法接近的隐蔽表位具有不同的特异性。综合我们的结果表明，我们能够成功分离和表征重组scFv，它们能够差异识别大肠杆菌热不稳定肠毒素B亚基的多种变性形式或组装中间体。
