Department of Biomolecular Engineering, Kyoto Institute of Technology, Japan.
J Immunol Methods. 2012 Nov 30;385(1-2):15-22. doi: 10.1016/j.jim.2012.07.021. Epub 2012 Aug 2.
In this study, we successfully developed a novel lectin enzyme-linked immunosorbent assay (lectin ELISA) for the detection of glycosides linked to carcinoembryonic antigen (CEA) as a model antigen using a scFv-immobilized hydrophilic polystyrene (phi-PS) plate and 12 different HRP-labeled lectins. Anti-CEA scFv genetically fused with a PS-tag at its C-terminus (scFv-PS) was successfully over-expressed in an insoluble fraction by cultivation of recombinant E. coli. A solid-phase refolding method was adopted to immobilize and refold scFv-PS on the surface of the phi-PS plate. Consequently, in sandwich ELISA using phi-PS plates immobilized with scFv and scFv-PS as well as Maxisorp™ plate with whole antibody (whole Ab), the highest sensitivity and S/N ratio were obtained from the scFv-PS-immobilized phi-PS plate as it was the antigen-binding domain with the highest surface immobilization density and remaining activity displayed on the phi-PS surface. During the lectin ELISA, high background signals were detected from the whole-Ab-immobilized Maxisorp™ plate, indicating that HRP-labeled lectins, particularly PHA-E, Con A, LCA, PSA, DSA, and MAA, directly recognized the glyco-chains of a whole Ab. However, a considerable amount of low background signals were detected from the scFv-PS-immobilized plate since the ligand antibody, namely scFv-PS, was produced by E. coli cells that had no potential for glycosylation during the process of post-transcriptional modification. Signals for glyco-chains conjugated with CEA were detectable using 6 kinds of HRP-labeled lectins, namely PHA-E, PHA-L, SBA, Con A, LCA, and DSA, with much higher sensitivities and S/N ratios. Thus, the lectin ELISA using scFv-PS as a ligand was considerably useful for detecting the glyco-chains of glycoproteins, including glyco-biomarkers, with much higher sensitivities and S/N ratios compared with a conventional whole Ab. By preparation of a phi-PS plate immobilized with different species of scFvs, this method is capable of the glyco-chain analysis of a number of glyco-biomarkers in the fields of clinical diagnosis and biochemical research.
在这项研究中，我们成功开发了一种新型凝集素酶联免疫吸附测定（lectin ELISA），用于检测与癌胚抗原（CEA）相连的糖苷作为模型抗原，使用 scFv 固定化亲水聚苯乙烯（phi-PS）板和 12 种不同的 HRP 标记的凝集素。在重组大肠杆菌的培养中，将 scFv 与 PS 标签在 C 末端融合的抗 CEA scFv（scFv-PS）成功地在不溶性部分中过表达。采用固相复性方法将 scFv-PS 固定并复性在 phi-PS 板的表面上。因此，在使用 phi-PS 板固定的 scFv 和 scFv-PS 以及 Maxisorp™板固定的全抗体（whole Ab）进行夹心 ELISA 时，从固定有 scFv-PS 的 phi-PS 板获得了最高的灵敏度和 S/N 比，因为它是具有最高表面固定密度和保留活性的抗原结合域在 phi-PS 表面上显示。在凝集素 ELISA 中，从固定有全抗体的 Maxisorp™板中检测到高背景信号，表明 HRP 标记的凝集素，特别是 PHA-E、Con A、LCA、PSA、DSA 和 MAA，直接识别整个 Ab 的糖链。然而，从固定有 scFv-PS 的板中检测到相当多的低背景信号，因为配体抗体，即 scFv-PS，是由在转录后修饰过程中没有糖基化潜力的大肠杆菌细胞产生的。使用 6 种 HRP 标记的凝集素，即 PHA-E、PHA-L、SBA、Con A、LCA 和 DSA，可检测到与 CEA 结合的糖链，灵敏度和 S/N 比均较高。因此，使用 scFv-PS 作为配体的凝集素 ELISA 对于检测糖蛋白，包括糖生物标志物的糖链非常有用，与传统的全抗体相比，具有更高的灵敏度和 S/N 比。通过制备固定有不同种 scFv 的 phi-PS 板，该方法能够在临床诊断和生化研究领域对多种糖生物标志物的糖链进行分析。
