Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag)
Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag)
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DOI：
10.1007/s00216-009-2999-y
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发表时间：
2009-10-01
影响因子：
4.3
通讯作者：
Kishimoto, Michimasa
中科院分区：
文献类型：
--
作者：
Kumada, Yoichi;Hamasaki, Kyoko;Kishimoto, Michimasa
通过重组大肠杆菌产生与聚苯乙烯结合肽（PS标签，（PS19-1; RAFIASRRIRRP，PS19-6; RIIIRRIRR））基因融合的单链抗体（scFv），用于直接和位点特异性地将scFv固定在具有高抗原结合活性的聚苯乙烯支持物上。成功地将人C-反应蛋白（CRP）特异性的融合PS标签的单链抗体（scFv-PS-tags）过表达为包涵体，并使用分批稀释法进行了复性。当scFv-PS标签在吐温20存在下固定在亲水性PS（phi-PS）板上时，它们显示出与疏水性PS（pho-PS）板上的全单克隆抗体（mAb）相当或更高的抗原结合活性，这是酶联免疫吸附测定（ELISA）的唯一方法。此外，当scFv-PS-tag在一步和两步ELISA中用作配体抗体时，减少了测定时间而不损失灵敏度。这些结果表明，PS-标签在phi-PS表面上的强烈和特异性附着防止了scFv构象变化，因此，scFv的高抗原结合活性得以保留。通过使用具有不同VH/VL对的PS-标签融合的scFv获得几乎相同的结果。因此，多种scFv可以通过PS标签的遗传融合功能化到phi-PS板上。在phi-PS板上具有高抗原结合活性的ScFv-PS-tags是比完整mAb更有用的配体抗体。因此，scFv-PS-tags可应用于临床诊断和蛋白质组学研究。
Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one-and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.