Site‐oriented immobilization of fusion antigen directed by an affinity ligand, and its validation in an immunoassay
Site‐oriented immobilization of fusion antigen directed by an affinity ligand, and its validation in an immunoassay
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DOI：
10.1002/sia.3712
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发表时间：
2011-10
影响因子：
1.7
通讯作者：
B. Feng;Yueping Luo;Fei Ge;Lu Wang;Liqun Huang;Youzhi Dai
中科院分区：
文献类型：
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作者：
B. Feng;Yueping Luo;Fei Ge;Lu Wang;Liqun Huang;Youzhi Dai
将蛋白质固定在固体表面并保持其生物活性是构建高效免疫分析的关键。在这项研究中，从噬菌体展示肽文库中筛选的聚苯乙烯（PS）表面的亲和力配体Lig1被遗传融合到嵌合抗原HCV的N/C端，可以被抗丙型肝炎病毒（HCV）的特异性抗体识别。用直接和间接酶联免疫吸附法（ELISA）比较了固定在PS表面的lig1融合HCV与原始HCV的免疫分析特性。结果表明，HCV - Lig1（与HCV - C端融合的Lig1）以位点导向的方式优先吸附在PS表面，并能很好地暴露特定的抗体结合位点，从而大大提高了检测灵敏度。原子力显微镜（AFM）图像显示，与原材料相比，HCV - Lig1以有序的状态排列在PS表面，其在界面结合过程中引起的构象和空间畸变要小得多。只要涂层抗原的特异性表位不位于其N端和C端，配体融合方法可能是定位蛋白固定的理想策略。版权所有©2010 John Wiley & Sons， Ltd
Immobilizing proteins on a solid surface in a site‐specific orientation and maintaining their bioactivity are crucial to the construction of high‐performance immunoassays. In this study, an affinity ligand for polystyrene (PS) surface screened from a phage display peptide library, named Lig1, was genetically fused to the N/C‐terminus of chimeric antigen HCV that could be recognized by specific antibodies against hepatitis C virus (HCV). Immunoassay characteristics of lig1‐fused HCVs immobilized on PS surface were compared to that of original HCV in both direct and indirect enzyme‐linked immunosorbent assay (ELISA). The results indicated that HCV‐Lig1 (Lig1 fused to HCV C‐terminus) was preferentially adsorbed on PS surface in a site‐oriented manner and would expose specific antibody‐binding sites well, which resulted in a substantial enhancement of detection sensitivity. AFM images showed that, compared to the original one, HCV‐Lig1 was arranged on PS surface in an ordered state and its conformational and steric distortions induced during the interfacial binding process were much slighter. As long as the specific epitope of a coating antigen is not located on both its N and C‐terminus, the ligand fusion approach could be an ideal strategy for site‐oriented protein immobilization. Copyright © 2010 John Wiley & Sons, Ltd.