Polyhydroxyalkanoate chip for the specific immobilization of recombinant proteins and its applications in immunodiagnostics
Polyhydroxyalkanoate chip for the specific immobilization of recombinant proteins and its applications in immunodiagnostics
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DOI：
10.1007/bf02931904
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发表时间：
2006-03-01
影响因子：
3.2
通讯作者：
Lee, SY
中科院分区：
文献类型：
--
作者：
Park, TJ;Park, JP;Lee, SY
在这项研究中，开发了一种新的策略，用于高选择性固定蛋白质，使用聚羟基链烷酸酯（PHA）解聚酶底物结合结构域（SBD）作为活性结合结构域。为确定该方法用于免疫诊断的适宜性，将抗B型肝炎病毒（HBV）前S2表面蛋白和抗严重急性呼吸综合征冠状病毒（SARS-CoV）囊膜蛋白的单链抗体（ScFv）分别与SBD融合，然后直接固定在PHA包被的玻片上。然后利用荧光标记的HBV抗原和抗SCVe抗体来检查PHA包被表面上的特异性相互作用。仅在点样位置检测到荧光信号，从而表明对其相应抗原/抗体的高度亲和力和选择性。此外，我们检测到少量的ScFv-SBD（2.7 ng/mL）和SCVe-SBD融合蛋白（0.6 ng/mL）。因此，这种使用PHA和SBD的微阵列平台技术似乎通常适用于免疫诊断，对生物分子或固体表面的合成或化学修饰没有特殊要求。
In this study, a novel strategy was developed for the highly selective immobilization of proteins, using the polyhydroxyalkanoate (PHA) depolymerase substrate binding domain (SBD) as an active binding domain. In order to determine the appropriacy of this method for immunodiagnostic assays, the single-chain antibody (ScFv) against the hepatitis B virus (HBV) preS2 surface protein and the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVe) were fused to the SBD, then directly immobilized on PHA-coated slides via microspotting. The fluorescence-labeled HBV antigen and the antibody against SCVe were then utilized to examine specific interactions on the PHA-coated surfaces. Fluorescence signals were detected only at the spotted positions, thereby indicating a high degree of affinity and selectivity for their corresponding antigens/antibodies. Furthermore, we detected small amounts of ScFv-SBD (2.7 ng/mL) and SCVe-SBD fusion proteins (0.6 ng/mL). Therefore, this microarray platform technology, using PHA and SBD, appears generally appropriate for immunodiagnosis, with no special requirements with regard to synthetic or chemical modification of the biomolecules or the solid surface.