Development of sandwich chemiluminescent immunoassay based on an anti-staphylococcal enterotoxin B Nanobody-Alkaline phosphatase fusion protein for detection of staphylococcal enterotoxin B
Development of sandwich chemiluminescent immunoassay based on an anti-staphylococcal enterotoxin B Nanobody-Alkaline phosphatase fusion protein for detection of staphylococcal enterotoxin B
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DOI：
10.1016/j.aca.2020.01.032
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发表时间：
2020-04-29
影响因子：
6.2
通讯作者：
Gao, Zhixian
中科院分区：
文献类型：
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作者：
Sun, Tieqiang;Zhao, Zunquan;Gao, Zhixian
本研究利用纳米抗体-碱性磷酸酶（Nb-ALP）融合蛋白建立了检测葡萄球菌肠毒素B（SE B）的夹心荧光免疫分析法（CLIA）。SEB结合纳米抗体从天然噬菌体展示文库获得，并且Nb-ALP融合蛋白被构建并作为用于在CLIA中检测抗体的热稳定的和潜在有效的物质获得。基于抗SEB单克隆抗体（mAb）和我们的融合蛋白Nb 37-ALP的夹心CLIA的工作范围为3.12-50.0 ng mL（-1），SC 50 = 8.59 +/- 0.37 ng mL（-1）。根据空白值加3个标准偏差，检测限为1.44 ng mL（-1）。为了深入了解SEB与Nb 37的相互作用，构建了SEB-Nb 37复合物的三维结构，并通过分子模拟和对接方法进行了验证。结果显示，Nb 37的互补决定区3（CDR 3）嵌入SEB的主要组织相容性复合体（MHC）和T细胞受体（TcR）结合位点形成的开口中，提示Nb 37可能影响MHC Ⅱ类分子和TcR对SEB的识别。Nb 37的CDR 3的精氨酸残基（Arg）101、Arg 102和苯丙氨酸残基（Phe）103可能有助于特异性结合，从而在它们与SEB之间形成六个盐桥。总之，就其特异性和灵敏度而言，所获得的抗SEB Nb-ALP似乎具有替代用于检测SEB的化学标记探针的潜力。(C)2020由爱思唯尔公司出版
In this study, sandwich chemiluminescent immunoassay (CLIA) for the detection of Staphylococcal enterotoxin B (SEB) was developed using nanobody-alkaline phosphatase (Nb-ALP) fusion protein. The SEB-binding nanobodies were obtained from a naive phage-display library and the Nb-ALP fusion protein was constructed and obtained as a thermally stable and potentially effective substance for detecting antibodies in CLIA. The working range of the sandwich CLIA based on anti-SEB monoclonal antibodies (mAbs) and our fusion protein, Nb37-ALP, was 3.12-50.0 ng mL(-1) with SC50 = 8.59 +/- 0.37 ng mL(-1). The limit of detection was 1.44 ng mL(-1) according to the blank value plus 3 standard deviations. In order to understand the interaction of SEB and Nb37 in depth, the 3D structure of the SEB-Nb37 complex was constructed and verified by molecular modeling and the docking method. The results showed that the complementary-determining region 3 (CDR3) of Nb37 embedded itself in the opening generated by the major histocompatibility complex (MHC) and T-cell receptor- (TcR) binding sites of SEB, indicating that Nb37 may affect the recognition of SEB by MHC class II molecules and the TcR. The arginine residue (Arg) 101, Arg102 and phenylalanine residue (Phe)103 of CDR3 in Nb37 may have contributed to specific binding to form six salt-bridges between these and SEB. In conclusion, in terms of their specificity and sensitivity, the obtained anti-SEB Nb-ALP appears to have the potential to replace chemically labeled probes for the detection of SEB. (C) 2020 Published by Elsevier B.V.