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Oriented Immobilization and Quantitative Analysis Simultaneously Realized in Sandwich Immunoassay via His-Tagged Nanobody

Liaoning Key Laboratory of Molecular Recognition and imaging, School of Bioengineering, Dalian University of Technology, No.2 Linggong Road, Dalian, Liaoning, 116023, China
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Academic Editor: José Manuel Herrero-Martínez
Molecules 2019, 24(10), 1890; https://doi.org/10.3390/molecules24101890
Received: 18 April 2019 / Revised: 8 May 2019 / Accepted: 13 May 2019 / Published: 16 May 2019
(This article belongs to the Special Issue Advances in Molecular Recognition Materials)
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Abstract

Despite the advantages of the nanobody, the unique structure limits its use in sandwich immunoassay. In this study, a facile protocol of sandwich immunoassay using the nanobody was established. In brief, β amyloid and SH2, an anti-β amyloid nanobody, were used as capture antibody and antigen, respectively. The SH2 fused with His-tag was first purified and absorbed on Co2+-NTA functional matrix and then immobilized through H2O2 oxidation of Co2+ to Co3+ under the optimized conditions. Then, 150 mM imidazole and 20 mM EDTA were introduced to remove the unbound SH2. The immobilized SH2 showed highly-sensitive detection of β amyloid. It is interesting that the quantification of the sandwich immunoassay was carried out by determining the His-tag of the detection nanobody, without interference from the His-tag of the capture nanobody. The immobilized SH2 detached exhibited outstanding stability during 30 days of storage. Taken together, His6-tag facilitated both the oriented immobilization of capture antibody and quantitative assay of detection antibody in sandwich immunoassay. We propose a facile and efficient sandwich immunoassay method that opens new avenue to the study of His-tagged protein interactions. View Full-Text
Keywords: sandwich immunoassay; nanobody; His-tag; oriented immobilization; β amyloid detection sandwich immunoassay; nanobody; His-tag; oriented immobilization; β amyloid detection
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
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Xu, L.; Cao, H.; Huang, C.; Jia, L. Oriented Immobilization and Quantitative Analysis Simultaneously Realized in Sandwich Immunoassay via His-Tagged Nanobody. Molecules 2019, 24, 1890.

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