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

• Published in: Molecules (Basel, Switzerland) Switzerland), 2019-05, Vol.24 (10), p.1890
• Main Authors: Xu, Li, Cao, Hanyu, Huang, Chundong, Jia, Lingyun
• Format: Article
• Language: English
• Subjects: Alzheimer's disease; Antibodies; Antigens; Carbon dioxide; Cobalt; Ethylenediaminetetraacetic acids; His-tag; Hydrogen peroxide; Imidazole; Immobilization; Immunoassay; Nanobodies; nanobody; oriented immobilization; Oxidation; Protein interaction; Proteins; Quantitative analysis; sandwich immunoassay; β amyloid detection
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules24101890

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 Co -NTA functional matrix and then immobilized through H O oxidation of Co to Co 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.