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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 2+ -NTA functional matrix and then immobilized through H 2 O 2 oxidation of Co 2+ to Co 3+ 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.

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