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

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.

Antibody‐Incorporated Nanomedicines for Cancer Therapy

“…[ 188 ] Typically, His‐tags are placed at the C‐ or N‐terminal of a protein because these terminals are less likely to disturb the binding affinity or activity of a protein molecule. [ 189 ] Reagents chelated to nickel or zinc can then be coordinated to the imidazoles in a His‐tag for protein purification, [ 190 ] immunoassay, [ 191 ] and nanoparticle functionalization. [ 192 ] Utilizing the His‐tag technique, recombinant antibodies or antibody fragments can be easily purified through immobilized metal affinity chromatography based on nickel ions for further conjugation.…”

Direct Immobilization of Engineered Nanobodies on Gold Sensors

“…In a recent example, nanobodies have been tagged with histidines, which served to couple the nanobody to cobalt-nitrilotriacetic acid metal-chelate beads. 7 …”

“…In a recent example, nanobodies have been tagged with histidines, which served to couple the nanobody to cobalt-nitrilotriacetic acid metal-chelate beads. 7 In spite of these and other efforts in the literature, 9−11 efficient, alternative immobilization methods are still needed to meet the requirements of a wide range of sensing applications. In this context, gold surfaces are widely employed as interfaces in various biochemical and chemical sensors because of their high electrical conductivity, unique optical properties, biocompatibility, and chemical stability.…”

“…Despite great advances in the nanobody technology, few approaches have been reported for the immobilization of nanobodies on sensing platforms. − Physical adsorption has been investigated for nanobody immobilization on gold nanoparticles which are used as immunoassay detection labels . While stable nanobody–gold nanoparticle conjugates can be generated, this requires careful consideration of the influence of the nanobody isoelectric point, pH, and ionic strength of the solution.…”

Functional comparison of paper-based immunoassays based on antibodies and engineered binding proteins

“…Antibodies have been commonly used as the affinity reagents in diagnostic tests; however, in recent years, alternative scaffolds have been investigated for use in diagnostic tests due to their desirable characteristics such as intrinsic thermal stability and ease of production. 14,15 In recent years, studies have begun reporting the use of nonantibody scaffolds in full sandwich assays using pairs of nanobodies, [16][17][18][19] affimers, 20,21 DARPins, 22 and the reducedcharge Sso7d variant (rcSso7d). [23][24][25][26][27][28][29][30][31] However, a direct functional comparison of antibodies and non-antibody scaffolds in an identical full sandwich immunoassay test format has yet to be conducted.…”