Multifunctional Nanobody Fusion Proteins in Immunoassays: Diverse Strategies for Enhanced Analytical Performance

Abstract

Nanobodies, derived from camelid heavy-chain antibodies, offer exceptional specificity, stability, and solubility, enabling rapid and sensitive immunoassays. Their small size makes them ideal scaffolds for genetic fusion with reporter proteins, functional peptides, and signal amplification modules. In contrast to conventional antibodies, nanobodies can be precisely engineered at the gene level, allowing directional conjugation and efficient incorporation of signal-enhancing elements. This tunability simplifies assay design and reduces processing steps. Additionally, recombinant expression systems enable stable, scalable, and standardized production of nanobody-based immunoreagents, ensuring reproducibility in diagnostics. In this review, we summarize recent advances in the engineering and application of nanobody fusion proteins in immunoassays, with a particular focus on three main strategies: signal reporting, site-oriented strategies, and multivalency. We highlight how these approaches enhance sensitivity, shorten assay time, and enable multiplex detection. Finally, we discuss future perspectives and remaining challenges in the design and application of nanobody-based platforms for diagnostics.

Keywords: Fusion protein; Immunoassay; Immunoreagents; Nanobody; Protein tag.

Fig.1

(A) Schematic diagram of dual-emission system based ratiometric fluoroimmunoassay for determination of 1-NAP based on several nanomaterials including SiNPs, AuNCs and MnO₂ nanosheet. Reprinted with permission from Ref. [50]. (B) Schematic diagram of enzyme cascade-amplified immunoassay based on Nb-ALP and MnO₂ nanoflakes for the determination of AFP. Reprinted with permission from Ref. [48]. (C) Schematic diagram of ratiometric fluorescent immunosensor for the detection of AFB₁. Reprinted with permission from Ref.[53]. (D) Schematic illustration of the immunosensor by utilizing the oxidase-mimicking activity of Ch-Pt NPs for the detection of fenitrothion. Reprinted with permission from Ref. [47].

Fig. 2.

(A) Design and working principle of the BRET nano Q-body and preparation of the paper device. Reprinted with permission from Ref. [65]. (B) Schematic diagram of the nanobody/NanoBiT system-mediated homogeneous bioluminescence immunosensor for detection of OTA. Reprinted with permission from Ref. [71]. (C) Scheme of the nanobody-based sandwich homogeneous split-luciferase assay for the rapid detection of human soluble epoxide hydrolase. Reprinted with permission from Ref. [72].

Fig. 3

(A) Production and identification of biotinylated AviTag-Nb fusion proteins. Schematic diagram of the construction of the expression plasmids for AviTag-Nb fusion proteins (a); Agarose gel electrophoresis analysis of the colony PCR (b); SDS-PAGE result (c); Identification of the different biotinylated AviTag-Nb fusions by icELISA (d). Reprinted with permission from Ref. [84]. (B) Schematic diagram of biotinNb and development of biotinNb LFIA(a); principle of targeted labeling of anti- procymidone btNb and detection of biotinNb-LFIA(b). Reprinted with permission from Ref. [82]. (C) Scheme of fluorescence immunoassay based on red-emission carbon dots quenching for fenitrothion detection. Reprinted with permission from Ref. [81].

Fig. 4

(A) Schematic representation comparing the random orientation of sdAb achieved by standard chemical cross-linking vs the highly oriented sdAb-SpyTag fusion capture surface obtained by first coating microspheres with SpyCatcher. Reprinted with permission from Ref. [91]. (B) Schematic representation of developing the double “Y” architecture by Nb-SpyTag/Im7 and SpyCatcher/CL7-tetramerizing helix and double “Y” assembly-based LFIA for detecting the SARS-CoV-2 N protein. Reprinted with permission from Ref.[93]. (C) Schematic representation of SpyCatcher-mediated nanobody-luciferase conjugates on a protein nanoscaffold based bioluminescence immunoassays for simultaneous detection for AFB₁ and OTA. Reprinted with permission from Ref. [94].

Fig. 5

(A) Characterization of fenobody architecture. Structural comparison of fenobody and ferritin (a), SDS-PAGE analysis of fenobody, ferritin, and nanobody (b), SEC profiles of self-assembled fenobody and ferritin (c), and TEM images showing the morphology of ferritin (d) and fenobody (e) particles. Reprinted with permission from Ref.[135]. (B) Schematic illustration of vector construction and sensitivity comparison among nanobody (2D), 2D-Nluc, and 2D-ferritin-Nluc formats. Reprinted with permission from Ref. [138].

Fig. 6

(A) Schematic diagram of nanobody multimerization strategy via C4bp fusion. Reprinted with permission from Ref. [146]. (B) Schematic diagram of the fabrication process of immunosensor based on AuNPs@ZIF-8 nano-composite and A1-C4bp heptamer. Reprinted with permission from Ref. [147]. (C) Scheme of one-step bioluminescent enzyme immunoassay for TBBPA by using NB/C4BP/Nluc heptamer. Reprinted with permission from Ref. [151]. (D) Schematic diagram for the construction of CM/FL-dLFI with AuNFs@sfGFP-C4bpα-Y4 probe. Reprinted with permission from Ref.[152].

Scheme 1.

Diverse nanobody fusion strategies for enhanced immunoassays