Nanobody-Nanoluciferase Fusion Protein-Enabled Immunoassay for Ochratoxin A in Coffee with Enhanced Specificity and Sensitivity

Abstract

Ochratoxin A (OTA), one of the best-known mycotoxins, causes problems concerning food safety with potential toxic effects in humans and animals. So, it is crucial to develop simple and sensitive methods for the detection of OTA. Herein, a nanoluciferase–nanobody fusion protein (Nb28-Nluc)-retaining antibody recognition and enzymatic activity was first prepared, which was then applied as a bifunctional tracer to construct a one-step bioluminescent enzyme-linked immunosorbent assay (BLEIA) for OTA in coffee samples. On the basis of Nb28-Nluc, the BLEIA can be completed with a one-step incubation and detection, with only a substrate replacement from 3,3′,5,5′-tetramethylbenzidine (TMB) to a Nluc assay reagent (Furimazine). Under the optimal experimental conditions, the proposed one-step BLEIA achieved a detection limit of 3.7 ng/mL (IC10) within 3 h. Moreover, the BLEIA method showed good repeatability and accuracy in the spike recovery experiments with recoveries of 83.88% to 120.23% and relative standard deviations (RSDs) of 5.2% to 24.7%, respectively. Particularly, the BLEIA displayed superior performances, such as fewer operations and more rapid and sensitive detection as compared with Nb28-based enzyme-linked immunosorbent assay. Therefore, the proposed one-step BLEIA has great potential for the sensitive and accurate screening of OTA in food samples.

Magnetic nanoparticle‐based immunosensors and aptasensors for mycotoxin detection in foodstuffs: An update

“…So far, multi‐class Nbs targeting different mycotoxins—for example, OTA (Xie et al., 2022), AFB 1 (Liu et al., 2020; Zuo, Yan et al., 2023), tenuazonic acid (Wang, Yang et al., 2022), FB 1 (Alsulami et al., 2021)—have been explored. In addition, the controllable recombination of Nbs with other biomolecules and the construction of multimeric Nbs multimerization are facilitate to prepare novel immunosensing systems for mycotoxin monitoring, including dual‐mode immunosensors (Zuo, Yan et al., 2023), chemiluminescent ELISA (Wang et al., 2023), bioluminescent ELISA (Bao et al., 2022), Nanobody/NanoBiT system‐based (Xie et al., 2022), and FRET‐based immunosensors (Su et al., 2022). Besides being utilized as the primary Abs to recognize targets, Nbs can also be applied as signal indicator‐labeled secondary Abs, which results in a significant enhancement of analytical sensitivity, which is the so‐called anti‐idiotypic Nbs by using mAbs as the immunogens (Cai et al., 2021; Tang et al., 2022).…”

Rapid and sensitive immunoassay for alpha-fetoprotein in serum by fabricating primary antibody–enzyme complexes using protein self-assembly

“…18–20 In the immunoassay field, nanobody can easily genetically fuse with various enzymes ( e.g. , horseradish peroxidase, 21 alkaline phosphatase, 22 and nano-luciferase 23 ) to develop PAECs. What's more, nanobody-based probes have higher probe intensity and faster binding rate in immunoassay compared with traditional intact antibodies.…”

“…17 Nanobody is the smallest known antigen-binding unit, which has attracted extensive attention in drug development, food safety, environmental monitoring, and other elds due to its nano-size, low cost, easy genetic manipulation, good water solubility, and high thermal stability. [18][19][20] In the immunoassay eld, nanobody can easily genetically fuse with various enzymes (e.g., horseradish peroxidase, 21 alkaline phosphatase, 22 and nano-luciferase 23 ) to develop PAECs. What's more, nanobody-based probes have higher probe intensity and faster binding rate in immunoassay compared with traditional intact antibodies.…”

“…The heptamer nanobody was used to develop an ultrasensitive competitive ELISA for the detection of OTA in coffee samples, and its limit of detection (LOD) was 175.56-fold lower than that of the monomer Nb28-based ELISA, which demonstrates that the nanobody multimerization based on protein self-assembly is an attractive strategy to increase the sensitivity of immunoassays. 23 To date, protein self-assembly has been widely used in nanobody multimerization to improve various properties of nanobodies, including affinity, stability, and half-life, but there are few studies on the application of protein self-assembly in the development of PAECs.…”