•Ab2β−Nb−AP has the potential to replace chemically-coupled probes.•Ab2β−Nb−AP is homogeneous enzyme-labelled antigen can be prepared reproducibly.•We developed a green and rapid one-step competitive enzyme immunoassay.•The sensitivity of one-step CLIA was 9-folds higher than two-step ELISA.A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB1 was developed. The anti-idiotypic nanobody–alkaline phosphatase (Ab2β−Nb−AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB1 were 2.69 and 0.35 ng mL−1, respectively, with a linear range of 0.93–7.73 ng mL−1. The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL−1, and the IC50 was 0.89 ± 0.09 ng mL−1 with a linear range of 0.29–2.68 ng mL−1. Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2β−Nb−AP fusion protein based one-step competitive immunoassay for monitoring FB1 contamination in cereals. The Ab2β−Nb−AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems.

•Nanobody is applied in detecting AFP in stead of traditional IgG antibody.•IPCR technology based on nanobody couple with high sesitivity, broad linear range.•The principles of AFP assays are applicable to detection other cancer biomarker.Immunoassay for cancer biomarkers plays an important role in cancer prevention and early diagnosis. To the development of immunoassay, the quality and stability of applied antibody is one of the key points to obtain reliability and high sensitivity for immunoassay. The main purpose of this study was to develop a novel immunoassay for ultrasensitive detection of cancer biomarker alpha-fetoprotein (AFP) based on nanobody against AFP. Two nanobodies which bind to AFP were selected from a phage display nanobody library by biopanning strategy. The prepared nanobodies are clonable, thermally stable and applied in both sandwich enzyme linked immunoassay (ELISA) and immuno-PCR assay for ultrasensitive detection of AFP. The limit detection of sandwich ELISA setup with optimized nanobodies was 0.48 ng mL−1, and the half of saturation concentration (SC50) value was 6.68±0.56 ng mL−1. These nanobodies were also used to develop an immuno-PCR assay for ultrasensitive detection of AFP, its limit detection values was 0.005 ng mL−1, and the linear range was 0.01–10,000 ng mL−1. These established immunoassays based on nanobodies were highly specific to AFP and with negligible cross reactivity with other tested caner biomarkers. Furthermore, this novel concept of nanobodies mediated immunoassay may provide potential applications in a general method for the ultrasensitive detection of various cancer biomarkers.

A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)–alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28–AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06–0.43 ng/mL. This assay was compared with LC–MS/MS, and the results indicated the reliability of Nb–AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.

•Nanobodies that bind to anti-DON antibody were isolated from a naïve library.•The nanobody-based ELISA was 18-fold more sensitive than conventional ELISA.•The selected nanobody was thermal stable and can be produced in large scale.•The molecular mechanism of DON antigen mimicry by nanobody was described.In this study, using mycotoxin deoxynivalenol (DON) as a model hapten, we developed a nanobody-based environmental friendly immunoassay for sensitive detection of DON. Two nanobodies (N-28 and N-31) which bind to anti-DON monoclonal antibody (MAb) were isolated from a naive phage display library. These nanobodies are clonable, thermally stable and mycotoxin-free products and can be served as coating antigen mimetics in heterologous immunoassay. The half inhibition concentration (IC50) of the immunoassay developed with N-28 and N-31 was 8.77 ± 0.41 ng mL−1 and 19.97 ± 0.84 ng mL−1, respectively, which were 18- and 8-fold more sensitive than the conventional coating antigen (DON-BSA) based immunoassay. In order to better understand the molecular mechanism of antigen mimicry by nanobody, the 3D structure of “nanobody (N-28) - anti-DON MAb” complex was presented and verified by molecular modeling and alanine-scanning mutagenesis. The results showed that hydrogen bond and hydrophobic interaction formed between Thr 102 – Ser 106 of N-28 and CDR H3 residues of anti-DON antibody may contribute to their binding. This novel concept of enhancing sensitivity of immunoassay for DON based on nanobody may provide potential applications in a general method for immunoassay of various food chemical contaminants.

•We evidenced that Ab2β Nbs can be used as a substitute for hapten-conjugates.•We developed and optimized a green and rapid Nb-ELISA for the determination of FB1.•The sensitivity of Nb-ELISA was improved 20-fold than that of the hapten-conjugate.•Ab2β Nb showed lower affinity than the hapten-conjugates to mAb.•Ab2β Nb was more fit for working as coating antigen than the hapten-conjugates.Nanobodies that are small and thermally stable, as well as have high expression level, are leading alternative to produce anti-idiotypic antibodies. These antibodies have the advantage of replacing mycotoxins and their conjugates for immunoassays. In this work, anti-fumonisin B1 (FB1) monoclonal antibody (mAb) was used as the target for biopanning from a naïve alpaca nanobody (Nb) phage display library. After three cycles of panning, one anti-idiotypic nanobody (Ab2β Nb) was isolated and subjected to a Nb-ELISA for the detection of FB1. Surface plasmon resonance was used to analyze the reaction kinetics between the Ab2β Nb and anti-FB1 mAb. The developed assay showed a half inhibitory concentration (IC50) of 0.95±0.12 ng/mL, a limit of detection of 0.15 ng/mL, a linear range of 0.27–5.92 ng/mL, and a low cross-reactivity toward FB2 of 4.93%. The sensitivity was enhanced approximately 20-fold compared with that of the chemosynthetic FB1–BSA conjugates. The equilibrium dissociation constant (KD) measured for Ab2β Nb: anti-FB1 mAb was 164.6 nM. The assay was compared with conventional ELISA (the commercial ELISA kit), and the results indicated the reliability of Ab2β Nb replacing the antigen–carrier protein conjugates. The use of biotechnology in developing the surrogate is an ideal strategy for replacing conventional synthesized antigens.

Phage display-mediated immuno-polymerase chain reaction (PD-IPCR) is an ultrasensitive detection technology that combines the advantages of immuno-PCR and phage display. The phage particle, which displayed antibody fragments including single-chain fragment variable (scFv), variable domain of heavy-chain antibodies (VHH), and antigen-binding fragment (Fab) on the surface can be directly used in IPCR, supplying both the detection antibody and deoxyribonucleic acid (DNA) template. In this work, we used ochratoxin A (OTA) as a model system to study the capacity of PD-IPCR in the detection of toxic small molecular weight compounds, especially mycotoxins. An alpaca-derived VHH library was constructed and subjected to four cycles of panning. In total, 16 clones with four unique sequences were selected by competitive binding with OTA. The clone VHH-28 resulted in the lowest 50% inhibitory concentration of 0.31 ng/mL in the phage enzyme-linked immunosorbent assay (ELISA) and was selected to develop the VHH phage-based real-time immuno-PCR (RT-IPCR). The detection limit of the VHH phage-based RT-IPCR was 3.7 pg/L, with a linear range of 0.01–1000 pg/mL. This method was compared with conventional ELISA, and validation results indicated the reliability of VHH phage-based RT-IPCR in the detection of OTA in cereal samples. This study provides a new idea for the ultrasensitive detection of mycotoxins and other toxic small molecular weight compounds.

•Aptamers were structurally modified and truncated to narrow down the binding region.•Functional loops are associated with various specific contacts with chemicals.•Aptamer fused by minimum functional structures with affinity increased.•The molecular interaction between DNA structure and chemicals has been profiled.•The developed approach could simultaneously detect the co-existing chemicals.An approach is developed to detect the organophosphorus pesticides via competitive binding to a recombinant broad-specificity DNA aptamer with a molecular beacon (MB), the binding of the MB to the aptamer results in the activation of a fluorescent signal, which can be measured for pesticide quantification. Aptamers selected via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) were structurally modified and truncated to narrow down the binding region of the target, which indicated that loops of the aptamer contributed different functions for different chemical recognition. Thereafter, a variant fused by two different minimum functional structures, was clarified with broad specificity and increased affinity. Further molecular docking and molecular dynamics simulations was conducted to understand the molecular interaction between DNA structure and chemicals. 3D modeling revealed a hot spot area formed by 3 binding sites, forces including hydrogen bonds and van der Waals interactions appear to play a significant role in enabling and stabilizing the binding of chemicals. Finally, an engineered aptamer based approach for the detection of organophosphorus pesticides was successfully applied in a test using a real sample, the limit of quantification (LOQ) for phorate, profenofos, isocarbophos, and omethoate reached 19.2, 13.4, 17.2, and 23.4 nM (0.005 mg L−1), respectively.

With the advantage of replacing mycotoxins and their conjugates, mimotopes have been applied to immunoassays, the most common of which were seleted from random phage displayed peptide libraries. However, these mimotopes were limited by the diversities of the peptide libraries. The aim of this study was to demonstrate that a variety of mimotopes can be obtained by constructing a second-generation peptide library. Using mycotoxin ochratoxin A as a model system, a dodecapeptide mimotope was isolated after panning the second-generation peptide library. The half inhibition concentration of the chemiluminescent enzyme-linked immunosorbent assay setup with this mimotope was 0.04 ng/mL, and the linear range was 0.006–0.245 ng/mL. The mimotope was also used to develop a qualitative dipstick assay with a cutoff level of 1 ng/mL. The method not only presents a high sensitivity but also contributes to the development of mimotope-based assays for mycotoxins avoiding the need of synthesizing toxic mycotoxin conjugates.