Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal.

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.

New approach for development of sensitive and environmentally friendly immunoassay for mycotoxin fumonisin B(1) based on using peptide-MBP fusion protein as substitute for coating antigen.

Here, on the basis of mimotope of small analytes, we demonstrated a new approach for development of sensitive and environmentally friendly immunoassay for toxic small analytes based on the peptide-MBP fusion protein. In this work, using mycotoxin fumonisin B1 (FB1) as a model hapten, phage displayed peptide (mimotope) that binds to the anti-FB1 antibody were selected by biopanning from a 12-mer peptide library. The DNA coding for the sequence of peptide was cloned into Escherichia coli ER2738 as a fusion protein with a maltose binding protein (MBP).

VHH phage-based competitive real-time immuno-polymerase chain reaction for ultrasensitive detection of ochratoxin A in cereal.

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.

Ochratoxin A mimotope from second-generation peptide library and its application in immunoassay.

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.

Application of mimotope peptides of fumonisin b1 in Peptide ELISA.

Anti-fumonisin B(1) (FB(1)) McAb 1D11 was used as the target for biopanning from a phage random loop-constrained heptapeptide library. After three cycles of panning, seven phages with three mimotope peptides were selected to mimic the binding of FB(1) to 1D11. After the identification of phage ELISA, the phage clone that showed the best linear range of detection was chosen for further research.