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

Abstract

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 gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double mutant gene. The Nb28-AP construct was transformed into E. coli BL21(DE3)plysS and soluble expression in bacteria was confirmed by SDS-PAGE 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 ng/mL 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.

Nanobody-based enzyme immunoassay for ochratoxin A in cereal with high resistance to matrix interference

“…Dilution of sample extract is commonly used to minimize matrix interference while a high dilution can cause lower assay sensitivity. Cereal samples (rice, oats and barley) that were found to be free of OTA by LC-MS/MS in our previous work [16] were subjected to matrix effects analysis. Diluted sample extract was used to prepare serial concentrations of OTA solution containing 20% methanol for Nb-ELISA.…”

“…For inter-assay, the recovery and RSD ranged from 85% to 99% and from 0.05 to 0.06, respectively (Table 1). Eight cereal samples naturally contaminated OTA tested in our previous work [16] were analyzed by Nb-ELISA and commercial ELISA kit, respectively. As seen in Table 2, the OTA content in samples tested by above two methods were in good agreement with each other.…”

“…The thermal stability is thought in part due to a disulfide bond between complementarity determining region (CDR) one and CDR three [9]. What’s more, the Nb also showed a higher resistance to cereal matrix interference than conventional antibody in ELISA, which performed better than VHH-phage and Nb-AP fusion in our previous work [16, 22]. This unique property of Nb can effectively avoid false negative results in sample screening.…”

“…After incubation for 10 min at room temperature, the lysate was centrifuged (15000g, 5 min) to separate supernatant containing the soluble proteins. The presence of nanobodies was detected by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot with rabbit anti-6×his tag IgG-HRP conjugate and TMB membrane peroxidase substrate [16]. …”

Enzymatic Hydrolysate-Induced Displacement Reaction with Multifunctional Silica Beads Doped with Horseradish Peroxidase–Thionine Conjugate for Ultrasensitive Electrochemical Immunoassay

“…7 Typically, the method usually consists of sandwichtype and competitive-type immunoassay formats. 8,9 Despite high sensitivity and specificity of the sandwich-type immunoassays, they have some disadvantages, e.g., a relatively long incubation time and expensive chemicals because of the use of two matched antibodies. 10 More unfavorably, the sandwich-type assays are unsuitable for quantitative monitoring of small molecules, e.g., mycotoxins, marine toxins, and food additives.…”