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

Abstract

A sensitive indirect competitive nanobody-based enzyme linked immunosorbent assay (Nb-ELISA) for ochratoxin A (OTA) with high resistance to cereal matrix interference was developed. Nanobodies against OTA (Nb15, Nb28, Nb32, Nb36) were expressed in E. coli cells and their thermal stabilities were compared with that of an OTA-specific monoclonal antibody 6H8. All nanobodies could still retain their antigen-binding activity after exposure to temperature 95°C for 5min or to 90°C for 75min. Nb28 that exhibited the highest sensitivity in ELISA was selected for further research. An indirect competitive ELISA based on Nb28 was developed for OTA, with an IC₅₀ of 0.64ng/mL and a linear range (IC₂₀-IC₈₀) of 0.27-1.47ng/mL. Cereal samples were analyzed following a 2.5 fold dilution of sample extracts, showing the good resistance to matrix interference of the Nb-ELSIA. The recovery of spiked cereal samples (rice, oats, barley) ranged from 80% to 105% and the Nb-ELISA results of OTA content in naturally contamined samples were in good agreement with those determined by a commercial ELISA kit. The results indicated the reliablity of nanobody as a promising immunoassay reagent for detection of mycotoxins in food matrix and its potential in biosensor development.

Keywords: Cereal; Immunoassay; Matrix interference; Nanobody; Ochratoxin A.

Fig. 1

(A) SDS-PAGE analysis of Nb28; (B) Western blot analysis of Nb28. Blots were stained with SYPRO Ruby protein gel stain or incubated with rabbit anti-6×his tag IgG/HRP conjugate and TMB membrane peroxidase substrate. Lane M: PageRuler unstained protein ladder (A) and spectrum multicolor broad-range protein ladder (B). Lane 1: Whole-cell extract under noninduced conditions. Lane 2: Whole-cell extract under induced conditions. Lane 3: Nb28 purified by high-capacity nickel IMAC resin.

Fig. 2

Nb-based indirect competitive ELISA standard curves. The error bars represent the standard deviation (n = 3).

Fig. 3

Binding kinetics of Nb15 to the OTA-OVA conjugate. Nbs (25 μg/mL) were immobilized onto the Ni-NTA biosensor (Cat. No.: 18-5101, ForteBio, CA) tips through the 6 × His tag. Then the probe was incubated with OTA-OVA conjugate at different concentrations (3, 10, 30, 90 nM) to determine the corresponding association and dissociation profiles of Nbs. Interferometry data were globally fit to a 1:1 binding model calculating the rate constants (k_a and k_d) and equilibrium dissociation constant K_D (K_D = k_d/k_a). The embedded table includes the value of k_a, k_d, K_D for nanobodies (Nb15, Nb28, Nb32, Nb36) to the OTA-OVA conjugate.

Fig. 4

Temperature stability of OTA-specific nanobodies Nb15, Nb28, Nb32, Nb36 and mAb 6H8. Nanobodies and mAb 6H8 were diluted to working conditions and incubated at increasing temperatures (25, 50, 65, 80, 95 °C) for 5 min (A) or at 90 °C for increasing times (0, 5, 15, 30, 45, 60, 75 min). After re-equilibated to RT, their reserved activity was determined by Nb-ELISA. The error bars represent the standard deviation (n = 3).

Fig. 5

Standard competitive inhibition curve for OTA Nb-ELISA under the optimized conditions. The error bars represent the standard deviation (n = 6).

Fig. 6

Standard curves of Nb-ELISA (A) and mAb-based ELISA (B) for OTA in rice, oats and barley samples after appropriate extration and dilution. Each assay was carried out in triplicate.