Nanobody-Alkaline Phosphatase Fusion Protein-Based Enzyme-Linked Immunosorbent Assay for One-Step Detection of Ochratoxin A in Rice

Abstract

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL^-1 and a limit of detection of 0.059 ng mL^-1, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice.

Keywords: alkaline phosphatase; immunoassay; mycotoxin; nanobody.

Figure 1

Characterization of Nb28-AP by SDS-PAGE. Lane M: Prestained protein ladder. Lane 1: Whole-cell protein before auto-induction. Lane 2: Whole-cell protein after auto-induction. Lane 3: Nickel sepharose column-purified Nb28-AP.

Figure 2

Thermal stability of Nb28 and Nb28-AP. Nb28 and Nb28-AP at working concontrations were incubated at serial temperatures (35, 45, 55, 65, 75, 85, 95 °C) for 5 min (A) or at 90 °C for various lengths of time (0, 5, 15, 30, 45, 60, 75 min) (B). After cooling to room temperature, the reserved activity of Nb28 and Nb28-AP was measured by an indirect ELISA. The error bars represent the standard deviation of three independent tests.

Figure 3

Effects of pH value (A), methanol (B), competitive time (C), and ionic strength (D) on the performance of Nb-AP-based one-step ELISA. The error bars represent the standard deviation of three independent tests.

Figure 4

Standard direct competitive inhibition curve of Nb-AP-based one-step ELSIA for OTA under the optimized conditions. The error bars represent the standard deviation of five independent tests.