One-Step Chemiluminescent Immunoassay for the Key Synthesis Protein of Ochratoxin A Using a Shark Single-Domain Antibody-Alkaline Phosphatase Fusion Protein.

Abstract

Basic leucine zipper (BZIP) proteins constitute one of the largest and most diverse families of transcription factors and play a crucial role in OTA biosynthesis by regulating the expression of genes within the biosynthetic gene cluster. As a key regulatory protein, BZIP holds promise as an early biomarker for identifying OTA-producing fungi before toxin accumulation occurs, thereby helping to prevent contaminated products from entering the food supply chain. Despite its potential significance, there have been no comprehensive reports describing the development of BZIP-specific antibodies. Herein, we generated a variable new antigen receptor-alkaline phosphatase (VNAR-ALP) fusion and developed a VNAR-ALP fusion-based chemiluminescent immunoassay (VACLIA) for BZIP detection. The optimized VACLIA exhibits a limit of detection of 0.058 μg/mL and a linear range of 0.3125-5 μg/mL, along with good selectivity. Recovery experiments conducted using spiked maize and coffee samples at different BZIP concentrations demonstrated high accuracy and reliability, with recoveries ranging from 102.50% to 111.39% and relative standard deviations not exceeding 12%. The detection of actual maize samples using liquid chromatography-tandem mass spectrometry showed a strong correlation with the results obtained from VACLIA, further validating the assay's effectiveness in real sample analysis. A comparative analysis of VACLIA with other global food safety monitoring systems highlighted its unique capability to identify potential hazards, provide timely alerts, and effectively mitigate OTA risk in food products. Overall, the developed VACLIA exhibited robust performance, high selectivity, and broad applicability in the detection of real samples, demonstrating its potential as a valuable tool for food safety monitoring.