Development of a Highly Sensitive and Specific Monoclonal Antibody Based on Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Zearalenone in Food and Feed Samples

Abstract

Zearalenone (ZEN) contamination in food and feed is prevalent and has severe effects on humans and animals post-consumption. Therefore, a sensitive, specific, rapid, and reliable method for detecting a single residue of ZEN is necessary. This study aimed to establish a highly sensitive and specific ZEN monoclonal antibody (mAb) and an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of ZEN residues in food and feed. The immunogen ZEN-BSA was synthesized via the amino glutaraldehyde (AGA) and amino diazotization (AD) methods and identified using 1H nuclear magnetic resonance (1H NMR), a high-resolution mass spectrometer (HRMS), and an ultraviolet spectrometer (UV). The coating antigens ZEN-OVA were synthesized via the oxime active ester (OAE), formaldehyde (FA), 1,4-butanediol diglycidyl ether (BDE), AGA, and AD methods. These methods were used to screen the best antibody/antigen combination of a heterologous icELISA. Balb/c mice were immunized with a low ZEN-BSA dose at long intervals and multiple sites. Suitable cell fusion mice and positive hybridoma cell lines were screened using a homologous indirect non-competitive ELISA (inELISA) and an icELISA. The ZEN mAbs were prepared by inducing ascites in vivo. The immunological characteristics of ZEN mAbs were then assessed. The standard curves of the icELISA for ZEN were constructed under optimal experimental conditions, and the performance of the icELISA was validated. The two ZEN-BSA immunogens (conjugation ratios, 11.6:1 (AGA) and 9.2:1 (AD)) were successfully synthesized. Four hybridoma cell lines (2B6, 4D9, 1A10, and 4G8) were filtered, of which 2B6 had the best sensitivity and specificity. The mAb 2B6-based icELISA was then developed. The limit of detection (LOD), the 50% inhibitive concentration (IC50), and the linear working range (IC20 to IC80) values of the icELISA were 0.76 μg/L, 8.69 μg/L, and 0.92–82.24 μg/L, respectively. The cross-reactivity (CR) of the icELISA with the other five analogs of ZEN was below 5%. Three samples were spiked with different concentrations of ZEN and detected using the icELISA. The average intra-assay recoveries, inter-assay recoveries, intra-assay coefficients of variations (CVs), and inter-assay CVs were 93.48–99.48%, 94.18–96.13%, 12.55–12.98%, and 12.53–13.58%, respectively. The icELISA was used to detect ZEN in various samples. The results were confirmed using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) (correlation coefficient, 0.984). The proposed icELISA was highly sensitive, specific, rapid, and reliable for the detection of ZEN in food and feed samples.

A sensitive monoclonal antibody-based ELISA integrated with immunoaffinity column extraction for the detection of zearalenone in food and feed samples

“…In the last twenty years, many ELISAs for the detection of ZEN in food and feed samples based on pAb or mAb have been developed. [19][20][21][22][23][24][25][26][27][28][29][30][31] As shown in Table 1, the IC 50 (or LOD) value achieved in our ELISA is about 1.6-308.1 times lower than those obtained in other ELISAs, which clearly demonstrates the high sensitivity of our ELISA. The high sensitivity of the newly produced mAb may be related to the coupling ratio in the immunogen (ZEN-BSA) and in the coating antigen (ZEN-OVA).…”

“…Also, a variety of polyclonal antibody ( pAb)-or monoclonal antibody (mAb)-based indirect competitive ELISAs (ic-ELISA) and direct competitive ELISAs (dc-ELISA) have been developed for the detection of ZEN in food and feed samples (Table 1). [19][20][21][22][23][24][25][26][27][28][29][30][31] However, establishing any kind of immunoassay is mainly dependent upon the quality of the antibody. Producing an antibody with the highest immunoaffinity recognition for a target analyte is always the main goal for immuno-analytical scientists to seek.…”

Preparation of a broad‐specificity antibody against zearalenone and its primary analogues and development of immunoassay of Coicis Semen and related products

“…As shown in Table 3, a detailed summary of previously reported ZEN antibodies that have been detected by ELISA methods, Wang, Wang et al. (2022) used the AGA method to synthesize immunogens to produce antibodies with higher specificity. Wang, Wang, Zhang, Jiang et al.…”

“…Immunization was performed every 2 weeks, for which the immunogen was emulsified with incomplete Freund's adjuvant. After three immunizations, blood was collected from the mice via the retroorbital venous plexus, and the serum titer and specificity were detected by ELISA (Wang, Qin et al., 2022). Mice with high titer and high specificity were selected for enhanced immunization via intraperitoneal injection of 200 µL immunogen.…”

Developments in mycotoxin analysis: an update for 2021-22

“…Research aimed at producing and testing high performance mAbs to improve immunoassay sensitivity and specificity is still ongoing. Wang et al (2022c), developed a high-quality anti-ZEN mAb starting from the synthesis of two new immunogens. The immunogen synthesis is a critical step in the preparation of highly specific antibodies, therefore specific attention was paid to this.…”