Preparation of Monoclonal Antibody against Deoxynivalenol and Development of Immunoassays.

Fusarium toxins are the largest group of mycotoxins, which contain more than 140 known secondary metabolites of fungi. Deoxynivalenol (DON) is one of the most important compounds of this class due to its high toxicity and its potential to harm mankind and animals and a widespread contaminant of agricultural commodities, such as wheat, corn, barley, oats, bread, and biscuits. Herein, a hybridoma cell 8G2 secreting mAb against DON was produced by fusing the splenocytes with a tumor cell line Sp2/0. The obtained mAb had a high affinity (2.39 × 109 L/mol) to DON. An indirect competitive Enzyme-Linked Immunosorbent Assay (ic-ELISA) showed that the linear range for DON detection was 3.125-25 µg/mL, and the minimum inhibitory concentration (IC50) was 18.125 µg/mL with a limit of detection (LOD) of 7.875 µg/mL. A colloidal gold nanoparticle (AuNP) with 20 nm in diameter was synthesized for on-site detection of DON within 10 min with vLOD of 20 µg/mL. To improve the limit of detection, the gold nanoflower (AuNF) with a larger size (75 nm) was used to develop the AuNF-based strip with vLOD of 6.67 µg/mL. Compared to the vLOD of a convectional AuNP-based strip, the AuNF-based strip was three times lower. Herein, three immunoassay methods (ic-ELISA and AuNP/AuNF-based strips) were successfully developed, and these methods could be applied for the DON detection in agricultural products.

Development of ELISA and Lateral Flow Immunoassays for Ochratoxins (OTA and OTB) Detection Based on Monoclonal Antibody.

Ochratoxins were important secondary metabolites secreted by fungi, and OTA and OTB are mainly significant mycotoxin, having toxic effects on humans and animals. Therefore, it is important to establish a rapid, sensitive, and precise method for ochratoxins detection and quantification in real samples. In this study, a stable monoclonal antibody (mAb) that recognizing both OTA and OTB toxins was employed for the establishment of indirect competitive ELISA (ic-ELISA), colloidal gold nanoparticles (CGNs), and nanoflowers gold strips (AuNFs) for detection of ochratoxins in real samples. A 6E5 hybridoma cell line stable secreting mAb against both OTA and OTB toxins was obtained by fusion of splenocytes with myeloma SP2/0 cells. The 6E5 mAb had a high affinity (3.7 × 108 L/mol) to OTA, and also showed similar binding activity to OTB. The optimized ic-ELISA resulted in a linear range of 0.06-0.6 ng/mL for ochratoxins (OTA and OTB) detection. The IC50 was 0.2 ng/mL and the limit of detection (LOD) was 0.03 ng/mL. The mean recovery rate from the spiked samples was 89.315 ± 2.257%, with a coefficient variation of 2.182%. The result from lateral flow immunoassays indicated that the LOD of CGNs and AuNFs were 5 and 1 µg/mL, respectively. All these results indicated that the developed ic-ELISA, CGNs, and AuNFs in this study could be used for the analysis of the residual of ochratoxins (OTA and OTB) in food and agricultural products.