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

Abstract

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 × 10 8 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.

Development of Immunochromatographic Strip for Detection of αB-VxXXIVA-Conotoxin Based on 5E4 Monoclonal Antibody

“…Colloidal gold nanoparticles (AuNPs) could easily be observed to conjugate with the antibody. The immunochromatographic strip test, based on colloidal gold nanoparticles, was successfully applied in the detection [ 21 ]. A schematic diagram of the immunochromatographic strip test is shown in Figure 2 , to explain the working mechanism.…”

Detection of αB-Conotoxin VxXXIVA (αB-CTX) by ic-ELISA Based on an Epitope-Specific Monoclonal Antibody

“…Moreover, complicated and tedious operation steps, high requirement of samples and detection practicability seriously limits their application in the detection of αB-CTX in real samples [ 19 ]. In contrast, immunoassays with high sensitivity, accuracy and adaptability have been widely used to detect the targets (toxins, pathogenic molecules and heavy metals) in different samples [ 20 , 21 , 22 ]. In the previous study, we developed ELISA and colloidal test strips based on monoclonal antibodies (mAb) against ω-CTX to detect ω-CTX MVIIA residue in Conus samples [ 5 ].…”

Comparative Assessment of Different Gold Nanoflowers as Labels for Lateral Flow Immunosensors

“…This principle provides quick and easy-to-obtain assay results and determines the practical demand for LFIA tests [18]. Many studies have successfully used GNFs as antibody carriers and detectable markers for LFIA using decreased LODs, usually from 4 to 10 times compared to LFIAs using common GNSs [19][20][21][22][23]. However, most of these works were limited by the consideration of a single GNF preparation without substantiating the nanoparticle size and shape requirements and the grounded choice of a method for their synthesis.…”