Preparation of an Immunoaffinity Column Based on Bispecific Monoclonal Antibody for Aflatoxin B1 and Ochratoxin A Detection Combined with ic-ELISA

Abstract

A novel and efficient immunoaffinity column (IAC) based on bispecific monoclonal antibody (BsMAb) recognizing aflatoxin B1 (AFB1) and ochratoxin A (OTA) was prepared and applied in simultaneous extraction of AFB1 and OTA from food samples and detection of AFB1/OTA combined with ic-ELISA (indirect competitive ELISA). Two deficient cell lines, hypoxanthine guanine phosphoribosyl-transferase (HGPRT) deficient anti-AFB1 hybridoma cell line and thymidine kinase (TK) deficient anti-OTA hybridoma cell line, were fused to generate a hybrid-hybridoma producing BsMAb against AFB1 and OTA. The subtype of the BsMAb was IgG1 via mouse antibody isotyping kit test. The purity and molecular weight of BsMAb were confirmed by SDS-PAGE method. The cross-reaction rate with AFB2 was 37%, with AFG1 15%, with AFM1 48%, with AFM2 10%, and with OTB 36%. Negligible cross-reaction was observed with other tested compounds. The affinity constant (Ka) was determined by ELISA. The Ka (AFB1) and Ka (OTA) was 2.43 × 108 L/mol and 1.57 × 108 L/mol, respectively. Then the anti-AFB1/OTA BsMAb was coupled with CNBr-Sepharose, and an AFB1/OTA IAC was prepared. The coupling time and elution conditions of IAC were optimized. The coupling time was 1 h with 90% coupling rate, the eluent was methanol–water (60:40, v:v, pH 2.3) containing 1 mol/L NaCl, and the eluent volume was 4 mL. The column capacities of AFB1 and OTA were 165.0 ng and 171.3 ng, respectively. After seven times of repeated use, the preservation rates of column capacity for AFB1 and OTA were 69.3% and 68.0%, respectively. The ic-ELISA for AFB1 and OTA were applied combined with IAC. The IC50 (50% inhibiting concentration) of AFB1 was 0.027 ng/mL, the limit of detection (LOD) was 0.004 ng/mL (0.032 µg/kg), and the linear range was 0.006 ng/mL~0.119 ng/mL. The IC50 of OTA was 0.878 ng/mL, the LOD was 0.126 ng/mL (1.008 µg/kg), and the linear range was 0.259 ng/mL~6.178 ng/mL. Under optimum conditions, corn and wheat samples were pretreated with AFB1-OTA IAC. The recovery rates of AFB1 and OTA were 95.4%~105.0% with ic-ELISA, and the correlations between the detection results and LC-MS were above 0.9. The developed IAC combined with ic-ELISA is reliable and could be applied to the detection of AFB1 and OTA in grains.

Determination of Aflatoxin B1 in Grains by Aptamer Affinity Column Enrichment and Purification Coupled with High Performance Liquid Chromatography Detection

“…For example, the European Union (EU) has set the maximum level of AFB 1 at 5.0 μg kg −1 in maize or rice, and 2.0 μg kg −1 in other cereals. In China, the mandated maximum level for AFB 1 ranges from 5.0 to 20 μg kg −1 in different cereals [ 7 ]. The presence of AFB 1 raises numerous health concerns in humans and livestock, as it exhibits mutagenic, teratogenic, carcinogenic, neurotoxic, and immunosuppressive properties [ 8 ].…”

Target-Induced Electrochemical Sensor Based on Foldable Aptamer and MoS2@MWCNTs–PEI for Enhanced Detection of AFB1 in Peanuts

“…At present, the determination methods of AFB1 reported at home and abroad mainly incorporate HPLC (high-performance liquid chromatography), , ELISA (enzyme linked immunosorbent assay), SPR (surface plasmon resonance), SERS (surface-enhanced Raman scattering), fluorescence, colorimetry assays, etc. Nevertheless, the poor portability, relatively high cost, and demand for a skilled operator have limited their applications for portable analysis.…”

Construction of Fluorescence Sensing Platform on the Basis of Molybdenum Disulfide Nanosheet for the Detection of AFB<sub>1</sub>

“…The current research achieves the rapid and sensitive quantitative detection of AFB 1 with specificity for serum and soybean milk. Aflatoxins are currently detected using thin-layer chromatography (LC)[23] [24], high performance liquid chromatography (HPLC)[25] [26][27] [28], and enzyme linked immunosorbent assays (ELISA)[29] [30]. The above detection methods have several drawbacks, including cumbersome operation, expensive detection instruments and equipment, and time-consuming and laborintensive issues.…”