| Summary: | A novel and efficient immunoaffinity column (IAC) based on bispecific monoclonal antibody (BsMAb) recognizing aflatoxin B<sub>1</sub> (AFB<sub>1</sub>) and ochratoxin A (OTA) was prepared and applied in simultaneous extraction of AFB<sub>1</sub> and OTA from food samples and detection of AFB<sub>1</sub>/OTA combined with ic-ELISA (indirect competitive ELISA). Two deficient cell lines, hypoxanthine guanine phosphoribosyl-transferase (HGPRT) deficient anti-AFB<sub>1</sub> hybridoma cell line and thymidine kinase (TK) deficient anti-OTA hybridoma cell line, were fused to generate a hybrid-hybridoma producing BsMAb against AFB<sub>1</sub> and OTA. The subtype of the BsMAb was IgG<sub>1</sub> via mouse antibody isotyping kit test. The purity and molecular weight of BsMAb were confirmed by SDS-PAGE method. The cross-reaction rate with AFB<sub>2</sub> was 37%, with AFG<sub>1</sub> 15%, with AFM<sub>1</sub> 48%, with AFM<sub>2</sub> 10%, and with OTB 36%. Negligible cross-reaction was observed with other tested compounds. The affinity constant (Ka) was determined by ELISA. The Ka (AFB<sub>1</sub>) and Ka (OTA) was 2.43 × 10<sup>8</sup> L/mol and 1.57 × 10<sup>8</sup> L/mol, respectively. Then the anti-AFB<sub>1</sub>/OTA BsMAb was coupled with CNBr-Sepharose, and an AFB<sub>1</sub>/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, <i>v</i>:<i>v</i>, pH 2.3) containing 1 mol/L NaCl, and the eluent volume was 4 mL. The column capacities of AFB<sub>1</sub> and OTA were 165.0 ng and 171.3 ng, respectively. After seven times of repeated use, the preservation rates of column capacity for AFB<sub>1</sub> and OTA were 69.3% and 68.0%, respectively. The ic-ELISA for AFB<sub>1</sub> and OTA were applied combined with IAC. The IC<sub>50</sub> (50% inhibiting concentration) of AFB<sub>1</sub> 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 IC<sub>50</sub> 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 AFB<sub>1</sub>-OTA IAC. The recovery rates of AFB<sub>1</sub> 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 AFB<sub>1</sub> and OTA in grains.
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