摘要：A novel and efficient immunoaffinity column (IAC) based on bispecific monoclonal antibody (BsMAb) recognizing aflatoxin B
1 (AFB
1) and ochratoxin A (OTA) was prepared and applied in simultaneous extraction of AFB
1 and OTA from food samples and detection of AFB
1/OTA combined with ic-ELISA (indirect competitive ELISA). Two deficient cell lines, hypoxanthine guanine phosphoribosyl-transferase (HGPRT) deficient anti-AFB
1 hybridoma cell line and thymidine kinase (TK) deficient anti-OTA hybridoma cell line, were fused to generate a hybrid-hybridoma producing BsMAb against AFB
1 and OTA. The subtype of the BsMAb was IgG
1 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
2 was 37%, with AFG
1 15%, with AFM
1 48%, with AFM
2 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
1) and Ka (OTA) was 2.43 × 10
8 L/mol and 1.57 × 10
8 L/mol, respectively. Then the anti-AFB
1/OTA BsMAb was coupled with CNBr-Sepharose, and an AFB
1/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 AFB
1 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
1 and OTA were 69.3% and 68.0%, respectively. The ic-ELISA for AFB
1 and OTA were applied combined with IAC. The IC
50 (50% inhibiting concentration) of AFB
1 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
50 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
1-OTA IAC. The recovery rates of AFB
1 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
1 and OTA in grains.
