Preparation of an Immunoaffinity Column Based on Bispecific Monoclonal Antibody for Aflatoxin B(1) and Ochratoxin A Detection Combined with ic-ELISA

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.