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

Foods

Disha Lu¹  Xu Wang¹  Lin Luo¹  Zhili Xiao¹  Ruijue Su¹  Yongjian Cheng¹  Hong Wang¹ 

[1] Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou 510642, China;

关键词: aflatoxin B₁;    ochratoxin A;    bispecific monoclonal antibody;    immunoaffinity column;    ic-ELISA;   

DOI  :  10.3390/foods11030335

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown