In this study, forchlorfenuron (CPPU) was coupled with succinic anhydride to yield a CPPU hapten (CPPU-COOH), and a high-affinity monoclonal antibody (mAb) that can specifically recognize CPPU was produced. Using this mAb as a recognition reagent, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) for CPPU was optimized, which exhibits an IC50 of 1.04 ng/mL, a limit of detection of 0.16 ng/mL, and a linear range of 0.31–3.43 ng/mL for CPPU. Cross-reactivity percentages with six analogues were all below 6%. The average recovery rates for cucumber and orange samples were from 85.23% to 119.14%. The analysis results of this icELISA showed good consistency with those from liquid chromatography mass spectrometry. These results suggest that the proposed icELISA provides a sensitive, specific, and reliable strategy for CPPU detection in food samples.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.