Preparation of a broad-specificity antibody against zearalenone and its primary analogues and development of immunoassay of Coicis Semen and related products.

Abstract

The purpose of this study was to prepare a highly sensitive and specific zearalenone (ZEN) monoclonal antibody, which was then used to develop an indirect enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold immunochromatographic assay (GICA). These techniques were used for the detection of Coicis Semen and related products (Coicis Semen flour, Yimigao, and Yishigao). Immunogens were synthesized by oxime active ester techniques and characterized via ultraviolet spectrophotometry. Immunogens were injected subcutaneously into the abdominal cavities and backs of mice. Using the prepared antibodies, we developed ic-ELISA and GICA rapid detection methods, which were then applied for the rapid detection of ZEN and its analogues from Coicis Semen and related products. For ic-ELISA, the half maximal inhibitory concentration (IC50 ) values for ZEN, α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL), and β-zearalanol (β-ZAL) were determined to be 1.13, 1.69, 2.06, 0.66, 1.20, and 0.94ng•mL-1 , respectively. For GICA, the cutoff values of ZEN, α-ZEL, β-ZEL, α-ZAL, and β-ZAL on test strips were 0.5ng•mL-1 in phosphate buffer saline (0.01M, pH 7.4), while ZAN was found to be 0.25ng•mL-1 . Furthermore, the cutoff values of test strips were between 10 and 20µg∙kg-1 in Coicis Semen and related products. The results of these two detection methods were in good agreement with results from liquid chromatography-tandem mass spectrometry. This study provides technical support for the preparation of broad-specificity monoclonal antibodies against ZEN and lays the foundation for the simultaneous detection of multiple mycotoxins from food and herbal medicines.