Preparation of highly sensitive monoclonal antibody against α-zearalanol based on the similar antigen determinant structure to zearalanone

Abstract

In this study, we report a new method to prepare highly sensitive monoclonal antibody against α-zearalanol (ZAL) based on a similar antigen determinant structure. Zearalanone (ZAN), structural analogs of ZAL, was modified by oximation to obtain ZAN-O. ZAN-O was then coupled with bovine serum albumin using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) to synthesise the artificial complete antigen ZAN-O-BSA. ZAN-O-BSA was used to immunise the BALB/c mice. The splenocytes of the immunised mice were fused with myeloma NS0 cells. During the process of cell fusion, ZAL was used as an inhibitor instead of ZAN to screen the hybridoma cell lines that can secrete monoclonal antibodies against ZAL. The sensitivity (half inhibitory concentration, IC50) of the prepared monoclonal antibody was 0.475 ng/ml, the limit of detection (LOD) was 0.050 ng/ml, the linear range of detection was 0.066-3.399 ng/ml, the affinity constant Kaff was 6.18×107 l/mol, the cross-reactivity rate with structural analogues, such as β-zearalanol, α-zearalenol, β-zearalenol, ZAN and zearalenone were 28.07, 13.16, 15.83, 60.28 and 7.95% respectively. The cross-reactivity with other mycotoxin and carrier proteins were all less than 0.05%. The prepared monoclonal antibody can be used to establish a highly sensitive immunoassay for the detection of ZAL.