Development And Application Of Monoclonal Antibodies Against Zearalenone

Zearalenone(ZEN) is a secondary metabolite mainly produced by members of Fusarium graminearum while growing in maize, grains and feed. It can exert estrogenic effects on the mammalian reproductive system by contaminating various foods, feeds and animal tissues. ZEN is a small active molecular which is regarded as a hapten. In our study, ZEN-bovine serum protein(BSA) conjugation and ZEN-ovalbumin(OVA) conjugation were preparared by chemical method. In ZEN-BSA, the molar ratio of zearalenone to BSA was approximate 6:1 which was determined spectrophotometrically. We found ZEN-BSA had good immunogenicity by detecting the sera antibodies of the immunized BALB/c mice.The 8-week-old female BALB/c mice were immunized four times with 50μg ZEN-BSA interval two weeks. Spleen cells of immunized BALB/c mice were fused with SP2/0-Ag-14 myeloma cells three days after boosted immunization via tail vein. Using ZEN -OVA, ZEN -BSA and BSA as screening antigen, three hybridomas secreting momoclonal antibodies(McAbs) against ZEN were obtained by indirect enzyme-linked immunosorbent assay(iELISA) and designated as 2C7, 3B10 and 3D5 respectively. The ascites titers of 2C7 and 3B10 were higher than 1:1 × 106 and 3D5 was only 1:10240 in iELISA with the antigen of ZEN-BSA. While the ascites titers of 2C7 and 3B10 were 1:1600 and 3D5 was higher than 1:1×106 by using the plates coated with ZEN-OVA. 2C7 and 3D5 belonged to IgM subtype, and 3B10 belonged to IgG2b subtype. By block ELISA detecting, we found that 2C7, 3B10 and 3D5 were specific to ZEN.Using HRP - ZEN conjugation as the competitive antigen, an competitive ELISAs(cELISAs) based on 2C7, 3B10 and 3D5 were developed as the ELISA kits for immunodetection of ZEN. The limit of detection of the ELISA kits based on 2C7 and3D5 were lng/ml and 25 ng/ml respectively, while the 3B10 was higher than 100ng/ml. The block ELISA kits based on 2C7 and 3D5 were developed and then developed the standard curves. Concluding from the curves, the limit of detection of them were 0.1 ng/ml and lng/ml respectively, and the linear range for ZEN by the methods were between 0.1 - 10ng/ml and between 1 - 100ng/ml respectively.cELIS A was easy to operate and better for ZEN rapid detection, while it was hard to obtain good ZEN-HRP conjugation, and HRP was easy to inactivate in conjugation process, therefore, it restricted the development of cELISA. Although block ELISA was labourious and time-consuming, it was more sensitive than cELISA and did not need conjugating ZEN to HRP. In conclusion, block-ELISA was a very promising method for ZEN immunodetection. The various factors and conditions for ELISA kits were to be explored, and the optimal reaction conditions need to be determined in further research.