Preparation Of Specific Monoclonal Antibodies And Establishment Of Immunoassay Of Deoxynivalenol

Deoxynivalenol (DON) is a trichothecene mycotoxin produced by Fusarium graminearum, a pathogen causing Fusarium Head Blight of wheat. It is necessary to establish a rapid and simple assay to detect DON. High affinity monoclonal antibodies (Mab) against DON were produced by cell fusion with 500 mg/mL Polyethylene Glycol 400, and cell sub-cloning in HAT (H: hypoxanthine, A: aminopterin; T: thymidine) culture medium for screening and limiting dilution. Hybridoma lines were screened for specificity to DON by Enzyme-linked Immunosorbent Assay (ELISA). One hybridoma cell line (3B2) secreting monoclonal antibody (MAb) against DON was produced by fusing mouse myeloma cells (SP 2/0) with spleen cells from BAL B / C mice which were immunized by the artificial antigen conjugated with Ovalbumin (OVA). The MAb obtained in this experiment could specifically react with DON without cross-reactivity to DON related compounds except 3-acetyldeoxynivalenol (3-Ac-DON), with the titres of ascitic fluids up to 1×10⁷ by indirect ELISA. Isotype and subclass of the monoclonal cell line (3B2) showed that it belonged to IgG1. The light chain of the MAb was identified to beκ. Ascites antibodies generated by hybridoma of 3B2 cells were purified. Inhibition rate studies showed that the detection limit of the ELISA was 11μg/L, IC₅₀ value being 29μg/L. The MAb can be used to prepare the reagents for analyzing DON residue.One of antibody secreted from hybridoma cell lines was selected to be used for further study. By the study of the three factors, including the condition of the concentration of methanol, the icon concentration, the block, it is eventually fixed for the ELISA assay conditions: the concentration of methanol is 8%, the concentration of 3-HS-DON-BSA is 2μg/mL; the ascitic fluids is diluted 1×10^-7 multiple; the block is 2% OVA. An assay curse is formed by OD450 and the negative logarithm of the DON concentration(μg/mL) as the two influencing factors, in the range of 0.01μg/mL-10μg/mL, the two factors represent an apparent linear relation, the equation is as follows: y=0.2396x + 0.3177, the R² reaches 0.9999 and the sensitivity reaches 11μg/mL, the intra-assay CV is 1.2%, the inter-assay CV is 4.7%, the recovery rate from standard DON is 1%, 90.73%,89.30%,86.80% and 80.12%, respectively.