Abstract:

Objective      To prepare monoclonal antibodies(McAbs) against deoxynivalenol（DON）and characterize their properties. Methods     Using a low-dose and longcycle immunization scheme, female BALB/c mice were immunized with DON coupled to bovine serum albumin(DON-BSA). Hybridoma cell lines were obtained by cell fusion. McAbs in the supernatant were determined by ELISA. Hybridoma cells which secreted McAbs to DON-BSA were cloned again and again until the McAb-positive rate reached 100%, and the specificity of McAbs to DON was further confirmed by competitioninhibition ELISA, and a large amount of McAbs was made by ascites in liquid paraffinprimed mice and purified by saturated ammonium sulfate. The subtype of the McAb was tested by a subtype identification ELISA kit. The protein concentration was tested by a BCA kit. The titer, work concentration for reference and affinity constant of the McAb were measured by ELISA. Results      The titer of antibodies in sera from Balb/c mice immunized by DON-BSAwas 1∶256000 and all had a strong cross-reaction with BSA. Hybridoma cells which secreted McAbs against DONBSA were screened by ELISA after cell fusion.  A hybridoma cell line secreting McAb against DON-BSA was established by 3 rounds of cloning and named 3G5. A large amount of McAbs was obtained by ascites production. The subtype of the McAb belongs to IgG1, the concentration of IgG was 6.06g/L; the McAb titer was 1∶ 500000, the McAb work concentration for reference was 1∶64000 and the McAb affinity constant was 1.62×109. The linear range of the competitive inhibition ELISA calibration curve was from 9.8 to 10000ng/mL, and the linear regression equation was Y=-0.2726X+0.2259(r=0.9309). Conclusion     The hybridoma cell line secreting McAbs against deoxynivalenol is obtained, and the prepared antibody showed high sensitivity and specificity to DON, so it can be used to produce a homemade DON-ELISA kit of high quality.

Key words: Deoxynivalenol; Monoclonal antibody; Characterization; Affinity constant