| Deoxynivalenol (DON) produced by Fusarium, is a mycotoxin, which is generally existed in food and feed. DON can cause disease in human and several animal species. Ingestion of contaminated grain or by products of exposed animals causes severe long-term illness, including acute toxicity, reproduction toxicity, neurotoxicity, and immunosuppression. Because of the importance of this toxin economically and from a food safety standpoint a variety of analytical techniques have been developed for detection of DON and related trichothecene mycotoxins in foods. The first methods developed for the determination of trichothecenes in grain were based on thin layer chromatography (TLC). Other commonly reported methods include Gas Chromatography(GC) High Pressure Liqaid Chromatography, (HPLC). Although sensitive and accurate, most of the chromatographic methods developed are laborious, expensive, time-consuming, and unsuitable for the analysis of many samples in a short time. They also require sophisticated equipment and extensive clean-up procedures.Antibody-based techniques provide a simple and economical alternative to instrumental methods for mycotoxin analysis. Immunochemical methods are gaining wide acceptance as they offer the advantage of sensitivity, specificity, rapidity, simplicity and cost effectiveness, which is important for routine testing.The McAb against DON and ciELISA method successfully developed based on the McAb. It set basis for the production of DON detection kit.1 According to molecular structure of DON. DON was coupled to protein carriers, bovine serum albumin(BSA) and ovalbuhnin(OVA) by CDI method and the mixed acid anhydride method in different reaction conditions respectively. The conjugate were identified by full-wavelengh ultraviolet scanner and SDS-PAGE, the result showed that the conjugate were successfully coupled. The conjugation molecular ratio of DON with BSA and OVA were about 13.16:1 and 10.53:1 respectively. The two artificial antiens supplied ideal materials for the perparation of McAb against DON.2. The BABL/c mice was immunized with 3-HS-DON-BSA.7 days later using the best immunized BALB/c for strengthen immunization, and then the monoclonal antibodies (McAb)against DON was studied. The results showed one hybridoma cell line named 3D6 was obtained, which grew stably and secreted high titer anti-DON McAb, the titers of anti-DON McAb in cell culture supernatants and ascites were 1:800 and 1:51200 respectively, the McAb against DON belongs to IgG1, the concentration of DON that inhibits 50% of antigen-antibody binding(the value of IC50)was 53μg L-1. The cross-reaction of the ascites to T-2 is 6.22%(less than 10%).3. To improve the stability of immunoassay analysis methods for DON residues detection, saturated ammonium sulfate method was employed to purify the anti-DON McAb in ascites, and the purified McAb proved to be with good purification and reactive ability identified by indirect ELISA. Using artificial antigen 3-HS-DON-OVA as coating antigen, an indirect competitive inhibition ELISA(ciELISA)method determination of DON was developed through the optimization of the dilution of coating antigen, anti-DON McAb, and reaction condition, and other components respectively. Then the competitive inhibitory indirect ELISA standard curve equation was:Y=-41.447X+134.64 (R2=0.9915). The detection limit was 10μg L-1. Samples with DON levels from 10 to 1000μg L-1. The coefficient of variation in group of this method is 0.61% while the coefficient of variaton among groups is 3.28%. It can be used to detection the DON in actual application. |
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