Study On Immunoassay For Fumonisin B1

Fumonisins are a group of mycotoxins which are toxic and carcinogenicity mainly produced by Fusarium moniliforme. It usually happens in grain crops like maize and maize-based food and animal feed. The toxins can cause many diseases such as leukoencephalomalacia in horses, pulmonary edema in pigs and esophageal cancer in humans. It becomes a serious threat to human and animal, so more and more people pay attention on it. Among fumonisins, B₁ is the most abundant and most toxic. In this study, a indirect competitive ELISA, chemiluminecense ELISA and gold colloidal immunochromatography assay were developed to analyze FB₁ in food samples.FB₁ must be connected to a vector protein in order to gain immunogenicity because it's a small molecule which can not stimulate immune response. FB₁ was conjugated using glutaraldhyde to keyhole limpet hemocyanin (KLH) for use as immunogen which was used to immunize New Zealand rabbits. According to the similar methods, FB₁ was connected to ovalbumin(OVA) for use as a coating antigen for the ELISAs. The titer of the antiserum was 1:9600, detected by indirect ELISA. The antiserum was purified by caprylic acid-Ammonium sulfate method and proteinG chromatography respectively. Between the two methods, it showed that the purified antibody by proteinG chromatography had higher purity and titer.Indirect competitive ELISA(IC-ELISA) and chemiluminescence ELISA(CL-ELISA) were established respectively on the base of purified anti-FB₁ antibody. On condition that antigen coated for 4h under 37℃, concentration of coating antigen was 0.5μg/mL, anti-FB₁ polyclonal antibody was diluted to 1:4000 and goat anti-rabbit IgG:HRP was diluted to 1:6000, the detection sensitivity of IC-ELISA was 0.41ng/mL and the detection range was 0.41-33.3ng/mL. Aflatoxin, deoxynivalenol and Zearalenone showed no cross-activity. In the CL-ELISA, the parameters were as follows: coating antigen was 0.5μg/mL, anti-FB₁ antibody was diluted to 1:5000, the goat anti-rabbit IgG:HRP was diluted to 1:40000. The detection sensitivity was 0.14ng/mL and the detection range was 0.14-33.3ng/mL. The two methods and FB₁ detection kit were used to detect FB₁ in samples and recoveries were all over 80% in the detection range respectively. Besides, a preliminary study was conducted on colloidal gold immunochromatography assay and a trip was got to detect FB₁ over 100ng/mL. Although it had a higher limit, it made spot detection easier.