Development Of Several Immunological Examination Methods Based On Monoclonal Antibodies For Detection Of 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, an indirect competitive ELISA, Immunomagnctic beads ELISA and chemiluminecense ELISA 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 ovalbumin(OVA) for use as a coating antigen for the ELISAs. According to the similar methods, FB1 was connected to keyhole limpet hemocyanin (KLH) for use as immunogen which was used to immunize 8-week-old BALB / c mouse to develop the monoclonal antibody. After three times of subcloning, 2 hybridoma cell lines stably secreted specific antibody against FB1 were screened by indirect competitive ELISA. The immunological sub-type IgG1κof the monoclonal antibody was determined. The antiserum was purified by proteinG chromatography respectively. The ascites monoclonal antibodies with titre l: 16000 were purified and characterized by SDS-PAGE. It showed that the purified antibody chromatography had higher purity and titer. AflatoxinB1, Chlorpromazine , streptomycin and Zearalenone showed no cross-activity with monoclonal antibodies.Indirect competitive ELISA(IC-ELISA) , Immunomagnctic beads ELISA(IMB-ELISA) and chemiluminescence ELISA(CL-ELISA) were established respectively on the base of specific antibody against FB1. ELISA is widely used detection method. There are two competition models in IC-ELISA: Direct antibody or antigen coated ELISA and indirect competition ELISA. IC-ELISA on condition that antigen coated for 4h under 37℃, concentration of coating antigen was 0.5μg/mL, anti-FB1 monoclonal antibody was diluted to 1:4000 and goat anti-rabbit IgG:HRP was diluted to 1:8000, the detection sensitivity of IC-ELISA was 0.44ng/mL and the detection range was 0.44-73.06ng/mL. Although IC-ELISA is time-consuming,the detection sensitivity and the detection range are better than others.Using immunomagnetic beads(IMBs) as solid-phase carrier coated KLH-FB1. In the IMB-ELISA, the parameters were as follows: Immunomagnetic beads was diluted to 1:20, anti-FB1 antibody was diluted to 1:4000, the goat anti-rabbit IgG:HRP was diluted to 1:10000. The detection sensitivity was 0.24ng/mL and the detection range was 0.54-26.3ng/mL. The good enrichment and fast reaction rate increase the detection sensitivity.In the CL-ELISA, the parameters were as follows: coating antigen was 0.5μg/mL, anti-FB1 antibody was diluted to 1:4000, the goat anti-rabbit IgG:HRP was diluted to 1:30000. The detection sensitivity was 0.13ng/mL and the detection range was 0.28-37.2ng/mL. This method has a good development prospect.The detection sensitivity of the three methods was better than FB1 detection kit. The three methods and FB1 detection kit were used to detect FB1 in samples and recoveries were all over 80% in the detection range respectively.