Prepration Of Artificial Antigen And Establishment Of Protein Chip For Determination Of Fumonisins B₁

Mycotoxins are secondary metabolism produced by mycetes, which wildly exist in kinds of grains, especial in corn and the produce of corn, and make them polluted. Fumonisin B1, mainly produced by several fungi, is harmful to human and animals, because of its acute toxicity and subacute toxicity, neurotoxicity, carcinogenicity and so on.People have been paying more and more attention to FBI in the worldwide, because the increasing threaten of FBI to human. There are several methods to detect FBI, including thin layer chromatography (TLC), High Performance Liquid Chromatography (HPLC), gas chromatography (GC), gas chromatography mass spectrometer (GC-MS) and so on.Protein chip technology is a new method which has been developed on proteomics and gene chip. The mechanism of protein chip is:both antigen and antibody are protein, use microarrayer high-density to fix the antigen, which can specific bind with antibody on the surface of glass slides or other carrier, followed by adding the samples and antibody, then added goat anti mouse antibody CY3. Eluted the useless protein, useing indirect competitive mechanism, when the test samples content is high, the antigen-specific antibody reaction is less (fluorescent secondary antibody is also less), afterwards use scanner and software analyze fluorescence signal and signal noise ratio, determine the content of test samples. Compared with traditional methods, Portein chip is miniaturization, integrated, high-pass quantization, high sensitivity, minimal sample requirements and so on.In this study, artificial antigen FB1-OVA was prepared, it was detected and determined concention, which will be useful for preparation of the monoclonal antibody against FB1 and FB1 protein chip. Based on indirect competition detection mechanism, a protein chip method was also constructed, and evaluated by using flourescence signal intensity and signal-noise ratio. Main contents and methods are as follows:1. FB1 were Coupled with carrier protein OVA by GA one-step method, then identified the coupled antigen (FB1-OVA) through SDS-PAGE. Finally, determined its concentration was 1.08mg·mL-1.2. For construction of the protein chip method, FB1-OVA was spotted on glass slide (25℃,75% humidity) by microarrayer first, after fixation and blocking, anti-FB1 antibody and goat anti mouse IgG-CY3 were added,.Finally, fluorescence signal was obtained on scanner at the wave of 553 nm, the collected images was processed by GenePixpro6.0 software. At last, a competitive inhibition curve was made, on the basis of analyzing the fluorescence signal and signal-noise ratio. The optimal concentration of FB1-OVA and anti-FB1 antibody were determined by orthogonal experiment, which were 1:40 dilution (25ug·mL-1) and 1:1200 dilution, respectively. Other optimized response conditions were: fixing at 4℃overnight, blocking with 1% BSA at 37℃for 1h, primary antibody at 37℃for 1 h,2 ug·mL-1 goat anti-mouse IgG-CY3 at 37℃for 1h.