Study On Establishing Simultaneous Detection For Three Kinds Of Mycotoxin By Membrane Immune-chip And Preparation Of Anti-GM Polyclonal Antibodies

1 Mycotoxins are toxic metabolites produced by some fungis, which has been encountered as a natural contaminant in foods and feeds. Once entering human's body, it may cause serious danger, such as deformity, mutation or cancer and so on. In pres-ent, the commonly used methods to detect mycotoxins are thin layer chromatography, high performance liquid chromatography(HPLC) and enzyme-linked immunosorbent assay(ELISA). Membrane immune-chip is a kind of protein-chip. It is a technique that involves antigen or antibody on membrane. This method is not only quick and multi-component detection, but also easily to interpret the results. So, this method is very suitable for large samples detected on site. Based on three prepared mycotoxins (AFB1, OTA and ZEN) artificial antigen and related monoclonal antibody, this research adopts similar indirect competitive ELISA operating mode, to do research into simultaneous detection of AFB1,OTA and ZEN on membrane immune-chip. The results are as follows:(1) Respectively study of AFB1, OTA and ZEN immunoassay on membraneTo PVDF membrane as the solid phase, using a simple spotting method for optimization of loading buffer, fixed method of coated antigen and blocking mode. The results was:Tris-HCl buffer(containing 10% glycerin and 0.1% trehalose, pH 8.5) as the loading buffer, incubating in wet chamber for 2h at 37℃, blocking with 5% skim milk for 1h at 37℃. On the basis of above, experiment was carried out for determination of the lattice concentration of AFB1, OTA and ZEN artificial antigen, and the response concentration of anti-AFB1,OTA and ZEN McAb, then the detection threshold of the three were determined. The results are as follows:lattice concentration of AFB1 antigen was 125μg/mL, concentration of anti-AFB1 McAb was 1/2000, the detection threshold of AFB1 was 2.0 ng/mL; lattice concentration of OTA antigen was 10μg/mL, concentration of anti-OTA McAb was 1/5000, the detection threshold of OTA was 2.0 ng/mL; lattice concentration of ZEN antigen was 40μg/mL, concentration of anti-ZEN McAb was 1/5000, the detection threshold of ZEN was 3.0 ng/mL.(2) The method of simultaneous detection of AFB1,OTA and ZEN by membrane immune-chip was establishedThis method was established basing on the first results. The detection threshold of the chip for AFB1/OTA/ZEN were 2.0/2.0/3.0 ng/mL. We found that the results was not significantly affected when 20% of the methanol concentration in buffers. The specificity of the membrane chip was proved by cross-reactivity with other mycotoxins such as deoxynivalenol and fumonision B1.The repeatability of the membrane chip was well conformed by several repeated and the the membrane chips were still in use when stored at 4℃for 90 days.(3) Comparison of membrane immune-chip and ELISA12 maize samples were both detected by the membrane immune-chip and Commercial ELSIA kits(AFB1,OTA and ZEN respectively).3 samples were AFB1 positive,4 samples were ZEN positive, of these 2 samples were interpretated as both AFB1 and ZEN positive, and OTA was not dectected. All of the results were in consistant with ELISA.2 Aminoglycosides antibiotics are frequently used antibiotics with broad spectrum of antibacterial activity, mostly including Gentamicin(GM), Streptomycin(SM) and Kanamycin(KM) etc. They are frequently added into feeds for the prevention or treatments of animal diseases. However, illegal administration will lead to residues in edible tissues, especially in the milk, and finally do latent harm to human health. So the timely test of the residue of these drugs is an effective way to prevent and control it. In order to establish the method of immunological detection of gentamicin, the research conducts the preparation for anti-gentamicin polyclonal antibodies. The results are as follows:The immunogen GM-BSA was synthesized by the coupling of gentamicin (GM) and bovine serum albumin (BSA) with carbodiimide as a coupling agent. The coated antigen GM-GA-OVA was synthesized with glutaraldehyde(GA) as a link arm. Both the antigen were identified successfully by SDS-PAGE. Balb/c mice were immunized with GM-BSA. By strengthening immunization four times, high titre(up to 1: 2.56×105)was obtained in all 9 mice which was detected by indirect ELISA. Indirect competitive ELISA was used to test the specificity of antiserum. Competitive inhibition curve indicates that the linear range of calibration curves was 1.0 ng/mL-100.0 ng/mL. The curve equation is y=-15.596 In (x)+85.847, coefficient correlation R2=0.9985, its IC50 is 10.2 ng/mL.