| Deoxynivalenol(DON),a secondary metabolite,is abundantly generated by Fusarium graminearum and widly contaminates cereal such as barley,wheat,corn and their products.China is one of the most seriously contaminated areas particularly in the rainy years.DON can cause people nausea, vomiting, stomach discomfort, reproductive system damaged and even with carcinogenicity, teratogenicity and mutagenicity. Therefore many methods detecting DON were established by researchers all over the world in recent years. Immunoassay become one of the most important way to detect DON because of its simple, fast, low cost, high specificity. However, immunoassays of small molecules such as DON usually need coating antigen or competitive antigen that traditional synthetic process usually need professional operators, complex separation and purification process and using a large number of organic reagents which have serious adverse effects on the health of operators and the protection of environment. At the same time,the using of DON at the synthetic process of antigen is also dangerous.This study, DON detection antigen, named D8-MBP, was biosynthesised by selecting from Ph.D.-12 Phage Display Peptide Library with anti-DON monoclonal antibody as target and prokaryotic expression. In addition, ELISA and GICA were developed for detecting DON in corn, wheat and feedstuff based on D8-MBP. It is proved by the experimental results that DON-BSA can be completely replaced by D8-MBP for immunoassay.1 Selection of DON mimotopesA phage clone, named D8, that can compete antibody with DON was obtained by three-round biopanning based on Ph.D.-12 Phage Display Peptide Library. During the biopanning, some restrict measures, for instance chaging the way of elution(the first round taking acid elution, the second and the third rounds taking competive elution), reducing the concentration of coating antibody(100 ?g/m L~50 ?g/m L),increasing the concentration of Tween-20(0.1% ~ 0.5%),changing blocking agent alternately(BSA?OVA) etc, were taken for obtaining high affinity clones. The half inhibition concentration(IC50) and linear range of phage-ELISA based on D8 were 44.70 ± 0.88 ng/m L and 8.24~231.19 ng/m L, respectively.2 Biosynthesis of DON antigenThe genes of D8 were amplified by PCR and cloned into PMAL-PIII for constructing the expression vector. And then, biosynthesis DON antigen was obtained by transforming the xpression vector into E.coli TB1, inducing by IPTG and purification.3 Building of ELISA and GICA based on D8-MBP3.1 Building of ELISA based on D8-MBPIonic strength and p H value of buffer solution of ELISA based on D8-MBP was optimized. The results showed that 7.4 of p H and 10 m M of ionic strength were the optimized condition. In addition, the activity of D8-MBP can not decrease obviously when it was heat treating in 37 ?for a long time but can decrease obviously when it was heat treating in 60 ? or 90 ? as time goes on. The half inhibition concentration(IC50),linear range and limit of detection of ELISA based on D8-MBP were 57.98±0.97 ng/m L,11.32~286.77 ng/m L,9.83 ng/m L respectively which was about two-fold sensitivity compare with DON-BSA with the same antibody.3.2 Building of GICA based on D8-MBPGold nanoflowers(Au NFs) were synthesized by two step method and characterized by transmission electron microscope(TEM).The diameter of Au NFs were about 75 nm.And then GICAbased on D8-MBP was established.The cut off value of GICA based on D8-MBP was 25 ng/m L which was two-fold sensitivity compare with DON-BSA(cut off value:50 ng/m L) with the same antibody.4 The actual sample testingCorn, wheat, feedstuff each of 15 samples that saved by our laboratory were analysed using D8-MBP-ELISA, D8-MBP-GICA and commercial ELISA kit, respectively. The results showed that the three methods were in high agreement with each other. More over, The coefficients of correlation R2 between D8-MBP-ELISA and commercial ELISA kit was 0.973. It was demonstrated that DON-BSA can be replaced in immunoassay. |
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