邻苯二甲酸酯类塑化剂免疫检测方法的研究-食品科学专业论文.docxVIP

ABSTRACTAn ABSTRACT An indirect competitive enzyme-·linked immunosorbent assay(ic·-ELISA)for the determination ofphthalate esters(PAEs)in food had been developed． For dimethyl phthalate(DMP)，the hapten 4-amino dimethyl phthalate(4一DMAP)was designed and synthesized．4-nitro dimethyl phthalate was as a material，the nitro group of which was reduced to obtain hapten 4-amino dimethyl phthalate(4-DMAP)．The hapten 4-DMAP was conjugated to bovine serum albumin(BSA)by diazotization to prepare immunogen 4-DMAP-BSA，then the hapten was conjugated to ovalbumin(OVA)by glutaraldehyde method to prepare coating antigen 4-DMAP-OVA．The antisera against dimethyl phthalate were obtained and characterized after immunizing four New Zealand white rabbits with immunogen 4-DMAP—BSA．The best antiserum was generated by rabbit No．2 with the highest titer l：256000 and the inhibition rate up to 87％to 1 000 pg／L DMP． By optimizing the working conditions of ELISA．we selected O．03 lxg／well coating concentration of coating antigen 4-DMAP—OVA，l：4000 4-DMAP-BSA antibody dilution，O．5％skim milk powder as blocking solution and pH 7．4 0．0 1 M PBS assay buffer to establish indirect competitive ELISA of DMP．The assay sensitivity(IC50)is 23．3 1+2．62 ng／mL and the limit of detection(ICl5)is 2．01+0．33 ng／mL．The cross-reactivity values of the antibody with structural analogs were less than 0．1％．Th．e experimental results from spiked liquor,vegetables and beverages samples at 1 00 ng／g(or ng／mL)，500 ng／g(or ng／mL)and 5000 ng／g(or ng／mL)indicated that the recoveries ranged from 84．90％to 1 1 9．1 7％，the variation coefficient(n=3)was less than 1 5％．The established ic—ELISA which was highly correlated with GC-MS(R=0．977)could be used to rapidly detect dimethyl phthalate in foods． For dicyclohexyl phthalate(DCHP)，the hapten 4-amino dicyclohexyl phthalate (4一DCHAP)was designed and synthesized．4-amino phthalic acid and cyclohexanol generated into 4-amino dicyclohexyl phthalate by esterification reaction．The hapten 4-DCHAP