The Establishment Of Enzyme-linked Immunosorbent Assay For Dimethyl Phthalate Artificial Antigens Peridium

Dimethyl phthalate (DMP) is a kind of compound of phthalate esters and widely used as plasticizer in our life, such as food packaging, medicare equipment, architectural decoration and agricultural formulating and all their like. Plenty of DMP can be released into the air while plasticisers are widely used, and then the DMP will be absorbed by human through breathing or diet and other ways. It has been proved that DMP is harmful to human, and regarded as a kind of environment pollutants in our country.Nowadays the DMP monitoring methods are mainly gas chromatography. high performance liquid chromatography and gas chromatography-mass spectrometry both at home and abroad. Although these methods could detect DMP content accurately, they have high requirement for instruments, and their preoperations are relatively complex and time-consuming. Then a rapid, simple, high sensitivity and low cost method must be found to detect DMP content in environment and articles for daily use in order to detect DMP pollution level more efficiently in the environment.Enzyme-linked immunosorbent assay has a lot of advantages compared with aforementioned methods, such as simple and rapid operation, tested samples is specificitied, the expensive equipments are needless and more than one sample can be detected at the scene.This research adopts diazotization to couple dimethyl-4-amino phthalate with bovine serum albumin and ovalbumin to synthesize artificial antigen. Through screening, choose DMAP-BSA immunogen immuned balb/c mice. After six immunizations, antibody with high titer and sensitivity could be obtained. Hybridoma cell strain could be filtrated out by using hybridoma technique which can secrete DMP antibody steadily, named 2C11, sandwich ELISA tested its hypotype was IgG2a. Expanding culture 2C11 cell, prepared DMP monoclonal antibody large-scale by using mice ascites, and purified the antibody with bitter-ammonium sulfate law. Optimized ELISA detection condition, the experimental result is as follow,5% skim milk powder acts as sealing agent, second antibody reaction temperature is 37℃, color-developing agent is OPD, create the ELISA standard curve of artificial antigen peridium as y=19.68Log(x)+20.71, correlation coefficient R2 is 0.993, DMAP-OVA peridium concentration is 0.5p.g/mL; the best dilution multiple of monoclonal antibody is 4000, linear detection range is between 0.92 and 1029.68μg/L, lowest detectable limit IC10 is 0.28μg/L. This study successfully demonstrated the antibody can satisfy to test DMP content in environment.