Establishment Of ELISA Assay And Development Of ELISA Test Kit For Detection Of PAEs

Phthalic acid esters (PAEs), which is now widely used as plasticizers in industrial production fields, when released in the environment, can be a great threat to human’s health and ecological system. As early as in1995, the World Health Organization (WHO) has announced that it is necessary to monitor PAEs to prevent of global environmental pollution. After the storm of plasticizer exposured in Taiwan, the detection of phthalate esters material has become a hot spot. Although the reported detection methods, such as fluorescence spectrophotometry, gas chromatography (GC), liquid chromatography (LC), high performance liquid chromatography(HPLC), liquid chromatography-mass spectrography (LC-MS), gas chromatography-mass spectrography (GC-MS), can finish the detection for PAEs at a certain level, but the procedure is verbose, and sensitivity is low. Therefore it is imperative to establish a method which is simple, rapid and sensitive to realize the real-time monitoring of PAEs. ELISA can be a very good solution to solve this problem, but the key is preparation of the corresponding antigen and antibody.This research is mainly divided into two parts. The first part the establishment of ELISA for detection of DMP residual. A hapten dimethyl4-aminophthalate (DMAP) which possesses the structural feature of DMP was synthesized via esterification and reduction with4-nitrophthalic acid and methanol. Then DMAP was linked to ovalbumin (OVA) and bovine serum albumin (BSA) through diazotization to make artificial antigen DMAP-OVA and DMAP-BSA. BALB/c mice were immunized with DMAP-OVA and the serum was collected for ELISA analysis after the fourth immunization. First phalanx titration was used to determine the optimum concentration of coating antigen and dilution multiple of antiserum, then an indirect competitive ELISA for DMP was developed and showed an IC50with311ng/mL, which proved that antibody is high-specificity, can be used for detection of DMP. It is also a guarantee for the subsequent cell fusion, screening of hybridoma cell and large-scale preparation of monoclonal antibody.The second part is the development of ELISA test kit for detection of DBP. First revived hybridoma cell line that secrete antibody aim at DBP, after three times’ screening by limiting dilution assays, a hybridoma cell line whit high sensitivity and high titer was got. In vivo induced ascites method and octylic acid ammonium sulfate methods were used to prepared and purified monoclonal antibodies of DBP. Then indirect competitive ELISA was optimized, such as the confining liquid, the concentration of BSA, reaction time and temperature, coloration system, BA-ELISA. Under the optimal conditions, an indirect competitive standard curve was established, the regression equation y=(A-D)/[1+(x/C)B)]+D with the value A=1.11, B=0.9, C=191, D=-0.05, correlation coefficient R2=0.9952, when the concentration of DBP within the scope from13ng/mL to1000ng/mL (IC2o～ICso), it showed good relationship between the natural logarithm of DBP concentration and inhibition rate, the minimum detection limit is6.25ng/mL. The results of this study can used for detection of DBP in environment and the mass production of ELIS A test kit.