Study On The Immunoassay For The Detection Of Dibutyl Phthalate

In this paper,polyclonal antibody against dibutyl phthalate was prepared,and an indirect eompetitive enzyme-linked immunosorbent assay for dibutyl phthalate was established.An up-conversion fluorescent labeled magnetic separation immunoassay method for dibutyl phthalate was established by using upconversion fluorescent nanomaterials and magnetic nanospheres.In this study,4-aminophthalic acid was used to synthesize hapten dibutyl 4-aminophthalate by esterification reaction,and the synthesized hapten was coupled with keyhole limpet hemocyanin(KLH)by diazotization reaction to prepare the immune antigen and coupled with bovine serum albumin(BSA)to prepare the coating antigen.An indirect competitive enzyme-linked immunosorbent assay(ELISA)for dibutyl phthalate was established using the purified antibodies.After optimizing the reaction conditions in the method,the optimal working conditions for the indirect competition ELISA of dibutyl phthalate were determined as follows:the coating amount of the coating antigen was 0.05?g/well,and the antibody was diluted 5000 times;the blocking solution was 0.5%skim milk powder,enzyme-labeled secondary antibody was diluted 20000 times.For the developed ELISA,the IC50 was 40.68 ?g/L and the limit of detection was 1.98 ?g/L,the method has good specificity for dibutyl phthalate,the method was applied to detect liquor,milk and edible oil samples,and the recoveries ranged from 84.57%to 102.4%.There was a good correlation(R2=0.9908)between the this ELISA and gas chromatography-mass spectrometry(GC-MS)for the detection of DBP in the spiked samples.In the experiment,the dibutyl 4-aminophthalate coating antigen was prepared by diazotization.A water-soluble up-converting fluorescent material was obtained by a thermal decomposition method and a ligand exchange method.An immunosensor probe was prepared by coupling coating antigen with polystyrene magnetic microspheres by an activated ester method,and a fluorescent signal probe was prepared by coupling up-conversion fluorescent nanomaterials with dibutyl phthalate antibody.After optimization,the immunosensor probe and dibutyl phthalate standard solution were added 40 ?L,the signal probe was added 50 ?L,and the reaction time was 30 min.An up-conversion fluorescent labeling magnetic separation immunoassay method for dibutyl phthalate was established under optimal working conditions,the detection limit of this method was 0.01?g/L,and the detection range was 0.01-20 ?g/L.The method has good specificity and has no obvious cross-reaction with other structural analogs of dibutyl phthalate.The method was applied to detect liquor,milk and edible oil samples,and the recoveries ranged from 85.57 to 98.79%.There was a good correlation(R2=0.9905)between the this method and gas chromatography-mass spectrometry(GC-MS)for the detection of DBP in the spiked samples.