Determination Of Phthalates Residues By An ELISA Immunoassay And Instrumental Analysis

Phthalic acid ester is an important kind of environmental hormone. It disturbs the hormone which maintains the balance and adjust the Production,the release,the shift the metabolism,the response and the elimination of the normal hormone in the growth process,or causes the unhealthy influence and changes the endocrine function in the descendant biology which is not injured in the body of the biology.Immunological analysis technique is an important method for detection of the small molecules residue. In this work, polycolonal antibody against the Di-n-octyl phthalate was prepared. A simple synthesis method of Di-n-octyl 4-nitrophthalate (DONP) and its hapten Di-n-octyl 4-aminophthalate (DOAP) preparation were described. A specific antibody immunized with hapten -BSA and a desirable coating antigen with a hapten were obtained. The coupling reaction was confirmed a success by UV-visible light spectrum scanning. The conjugation rates of immunogen and coating antigen was 18 and 7.5 respectively.Polyclonal antibody DOP-antibody was generated against the coupled DOP -BSA, and their antiserum titer reached 48000, 12000, respectively. A general and specific competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of the DOP was developed. Using the designated antibody and coating antigen, a competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed and the sensitivity of ic-ELISA showed that the IC50 was 19.2ng/mL,the LOD was 1.9ng/mL and the linear range was 5-75ng/ml.The cross-reaction rate to the other phthalate was tested. The cross-reaction rates were 65.5%,22.6%,17.6% and 21.2% to DONP,DOAP,DEHP,DHP and DBP,respectively.A multi-residue method was developed for the confirmation and quantification of nine kinds of phthalates in PVC by HPLC-ESI-MS/MS. The mean recovery for each analyzes range from 65.28%~98.33%, with the relative standard deviations from 5.35%~11.22%. The limits of detection of DMP,DEP,DBP,DPP,BBP,DCHP,DEHP,DOP,DIDP were 15,7,20～50,5,5,5,20,15,9μg/kg,respectively.