The Study Of Fluorescence Immunoassay For Diisobutyl Phthalate

Diisobutyl phthalate,acting as one kind of auxiliaries in material processing, belongs to Phthalate esters(PEs). Its large scale use in plastic products processing began in the 1950s. With the aid of it, many material processing properties such as ductility and durability could be improved. This chemical compound has been frequently used in food packing, toys, medical application, household and personal care products in the absence of relatively perfect toxicology studies. In recent years, a large number of toxicological studies have indicated that:diisobutyl phthalate is one kind of environmental estrogens and has adverse effect on the body’s normal development because it can affect secretions exerting normal function such as synthesis, release, transport and binding in the body. Therefore, it is very important and meaningful to establish a immunological analysis method to realize rapid detection of diisobutyl in some food contamination.Up to now, the major analysis methods applied to detect DiBP are high-performance liquid chromatography (HPLC) and gas chromatography (GC), generally combining with mass spectrometry (MS). Although these analytical techniques give accurate concentrations of DiBP in water or food samples and their limit of detection are low, they are time-consuming, need expensive instruments and skilled personnel, and are unsuitable for analysis of many samples.Therefore, it is necessary to develop a simple,rapid, reliable, sensitive and low price analysis method for DiBP.immunoassay has potential to be used for rapid and simple determination of DiBP due to their ease of use, low, selectivity.In this paper, with the aid of a structural analogue 4-nitro-phthalic acid as an intermediate, haptens were obtained after two or three steps of chemical reactions including esterification, hydrogenation and amidation. And the haptens all remain diisobutyl phthalate structural characteristics but modifying amino group or carbonyl group. Hereafter, the hapten with amino group was conjugated with carrier proteins carrier protein bovine serum albumin (BSA) or chicken serum albumin (OVA) by using diazotization method to prepare immunogen and coating antigen, respectively. After immunizing rabbits with immunogen, polyclonal antibodies against diisobutyl phthalate was obtained. Immunoassay methods for diisobutyl phthalate was established by using a high sensitivity antibody.Under the optimized conditions, the quantitative working range of the icFIA was from 10.47 to 357.06 ng/mL (R2=0.991) with the limit of detection of 5.82 ng/mL. The indirect fluorescence immunoassay method was applied to determine DiBP contamination in edible oil samples. For one thing,the specificity test results show that other similar phthalates do not interfere significantly in the analysis, the cross-reactivity was less than 15%. The recovery was 79 to 102%. What is more, the reliability of the icFIA was validated by GC-MS. It is indicated that the developed icFIA was suitable for monitoring of DiBP in some food samples.