Study On Enzyme-linked Immunosorbent Assay For Dibutyl Phthalate

Dibutyl phthalate, commonly referred to as DBP, is predominantly used as a plasticizer in nitrocellulose lacquers to make them flexible. It also can be used in polyvinyl chloride and polyvinyl acetate plastics. Meanwhile, DBP is one kind of industrial raw materials for anti-foaming agent, latex adhesives and textile fiber lubricants. It is also used as a solvent for dyes and pesticides. These materials are used to make many products that we use every day such as plastics, paints, glue, insect repellents, perfume, hair spray, nail polish and so on. DBP can enter the environment through releases from factories that make or use it and from household items containing it. Because DBP is not bound to the final product, it can move out of plastic materials into the environment over long periods of time. Therefore, DBP is widespread in the environment. Humans can be exposed to DBP through air, water, food, or skin contact with plastics that have DBP in them. In recent years, DBP is considered to be an environmental endocrine disruptor, with reproductive toxicity, developmental toxicity and potential carcinogenic effects.In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, methods for DBP determination must be developed. Several methods have been reported for the determination of DBP using a variety of techniques, including gas chromatography coupled with mass spectroscopy (GC-MS) and high performance liquid chromatography (HPLC). Although the chromatographic techniques provide a low level of detection for phthalates, they are time consuming and have high instrumentation costs. On the contrary, immunoassay especially enzyme-linked immunosorbent assay (ELISA) is a fast, simple, and economic analytical method. Because of its strong selectivity and sensitivity, efforts for sample cleanup can be reduced to a minimum, which makes the immunoassays highly convenient tools for high throughput studies for a large number of samples in a short period of time.In this study, a hapten dibutyl 4-aminophthalate (DBAP) which possesses the structural feature of DBP was synthesized via esterification and reduction with 4-nitrophthalic acid and butanol. Then DBAP was linked to ovalbumin (OVA) or bovine serum albumin (BSA) through diazotization to make artificial antigen DBAP-OVA or DBAP-BSA. BALB/c mice were immunized with DBAP-OVA and antibody they produced with high titer and high sensitivity. One hybridoma cell line secreting stable anti-DBP antibody was produced and screened out by lymphocyte hybridoma technique. The chromosome number of hybridoma was 96-105. The antibody subtype was IgG1. A large number of monoclonal antibody was produced by mouse ascites method and purified with caprylic acid-ammonium sulfate. The purity and recovery of monoclonal antibody were 91.1% and 68.3%, respectively. OVA as a blocking reagent, competitive reaction system was 0.01 mol/L pH6.8 PBS containing 10% DMF,1.6% NaCl, and 0.1% BSA, and 1.5 h of competitive reaction at 25℃were considered to be optimal in ELISA. An artificial antigen-coated indirect competitive ELISA for DBP was developed and showed a linear detection range of 11.56～863.72μg/L with a limit of detection (LD) of (9.21±1.28)μg/L. In addition, a novel ELISA format employing direct coating of hapten on microtiter plates is established for the detection of DBP. PS surface was first carboxylated by permanganate oxidation in diluted sulfuric acid and then DBAP was immobilized on the surface of microtiter plates by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). This assay demonstrated high sensitivity. Linear detection ranged of 2.16～155.92μg/L. LD for DBP equaled to 0.93μg/L, which was 10 times lower than LD of artificial antigen-coated indirect competitive ELISA for DBP. Low cross-reactivities were observed for some structurally related phthalates except for DBAP and butyl benzyl phthalate (BBP) (21.5% and 27.1%, respectively). The average recoveries of DBP from fortified tap water and East Lake water samples were in ranges of 90.5%～102.7% and 92.4～100.5%, respectively. The developed hapten-coated indirect competitive ELISA format can be used as a convenient quantitative tool for the sensitive and accurate monitoring DBP in aquatic environment and certain cosmetic samples.