Highly Sensitive Immunoassay Methods For The Detection Of Di-(2-Ethylhexyl) Phthalate And Benzyl Butyl Phthalate

Since the 2011 Taiwan plasticizing agent event, a variety of liquors were detected with critically excessive plasticizer in 2012, and then the forbidden plasticizer was found in PVC fresh-keeping film in 2013, the plasticizer received much attention of people, and indicated that the problem of safety in foods and household products. Di-(2-ethylhexyl)phthalate(DEHP) and benzyl butyl phthalate(BBP) are two kinds of most commonly used plasticizer, but with high toxicity. Exposure to DEHP or BBP for long-term can results serious health problems.Therefore, it is particularly important to develop the methods which can detect the phthalate esters simply and quickly. In this paper, two highly sensitive enzyme-linked immunosorbent assays based on antigen-coated plate format for DEHP and BBP in infant supplies and food samples,respectively.The polyclonal antibodies were raised against di-(2-ethylhexyl)4-amino phthalate(4-DEHAP) or butyl benzyl 4-aminophthalate(4-BBAP) conjugated to bovine serum albumin(BSA) by the amino diazotization method. Coating antigen was prepared by 4-DEHAP or4-BBAP conjugated to ovalbumin(OVA) using the same procedure.Under the optimal experimental conditions, the dc-ELISA had a detection limit about 0.0042ng/mL, with a quantitative working range of 10-3-103ng/mL(R2=0.998). The cross-reactivity with other structurally related phthalate esters was below 1%. The recoveries of DEHP ranged from 80.8% to 119.2% indicated that the method was successfully applied to the determination of DEHP in infant supplies. And the BA-ELISA has a linear working range of 10-4-102ng/mL(R2=0.997) with a limit of detection of 0.0007ng/mL. Little cross-reactivity(<0.01%) to structurally related phthalates was observed. The method was successfully applied to the determination of BBP in sports drink, tea drink, fruit juice, milky tea,carbonated beverage, and mineral water, without purification or preconcentrations. The recoveries were obtained in the range of 81.9% to119.1%. The results suggested that the developed dc-ELISA and BA-ELISA would be simple, sensitive, and specific methods for rapid monitoring of DEHP and BBP in infant supplies and foods samples. The content and innovation of this work have been described in detail and summarized as follows:1. Synthesis of Hapten(4-BBAP)A novel hapten was synthesized using a hydrolysis reaction, an esterification reaction, and a reduction reaction. Briefly, 4-nitrophthalicanhydride and anhydrous n-butyl alcohol were mixed and heated to120°C. The pH was adjusted to approximately 8 to 8.5 by using NaOH and Na2CO3. PEG-600 was added to the stirring mucus. The solution then reacted for 3h at 100°C. Butyl benzyl 4-nitrophthalate(4-BBNP) was obtained. 4-BBNP was added to a stirred solution of benzene and concentrated hydrochloric acid. Then, zinc dust was added. After reacting for 12 h at room temperature, 125 ml icy distilled water was added and the pH of the mixture was adjusted to approximately 9 to10 using 1 M Na OH.The mixture was then transferred to a separating funnel and the benzene layer was removed. The combined benzene extracts were distilled under reduced pressure to obtain the pale yellow crude product. The crude product was recrystallized from ethanol, 4-BBAP was obtained. The products were characterized by 1H NMR and 13 C NMR.2. Preparation of the polyclonal antibodies4-DEHAP or 4-BBAP was covalently attached to BSA and OVA by diazotization method. 4-DEHAP or 4-BBAP was coupled to BSA for the immunogen or OVA for the coating antigen. The structures of all conjugates were detected by UV-vis spectrophotometer. The immunogen(DEHAP-BSA or BBAP-BSA conjugates) was used to immunize male New Zealand white rabbits. The polyclonal antibodies were obtained when the titers were 1:64000.3. A highly sensitive direct competitive enzyme-linked immunosorbent assay for the detection of DEHP in infant suppliesA sensitive and specific dc-ELISA method was studied for the detection of DEHP based on antigen-coating format. The conjugates of the antibody with horseradish peroxidase(HRP) were used as the detection probe. Under the optimal conditions, the regression equation was Y=0.6470-0.034lgc(R2=0.998), Y is the absorbance at 490 nm, c is the concentration of DEHP. The assay had a detection limit(LOD)about 0.0042ng/mL, with a quantitative working range of 10-3-103ng/mL.This method was successfully applied to the determination of DEHP in infant supplies. It was simple, rapid, selective, highly sensitive and cost-effective.4. Determination of BBP in food by biotin-streptavidin amplified enzyme-linked immunosorbent assayA sensitive BA-ELISA method for the determination of BBP in food was reported in this paper. The biotin-conjugated goat anti-rabbit IgG and HRP-conjugated streptavidin were used as the detection probe. After optimization, the regression equation was Y=0.803-0.068lgc(R2=0.997),Y is the absorbance at 490 nm, c is the concentration of BBP. The detection limit of the method was 0.0007ng/mL, with a quantitative working range of 10-4-102ng/mL. The recoveries obtained ranged from81.9% to 119.1%. This method was successfully applied to determine the BBP in food samples using a simple and rapid extraction procedure.Compared with the chromatography the BA-ELISA method was simple,low cost, highly sensitive and specific.