Development of highly specific fluorescence immunoassay and enzyme-linked immunosorbent assay for detection of dimethyl phthalate in water samples

Abstract

A direct competitive fluorescence immunoassay (dc-FIA) and a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for the screening of dimethyl phthalate (DMP) in water samples were developed. The immunoassays utilise polyclonal antibodies against DMP raised in rabbits. The anti-DMP antibodies were linked to horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC). Under the optimal experimental conditions, the dc-ELISA has a linear working range of 0.1Á2000 ng/ml (R 2 00.993) with a limit of detection of 0.09 ng/ ml. In the dc-FIA, the linear working range was 0.05Á30 ng/ml (R 2 00.996), and the limit of detection was 0.02 ng/ml, which is approximately four-fold more sensitive than the dc-ELISA using the same antibody and coating antigen. The results show low cross-reactivity with other structurally related compounds. The proposed methods are successfully applied to determine the DMP contaminants with a simple extraction procedure, and good recoveries were obtained.

Determination of Dimethyl Phthalate in Environment Water Samples by a Highly Sensitive Indirect Competitive ELISA

“…As shown in Fig. 3, the result indicated that the maximum absorbance was obtained with pH 7.4; it is similar with the result of other immunoassay methods [23,26]. Thus, pH 7.4 PBS buffer solution was chosen for our further study.…”

“…4, the absorbance intensity was maximal with the sodium chloride concentration of 0.15 mol/L. This result was in agreement with our previous works [23][24][25]. Hence, it was chosen as the best concentration of sodium chloride in PBS buffer solution for this assay.…”

“…Hence, for the DMP, the conjugation of dimethyl 4-aminophthalate coupled to BSA via amino diazotization linkage was used as the immunogen [23]. The production was able to stimulate the immune response in animal.…”

“…The rabbit polyclonal antibody was prepared by immunization with DMAP-BSA and purified by a modified saturated ammonium sulfate method in our previous work [23]. The titer of the polyclonal antibody used in this study was 1:64,000 obtained by ic-ELISA.…”

“…Finally, dialyzed 12 h use phosphate buffer of pH 7.2. The mole ratio of HRP with goat anti-rabbit IgG was 1.66 according to the equation [23]. The conjugation was stored at −20°C until used.…”