An ultra-sensitive monoclonal antibody-based enzyme-linked immunosobent assay for dibutyl phthalate in human urinary

Dibutyl phthalate (DBP) has been extensively used as a plasticizer in many daily products, which is highly toxic to human, notably affecting the reproductive and developmental function. As the previous method is expensive, time-consuming, low sensitivity and just focused on the environment. Present study was aimed to establish an ultra sensitive and simple method based on good quality monoclonal antibody, applying to evaluate excretion level of DBP in urine samples of Chinese population directly. A monoclonal antibody was generated and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mouse. The mouse was previously immunized using a specially designed amino derivative of DBP conjugated with bovine serum albumin (BSA) as immunogen. Cross reactivity values of the monoclonal antibody against DBP, di-isobutyl phthalate (IMP) were observed 100% and 1.25%, while for dimelhyl phthalate (DMP), butyl benzyl phthalate (BBP) and didecyl phthalate (DDP) the values were <0.06%. The standard curve was constructed at 0-50 ng mL(-1) and good linearity (R-2 = 0.994) was achieved. The observed IC50 (7.34 ng mL(-1)) and LOD (0.06 ng mL(-1)) values was improved 1000-fold to polyclonal antibody and 5-fold to other monoclonal antibodies. A total 1246 urine samples were analyzed and the detection frequency of DBP was observed 72.874 by ic-ELISA. The 95th percentile and mean concentration of DBP were 12.07 and 3.00 ng mL(-1). Acceptable recovery rates of DBP were 97.8-114.3% and coefficients variation 5.93-11.09%. The concentrations of DBP in females were found significantly higher (p < 0.05) than males. Similarly, the DBP in middle aged and low educated individuals was found higher (p < 0.001) than the others. Considering the adverse health effects, DBP internal exposure in the Chinese population should be reduced. The ic-ELISA method has been proved as a cost effective, specific, and highly sensitive screening tool to detect DBP in urinary samples. (C) 2015 Elsevier B.V. All rights reserved.