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 (DIBP) were observed 100% and 1.25%, while for dimethyl 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 72.87% 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.