Preparation Of Monoclonal Antibodies And Development Of ELISA For Mono-butyl Phthalate (MBP)

Phthalate esters (PAEs) are one of the most important environmental hormoneand widely used as plasticizers in polyvinyl chloride (PVC) resins to improveflexibility and resilience. For its low water solubility, high octanol-water partitioncoefficient and relatively stablity in the natural environment, PAEs have becomeubiquitous environmental pollutants in natural water, soil, air and food. Moreover, themetabolites of PAEs also can be detected in human serum and plasma. Butyl benzylphthalate (BBP), as a kind of PAEs, is one of the most widespread phthalateplasticizer usually found in food packing, preservative film, carrier of pesticide pipes,vinyl floor tiles, adhesives and many daily products. Several studies reported thatBBP had reproductive and developmental toxicity in human and animal bodies.Humans are exposed to BBP through ingestion, inhalation, and dermal contact. BBPcan be hydrolyzed to mono-butyl phthalate (MBP) after entering human body. MBP isconsidered to be the active metabolite responsible for adverse effects and possessmore reproductive and developmental toxicity than BBP. It has an inversedose-response relationship with sperm motility and concentration. In addition, MBPalso can alter the levels of inhibin B and FSH (follicle-stimulating hormone) in human.At present, the level of human body exposure to PAEs mainly based on primarymetabolism product of PAEs in the urine, which are used as biological markers andthe study of MBP in the urine provides important information for evaluating thehuman body exposure to PAEs and the effect of PAEs on human health.At present, the mostly used method to detect MBP is high-performance liquidchromatography (HPLC). Up to date, we do not find any report on immunologicmethod to detect MBP. The immunogens were prepared by conjugating MBP to BSAat different conjugate ratios based on mixed anhydride method. The mice wereimmunized by three different kinds of immunizations. We chose the mice which were immunized by MBP-BSA with conjugate ratio of14.7via food pad injection to cellfusion. A hybridoma cell line named2F7was obtained via colony hybridization. ThemAb against MBP secreted by2F7had an isotype of IgG1, and showed an affinity of1.93×10~8and the CR values of the mAb with other analogues of MBP were not morethan5%, except for BBP (11.5%). The high affinity and IgG characteristics of themAb against MBP could be used for MBP detection in the future. We developed theic-ELISA and optimize the procedures. The equation of linear regression of MBP wasy=-8.9563x+109.77，the correlation coefficient R~2=0.9947, the lowest limitdetection was0.8ng/mL, a linear range was0.8-1000ng/mL. The differentconcentration of the standard MBP were added into the urine samples, the recoverieswere116.18%,110.22%,99.78%,98.02%and100.02%respectively. It wouldprovide foundations for the development of MBP ELISA test kits and colloidal goldtest strip.