Development of fluorescence polarization immunoassay to detect dibutylphthalate (dbp) and its active metabolite monobutylphthalate (mbp) in environmental samples
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Faculty of Chemistry, M. V. Lomonosov Moscow State University, Moscow, Russia
A. N. Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia
Publication date: 2022-05-27
Public Health Toxicol 2022;2(Supplement 2):A6
Dibutylphthalate (DBP) is widely used in cosmetics, printing inks, and pharmaceutical coatings as a plasticizer to impart softness, strength, flexibility, and elasticity to plastic products. DBP is one of the most widely studied phthalates and it has been found that when ingested, DBP and its active metabolite monobutylphthalate (MBP) can cause harm to the hormonal system, liver and lungs. Therefore, the development of a simple, fast and cost-effective method for the immunoassay of phthalates is urgently needed. Promising methods for the determination of phthalates for quality control of food products and environmental objects are immune methods. The method of fluorescence polarization immunoassay (FPIA) makes it possible to determine low molecular weight analytes in a homogeneous medium without separation with high specificity and sensitivity, has greater accuracy, stability of reagents, as well as the speed and simplicity of analysis. The aim of this work is to obtain immunoreagents and develop a rapid method for the determination of DBP and its metabolite MBP in water, using the FPIA method. For the development of FPIA phthalates, we used rabbit polyclonal antibodies obtained against the conjugate of BSA with an amino derivative of DBP by the diazo method (Ab-DBP) or against the conjugate of cationized BSA with MBP by the carbodiimide method (Ab-MBP). Fluorescein-labeled tracers were synthesized from the amino derivative DBP or MBP, activated by NHS at the carboxyl group, with various fluorescein-containing labels (FITC, DTAF, EDF, AMF, GAF). The resulting immunoreagents: antibodies and tracers were studied for binding kinetics. It was shown that antibodies to MBP (Ab-MBP) after 3 and 4 cycles of immunization bind well to the MBP-GAF tracer, but the time to reach equilibrium is about 90 min. Calibration dependences were constructed, FPIA for MBP determination was optimized, and the analytical characteristics of the method were determined (detection limit 5 µg/mL, linear range 10–100 µg/mL). Cross-reactivity studies have shown that Ab-MBPs are highly specific to MBPs only. When developing FPIA to determine DBP, the time to establish equilibrium in the system was only 15 min, the matched pair of Ab-DBP immunoreagents and the DBP-FITC tracer showed a limit of detection on the calibration curve of 3 µg/mL and a linear range of 4–13 µg/mL. Total time for FPIA performance is 1 min. Using the developed FPIA method, water samples taken from the Moscow River and from a water reservoir in the Moscow region were tested for the possibility of determining phthalates.
This study was financially supported by the Russian Science Foundation (Grant number: 21-14-00306).