Environmental Context.Benzene, toluene, ethylbenzene, and xylenes (BTEX) are used as solvents in paints and coatings and are constituents of petroleum products. BTEX can contaminate air, water or soil and is toxic; benzene, in particular, is a recognized human carcinogen. Most existing methods for detecting BTEX are time-consuming, complicated and very expensive for routine screening. A rapid immunoassay of BTEX is presented that greatly simplifies environmental monitoring of water contamination. Abstract.For the rapid screening of BTEX (benzene, toluene, ethylbenzene, xylenes), a fluorescence polarization immunoassay (FPIA) was developed using the fluorescence polarization analyzer Abbott TDx. Several fluorescence-labelled tracers were synthesized by binding ethylenediamine fluorescein thiocarbamyl (EDF) to various substituted phenylcarboxylic acids. The binding of 27 tracers with two antisera that can be considered as class-specific for BTEX was investigated to select optimal tracer–antibody pairs. Significant differences were found in binding, titer, sensitivity, and assay kinetics. A best pair of anti-BTEX antiserum and EDF-labelled p-tolylacetic acid tracer was selected for FPIA. To simplify the method, an immunocomplex single reagent was prepared to detect BTEX by a one-step FPIA. One-step FPIA is a rapid homogeneous type of immunoassay that has only one pipetting step, does not need separation of free and bound analyte and can be performed at room temperature. The within-run coefficient of variation was ranged between 3.4% and 5.7%. Furthermore, if the measurement can be done at constant temperature, standards for every assay run are unnecessary. Cross-reactivity studies of petroleum compounds and a BTEX mixture indicated that p-xylene was most reactive with a limit of detection (LOD) of 0.22 µg mL−1 in 50 µL of sample. The LOD for toluene and benzene was 2.1 and 11 µg mL−1 respectively. The immunocomplex single reagent has proven to be significantly more stable than the corresponding solutions of antibody and tracer.

Progesterone has been known to play a significant role in the facilitation of implantation and in the maintenance of pregnancy. This critical influence of progesterone is most likely due to its well-known ability to inhibit uterine contractility and to suppress the immune response to the fetus. 1,2) During pregnancy as well as the luteal phase of the menstrual cycle after ovulation, progesterone is essential for reproductive function.3,4) Therefore, progesterone can be utilized as a marker for the diagnosis of early pregnancy. Antibodies that bind specifically to progesterone are essential in developing immunoassay methods to measure the progesterone level.Fluorescence polarization immunoassay (FPIA) is based on the principles of two technologies: competitive antibody binding and fluorescence polarization. The competitive antibody-binding assay is based on the competition between free progesterone and fluorescence-labeled progesterone tracer for the binding site on the antibody. The fluorescence polarization assay is based on the principle that molecules rotate in liquid and the rate of rotation is related to the size of the molecule. The disturbance of polarized fluorescence is higher in a small fluorescence-labeled antigen (tracer) than in antibody-bound tracer. When a small fluorescence molecule is excited with polarized light, its rapid Brownian rotation leads to randomization of the molecular orientation before fluorescence emission occurs, and the polarization becomes low. If the molecule binds to a large specific antibody (MW 150000 Da), rotation slows and the emitted fluorescence retains substantial polarization. Hence the degree of antibody binding may be monitored by measurement of fluorescence polarization without physical separation steps.Because of the dependence on molecular size in FPIA, it is particularly suited to the assay of small haptens such as steroids and drugs. Its major applications are in clinical diagnostics, especially in the areas of therapeutic drug monitoring and drug-abuse screening.5,6) A fully automated instrument, the Abbott TDx analyzer, incorporating a polarization fluorometer and a versatile diluter-dispenser has been introduced. For progesterone-related investigations, several hybridoma cell lines producing monoclonal antibodies against immunogens of conjugation-site specific progesterone have been established, as previously described. 7,8) Several immunoassays were reported such as enzyme immunoassay (EIA), 7) cytometric fluoroimmunoassay (CFIA), 9) visual membrane EIA, 8) and FPIA to measure progesterone levels. 10)In this investigation, an immunocomplex single reagent (SR) is developed to simplify the FPIA procedure (one-step FPIA) and its performance characteristics were evaluated in determining progesterone levels. MATERIALS AND METHODS Instruments and ChemicalsProgesterone, progesterone-3-(O-carboxymethyl)oxime (P-3CMO), 11a-hemisuccinyloxyprogesterone (P-11HS), N-hydroxysuccinimide (NHS), dimethylformamide (DMF), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-hydroch...