Synthesis of Steroid Tracers by an Oxime Ligation Method and Their Use in Fluorescent Polarisation Immunoassay.

ctive: The development of a fast and reliable method for the quantitative evaluation of steroid hormones is very relevant for scientific and clinical research. The goal of this study was to develop a new universal method for the synthesis of fluorescent tracers from carbonyl-containing biomolecules by oxime ligation. Methods: A method for the synthesis of ketosteroid oxime conjugates with a fluorescein (6-FAM) derivative connected via a short bifunctional aminooxypropyl linker is proposed. Oxime stereoisomers were separated by preparative RP TLC and HPLC. The conjugates were used as tracers for the fluorescence polarization immunoassay (FPIA) method with monoclonal antibodies. Results and Discussion: Parameters of the binding of the tracers with monoclonal antibodies (mAbs) to progesterone were studied. The tracers demonstrated high efficiency for the measurement of steroid hormone concentration, with the detection limit of progesterone being lower than that for a previously described analog. The Z-isomer was found to be more sensitive in FPIA compared to the E-isomer. Conclusions: Thus, we have developed an effective and universal approach to the synthesis of fluorescent conjugates with a linker length optimal for immunoassay, which improves their analytical characteristics and simplifies their preparation. The synthesized compounds have potential for practical use in clinical diagnostics and the analysis of environmental objects. [ABSTRACT FROM AUTHOR]