A new one-step antigen heterologous homogeneous fluorescence immunoassay for progesterone detection in serum

•A fast and simple one-step assay for the detection of progesterone was developed.•Usage of an estradiol tracer made the assay much faster and lowered the detection limit.•The test duration was about 12 min.•The detection limit was 0.1 µg/L in buffer and 5 µg/L in human serum.•A novel dye/quencher pair was used (FEW S2314/DY800).

A new homogeneous immunoassay for the detection of progesterone was developed to measure its concentration in human serum. We utilized the weak cross-reactivity of a monoclonal anti-progesterone antibody to an analog molecule (in this case β-estradiol) to create a mixture, in which the fluorescence-labeled antibody (AbF) and quencher-labeled BSA-estradiol (eBSAq) were at optimized equilibrium. At this stage, most antibodies were bound to eBSAq and the fluorescence of AbF was quenched. After adding samples containing free progesterone to the system, these would replace the eBSAq at the antigen-binding site. The fluorescence would be released. In contrast to conventional competitive immunoassays, the fluorescence signal increases with increasing progesterone concentration, greatly simplifying detection and calibration. The performance of the assay was very simple; there was only one mixing step; and other hormones like testosterone, estradiol or dehydroepiandrosterone (DHEA) do not interfere the assay. A wide linear range from 0.1 µg/L to 100 µg/L was achieved in buffer, with a LOD of 0.1 µg/L. In human serum the LOD was 5 µg/L, and the linear range was 5–500 µg/L. For this assay it is important to find the right combination of antibody and cross-reactive antigen. If such a combination could be defined, it is conceivable to apply this assay to a wide range of analytes.

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