Competitive immunoassay of progesterone by microchip electrophoresis with chemiluminescence detection

A sensitive and rapid homogeneous immunoassay method based on microchip electrophoresis-chemiluminescence detection (MCE-CL) using luminol-hydrogen peroxide as chemiluminescence system catalyzed by horseradish peroxidase (HRP) was developed for the determination of progesterone (P). The assay was based on the competitive immunoreactions between HRP-labeled P antigen (HRP-P) and P with a limited amount of anti-P mouse monoclonal antibody (Ab), and MCE separation of free HRP-P and HRP-P-Ab immunocomplex followed by CL detection. The effect of various factors such as conditions for the CL reaction, MCE and incubation time for the immunoreactions were examined and optimized. Under optimal assay conditions, the MCE separation was accomplished within 80 s. The linear range of detection for P was 8–800 nM with a detection limit of 3.8 nM (signal/noise ratio = 3). This present method has been applied to determine P in human serum samples from normal and pregnant women. The result indicates that the proposed MCE-CL based homogeneous immunoassay method can serve as an alternative tool for clinical assay of P.