Abstract
A high-throughput, simple and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the clinical determination of progesterone in human serum, using luminol-hydrogen peroxide as chemiluminescence system catalyzed by horseradish peroxidase (HRP). The solid phase of anti-progesterone antibody was prepared by immunoreaction between anti-progesterone polyclonal antibody and donkey anti-rabbit IgG, i.e. second antibody, which had been physically absorbed on the wells of polystyrene microplate and was used as a universal solid phase. The effect of various factors, such as the dilution of immunoreagents, chemiluminescence substrate, chemiluminescence reaction time and incubation condition were examined and optimized. The optimal dilutions of anti-progesterone antibody and HRP-progesterone conjugate were 1:10000 and 1:15000, respectively. The II substrate was chosen and the luminescence was determined after 10 min incubation. The immunoreacted sample was incubated in water bath at 37°C for 1 h. The assay was evaluated with sensitivity as low as 0.08 μg l −1. The relative standard deviation (RSD) was less than 15% for both intra- and inter-assay precision. The recoveries of three different spiked concentration samples were 101%, 101% and 94.4%, respectively. This proposed method has been successfully applied to the clinical evaluation of progesterone in 36 human sera. The results showed a good correlation with the accredited radioimmunoassay (RIA) with a correlative coefficient of 0.9502.
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