Add time:08/08/2019         Source:sciencedirect.com

For the determination of prostaglandin (PG) D2 produced by cultured cells in response to external stimuli, immunological methods would be convenient and useful. However, PGD2 is unstable under the physiological conditions, so that it has been difficult to get a specific antibody for the parent PGD2. In an attempt to get a specific antibody for PGD2, we tried to prepare monoclonal antibodies for 11-deoxy-11-methylene-PGD2, a novel, chemically stable, isosteric analogue of PGD2. We successfully cloned a hybridoma cell line secreting a monoclonal antibody reacting specifically with the parent PGD2. To develop the enzyme-linked immunosorbent assay (ELISA) for PGD2, the immobilized antigen using the stable PGD2 derivative was immunoreacted in a competitive manner with the monoclonal antibody in presence of free PGD2. The optimization of the assay provided a sensitive calibration curve for PGD2 from 0.32 pg to 0.18 ng with a value of 7.6 pg at 50% displacement. PGD2 was almost stable during the ELISA condition. The developed assay method was useful for applying to the direct determination of PGD2 in the culture medium of mouse 3T3-L1 adipocytes. The incubation of PGD2 in the maturation medium of adipocytes at 37 °C caused the chemical conversion into PGJ2 derivatives. The conversion became more evident after 6 h of the incubation. These findings indicate the importance of considering the optimal time for collecting the samples to be determined for PGD2 before the conversion starts to occur.

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