Abstract
Prostaglandin (PG) D2 can be produced in adipocytes and dehydrated to PGs of J2 series, including Δ12-PGJ2 and 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2), which serve as pro-adipogenic prostanoids through the activation of peroxisome proliferator-activated receptor γ. To accomplish the quantification of Δ12-PGJ2 in the cell culture system of adipocytes, the present study aimed to develop a sensitive and specific immunological assay for Δ12-PGJ2. Here, we established a cloned hybridoma cell line secreting a monoclonal antibody specifically recognizing Δ12-PGJ2 and utilized for the development of its solid-phase enzyme-linked immunosorbent assay (ELISA). The immobilized antigen using a conjugate of Δ12-PGJ2 and γ-globulin was competitively allowed to react with the monoclonal antibody in the presence of free Δ12-PGJ2. The assay provided a sensitive calibration curve for Δ12-PGJ2, allowing us to determine a range from 0.16 pg to 0.99 ng with a value of 13 pg at 50% displacement in one assay. The monoclonal antibody showed almost no cross-reactivity with other related prostanoids since PGJ2 and 15d-PGJ2 were only recognized with much lower values of 0.5% and 0.2%, respectively. The accuracy for determining Δ12-PGJ2 in the culture medium of adipocytes was confirmed by measurement after the culture medium was fortified with known amounts of authentic Δ12-PGJ2 in a range from 10 to 200 pg/mL. The application of our ELISA revealed that the formation of Δ12-PGJ2 became more pronounced after several hours of incubation of PGD2 at 37°C in fresh maturation medium of cultured adipocytes. Furthermore, we provide evidence for the increased ability of cultured adipocytes to synthesize endogenous Δ12-PGJ2 during the progression of adipogenesis. These results indicate the reliability and usefulness of our solid-phase ELISA for stable Δ12-PGJ2, reflecting the biosynthesis of unstable PGD2 in the culture system of adipocytes.
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