A monoclonal antibody specific for Δ 12-prostaglandin J 2 and its utilization in the immunological assay in cell culture system of adipocytes

摘要

Prostaglandin (PG) D 2 can be produced in adipocytes and dehydrated to PGs of J 2 series, including Δ 12- PGJ 2 and 15-deoxy-Δ 12,14-PGJ 2 (15d-PGJ 2), which serve as pro-adipogenic prostanoids through the activation of peroxisome proliferator-activated receptor γ. To accomplish the quantification of Δ 12-PGJ 2 in the cell culture system of adipocytes, the present study aimed to develop a sensitive and specific immunological assay for Δ 12-PGJ 2. Here, we established a cloned hybridoma cell line secreting a monoclonal antibody specifically recognizing Δ 12-PGJ 2 and utilized for the development of its solid-phase enzyme-linked immunosorbent assay (ELISA). The immobilized antigen using a conjugate of Δ 12-PGJ 2 and γ-globulin was competitively allowed to react with the monoclonal antibody in the presence of free Δ 12-PGJ 2. The assay provided a sensitive calibration curve for Δ 12-PGJ 2, allowing us to determine a range from 0.16a?‰ pg to 0.99a?‰ ng with a value of 13a?‰pg at 50% displacement in one assay. The monoclonal antibody showed almost no cross-reactivity with other related prostanoids since PGJ 2 and 15d-PGJ 2 were only recognized with much lower values of 0.5% and 0.2%, respectively. The accuracy for determining Δ 12-PGJ 2 in the culture medium of adipocytes was confirmed by measurement after the culture medium was fortified with known amounts of authentic Δ 12-PGJ 2 in a range from 10 to 200a?‰pg/mL. The application of our ELISA revealed that the formation of Δ 12-PGJ 2 became more pronounced after several hours of incubation of PGD 2 at 37°C in fresh maturation medium of cultured adipocytes. Furthermore, we provide evidence for the increased ability of cultured adipocytes to synthesize endogenous Δ 12-PGJ 2 during the progression of adipogenesis. These results indicate the reliability and usefulness of our solid-phase ELISA for stable Δ 12-PGJ 2, reflecting the biosynthesis of unstable PGD 2 in the culture system of adipocytes.