Generation of monoclonal antibody against 6-Keto PGF_1α and development of ELISA for its quantification in culture...

Prostacyclin or prostaglandin I₂ (PGI₂), a metabolite of arachidonic cyclooxygenase pathway, has been demonstrated as an effector of adipocyte differentiation. However, due to its instability in biological fluid, it is difficult to evaluate the role of PGI₂ in regulating adipocyte differentiation in different stages in culture. Therefore, this study aimed to establish a simple and rapid method for the production of monoclonal antibody against 6-Keto PGF₁α, a stable PGI₂ metabolite, and its quantification to determine the role of PGI₂ in culture medium. Eight-week-old female BALB/c mice were immunized with the hapten of 6-Keto PGF₁α and BSA for several weeks until a higher antibody titer (absorbance value > 0.9 at 1000-times dilution) against 6-Keto PGF₁α was found. Then, fusion of antibody-producing spleen lymphocytes with SP-2 myeloma cells and thymocytes was performed and cultured in HAT-medium supplemented with hypoxanthine, aminopterin, and thymine. Specific antibody-producing cells (M2-A4-B8-D10) against 6-Keto PGF₁α were identified and separated. A standard ELISA calibration curve was developed with 100% reactivity for 6-Keto-PGF 1 α ranging from 0.26 pg to 6.44 ng corresponding to 90% and 10% of the maximum binding capacity for the immobilized antigen respectively. This method can easily be applied to monitor PGI₂ regulation in different stages of cultured adipocytes to reveal the regulatory roles of PGI₂ in maintaining homeostasis and adipocyte differentiation.