Characterization of Monoclonal Antibody Reactive with 10-Hydroxy-12(Z)-octadecenoic Acid (10-ohoda) and Its Demonstration in Cultured Human Macrophages

摘要

10-Hydroxy-12(Z)-octadecenoic acid (10-OHODA) has an inhibitory effect on the tension of papillary muscles in isolated guinea-pig hearts. To establish an immunoassay for 10-OHODA a mouse monoclonal antibody (MoAb), YM-1, was produced. In order to evaluate the ability of this MoAb to recognize various 10-OHODA analogs including leukotoxin (9,10-epoxy-12-octadecenoic acid, LTx), a sensitive enzyme-linked immunosorbent assay (ELISA) was developed using the avidin-biotin complex (ABC). The detection limit for 10-OHODA was as low as 0.5 ng in this system. In order to demonstrate the presence of 10-OHODA in living cells, macrophages derived from the human leukemia cell line THP-1 by adding 160nM phorbol 12-myristate 13-acetate (PMA) were exposed to 95 O2, and 5 CO2for 24 h. 10-OHODA and other fatty acids were extracted from the exposed macrophages with diethylether after phospholipase A2treatment. The 10-OHODA content was determined using the new ELISA, and 18.5 ng 10-OHODA was detected in the macrophages exposed to the high oxygen concentration (1 #xD7; 106cells).