Characterization of monoclonal antibody reactive with 10-hydroxy-12(Z)-octadecenoic acid (10-OHODA) and its demonstration in cultured human macrophages.
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% CO2 for 24 h. 10-OHODA and other fatty acids were extracted from the exposed macrophages with diethylether after phospholipase A2 treatment. 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 x 10(6) cells).
Yamada
Y
Y
Department of Forensic Medicine, Faculty of Medicine, University of Tokyo, Japan.
Nagao
M
M
Takatori
T
T
Iwase
H
H
Nakajima
M
M
Iwadate
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D008264:Macrophages / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D008956:Models, Chemical; D009829:Oleic Acids / Q000032:analysis* / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D009829:Oleic Acids; C040430:10-hydroxy-12-octadecenoic acid