Development of enzyme-linked immunosorbent assay for prostaglandin D2 using the stable isosteric analogue as a hapten mimic and its application.
For the determination of prostaglandin (PG) D(2) produced by cultured cells in response to external stimuli, immunological methods would be convenient and useful. However, PGD(2) is unstable under the physiological conditions, so that it has been difficult to get a specific antibody for the parent PGD(2). In an attempt to get a specific antibody for PGD(2), we tried to prepare monoclonal antibodies for 11-deoxy-11-methylene-PGD(2), a novel, chemically stable, isosteric analogue of PGD(2). We successfully cloned a hybridoma cell line secreting a monoclonal antibody reacting specifically with the parent PGD(2). To develop the enzyme-linked immunosorbent assay (ELISA) for PGD(2), the immobilized antigen using the stable PGD(2) derivative was immunoreacted in a competitive manner with the monoclonal antibody in presence of free PGD(2). The optimization of the assay provided a sensitive calibration curve for PGD(2) from 0.32 pg to 0.18 ng with a value of 7.6 pg at 50% displacement. PGD(2) was almost stable during the ELISA condition. The developed assay method was useful for applying to the direct determination of PGD(2) in the culture medium of mouse 3T3-L1 adipocytes. The incubation of PGD(2) in the maturation medium of adipocytes at 37 degrees C caused the chemical conversion into PGJ(2) derivatives. The conversion became more evident after 6 h of the incubation. These findings indicate the importance of considering the optimal time for collecting the samples to be determined for PGD(2) before the conversion starts to occur.
Mazid
Md Abdul
MA
Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504, Japan.
Nishimura
Kohji
K
Nagao
Kohjiro
K
Jisaka
Mitsuo
M
Nagaya
Tsutomu
T
Yokota
Kazushige
K
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002138:Calibration; D002460:Cell Line; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned / Q000737:chemistry; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay; D006241:Haptens; D006825:Hybridomas / Q000166:cytology; D051379:Mice; D015230:Prostaglandin D2 / Q000031:analogs & derivatives* / Q000032:analysis* / Q000276:immunology; D012680:Sensitivity and Specificity; D013696:Temperature
D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D006241:Haptens; D015230:Prostaglandin D2