A particle concentration fluorescence immunoassay for prostaglandin D synthase in the rat central nervous system.
A solid phase, particle concentration fluorescence immunoassay (PCFIA) was developed for the measurement of prostaglandin (PG) D synthase in the 100,000g supernatant of various regions of the rat central nervous system. In this assay, the enzyme (in the range of 1-25 micrograms protein of brain supernatant or 1-100 ng of the purified enzyme) is attached to submicrometer carboxypolystyrene beads coated with polyclonal anti-rat brain PGD synthase IgG. The total particle-bound enzyme is assayed with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-PGD synthase IgG after incubation for 1 h. The optimum assay condition was obtained when carboxyl particles coated with ca. 500 micrograms/ml of polyclonal IgG at pH 5.0 and 5 micrograms/ml of FITC-IgG were used. No significant fluorescence was observed when FITC conjugates or carboxyl particles were prepared using IgG from nonimmunized rabbits. Heat treatment of the brain supernatant decreased the specific binding of the enzyme in parallel with the loss of enzyme activity, indicating that the denatured enzyme is not recognized by this assay method. The PGD synthase immunoreactivity was widely distributed in the brain regions and was highest in the paraflocculus. Although slight discrepancy was observed between the concentration by PCFIA and the enzyme activity measured by using [14C]PGH2 in some brain regions, there is a considerable correlation (0.727) between the values by both methods in the same brain regions. The PCFIA now developed showed higher sensitivity (around 10 times), greater reliability, and larger number of samples measurable at once than the radio-TLC assay using [14C]PGH2. This method could provide valuable information concerning the regulatory mechanisms of PGD synthase.
Islam
F
F
Hayaishi Bioinformation Transfer Project, Research Development Corporation of Japan, Osaka.
Urade
Y
Y
Watanabe
Y
Y
Hayaishi
O
O
D000818:Animals; D000906:Antibodies; D001921:Brain / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D054834:Lipocalins; D009900:Optic Nerve / Q000201:enzymology; D009928:Organ Specificity; D010902:Pituitary Gland / Q000201:enzymology; D011489:Protein Denaturation; D051381:Rats; D012584:Sciatic Nerve / Q000201:enzymology; D013116:Spinal Cord / Q000201:enzymology*; D013861:Thiocyanates
D000906:Antibodies; D005452:Fluoresceins; D005456:Fluorescent Dyes; D054834:Lipocalins; D013861:Thiocyanates; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D016650:Fluorescein-5-isothiocyanate