Highly sensitive fluorimetric enzyme immunoassay for prostaglandin H synthase solubilized from cultured cells.
A highly sensitive fluorimetric enzyme immunoassay was developed for prostaglandin H (PGH) synthase, an intrinsic membrane protein, using n-octyl beta-D-glucopyranoside for solubilization of the synthase from the endoplasmic reticulum membrane. An anti-PGH synthase IgG-coated polystyrene ball was reacted with a PGH synthase standard or crude detergent extract of cells, washed to remove the detergent, and then incubated with anti-PGH synthase Fab'-beta-D-galactosidase conjugate. The bound beta-D-galactosidase activity was quantitated with 4-methylumbelliferyl-beta-D-galactoside, a fluorogenic substrate. The detection limit for the PGH synthase standard was found to be 1.0 pg/assay. The sensitivity of this assay is increased by about 2-3 orders of magnitude over those of previously reported radioimmunoassay or colorimetric enzyme immunoassay. The high sensitivity of the fluorimetric enzyme immunoassay allowed reliable detection of low levels of human PGH synthase present in small samples of cultured cells. Our studies with this fluorimetric immunoassay for the synthase developed a general method for determination of membrane-bound proteins at ultrasensitive levels.
Ruan
K H
KH
Department of Internal Medicine, University of Texas Medical School, Houston 77030.
Kulmacz
R J
RJ
Wilson
A
A
Wu
K K
KK
D000818:Animals; D000918:Antibody Specificity; D002478:Cells, Cultured / Q000201:enzymology; D004730:Endothelium, Vascular / Q000201:enzymology; D005470:Fluorometry / Q000379:methods*; D005960:Glucosides; D006027:Glycosides; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives; D007124:Immunoenzyme Techniques; D008565:Membrane Proteins / Q000032:analysis; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis* / Q000302:isolation & purification; D012756:Sheep; D001616:beta-Galactosidase / Q000032:analysis
C030561:4-methylumbelliferyl glycosides; D005960:Glucosides; D006027:Glycosides; D008565:Membrane Proteins; C018619:octyl-beta-D-glucoside; D006923:Hymecromone; D011451:Prostaglandin-Endoperoxide Synthases; D001616:beta-Galactosidase