A radioreceptor binding assay for platelet-activating factor (PAF) using membranes from CHO cells expressing human PAF receptor.
A simple and reproducible radioreceptor assay (RRA) has been developed using membranes from CHO cells which can stably express human platelet-activating factor (PAF) receptor. The CHO cells expressing the PAF receptor, termed CHO.1F8, showed a significant intracellular Ca2+ response to PAF, and the same binding properties to [3H]WEB 2086, a PAF antagonist, as reported (Kd, 13.6 +/- 1.9 nM; Bmax, 2.5 +/- 0.4 pmol/mg protein (n = 6)). A competitive binding assay was done using the CHO.1F8 cell membranes and [3H]WEB 2086. The minimum detectable dose of PAF was 0.3 nM (approximately 30 pg per well) and the assay was highly specific for PAF. This method makes it possible to handle large numbers of samples rapidly and simultaneously, since the receptor membrane is prepared in advance and the binding assay can be completed within 3 h. Using this method, we have determined the production and cell association of PAF in human neutrophils.
Aoki
Y
Y
Life Science Research Laboratory, Japan Tobacco Inc., Kanagawa, Japan.
Nakamura
M
M
Kodama
H
H
Matsumoto
T
T
Shimizu
T
T
Noma
M
M
D000818:Animals; D016466:CHO Cells / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008055:Lipids / Q000302:isolation & purification; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000737:chemistry / Q000187:drug effects; D010972:Platelet Activating Factor / Q000032:analysis* / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011869:Radioligand Assay / Q000379:methods*; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results
D008055:Lipids; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; C039351:platelet activating factor receptor; D009240:N-Formylmethionine Leucyl-Phenylalanine