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A sensitive and specific radioimmunoassay for platelet-activating factor
1Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082.
A platelet-activating factor (PAF) analog with a reactive omega-aldehyde group at the sn-1 position was synthesized. The hapten-thyroglobulin conjugate was used to immunize rabbits to produce specific antibodies to PAF. The purified immunoglobulin G (IgG) fraction was found to bind stereo-specifically to tritiated PAF and to crossreact minimally with lysoPAF, plasmalogens, and other phospholipids. The radioimmunoassay detected as little as 20 pg of PAF per assay tube and was used to explore agonist-induced synthesis of PAF in rabbit neutrophils. Calcium ionophore A23187 at 1 microM induced PAF synthesis peaking at 2 min and reaching basal levels after 5 min. N-Formyl-Met-Leu-Phe (FMLP) at 0.1 microM also stimulated rapid synthesis and degradation of PAF with a peak at 5 min. Both A23187 and FMLP stimulated PAF synthesis in a dose-dependent manner. The radioimmunoassay should be applicable to the quantitation of PAF in biological samples.
A platelet-activating factor (PAF) analog with a reactive omega-aldehyde group at the sn-1 position was synthesized. The hapten-thyroglobulin conjugate was used to immunize rabbits to produce specific antibodies to PAF. The purified immunoglobulin G (IgG) fraction was found to bind stereo-specifically to tritiated PAF and to crossreact minimally with lysoPAF, plasmalogens, and other phospholipids. The radioimmunoassay detected as little as 20 pg of PAF per assay tube and was used to explore agonist-induced synthesis of PAF in rabbit neutrophils. Calcium ionophore A23187 at 1 microM induced PAF synthesis peaking at 2 min and reaching basal levels after 5 min. N-Formyl-Met-Leu-Phe (FMLP) at 0.1 microM also stimulated rapid synthesis and degradation of PAF with a peak at 5 min. Both A23187 and FMLP stimulated PAF synthesis in a dose-dependent manner. The radioimmunoassay should be applicable to the quantitation of PAF in biological samples.