Development of a sensitive and specific 14C-serotonin release assay for platelet-activating factor (PAF) in human...

A modified, sensitive and specific serotonin release assay has been developed for the routine determination of PAF in human neutrophils. PAF-mediated release of the serotonin from rabbit platelets served as the principle of the bioassay. Prior to the assay, the sample containing PAF underwent a three-step extraction and purification process, namely Bligh and Dyer extraction, high-performance liquid chromatography (HPLC) purification and an additional post-HPLC Bligh and Dyer extraction. The overall recovery after the three-step extraction and purification process was never less than 70%. Rabbit platelets were labeled with 14C-serotonin and gel-filtered on Sephadex CL-2B column before using for the PAF assay. A standard curve was prepared for the serotonin release with known amounts of PAF between 10 and 1000 fmol. The sensitivity of the assay was 10 fmol/tube, which still caused approximately 5% of the maximum serotonin release. Our method is simple, accurate, sensitive and rapid for routine measurements of PAF in human neutrophils.