Development of a sensitive and specific 14C-serotonin release assay for platelet-activating factor (PAF) in human neutrophils.
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.
Müller
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S
Department of Gynecological Endocrinology, Free University Berlin, Germany.
Nigam
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D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D009504:Neutrophils / Q000737:chemistry*; D010972:Platelet Activating Factor / Q000032:analysis* / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D011817:Rabbits; D012680:Sensitivity and Specificity; D012701:Serotonin / Q000378:metabolism
D010972:Platelet Activating Factor; D012701:Serotonin; D000001:Calcimycin