Müller S, Nigam S
Department of Gynecological Endocrinology, Free University Berlin, Germany.
J Lipid Mediat. 1990 Nov-Dec;2(6):329-41.
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.
已开发出一种改良的、灵敏且特异的血清素释放测定法，用于常规测定人中性粒细胞中的血小板活化因子（PAF）。PAF介导的兔血小板血清素释放用作生物测定的原理。在测定之前，含有PAF的样品经过三步提取和纯化过程，即布利格和戴尔提取、高效液相色谱（HPLC）纯化以及HPLC后额外的布利格和戴尔提取。三步提取和纯化过程后的总回收率从未低于70%。兔血小板用14C-血清素标记，并在用于PAF测定之前在Sephadex CL-2B柱上进行凝胶过滤。用10至1000 fmol的已知量PAF制备血清素释放的标准曲线。该测定法的灵敏度为10 fmol/管，这仍可引起约5%的最大血清素释放。我们的方法对于人中性粒细胞中PAF的常规测量而言简单、准确、灵敏且快速。
