Reimert C M, Minuva U, Kharazmi A, Bendtzen K
Laboratory of Medical Allergology, University Hospital, Copenhagen, Denmark.
J Immunol Methods. 1991 Jul 26;141(1):97-104. doi: 10.1016/0022-1759(91)90214-z.
Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN) is one of the cationic proteins found in the granules of the human eosinophilic granulocytes. EPX was purified from extracts of granules isolated from blood buffy coat cells of healthy donors. Polyclonal anti-EPX antibodies were subsequently raised in rabbits. The anti-EPX-antibodies raised in rabbits showed no reactivity with other proteins in the granule extract. The sandwich ELISA utilized the biotin/avidin amplification system and measured EPX over the range of 60-2000 pg/ml. The intra- and interassay coefficients of variation were 6.5% and 8.2%, respectively, and the mean recoveries of 25 and 50 pg of purified EPX added to diluted serum samples were 106 +/- 16% (mean +/- SD; n = 12) and 112 +/- 14%, respectively. Using this assay we found high amounts of EPX in normal human urine (U-EPX). U-EPX was purified by a two step procedure involving affinity chromatography on heparin Sepharose and size exclusion chromatography on Sephadex G-50 superfine. Extracted EPX and U-EPX had ribonuclease activity and comigrated on agarose electrophoresis. They also showed immunological identity when evaluated with rabbit anti-EPX antibodies, but they differed slightly on SDS-PAGE probably due to differences in glycosylation. Our results support the findings that EPX/EDN is identical to a nonsecretory ribonuclease isolated from urine.
嗜酸性粒细胞蛋白X/嗜酸性粒细胞衍生神经毒素（EPX/EDN）是在人类嗜酸性粒细胞颗粒中发现的阳离子蛋白之一。EPX是从健康供体血液血沉棕黄层细胞分离出的颗粒提取物中纯化得到的。随后在兔体内制备了多克隆抗EPX抗体。兔体内产生的抗EPX抗体与颗粒提取物中的其他蛋白质无反应性。夹心ELISA利用生物素/抗生物素蛋白放大系统，在60 - 2000 pg/ml范围内检测EPX。批内和批间变异系数分别为6.5%和8.2%，向稀释血清样本中添加25 pg和50 pg纯化EPX的平均回收率分别为106±16%（平均值±标准差；n = 12）和112±14%。使用该检测方法，我们发现正常人尿液（U-EPX）中含有大量EPX。U-EPX通过两步法纯化，包括在肝素琼脂糖上进行亲和层析和在Sephadex G-50超细凝胶上进行尺寸排阻层析。提取的EPX和U-EPX具有核糖核酸酶活性，在琼脂糖电泳上迁移情况相同。用兔抗EPX抗体评估时，它们也显示出免疫学同一性，但在SDS-PAGE上略有不同，可能是由于糖基化差异。我们的结果支持了EPX/EDN与从尿液中分离出的一种非分泌性核糖核酸酶相同的发现。
