Paul P, Rothmann S A, Zydiak L, Beck G J
Exp Hematol. 1987 May;15(4):382-8.
The reproducibility and utility of an in vitro bioassay for erythropoietin were evaluated, and modifications, including the use of frozen target cells, were made. The assay, based on 3H-thymidine incorporation into phenylhydrazine-treated mouse spleen cells, showed a dose-response curve from 0.2 to 20 mU of sheep plasma or human urinary erythropoietin, with a 50-100-fold increase in 3H-thymidine incorporation. The dose-response curve, using purified, recombinant erythropoietin was identical to that from material obtained from other sources. The use of frozen cells simplified the methodology, reduced the time for assay preparation, and lowered costs. The freezing process resulted in 15% attrition of the cells, but after one year of storage, this level was not increased nor was the response to erythropoietin diminished. The nonspecificity of the assay was reduced by supplementing the cells with critical nutrients, depleting fetal bovine serum of endogenous erythropoietin, and reducing solute variability by ultrafiltration and reconstitution of samples with culture medium. Heat inactivation diminished inhibitors in sera samples, but affinity chromatography using wheat-germ lectin-Sepharose was most effective for extracting erythropoietin and reducing nonspecific effects. The assay is sensitive, can be used to measure multiple samples rapidly, and should prove useful for complementing immunoassay techniques.
对促红细胞生成素的体外生物测定法的可重复性和实用性进行了评估，并进行了改进，包括使用冷冻靶细胞。该测定法基于将3H-胸腺嘧啶核苷掺入经苯肼处理的小鼠脾细胞，对0.2至20 mU的绵羊血浆或人尿促红细胞生成素呈现剂量反应曲线，3H-胸腺嘧啶核苷掺入量增加了50至100倍。使用纯化的重组促红细胞生成素得到的剂量反应曲线与从其他来源获得的物质的曲线相同。使用冷冻细胞简化了方法，减少了测定准备时间并降低了成本。冷冻过程导致细胞损耗15%，但储存一年后，该水平没有增加，对促红细胞生成素的反应也没有减弱。通过向细胞补充关键营养素、去除胎牛血清中的内源性促红细胞生成素以及通过超滤和用培养基重构样品来降低溶质变异性，降低了测定法的非特异性。热灭活减少了血清样品中的抑制剂，但使用麦胚凝集素-琼脂糖的亲和色谱法对于提取促红细胞生成素和减少非特异性效应最为有效。该测定法灵敏，可用于快速测量多个样品，并且应证明对补充免疫测定技术有用。
