SERUM ERYTHROPOIETIN BIOASSAY USING A NEW CELL LINE

说明(调查人员摘要)：在此第一阶段提案中，计划 从红白血病小鼠肿瘤中分离细胞系的探索 这是来自红白血病小鼠肿瘤的高度敏感株 对促红细胞生成素高度敏感。他建议利用这条线路来 建立人促红细胞生成素灵敏、特异的生物测定方法 血清。汉金斯博士的初步研究表明，他已经分离出一条 它对极低水平的促红细胞生成素很敏感。这条线路，HC24 现在已经连续播放了超过120段，超过18段 一个月的期间。它似乎对其他各种疾病不敏感 生长因子包括IL-1、IL-2、IL-3、GM-CSF或通过口口相传(Dr。 S.Ruscetti)、IL-4、IL-5、IL-6和G-CSF。这项特定研究的设计 是分离和鉴定稳定的、对EPO高度敏感的克隆细胞 来自非肠道HC管路的管路。他将使用一种氚胸苷 克里斯特尔描述的测定促红细胞生成素反应的摄取程序 但也将评估使用四甲基偶氮唑盐底物分析的可能性 替换氚胸苷。他会比较这些方法，他会 体外测定促红细胞生成素生物学作用的可行性 人类血清的存在。他将研究人类血清的作用。 关于抑制细胞和他会用抗体耗尽样本去除 EPO查看活动是否已删除。他会去寻找 人血清对促红细胞生成素定量的影响及能力分析 在非常小的体积中检测到非常低的水平。

DESCRIPTION (Investigator's Abstract): In this Phase I proposal, plans to exploit the isolation of a cell line from erythroleukemic murine tumors which is highly sensitive line from erythroleukemic murine tumors which is highly sensitive to erythropoietin. He proposes to utilize this line to develop a sensitive and specific bioassay for erythropoietin in human serum. Dr. Hankins' preliminary work has shown that he has isolated a line which is sensitive to very low levels of erythropoietin. This line, HC24 has now been carried continuously for more than 120 passages over an 18 month period. It does not appear to be sensitive to a variety of other growth factors including IL-1, IL-2, IL-3, GM-CSF or by word of mouth (Dr. S. Ruscetti) IL-4, IL-5, IL-6 and G-CSF. The design of this specific study is to isolate and characterize stable, highly epo sensitive clonal cell lines from the parenteral HC lines. He will use a tritiated thymidine uptake procedure described by Krystal to assay response to erythropoietin but will also evaluate the possibility of using an MTT substrate assay to replace tritiated thymidine. He will compare these methods and he will determine the feasibility of assaying biologic action of epo in-vitro in the presence of human serum. He will look for the effects of human serum on inhibiting cells and he will use antibody depletion of samples removing epo to see whether or not activity is removed. He will look for the effects of human serum on quantitating epo and analyze the ability to detect very low levels in very small volumes.