CLINICAL HEMOPOIETIN ASSAY BY RECEPTOR LIGAND BINDING

描述：目前用于临床定量细胞因子的测定， 如白细胞介素、集落刺激因子和促红细胞生成素 (Epo)是基于单克隆或多克隆抗体识别的 激素的RIA或ELISA的形式。 这种方法需要很多 时间和精力来开发纯化的，高亲和力的抗体，但 这些测定相对不敏感，几乎不能检测 正常血液样本中的激素 它们只对病人有用 促红细胞生成素水平升高 此外，这些分析不能 区分生物活性分子和非活性分子。 的 我们实验室和其他实验室最近克隆了许多生长因子受体 允许细胞因子测定的新方法。 利用配体结合 细胞因子受体的胞外结构域代替抗体， 传统的分析将允许鉴定和定量 生物活性受体结合分子。 由于基于受体的测定具有更高的特异性， 可推广到所有受体配体。 在该提案的第一阶段， 本申请人制备了鼠Epo受体并引发Epo 结合分析 结果表明，开发研制高性能的 基于其与EpoR的相互作用测定生物活性Epo。 在第二阶段，他们将产生重组人EpoR，建立其 Epo结合特性，并将其用于开发临床Epo检测。 这些 反过来，研究将建立一种方法， 表达和纯化其他细胞因子受体并开发 生物活性白细胞介素和集落的临床测定 刺激分子

DESCRIPTION: Current assays for clinical quantitation of cytokines such as the interleukins, colony stimulating factors, and erythropoietin (Epo) are based on monoclonal or polyclonal antibody recognition of the hormone in the form of RIAs or ELISAs. This approach requires much time and effort to develop purified, high affinity antibodies, yet these assays are relatively insensitive, and barely allow detection of the hormone in normal blood samples. They are useful only for patients with elevated Epo levels. Additionally, these assays can not distinguish biologically inactive molecules from active ones. The recent cloning of many growth factor receptors in our lab and others permits a new approach to cytokine assays. Using the ligand-binding exoplasmic domains of cytokine receptors instead of antibodies in traditional assays would allow for identification and quantitation of biologically active receptor binding molecules.In addition to the greater specificity of receptor based assays, this approach is easily generalizable to all receptor ligands. In Phase I, of this proposal, the Applicants prepared the murine Epo receptor and initiated an Epo binding assay. The results indicate that it is feasible to develop the assay for biologically active Epo based on its interaction with EpoR. In Phase II, they will generate recombinant human EpoR, establish its Epo binding properties and use it to develop clinical Epo assay. These studies will, in turn, establish methods which will make economically feasible to express and purify other cytokine receptors and develop clinical assays for biologically active interleukins and colony stimulating molecules.