CORE--RADIOIMMUNOASSAY OF AND PRODUCTION OF ANTISERA

本项目的重点是激活素/抑制素及其结合蛋白、受体和下游调节剂在调节生殖功能、细胞生长和发育中的作用。该计划的第一个目标是描述激活素-激活素/受体复合体的结构以及激活素、激活素受体和结合蛋白以及下游调控分子之间的物理相互作用。第二个目的是探索这些分子在细胞和系统水平上的生理和病理生理学意义，并研究蛋白质表达和分泌的调控以及作用方式。这些研究需要高亲和力、高滴度、适当特异性的抗体用于免疫沉淀、免疫印迹放射免疫分析(RIA)、免疫组织化学和/或免疫中和技术。该核心将生产和鉴定免疫中和技术所需的多克隆抗血清。该中心将生产和鉴定本计划项目所需的多克隆抗血清，以进行结构、功能和机制研究。我们将使用与载体蛋白连接的合成肽作为免疫原，开发针对根据cdna序列或从纯化天然产品的胰酶片段的氨基酸序列预测的基因产品的抗体。或者，使用体外细胞表达系统生产的、由个别项目提供的蛋白质将直接注射到宿主动物中，以生产抗血清。为了减少与目的蛋白以外的抗原的交叉反应，选定的抗血清将使用认知肽、重组蛋白或融合蛋白共价结合到琼脂糖凝胶上进行免疫亲和纯化。该计划中的项目提出的生理学和药理学研究在很大程度上取决于我们测量各种生物液中相关激素的能力。核心将提供脑下垂体促性腺激素和性类固醇的RIA。核心负责确保这些化验的质量控制。核心将由C.Rivier博士(对生物流体的RIA分析，10%的工作)和W.Vale博士(抗体生产，3%的工作)全面负责。核心B的抗血清生产和纯化部分的日常运作将在J.沃恩的监督下进行(10%的工作)。

This Program Project focuses on the role of activins/inhibins and their binding proteins, receptors and downstream modulators in the regulation of reproductive function, cellular growth and development. The first aim of this Program is to characterize the structure of activin-activin/receptor complexes and physical interactions between activin, its receptors and binding proteins, and downstream regulatory molecules. The second aim is to explore the physiologic and pathophysiologic significance of these molecules at the cellular and system levels and to study the control of protein expression and secretion as well as modes of action. These studies require high affinity, high titer antibodies of appropriate specificity for immunoprecipitation, immunoblot radioimmunoassay (RIA), Immunohistochemistry, and/or immunoneutralization techniques. This Core will produce and characterize polyclonal antisera needed by the immunoneutralization techniques. This Core will produce and characterize polyclonal antisera needed by the Projects of this Program to carry out structural, functional and mechanistic studies. We will use synthetic peptides conjugated to carrier proteins as immunogens to develop antibodies directed towards gene products predicted from the cDNA sequences or from amino acid sequencing of tryptic fragments of purified natural products. Alternatively, proteins produced using in vitro cell expression systems and provided by individual Projects will be injected directly into host animals for production of anti-sera. To reduce cross-reactivity with antigens other than the protein of interest, selected antisera will be immunoaffinity purified using the cognitive peptide, recombinant protein, or fusion protein covalently bound to an agarose- based gel. Physiologic and pharmacologic studies proposed by the Projects in this Program are critically dependent upon our ability to measure relevant hormones in a variety of biological fluids. The core will provide RIAs of pituitary gonadotropic hormones and sex steroids. The Core is responsible for ensuring quality control of these assays. The core will be under the overall responsibility of Drs. C. Rivier (RIA analysis of biological fluids, 10% effort) and W. Vale (antibody production, 3% effort). Day to day operation of the antisera production and purification component of Core B will be under the supervision of J. Vaughan (10% effort).