A MAB BASE SANDWICH ASSAY FOR AMYLIN & PLASMA

拟议研究的目标是生产诊断试剂和/或 通过生产单克隆性糖尿病治疗试剂 多肽荷尔蒙的抗体，胰淀素。胰淀素是一种新发现的 一种能有效控制葡萄糖代谢的多肽激素和 直接测量血浆中的多肽水平将是进一步 了解和量化荷尔蒙在 I型和II型糖尿病的疾病过程。单抗 试剂将保证高亲和力抗体的稳定供应 针对淀粉蛋白上的不同表位，ii)用于制造 针对胰淀素的同位素或酶结合抗体，以及，iii)是 这对建立胰淀素的夹心测定法很有用。 在第一阶段，我们将对产生的单抗进行鉴定 对于：i)对胰淀素的专一性，ii)表观亲和常数，iii)表位 使用多肽片段的作图，IV)与其他 单抗和/或可用的多克隆抗血清，v) 免疫球蛋白亚型。在第11阶段，我们将使用这些试剂来i) 建立一种检测胰淀素的夹心法，它)构建一根亲和柱 从人血浆中分离纯化胰淀素并进一步鉴定；iii)鉴定 阻断胰淀素与其受体结合的抗体并使用这些 体内调节胰淀素作用的抗体：IV)表征 胰淀素的代谢产物。

The goal of the proposed research is to generate diagnostic reagents and/or therapeutic reagents for diabetes through production of monoclonal antibodies to the peptide hormone, amylin. Amylin is a newly discovered peptide hormone that is active in the control of glucose metabolism and the measurement of peptide levels directly in plasma will be key to further understanding and quantitating the role that the hormone plays in the disease processes of Type I and Type II diabetes. Monoclonal antibody reagents will i) assure stable supplies of high affinity antibodies specific to different epitopes on the amylin protein, ii) be used to make isotope or enzyme conjugated antibodies specific for amylin and, iii) be useful for the development of a sandwich assay for amylin. During Phase I, we will characterize the monoclonal antibodies produced for: i) specificity to amylin, ii) apparent affinity constant, iii) epitope mapping using peptide fragments, iv) ability to form a complex with other monoclonal antibodies and/or available polyclonal antisera, v) immunoglobulin subtype. In Phase 11 we will use these reagents to i) develop a sandwich assay for amylin-, it) construct an affinity column to purify and further characterize amylin from human plasma; iii) identify antibodies that block amylin binding to its receptor and use these antibodies to modulate amylin action in vivo: iv) characterize the metabolic products of amylin.