A Novel Apoptosis Assay with Antibodies to ssDNA

描述（由申请人提供）：在本项目第一阶段所做的工作 一项研究表明，甲酰胺诱导DNA的选择性变性 在凋亡细胞中。甲酰胺的这种新的影响， 凋亡DNA结合单克隆抗体检测变性DNA 抗单链DNA抗体（MAb），使开发一种 灵敏、特异的细胞凋亡检测方法。主要 拟议的第二阶段项目的目标是开发我们的甲酰胺单克隆抗体检测， 可能的商业应用。为此，我们将开发分析 福尔马林固定的组织切片染色方案 石蜡包埋的档案材料、冷冻组织切片和细胞学 准备工作 用于商业用途的甲酰胺单克隆抗体检测试剂盒的生产将是主要的 第二阶段项目的目标。为编制资料袋而提议的工作将 包括抗ssDNAMAb产生和结合，最佳的 试剂盒的方案和试剂以及试剂盒稳定性的评价 组件在不同的储存和运输条件下。发展 甲酰胺单克隆抗体测定法在评价和预测 在实体瘤的治疗反应中，我们建议：a）评估 在化学敏感性和凋亡诱导之间，B）测量凋亡 在用抗血管生成因子处理的肿瘤的内皮中，和c）研究 用特异性凋亡诱导疗法（例如， 死亡配体TRAIL）。基于甲酰胺-单克隆抗体技术的高通量ELISA 将发展成为一种新的、快速的大批量筛选药物的方法 因为它们的凋亡和抗凋亡活性。 拟定商业应用： 预期所提出的测定法具有重要的商业应用价值， 提供细胞和组织中细胞凋亡的灵敏、特异和通用检测 切片，并使高通量筛选药物的凋亡和 抗凋亡活性。

DESCRIPTION (Provided by Applicant): Work performed during the Phase I of this project has demonstrated that formamide induces selective denaturation of DNA in apoptotic cells. This novel effect of formamide on the stability of apoptotic DNA combined with the detection of denatured DNA with monoclonal antibody (MAb)against single-stranded DNA, has made it possible to develop a sensitive and specific assay for the identification of apoptosis. The major goal of the proposed Phase II project is to develop our formamide-MAb assay for possible commercial applications. For this purpose, we will develop assay protocols for staining of histological sections from formalin-fixed paraffin-embedded archival material, frozen tissue sections and cytological preparations. Production of a formamide-MAb assay kit for commercial use will be the major goal of the Phase II project. Work proposed for the development of the kit will involve production and conjugation of anti-ssDNA MAb, selection of optimal protocols and reagents for the kit and evaluation of the stability of the kit components under variable storage and shipping conditions. To develop applications of formamide-MAb assay for the evaluation and prediction of therapeutic response in solid tumors, we propose to: a) evaluate relation between chemosensitivity and the induction of apoptosis, b) measure apoptosis in endothelium of tumors treated with antiangiogenesis factors and c) study apoptosis in tumors treated with specific apoptosis-inducing therapy (e.g. death ligand TRAIL). High-throughput ELISA based on formamide-MAb technique will be developed as a new and rapid method for high-volume screening of drugs for their apoptotic and anti-apoptotic activity. PROPOSED COMMERCIAL APPLICATION: Significant commercial applications could be expected for the proposed assay, which provides sensitive, specific and universal detection of apoptosis in cells and tissue sections and makes possible high-throughput screening of drugs for apoptotic and anti-apoptotic activity.