A Novel Apoptosis Assay with Antibodies to ssDNA

描述(由申请人提供)：在本项目第一阶段完成的工作 项目已经证明，甲酰胺可以诱导DNA选择性变性。 在凋亡细胞中。甲酰胺对乳清蛋白稳定性的新影响 细胞凋亡性DNA与变性DNA的联合检测 针对单链dna的抗体(Mab)使研制出一种 灵敏、特异的细胞凋亡鉴定方法。少校 拟议的第二阶段项目的目标是开发我们的甲酰胺单抗检测方法，用于 可能的商业应用。为此，我们将开发一种检测方法 福尔马林固定的组织切片染色规程 石蜡包埋档案材料、冰冻组织切片和细胞学 准备工作。 主要是生产用于商业用途的甲酰胺-单抗检测试剂盒。 二期工程的目标。拟议的试剂盒开发工作将 包括抗单链DNA单抗的制备和偶联，最佳单抗的筛选 试剂盒的操作规程和试剂及试剂盒的稳定性评价 在不同的储存和运输条件下的组件。发展，发展 甲酰胺-单抗试验在血吸虫病评价和预测中的应用 实体瘤的治疗反应，我们建议：a)评估 化疗敏感性和诱导细胞凋亡之间的关系，b)检测细胞凋亡 抗血管生成因子治疗肿瘤血管内皮细胞及c)研究 用特定的细胞凋亡诱导疗法治疗的肿瘤中的细胞凋亡(例如 死亡配体踪迹)。基于甲酰胺单抗技术的高通量酶联免疫吸附试验 将发展成为一种新的、快速的药物大容量筛选方法 具有促凋亡和抗细胞凋亡的活性。 建议的商业应用： 拟议的检测方法有望有重大的商业应用，它 提供对细胞和组织中的凋亡的敏感、特异和普遍的检测 切片，并使高通量筛选药物的凋亡和 抗细胞凋亡活性。

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.