DEVELOPMENT OF IMMUNO-PCR NEURODIAGNOSTIC KITS

描述(改编自申请者摘要)：病原生物为 被认为是许多感染的罪魁祸首，神经退行性变和AS 以及一些类型的神经精神疾病。神经活性检测 病原体在早期阶段将是诊断中的关键因素。 以及治疗任何可能的神经病理后果。酶连结 免疫吸附试验和聚合酶链式反应已经 单独用作特定的微生物检测方法。在现在 项目中，我们提出了一种病原体检测技术，该技术 结合了酶联免疫吸附试验和聚合酶链式反应的优点。预计这将导致 在一种高效、可重复的方法中检测到非常小的 病原体的数量，甚至在它们可以引发正常宿主免疫之前 回应。该项目将分两个阶段进行。在第一阶段，该方法 将通过测量具有以下特性的蛋白质和多肽来标准化 在脑功能或病理学方面特别重要。这将使用以下工具完成 要么是特异性和丰富表达它们的脑细胞，要么是在 异源细胞，如淋巴细胞，在这些细胞中它们的表达非常低 水平，通过一种被称为“非法转录”的现象。在第二阶段，我们 将扩展在阶段1中开发的协议以检测和量化 测量一些在人类群体中常见的病原体和 这是公共卫生当局非常关注的问题。四种不同的 免疫-聚合酶链式反应的方法将根据(A)基于电泳法和 DNA扩增产物的溴化乙锭染色及条带密度测定 获得：(B)测量在反应后产生的荧光 与染料PicoGreen的混合；反应后的比色法 地高辛-碱性磷酸连接到DNA的显色底物；以及 (D)荧光作用产生的络合物的荧光测定法 DNA-地高辛配基上的物质(AttoPhos)。 建议的商业应用： (A)开发快速、高灵敏、早期检测神经病原体试剂盒 人体和动物组织和体液；(B)评价疫苗的效力， 通常用于有感染风险的人；(C)免疫反应的评估 在初次感染或免疫之后，以及(D)可能的身份识别和 对导致特发性神经病变的微生物的治疗。

DESCRIPTION (adapted from applicant's abstract): Pathogenic organisms are believed to be responsible for a number of infections, neurodegenerative and as well as some types of neuropsychiatric diseases. Detection of neuroactive pathogens, at the earliest stages, would be a critical factor in the diagnosis and treatment of any possible neuropathological consequences. Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are already being used separately as specific microbial detection methods. In the present project, we propose to develop a technology of pathogen detection, which combines the advantages of ELISA and those of PCR. This is expected to result in a highly efficient, reproducible method for the detection of very small numbers of pathogens, even before they can elicit the normal host immune response. The project will be carried out in two phases. In phase 1, the method of immuno-PCR will be standardized by measuring proteins and peptides that have special importance in brain function or pathology. This will be done using either brain cells where they are specifically and abundantly expressed, or in heterologous cells, such as lymphocytes, where they are expressed at very low levels, by a phenomenon known as "'illegitimate transcription". In phase 2 we will extend the protocols developed in phase 1 to detect and quantitatively measure some of the pathogens that are commonly found in human populations and which are of great concern to public health authorities. Four different approaches to immuno-PCR will be compared, based (a) on electrophoresis and ethidium bromide staining of the amplified DNA and densitometry of the band obtained: (b) measurement of fluorescence produced following reaction of the PCR mixture with the dye, PicoGreen; colorimetry following reaction of chromogenic substrate with digoxigenin-alkaline phosphate ligated to DNA; and (d) fluorometry of the complex produced by the action of a fluorescent substance (AttoPhos) on the DNA-digoxigenin complex. PROPOSED COMMERCIAL APPLICATION: (a) Development of rapid and highly sensitive, early detection kits for neuropathogens in human and animal tissues and body fluids; (b) Evaluation of the efficiency of vaccines, commonly used in persons at risk for infection; (c) Evaluation of the immune reaction subsequent to primary infection or immunization, and (d) possible identification and treatment of microorganisms responsible for idiopathic neuropathogenesis.