DEVELOPMENT OF IMMUNO-PCR NEURODIAGNOSTIC KITS

描述（改编自申请人摘要）：病原微生物是 被认为是负责一些感染，神经退行性疾病， 以及某些神经精神疾病。神经活性物质检测 病原体，在最早的阶段，将是诊断的关键因素， 和治疗任何可能的神经病理学后果。酶联 免疫吸附测定（ELISA）和聚合酶链反应（PCR）已经 作为特异性微生物检测方法单独使用。本 项目，我们建议开发一种病原体检测技术， 结合了ELISA和PCR的优点。预计这将导致 在一种高效、可重复的方法中， 许多病原体，甚至在它们能够引发正常宿主免疫之前， 反应该项目将分两个阶段进行。在第一阶段，该方法 免疫PCR的标准化将通过测量蛋白质和肽， 在大脑功能或病理学中具有特殊重要性。这将使用 无论是脑细胞，在那里他们是专门和丰富的表达，或在 异源细胞，例如淋巴细胞，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 将扩展第一阶段开发的协议，以检测和定量 测量一些在人群中常见的病原体， 这是公共卫生当局非常关注的问题。四个不同 将比较免疫PCR的方法，基于（a）电泳和 扩增DNA的溴化乙锭染色和条带的密度测定 获得：（B）测量以下物质反应后产生的荧光： 与染料PicoGreen的PCR混合物; 具有连接至DNA的地高辛配基-碱性磷酸盐的显色底物;和 (d)通过荧光试剂的作用产生的络合物的荧光测定法 在DNA-洋地黄毒苷复合物上的物质（AttoPhos）。 拟定商业应用： (a)神经病原体快速、高灵敏、早期检测试剂盒的研制 人和动物组织和体液;（B）评价疫苗的效力， 通常用于有感染风险的人;（c）评估免疫反应 在初次感染或免疫接种后，以及（d）可能的识别和 治疗导致特发性神经发病的微生物。

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.