RAPID IDENTIFICATION OF MYCOPLASMAS WITH IMMUNOENZYMES

我们提出的这个项目是为了检验技术 一种新型支原体鉴定系统的可行性。 支原体是一种类似细菌的微生物，可引起人类和动物疾病 和污染细胞培养，是无处不在的。他们必须被识别以 属和种的水平，以确定其来源并帮助 制定有效的治疗和预防措施。我们的免疫酶 鉴定系统将使用在人工介质上生长的菌落作为 抗原性物质来源。菌落将被吸附到小的合成材料上 促进免疫酶反应的圆盘。他们将被安置在一个 特殊设计的多孔材料夹持器(“微过滤器”)，便于进入 处理和真空中介漂洗。从特定的电池中提取的样本 将抗血清添加到圆盘中孵化。抗原-抗体 将通过生化检测来检测识别试剂的反应 涉及蛋白A、生物素、亲和素和酶标记物的相互作用。 这种类型的序列为每个抗原创造了大量的标记分子 分子。这种类型的放大使这样的测试多了几个对数 比使用中的旧化验方法(如荧光抗体)更灵敏。添加 适当的底物(例如，二氨基联苯胺 过氧化物酶)将导致显色最终产物的大体可见的形成 在附着在盘上的菌落上和在菌落中。反应的程度将是 用我们自己设计的视频密度计进行定量。硬拷贝 打印结果将用于证实身份。整个系统 (协议、磁盘、微过滤器、电子密度计)代表了一种新的 以及独特的识别抗原颗粒的方法。 这项技术结合了快速、简单和灵敏。数据将 客观、定量，并与自动化方法和 计算机分析和检索。这一概念具有直接性和即时性 应用于普遍存在的支原体感染和 污染。它还具有用于其他生物医学领域的潜力。 例如细菌、酵母菌和真菌的快速物种形成。这个项目， 为SBIR计划开发，与计划描述一致 NIAID微生物学与传染病，“创新”一节 生物医学技术“。

The project which we propose is designed to examine the technical feasibility of a new type of identification system for mycoplasmas. Mycoplasmas, bacteria-like microbes which cause human and animal disease and contaminate cell cultures, are ubiquitous. They must be identified to the genus and species level in order to identify their source and to aid in developing effective therapy and prevention measures. Our immunoenzyme identification system will use colonies grown on artificial medium as a source of antigenic material. Colonies will be adsorbed to small synthetic disks to facilitate immunoenzyme reactions. They will be placed in on a specially-designed holder of porous material ("microfilter") for ease in handling and vacuum-mediated rinsing. Samples from a battery of specific antisera will be added to the disks and incubated. The antigen-antibody reaction which identifies the agent will be detected through biochemical interactions involving protein A, biotin, avidin, and an enzyme marker. This type of sequence creates a multitude of marker molecules per antigen molecule. This type of amplification makes such a test several logs more sensitive than older assays in use (e.g., fluorescent antibody). Addition of the appropriate substrate (e.g., diaminobenzidine in the case of peroxidase) will cause grossly-visible formation of chromogenic endproduct on and in the colonies adherent to the disk. The degree of response will be quantitated with a videodensitometer of our own design. Hard copy printout will serve to substantiate the identification. The entire system (protocol, disks, microfilter, electronic densitometer) represents a new and distinctive approach to the identification of antigenic particulates. This technique incorporates speed, simplicity, and sensitivity. Data will be objective, quantitative, and compatible with automated methods and computer analysis and retrieval. This concept has direct and immediate appliction to the widespread problem of mycoplasma infection and contamination. It also has the potential for use in other biomedical areas such as the rapid speciation of bacteria, yeasts and fungi. This project, developed for the SBIR program, is consistent with the Program Description of NIAID Microbiology and Infectious Diseases, section under "Innovative Biomedical Technologies".