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ENZYME IMMUNOASSAYS FOR THE DIAGNOSIS OF BLASTOMYCOSIS

用于诊断芽生菌病的酶免疫分析

基本信息

批准号：
3436588
负责人：
Gene M Scalarone
金额：
$ 5.28万
依托单位：
IDAHO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-09-30 至 1988-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3436588
关键词：
Blastomyces dermatitidis antifungal antibody antiserum blastomycosis communicable disease diagnosis diagnostic tests enzyme linked immunosorbent assay fungal antigens gel electrophoresis immunologic assay /test microorganism classification microorganism immunology respiratory infections serology /serodiagnosis

项目摘要

Blastomycosis, a human systemic fungal disease caused by the organism Blastomyces dermatitidis, exists primarily in the southeastern and south central United States. Infection results from the inhalation of the fungal spore into the lung and a disease similar to tuberculosis is produced. The immunological diagnosis of blastomycosis is extremely difficult at the present time due to the unavailability of reliable antigens and the lack of specificity and sensitivity of the serological assays for serum antibodies. During the past three years our laboratory has developed sensitive procedures for the detection of immune responses associated with the fungal diseases histoplasmosis and coccidioidomycosis, which are similar to blastomycosis. It is proposed that this technique, the enzyme-linked immunosorbent assay (ELISA), will be adapted and optimized for the immunodiagnosis of blastomycosis. Yeast-phase antigens will be prepared from several strains of B. dermatitidis and compared with commercially available Blastomyces complement-fixation/immunodiffusion antigens using electrophoretic separation techniques and immunoblotting methods in an effort to determine antigenic composition. Studies will then be performed on purification, optimization and utilization of these antigens in ELISA and DOT-ELISA procedure to detect antibodies (IgG, IgM, IgA) in sera from patients with blastomycosis and other systemic fungal diseases. In addition comparative evaluations will be performed using different enzyme systems, different substrates and the avidin-biotin system in an effort to develop a highly specific and sensitive assay for the definitive diagnosis of blastomycosis. This proposed research work has the potential for future expansion to include (1) use of the antigens as skin-testing reagents to detect delayed dermal hypersensitivity (2) use of the reagents to produce polyclonal/monoclonal antibodies that could be used in ELISA procedures to detect antigens and (3) use of the antigens in vaccine preparations for the prevention of blastomycosis.

芽孢霉病是一种人类系统性真菌病，由皮炎芽孢杆菌，主要存在于美国东南部和中南部。感染真菌孢子吸入肺部的结果并产生一种类似于结核病的疾病。这个芽生菌病的免疫学诊断极其困难目前由于无法获得可靠的抗原和缺乏特异性和敏感性血清抗体的血清学检测。在过去三年中多年来，我们的实验室已经开发出敏感的程序与真菌相关的免疫反应的检测组织胞浆菌病和球孢子菌病，它们是类似于芽生菌病。有人提出，这种技术，将采用酶联免疫吸附试验(ELISA) 并对芽生菌病的免疫诊断进行了优化。酵母期抗原将由几个菌株制备皮炎假单胞菌及其与市售的比较芽孢霉菌补体结合/免疫扩散抗原电泳法分离技术与免疫印迹方法测定抗原性成分。然后将对提纯、优化及其在酶联免疫吸附试验和斑点酶联免疫吸附试验中的应用流行性出血热患者血清中抗体(Ig G、Ig M、Ig A)的检测程序芽生真菌病和其他系统性真菌病患者疾病。此外，还将进行比较评价使用不同的酶系统、不同的底物和亲和素-生物素系统，努力开发一种高度特异度和敏感度测定用于明确诊断芽生菌病。本文提出的研究工作具有很大的发展潜力。扩大到包括(1)将抗原用作皮肤测试检测迟发性皮肤超敏反应的试剂(2) 产生多克隆/单克隆抗体的试剂可用于酶联免疫吸附试验检测抗原和(3) 抗原在预防疫苗制剂中的应用芽生真菌病。