ENZYME INNUNOASSAYS FOR THE DIAGNOSIS OF BLASTOMYCOSIS

芽生菌病诊断的酶免疫分析

• 批准号: 3135029
• 负责人: Gene M Scalarone
• 金额: $ 6.27万
• 依托单位: IDAHO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-30 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3135029
• 关键词: Blastomyces dermatitidis; alkaline phosphatase; animal tissue; antibody specificity; antifungal antibody; antiserum; biotin; blastomycosis; delayed hypersensitivity; diagnosis design /evaluation; electrophoresis; enzyme linked immunosorbent assay; fungal antigens; guinea pigs; human tissue; immunoglobulin A; immunoglobulin G; immunoglobulin M; laboratory rabbit; microorganism culture; protein purification; serum; spectrometry; urease; western blottings

Blastomycosis, a systemic granulomatous disease caused by the dimorphic fungal organism Blastomyces dermatitidis, exists in southeastern, southcentral and midwestern areas of the United States. Following inhalation of the infectious particle a wide variety of pulmonary and extrapulmonary manifestations may result. The diagnosis of human and canine blastomycosis has presented a challenge to clinicians for many years. In some cases cultural or histologic studies allow for rapid isolation and identification of the agent, but in other instances the procedures may be time consuming and may not provide evidence necessary for diagnosis. Immunodiagnostic assays generally provide a more rapid diagnosis, but problems related to specificity and sensitivity have been attributed to the currently used complement fixation (CF) and immunodiffusion (ID) tests. Recent studies in our laboratory (supported by an NIH AREA grant) using the enzyme-linked immunosorbent assay (ELISA) to detect anti-b-dermatitidis antibodies in human and dogs have provided encouraging results with respect to continued development of the assay. We have developed a new yeast cell lysate method for the preparation of antigens from various strains of B. dermatitidis and the reagents so produced have provided excellent results in the ELISA that were superior to results obtained with commercial B. dermatitidis antigens. Our work has indicated a need for an extensive study to optimize the assay and will be aimed at performing comparative evaluations concerned with (1) antigen preparation and characterization methods and (2) the development of antibody and antigen detection methods. The broad, long term objectives are concerned with comparative identification, isolation and purification of the active antigenic components of B. dermatitidis and to subsequently use the antigens and antibodies produced from them in ELISA or other assays to detect antibodies and antigens in humans and animals with blastomycosis. Yeast and mycelial phase lysate antigens will be prepared from human and canine isolates and compared with commercial B. dermatitidis commercial reagents with respect to antigenic composition (reactive and cross- reactive components) as determined by electrophoretic and immunoblotting methods. The lysate antigens and commercial reagents will be compared in ELISA procedures (microplate and nitrocellulose membrane) as determined by electrophoretic and immunoblotting methods. The lysate antigens and commercial reagents will be compared in ELISA procedures (microplate and nitrocellulose membrane) and an optimal antigen will be developed for the detection of antibody (IgG,IgM,IgA) associated with human and canine disease. Monospecific polyclonal antibodies will be prepared from the optimal antigen and utilized as probes for antigen detection. The ultimate aim of this project is to develop sensitive and specific methods to detect antibodies and antigens in humans and animals with blastomycosis.

芽生真菌病，一种由二晶型引起的系统性肉芽肿性疾病 真菌生物皮炎芽孢杆菌，存在于东南部， 美国中南部和中西部地区。跟随 吸入具有感染性的颗粒物可引起多种肺部和 可能会导致肺外表现。人类和人类疾病的诊断 犬芽孢菌病对许多临床医生来说是一个挑战。 好几年了。在某些情况下，文化或组织学研究允许快速 代理的隔离和标识，但在其他情况下 程序可能很耗时，而且可能不提供必要的证据 用于诊断。免疫诊断分析通常提供更快速的 诊断，但与特异性和敏感性相关的问题 归因于目前使用的补体结合(CF)和 免疫扩散(ID)试验。我们实验室的最新研究(支持 由美国国立卫生研究院地区拨款)使用酶联免疫吸附试验 (酶联免疫吸附试验)检测人和狗的抗乙型皮炎抗体 在持续发展方面提供了令人鼓舞的结果 化验结果。我们开发了一种新的酵母菌细胞裂解物方法 不同皮炎杆菌株抗原的制备及免疫原性研究 如此生产的试剂在ELISA法中提供了良好的结果 优于商业皮炎杆菌的检测结果 抗原。我们的工作表明有必要进行广泛的研究 优化化验方法，并将致力于进行比较 与(1)抗原制备和表征有关的评价 方法和(2)抗体和抗原检测的发展 方法：研究方法。广泛的、长期的目标是比较 活性抗原性的鉴定和分离纯化 皮炎杆菌的成分，并随后使用这些抗原和 在酶联免疫吸附试验或其他检测中产生的抗体 芽孢菌病患者和动物体内的抗体和抗原。酵母菌 菌丝相裂解抗原将从人和 犬的分离株及其与商品皮炎杆菌的比较 关于抗原成分的试剂(反应性和交叉性 活性成分)通过电泳法和免疫印迹法检测 方法：研究方法。将对裂解抗原和商业试剂进行比较 在酶联免疫吸附试验中(微孔板和硝酸纤维素膜) 用电泳法和免疫印迹法检测。裂解物 抗原和商业试剂将在酶联免疫吸附试验程序中进行比较 (微孔板和硝酸纤维素膜)，最佳抗原为 开发用于检测与以下疾病相关的抗体(Ig G、Ig M、Ig A) 人类和犬类疾病。单特异性多克隆抗体将是 从最佳抗原制备并用作抗原探针 侦测。该项目的最终目标是开发敏感和 检测人和动物体内抗体和抗原的特殊方法 患有芽孢霉病。