Aspergillosis: Engineering Antibody for Diagnosis

描述（由申请人提供）：真菌病原体烟曲霉（Aspergillus fumigatus）主要在免疫功能紊乱的患者中引起侵袭性疾病，最常见的是严重中性粒细胞减少症或器官移植后。在过去三十年中，侵袭性曲霉病的发病率稳步上升，目前的死亡率在45-90%之间。长期以来，烟曲霉引起的侵袭性疾病的诊断一直存在问题，因为培养物的生长和菌丝的显示都是明确诊断所必需的。2003年，美国食品和药物管理局批准了一种诊断测试，用于检测血清中的烟曲霉半乳甘露聚糖。目前的工作重点是了解该测试的最佳用途和局限性。然而，由于灵敏度和特异性的限制，这种分析可能不是最佳的。本实验中使用的单克隆抗体是用标准杂交瘤技术制备的。最近引入的抗体工程技术已经允许产生具有10,000倍亲和力增强的抗体试剂。更高亲和力的试剂可能允许开发更敏感的检测试验，并且可以选择增强特异性。本课题的目标是开发半乳甘露聚糖检测的第二代抗体试剂。该方法将诱变单克隆抗体结合烟曲霉半乳甘露聚糖。提出了两个具体目标：1。确定半乳甘露聚糖结合单克隆的分子特征

DESCRIPTION (provided by the applicant): The fungal pathogen Aspergillus fumigatus causes invasive disease predominantly in patients with derangements in immune function, most often severe neutropenia or following organ transplantation. The incidence of invasive aspergillosis has risen steadily over the past three decades and mortality rates currently range from 45-90%. The diagnosis of invasive disease due to A. fumigatus long has been problematic, as both growth in culture and demonstration of hyphae are required for definitive diagnosis. In 2003, a diagnostic test was approved by the U.S. Food and Drug Administration for detection of A. fumigatus galactomannan in serum. Current efforts are focused on understanding the best use and limitations of this test. However, this assay likely is not optimal because of limitations in both sensitivity and specificity. The monoclonal antibody used in this assay was developed with standard hybridoma technology. More recently introduced techniques for antibody engineering have allowed the generation of antibody reagents with 10,000 fold enhancement of affinity. Higher affinity reagents may allow for development of more sensitive detection assays and can be selected for enhanced specificity. The goal of this proposal is to develop second generation antibody reagents for galactomannan detection. The approach will be to mutagenize monoclonal antibodies we have made that bind A. fumigatus galactomannan. Two specific aims are proposed: 1. to determine the molecular characteristics of galactomannan-binding monoclonal antibodies to be used for generating higher affinity, more selective reagents; and 2. to determine whether higher affinity, more selective reagents can be created through antibody engineering. We expect that these studies will provide new knowledge regarding the application of antibody engineering techniques to affinity enhancement of Ab reagents that bind carbohydrate antigens. The availability of reagents that have potential to improve both sensitivity and specificity of galactomannan detection would be a significant advance.