Aspergillosis: Engineering Antibody for Diagnosis

描述（由申请方提供）：真菌病原体烟曲霉主要在免疫功能紊乱的患者中引起侵袭性疾病，最常见的是重度中性粒细胞减少症或器官移植后。侵袭性曲霉病的发病率在过去三十年中稳步上升，目前死亡率为45 - 90%。对A.烟曲霉长期以来一直是个问题，因为确定性诊断需要培养物中的生长和菌丝的显示。2003年，美国食品和药物管理局批准了一项诊断测试，用于检测A。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，该测定可能不是最佳的。本试验中使用的单克隆抗体采用标准杂交瘤技术开发。最近引入的抗体工程技术已经允许产生亲和力增强10，000倍的抗体试剂。更高亲和力的试剂可以允许开发更灵敏的检测测定，并且可以被选择用于增强的特异性。本提案的目标是开发第二代半乳甘露聚糖检测抗体试剂。方法将是诱变我们制造的结合A的单克隆抗体。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抗体的分子特征， 用于产生更高亲和力、更有选择性的试剂的抗体;和2.以确定是否可以通过抗体工程产生更高亲和力、更有选择性的试剂。我们期望这些研究将提供关于抗体工程技术应用于结合碳水化合物抗原的Ab试剂的亲和力增强的新知识。有可能提高半乳甘露聚糖检测的灵敏度和特异性的试剂的可用性将是一个重大的进步。

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.