: 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.
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