Development of anti-Aspergillus single chain Abs as serum and imaging diagnostics

开发抗曲霉单链抗体作为血清和成像诊断

• 批准号: 8413658
• 负责人: MICHAEL D GUNN
• 金额: $ 46.25万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8413658
• 关键词: Adverse effects; Affinity; Animals; Antibodies; Antibody Dissociations; Antibody Specificity; Antigens; Aspergillosis; Aspergillus; Avidity; Bacteriophages; Binding; Biodistribution; Biological Assay; Biology; Blood; Cell surface; Cells; Characteristics; Clinical; Collaborations; Complementarity Determining Regions; Data; Detection; Development; Diagnosis; Diagnostic; Diagnostic Imaging; Diagnostic Reagent; Diagnostic tests; Disease; Engineering; Enzyme-Linked Immunosorbent Assay; Fungal Typing; Goals; Grant; Human; Human Cloning; Image; Imaging Techniques; Immunocompromised Host; Incidence; Individual; Infection; Kinetics; Laboratories; Lead; Libraries; Light; Methodology; Methods; Modality; Modeling; Mus; Mutagenesis; Mutate; Mutation; Mycoses; Nucleic Acids; Patients; Phage Display; Pre-Clinical Model; Process; Radiolabeled; Rattus; Reagent; Recombinant Antibody; Recombinants; Recording of previous events; Risk; Sampling; Science; Screening procedure; Sensitivity and Specificity; Serum; Specificity; Surface Antigens; Testing; Tissues; Variant; base; clinical application; clinically relevant; complementarity-determining region 3; cross reactivity; density; design; diagnostic accuracy; experience; fungus; galactomannan; improved; mortality; mutant; novel; pathogen; radiotracer; single photon emission computed tomography; success; tumor

Project Summary Invasive aspergillosis (IA) is the most common filamentous fungal infection in immunocompromised patients. Mortality in patients diagnosed with this condition ranges from 70% to 90% at one year. Current methods to diagnose IA include imaging techniques and the detection of antigens, anti-Aspergillus antibodies, or Aspergillus nucleic acids. Unfortunately, current tests have only a poor to moderate accuracy for the diagnosis of invasive aspergillosis in immunocompromised patients. Here, we propose to develop highly sensitive and specific recombinant anti-aspergillus single-chain antibodies (scFvs) and using these as the basis for new and accurate diagnostic assays. In the R21 portion of this grant, our goal is to generate high affinity monomeric or multimeric aspergillus-specific scFvs and develop these reagents to the extent that their utility as potential diagnostics is demonstrated. In Aim 1, we will identify aspergillus- specific phage scFv clones by screening na¿ve human scFv phage libraries. In Aim 2, we will identify scFvs that detect circulating aspergillus antigens. In Aim 3, we will express and purify these clones as monomeric scFvs and various multimeric constructs and validate their binding activities. In Aim 3, we will identify scFv pairs that can be used as capture and detection reagents for ELISA assays and diagnostic Ab arrays. In Aim 4, we will generate radiolabeled aspergillus-binding reagents and identify those with the highest affinity, stability, and target density. The R33 portion of this grant will involve three aspects. First, we will improve the sensitivity and specificity of our recombinant antibodies by generating and screening randomly mutated variants of these antibodies. Second, we will development these recombinant antibodies as serum diagnostics using either standard ELISA assays or antibody arrays. Third, we will develop methods to detect Aspergillus infection via the use of recombinant antibody imaging.

项目总结