Circulating Cell-free Antigens for Monitoring of Machining Fluids

用于监测加工液的循环无细胞抗原

• 批准号: 9979088
• 负责人: Jagjit S Yadav
• 金额: $ 21.5万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2022-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979088
• 关键词: Circulating; Cell; free; Antigens; Monitoring

Project summary (Abstract) Mycobacteria colonizing industrial metalworking fluids (MWFs) are recognized causal antigens of the occupational lung disease hypersensitivity pneumonitis (HP) and related respiratory symptoms in machinists. Currently there are no immunoassays for routine monitoring of these industrial process fluids for the offending antigens for exposure assessment. This is primarily due to the lack of knowledge of specific mycobacterial antigens prevalent in these fluids. Considering the fact that intact cell count may underestimate the total antigen load for mycobacteria (due to the accumulating cell-free antigens) especially during prolonged circulation of these fluids, monitoring for the antigens may be more informative. Therefore, the overall goal of this study is to identify major circulating cell-free (cf) mycobacterial antigens prevalent in these fluids and target them for development of antigen monitoring assay for exposure assessment. In this context, assays targeting whole antigens may lack specificity because of presence of multiple and cross-reactive epitopes in individual antigens, necessitating identification of specific key epitopes as detection antibody targets. This led us to hypothesize that identification of mycobacterial antigenic proteins selectively expressed and accumulated in machining fluids as dominant cell-free antigens and their key epitopes could form the basis for an informative antigen monitoring in these industrial fluids by serving as assay targets in a multiplex immunoassay, offering an early and reliable exposure assessment strategy in the machining industry. The specific aims are to: (1). Identify circulating cell-free mycobacterial antigens (and their key epitopes) predominant in field MWF; (2). Designing of a multiplex immunoassay platform for routine fluid monitoring targeting the cf mycobacterial antigens (epitopes) in industrial in-use MWF. Candidate antigens will be identified using immunoproteomic approach based on shotgun proteomics and their key epitopes identified. In parallel efforts, an ELISA-based immunoassay will be optimized for MWF matrix and expanded to a multiplex immunoassay using key identified epitopes and antibodies; the assay will be validated for rapid and sensitive detection of circulating cf antigens in industrial MWF samples for mycobacterial exposure assessment. The resulting information will further the NORA’s objectives and NIOSH’s r2p initiative by providing a set of tools for HP-causative antigen exposure assessment in machining fluids. These outcomes will facilitate future epidemiological studies and development of intervention strategies for exposures to these industrial process fluids in long-term.

项目概要 工业金属加工液 (MWF) 中定殖的分枝杆菌是公认的致病抗原 职业性肺病、过敏性肺炎（HP）和机械师的相关呼吸道症状。 目前还没有免疫分析方法可以对这些工业过程流体中的有害物质进行常规监测 这主要是由于缺乏对特定分枝杆菌的了解。 考虑到完整细胞计数可能会低估总数。 分枝杆菌的抗原负荷（由于无细胞抗原的积累），尤其是在长时间 因此，对这些液体的循环进行抗原监测可能会提供更多信息。 这项研究旨在确定这些液体中普遍存在的主要循环无细胞 (cf) 分枝杆菌抗原并靶向 他们开发了用于暴露评估的抗原监测测定，在这种情况下，测定靶向。 由于个体中存在多个交叉反应表位，整个抗原可能缺乏特异性 抗原，需要鉴定特定的关键表位作为检测抗体目标。 致力于鉴定选择性表达和积累的分枝杆菌抗原蛋白 在加工流体中作为主要的无细胞抗原及其关键表位可以构成 通过作为多重检测中的信息检测目标来监测这些工业液体中的抗原 免疫测定，为机械加工行业提供早期且可靠的暴露评估策略。 具体目标是： (1) 鉴定循环无细胞分枝杆菌抗原（及其关键表位）。 现场MWF的主导技术；（2）设计用于常规液体监测的多重免疫分析平台。 针对工业使用的 MWF 中的 cf 分枝杆菌抗原（表位）将是候选抗原。 使用基于鸟枪蛋白质组学的免疫蛋白质组学方法进行鉴定，并鉴定了其关键表位。 同时，基于 ELISA 的免疫分析将针对 MWF 基质进行优化，并扩展到多重分析 使用关键的已识别表位和抗体进行免疫测定；该测定将被验证快速且灵敏 检测工业金属加工液样品中的循环 cf 抗原以进行分枝杆菌暴露评估。 由此产生的信息将通过提供一套工具来促进 NORA 的目标和 NIOSH 的 r2p 倡议 这些结果将有助于未来对加工液中 HP 致病抗原暴露的评估。 流行病学研究和针对这些工业过程暴露的干预策略的制定 长期液体。