Circulating Cell-free Antigens for Monitoring of Machining Fluids

项目摘要（摘要） 定植于工业金属加工液（MWF）中的分枝杆菌是公认的致病性抗原， 机械师职业性肺病过敏性肺炎（HP）及相关呼吸道症状。 目前还没有免疫测定法用于常规监测这些工业过程流体中的有害物质。 暴露评估的抗原。这主要是由于缺乏对特定分枝杆菌的了解， 这些体液中普遍存在的抗原。考虑到完整细胞计数可能会低估 分枝杆菌的抗原负荷（由于积累的无细胞抗原），特别是在长时间 这些液体的循环，监测抗原可能会提供更多信息。因此，总体目标是 本研究旨在鉴定这些体液中流行的主要循环无细胞（CF）分枝杆菌抗原， 他们开发了抗原监测检测方法，用于暴露评估。在这种情况下，靶向 全抗原可能缺乏特异性，因为在个体中存在多个和交叉反应性表位， 抗原，需要鉴定作为检测抗体靶标的特异性关键表位。这导致我们 假设鉴定选择性表达和积累分枝杆菌抗原蛋白 在加工液中作为主要的无细胞抗原及其关键表位可以形成一个基础， 在这些工业流体中作为检测目标， 免疫分析，为机械加工行业提供早期可靠的暴露评估策略。的 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） 在田间MWF中占优势;（2）.用于常规体液监测的多重免疫分析平台的设计 靶向工业用MWF中的cf分枝杆菌抗原（表位）。候选抗原将是 使用基于鸟枪蛋白质组学的免疫蛋白质组学方法鉴定它们，并鉴定它们的关键表位。在 平行的努力，基于ELISA的免疫测定将优化MWF矩阵，并扩大到多重 使用关键识别表位和抗体的免疫测定法;将验证该测定法的快速和灵敏度 检测用于分枝杆菌暴露评估的工业MWF样品中的循环cf抗原。的 由此产生的信息将进一步推动诺拉的目标和NIOSH的r2 p倡议，通过提供一套工具， 加工液中HP致病抗原暴露评估。这些成果将促进未来 进行流行病学研究，并制定针对接触这些工业过程的干预战略 液体长期。

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.