Circulating Cell-free Antigens for Monitoring of Machining Fluids

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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.