Title: Validation of Assays for Inflammatory Mediators in Exhaled Breath Condensate Short title: Validation of Assays in Exhaled Breath Condensate Keywords: Exhaled Breath Condensate, Interleukin 8, Leukotriene B4, Secretory leukoprotease inhibitor, Alpha-1-Antitrypsin, Myeloperoxidase

The use of exhaled breath condensate as a tool for non-invasive assessment of lung inflammation is becoming commonplace. Many authors use commercial ELISA kits to measure inflammatory mediators within EBC. However, the very low concentrations of mediators within EBC are often below the commercially validated concentration range of the relevant ELISA and crucially the linear part of the sigmoid standard curve. This study seeks to validate a series of assays for use in EBC and to compare the results in EBC with those from matched sol-phase sputum samples. The following mediators: Leukotriene B4, interleukin 8, secretory leukoprotease inhibitor, alpha-1-antitrypsin were measured by ELISA. Myeloperoxidase was measured by chromogenic substrate assay. Mediator concentrations reached lower limit of quantification in only one assay (alpha-1-antitrypsin) in 19.6% of subjects, whilst mediator concentrations reached lower limit of detection in three assays (leukotriene B4, interleukin 8 and alpha-1-antitrypsin in 31%, 6.5% and 61% of subjects respectively). No significant correlations were present between any mediators in exhaled breath condensate and sol-phase sputum. These results indicate that care must be exercised when interpreting mediator measurements in exhaled breath condensate and that assays must be validated at concentrations relevant to those found within the biological fluid.