No adverse effects, such as redness or itching were observed, and thus we conclude that TAP is a highly convenient and versatile new diagnostic tool for non-invasive biomarker measurements from skin that is safe to use. Methods Antibodies Human GRO-? (CXCL-2) ELISA Development Kit (Cat. patients. and captured IL-1, IL-1RA, CXCL-1/2 and hBD-1 was subsequently analyzed similar to the method used for quantitative analyses of proteins captured directly from skin using TAP. All four methods were capable of detecting IL-1, IL-1RA and hBD-1 from skin, with the exception of Ivachtin the ELISA method used for hBD-1 (see Figure?7, panels A, B and D). In contrast, none of the methods were capable of detecting CXCL-1/2 (see Figure?7, panel C). Clear differences were observed in the efficacy of protein measurements from the skin. TAP was by far the most sensitive method used, yielding apparent concentration values 20- (IL-1) and 15- (IL-1RA) fold in excess of the values found using a combination of skin lavage and ELISA. Using TAP capture antibody micro-arrays for the analyses of IL-1, IL-1RA, CXCL-1/2 and hBD-1 from skin lavage Ivachtin yielded only marginal improvements in detection efficacy over ELISA. Since TAP uses the same micro-arrays, this suggests that the greater efficacy of TAP may be related, Ivachtin at least in part, to differences in the concentration of skin proteins in skin lavage and in the fluid on the interface between micro-array and skin in the TAP procedure. Then again, applying the capture antibody micro-arrays in skin lavage proved approximately 8- (IL-1) and 3.5- (IL-1RA) times more efficient than applying the same skin lavage to capture antibody micro-arrays em in vitro /em , suggesting that capturing proteins directly from skin, as performed during the TAP procedure and during the capturing proteins using micro-arrays em in situ /em , is far more efficient than capturing of skin proteins from skin lavage em in vitro /em . Open in a separate window Figure 7 Measurements of IL-1 , IL-1RA, CXCL-1/2 and hBD-1 from healthy skin using TAP and from skin lavage techniques. TAP measurements of IL-1, IL-1RA, CXCL-1/2 and hBD-1 from healthy skin were analyzed and compared with three different methods using skin lavage. See text for details. Signal intensities for the different methods were compared to signals obtained using fixed concentrations of recombinant IL-1, IL-1RA, CXCL-1/2 and hBD-1 captured in solution. In the graphs, it is clearly visible that TAP is the most sensitive method Rabbit Polyclonal to APLF for the detection of IL-1 (panel A), IL-1RA (panel B) and hBD-1 (panel D) on skin. CXCL-1/2 (panel C) could not be detected by any of the methods. Each data point in the graphs represents the results of two spots analyzed on 20 TAPs (N?=?10). Y-axis: Apparent concentration of IL-1, IL-1RA, CXCL-1/2 or hBD-1 on skin in ng/ml. X-axis: Incubation time on skin in minutes. Error bars on graph present standard deviation of analyzed data points (N?=?10). Discussion Skin biomarker measurements have enormous value for detailed assessment of skin conditions, both for clinical applications and in skin care. Biomarkers secreted on skin can be used to assess the condition of skin at a given time-point, but may also be used to assess how a given skin condition may progress in time, or how skin may react to treatments. Skin biomarker measurements are usually performed via invasive methods that are unpleasant for patients and that require specialised laboratories for analyses. In recent years, methods have been developed for the analyses of skin biomarkers by non-invasive methods, based on the extraction of biomarkers from the surface of pores and skin. Using skin-lavage to draw out biomarkers from the surface of pores and skin, Portugal-Cohen em et al /em . have shown clear variations in expression levels of pores and skin hydrophilic biomarkers, including cytokines (IL-1, TNF- and IL-6) and antioxidants (uric acid, total antioxidant scavenging capacity), between lesional and non-lesional pores and skin in individuals with atopic dermatitis and psoriasis [7, 36]. Similarly, Portugal-Cohen.