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Development of TAP, a non-invasive test for qualitative and quantitative measurements of biomarkers from the skin surface.

Orro K, Smirnova O, Arshavskaja J, Salk K, Meikas A, Pihelgas S, Rumvolt R, Kingo K, Kazarjan A, Neuman T, Spee P - Biomark Res (2014)

Bottom Line: The Transdermal Analyses Patch (TAP) is a novel molecular diagnostic tool that has been developed to capture biomarkers directly from skin, which are quantitatively analyzed in spot-ELISA assays.Optimisation of protocols for TAP production and biomarker analyses makes TAP measurements highly specific and reproducible.In measurements of interleukin-1α (IL-1α), IL-1 receptor antagonist (IL-1RA) and human β-defensin (hBD-1) from healthy skin, TAP appears far more sensitive than skin lavage-based methods using ELISA.

View Article: PubMed Central - PubMed

Affiliation: FibroTx LLC, Mäealuse 4, 12918 Tallinn, Estonia.

ABSTRACT

Background: The skin proteome contains valuable information on skin condition, but also on how skin may evolve in time and may respond to treatments. Despite the potential of measuring regulatory-, effector- and structural proteins in the skin for biomarker applications in clinical dermatology and skin care, convenient diagnostic tools are lacking. The aim of the present study was to develop a highly versatile and non-invasive diagnostic tool for multiplex measurements of protein biomarkers from the surface of skin.

Results: The Transdermal Analyses Patch (TAP) is a novel molecular diagnostic tool that has been developed to capture biomarkers directly from skin, which are quantitatively analyzed in spot-ELISA assays. Optimisation of protocols for TAP production and biomarker analyses makes TAP measurements highly specific and reproducible. In measurements of interleukin-1α (IL-1α), IL-1 receptor antagonist (IL-1RA) and human β-defensin (hBD-1) from healthy skin, TAP appears far more sensitive than skin lavage-based methods using ELISA. No side-effects were observed using TAP on human skin.

Conclusion: TAP is a practical and valuable new skin diagnostic tool for measuring protein-based biomarkers from skin, which is convenient to use for operators, with minimal burden for patients.

No MeSH data available.


Related in: MedlinePlus

Measurements of recombinant IL-1RA. Measurements of recombinant IL-1RA, using 0.75 (triangles), 1.5 (squares) and 2.25 (diamonds) nanogram of anti-IL-1RA capturing antibody printed on nitrocellulose membranes, and a combination of either 500 or 166.67 ng/ml anti-IL-1RA detection antibody and 10% or 20% amplification reagents in spot-ELISA. Each data point consists of 10 measurements (N = 10). Each line presents an average mean of measurements obtained from duplicate. X-axis: Recombinant IL-1RA in ng/ml. Y-axis: Staining intensity defined as the mean pixel intensity measured on a 0–255 grey scale.
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Fig4: Measurements of recombinant IL-1RA. Measurements of recombinant IL-1RA, using 0.75 (triangles), 1.5 (squares) and 2.25 (diamonds) nanogram of anti-IL-1RA capturing antibody printed on nitrocellulose membranes, and a combination of either 500 or 166.67 ng/ml anti-IL-1RA detection antibody and 10% or 20% amplification reagents in spot-ELISA. Each data point consists of 10 measurements (N = 10). Each line presents an average mean of measurements obtained from duplicate. X-axis: Recombinant IL-1RA in ng/ml. Y-axis: Staining intensity defined as the mean pixel intensity measured on a 0–255 grey scale.

Mentions: Multiplex TAP protein measurements from skin require optimal analytic conditions to secure specific and sensitive measurements as well as a dynamic range that allows protein measurements from different skin conditions. IL-1α, IL-1RA, the combination of CXCL-1 and -2 (referred to as CXCL-1/2) and hBD-1 were chosen as model skin analytes based on literature reports describing that these proteins are expressed in healthy skin, and/or that these proteins have altered expression patterns in lesional (e.g. due to inflammation) or damaged (e.g. due to UV exposure) skin [7, 10–12, 18, 22, 23, 40–43]. For TAP micro-array development, Whatman Protran BA 85 nitrocellulose (0.45 μm porosity) strips were printed with three different amounts of capturing IL-1α, IL-1RA, CXCL-1/2 or hBD-1 antibodies (see Methods for details). Strips were subsequently incubated with different concentrations of recombinant IL-1α, IL-1RA, CXCL-2 or hBD-1. Following washing, strips were incubated with two different concentrations of anti-IL-1α, IL-1RA, CXCL-1/2 and hBD-1 detection antibodies and bound IL-1α, IL-1RA, CXCL-1/2 and hBD-1 were subsequently visualised using two different concentrations of amplification solution and quantitatively analyzed as described. As shown in Figure 4, exemplified by the data obtained for IL-1RA, the major variable that influenced the sensitivity as well as the dynamic range of recombinant IL-1RA measurements was the amount of capture antibody printed on the nitrocellulose strips. The highest amount of anti-IL-1RA used, 2.25 ng per printed spot, yielded the highest sensitivity, signal strength and dynamic range. The highest amount of anti-IL-1RA capture antibodies used, in combination with 10-fold diluted amplification reagents yielded the highest curve fit (R2 > 0.99). For the detection of IL-1RA from skin, 2.25 ng capture anti-IL-1RA per spot was chosen for printing TAP micro-arrays, in combination with a spot-ELISA protocol using 166.67 ng/ml detection anti-IL-1RA and a 1/10 fold dilution of amplification reagent. Optimal use of reagents for detection of IL-1α, CXCL-1/2 and hBD-1 were determined in a similar fashion.Figure 4


Development of TAP, a non-invasive test for qualitative and quantitative measurements of biomarkers from the skin surface.

Orro K, Smirnova O, Arshavskaja J, Salk K, Meikas A, Pihelgas S, Rumvolt R, Kingo K, Kazarjan A, Neuman T, Spee P - Biomark Res (2014)

Measurements of recombinant IL-1RA. Measurements of recombinant IL-1RA, using 0.75 (triangles), 1.5 (squares) and 2.25 (diamonds) nanogram of anti-IL-1RA capturing antibody printed on nitrocellulose membranes, and a combination of either 500 or 166.67 ng/ml anti-IL-1RA detection antibody and 10% or 20% amplification reagents in spot-ELISA. Each data point consists of 10 measurements (N = 10). Each line presents an average mean of measurements obtained from duplicate. X-axis: Recombinant IL-1RA in ng/ml. Y-axis: Staining intensity defined as the mean pixel intensity measured on a 0–255 grey scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362816&req=5

Fig4: Measurements of recombinant IL-1RA. Measurements of recombinant IL-1RA, using 0.75 (triangles), 1.5 (squares) and 2.25 (diamonds) nanogram of anti-IL-1RA capturing antibody printed on nitrocellulose membranes, and a combination of either 500 or 166.67 ng/ml anti-IL-1RA detection antibody and 10% or 20% amplification reagents in spot-ELISA. Each data point consists of 10 measurements (N = 10). Each line presents an average mean of measurements obtained from duplicate. X-axis: Recombinant IL-1RA in ng/ml. Y-axis: Staining intensity defined as the mean pixel intensity measured on a 0–255 grey scale.
Mentions: Multiplex TAP protein measurements from skin require optimal analytic conditions to secure specific and sensitive measurements as well as a dynamic range that allows protein measurements from different skin conditions. IL-1α, IL-1RA, the combination of CXCL-1 and -2 (referred to as CXCL-1/2) and hBD-1 were chosen as model skin analytes based on literature reports describing that these proteins are expressed in healthy skin, and/or that these proteins have altered expression patterns in lesional (e.g. due to inflammation) or damaged (e.g. due to UV exposure) skin [7, 10–12, 18, 22, 23, 40–43]. For TAP micro-array development, Whatman Protran BA 85 nitrocellulose (0.45 μm porosity) strips were printed with three different amounts of capturing IL-1α, IL-1RA, CXCL-1/2 or hBD-1 antibodies (see Methods for details). Strips were subsequently incubated with different concentrations of recombinant IL-1α, IL-1RA, CXCL-2 or hBD-1. Following washing, strips were incubated with two different concentrations of anti-IL-1α, IL-1RA, CXCL-1/2 and hBD-1 detection antibodies and bound IL-1α, IL-1RA, CXCL-1/2 and hBD-1 were subsequently visualised using two different concentrations of amplification solution and quantitatively analyzed as described. As shown in Figure 4, exemplified by the data obtained for IL-1RA, the major variable that influenced the sensitivity as well as the dynamic range of recombinant IL-1RA measurements was the amount of capture antibody printed on the nitrocellulose strips. The highest amount of anti-IL-1RA used, 2.25 ng per printed spot, yielded the highest sensitivity, signal strength and dynamic range. The highest amount of anti-IL-1RA capture antibodies used, in combination with 10-fold diluted amplification reagents yielded the highest curve fit (R2 > 0.99). For the detection of IL-1RA from skin, 2.25 ng capture anti-IL-1RA per spot was chosen for printing TAP micro-arrays, in combination with a spot-ELISA protocol using 166.67 ng/ml detection anti-IL-1RA and a 1/10 fold dilution of amplification reagent. Optimal use of reagents for detection of IL-1α, CXCL-1/2 and hBD-1 were determined in a similar fashion.Figure 4

Bottom Line: The Transdermal Analyses Patch (TAP) is a novel molecular diagnostic tool that has been developed to capture biomarkers directly from skin, which are quantitatively analyzed in spot-ELISA assays.Optimisation of protocols for TAP production and biomarker analyses makes TAP measurements highly specific and reproducible.In measurements of interleukin-1α (IL-1α), IL-1 receptor antagonist (IL-1RA) and human β-defensin (hBD-1) from healthy skin, TAP appears far more sensitive than skin lavage-based methods using ELISA.

View Article: PubMed Central - PubMed

Affiliation: FibroTx LLC, Mäealuse 4, 12918 Tallinn, Estonia.

ABSTRACT

Background: The skin proteome contains valuable information on skin condition, but also on how skin may evolve in time and may respond to treatments. Despite the potential of measuring regulatory-, effector- and structural proteins in the skin for biomarker applications in clinical dermatology and skin care, convenient diagnostic tools are lacking. The aim of the present study was to develop a highly versatile and non-invasive diagnostic tool for multiplex measurements of protein biomarkers from the surface of skin.

Results: The Transdermal Analyses Patch (TAP) is a novel molecular diagnostic tool that has been developed to capture biomarkers directly from skin, which are quantitatively analyzed in spot-ELISA assays. Optimisation of protocols for TAP production and biomarker analyses makes TAP measurements highly specific and reproducible. In measurements of interleukin-1α (IL-1α), IL-1 receptor antagonist (IL-1RA) and human β-defensin (hBD-1) from healthy skin, TAP appears far more sensitive than skin lavage-based methods using ELISA. No side-effects were observed using TAP on human skin.

Conclusion: TAP is a practical and valuable new skin diagnostic tool for measuring protein-based biomarkers from skin, which is convenient to use for operators, with minimal burden for patients.

No MeSH data available.


Related in: MedlinePlus