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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 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.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 IL-1α, IL-1RA, CXCL-1/2 and hBD-1 from healthy skin. TAP’s containing capture antibody micro-arrays coated with anti-IL-1α, -IL-1RA, -CXCL-1/2 and -hBD-1 capture antibodies were incubated on skin for 1, 5, 15 or 30 minutes. IL-1α, IL-1RA, CXCL-1/2 and hBD-1 captured from skin were analyzed in spot-ELISA and signals were quantified by determining the pixel intensities of digitized spots. Signal intensities were compared to signals obtained using fixed concentrations of recombinant IL-1α, IL-1RA, CXCL-1/2 and hBD-1 captured in solution. In the graph, the apparent average concentration of IL-1α (Panel A), IL-1RA (Panel B) CXCL-1/2 (Panel C) and hBD-1 (Panel D) on skin of three persons has been plotted against incubation time on skin. Each data point in the graphs represents results of three spots analyzed on 6 TAP’s (N = 18). Y-axis: Apparent concentration of IL-1α, 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 = 18).
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Fig5: Measurements of IL-1α, IL-1RA, CXCL-1/2 and hBD-1 from healthy skin. TAP’s containing capture antibody micro-arrays coated with anti-IL-1α, -IL-1RA, -CXCL-1/2 and -hBD-1 capture antibodies were incubated on skin for 1, 5, 15 or 30 minutes. IL-1α, IL-1RA, CXCL-1/2 and hBD-1 captured from skin were analyzed in spot-ELISA and signals were quantified by determining the pixel intensities of digitized spots. Signal intensities were compared to signals obtained using fixed concentrations of recombinant IL-1α, IL-1RA, CXCL-1/2 and hBD-1 captured in solution. In the graph, the apparent average concentration of IL-1α (Panel A), IL-1RA (Panel B) CXCL-1/2 (Panel C) and hBD-1 (Panel D) on skin of three persons has been plotted against incubation time on skin. Each data point in the graphs represents results of three spots analyzed on 6 TAP’s (N = 18). Y-axis: Apparent concentration of IL-1α, 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 = 18).

Mentions: TAP containing anti-IL-1α, -IL-1RA, -CXCL-1/2 and -hBD-1 capturing antibody micro-arrays were applied to normal, non-sun exposed skin on the inside of the lower arm of healthy volunteers (N = 3) for 1, 5, 15 or 30 minutes. Captured IL-1α, IL-1RA, CXCL-1/2 and hBD-1 were subsequently visualised using anti-IL-1α, -IL-1RA, -CXCL-1/2 and -hBD-1 detection antibodies in spot-ELISA, and quantitatively analyzed, as described. As shown in Figure 5, IL-1α, IL-1RA and hBD-1 could be efficiently detected on normal skin using TAP, whereas CXCL-1/2 was not detectable. Maximal signal strength was obtained following 15 minutes incubation on skin, however, no statistically significant differences were observed between 5, 15 and 30 minutes. An incubation time of 20–30 minutes was used in following experiments, to minimize variability in measurements due to differences in incubation times on skin.Figure 5


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 IL-1α, IL-1RA, CXCL-1/2 and hBD-1 from healthy skin. TAP’s containing capture antibody micro-arrays coated with anti-IL-1α, -IL-1RA, -CXCL-1/2 and -hBD-1 capture antibodies were incubated on skin for 1, 5, 15 or 30 minutes. IL-1α, IL-1RA, CXCL-1/2 and hBD-1 captured from skin were analyzed in spot-ELISA and signals were quantified by determining the pixel intensities of digitized spots. Signal intensities were compared to signals obtained using fixed concentrations of recombinant IL-1α, IL-1RA, CXCL-1/2 and hBD-1 captured in solution. In the graph, the apparent average concentration of IL-1α (Panel A), IL-1RA (Panel B) CXCL-1/2 (Panel C) and hBD-1 (Panel D) on skin of three persons has been plotted against incubation time on skin. Each data point in the graphs represents results of three spots analyzed on 6 TAP’s (N = 18). Y-axis: Apparent concentration of IL-1α, 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 = 18).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Measurements of IL-1α, IL-1RA, CXCL-1/2 and hBD-1 from healthy skin. TAP’s containing capture antibody micro-arrays coated with anti-IL-1α, -IL-1RA, -CXCL-1/2 and -hBD-1 capture antibodies were incubated on skin for 1, 5, 15 or 30 minutes. IL-1α, IL-1RA, CXCL-1/2 and hBD-1 captured from skin were analyzed in spot-ELISA and signals were quantified by determining the pixel intensities of digitized spots. Signal intensities were compared to signals obtained using fixed concentrations of recombinant IL-1α, IL-1RA, CXCL-1/2 and hBD-1 captured in solution. In the graph, the apparent average concentration of IL-1α (Panel A), IL-1RA (Panel B) CXCL-1/2 (Panel C) and hBD-1 (Panel D) on skin of three persons has been plotted against incubation time on skin. Each data point in the graphs represents results of three spots analyzed on 6 TAP’s (N = 18). Y-axis: Apparent concentration of IL-1α, 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 = 18).
Mentions: TAP containing anti-IL-1α, -IL-1RA, -CXCL-1/2 and -hBD-1 capturing antibody micro-arrays were applied to normal, non-sun exposed skin on the inside of the lower arm of healthy volunteers (N = 3) for 1, 5, 15 or 30 minutes. Captured IL-1α, IL-1RA, CXCL-1/2 and hBD-1 were subsequently visualised using anti-IL-1α, -IL-1RA, -CXCL-1/2 and -hBD-1 detection antibodies in spot-ELISA, and quantitatively analyzed, as described. As shown in Figure 5, IL-1α, IL-1RA and hBD-1 could be efficiently detected on normal skin using TAP, whereas CXCL-1/2 was not detectable. Maximal signal strength was obtained following 15 minutes incubation on skin, however, no statistically significant differences were observed between 5, 15 and 30 minutes. An incubation time of 20–30 minutes was used in following experiments, to minimize variability in measurements due to differences in incubation times on skin.Figure 5

Bottom Line: 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.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