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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 IL-1α and IL-1RA from three different skin regions on healthy volunteers on five consecutive days. TAP’s containing capture antibody micro-arrays coated with anti-IL-1α and -IL-1RA capture antibodies were incubated on skin of the inner side of the lower arm (‘Forearm’), cheek (‘Face’) or collar bone (‘Neck’) regions of ten healthy volunteers for 20 minutes. The capturing procedure was repeated on the four following days on exact the same regions, at around the same time-point of day. IL-1α and IL-1RA 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 and IL-1RA, captured in solution. In graph A, the apparent average concentrations of IL-1α black bars and IL-1RA (white bars) have been plotted for the three different body regions. In graph B, the apparent average concentrations of IL-1α black bars and IL-1RA (white bars) have been plotted for the three different body regions for each of the ten persons (1–10 in graphs) analysed. Y-axis: Apparent concentration of IL-1α and IL-1RA on skin in ng/ml. X-axis: Individual participants labeled 1–10. Error bars in graph A represent the standard deviations from average of combined measurements in the 10 healthy volunteers. Error bars in graph B, represent the standard deviations from the average of measurements from five different days for each of the healthy volunteers.
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Fig6: Measurements of IL-1α and IL-1RA from three different skin regions on healthy volunteers on five consecutive days. TAP’s containing capture antibody micro-arrays coated with anti-IL-1α and -IL-1RA capture antibodies were incubated on skin of the inner side of the lower arm (‘Forearm’), cheek (‘Face’) or collar bone (‘Neck’) regions of ten healthy volunteers for 20 minutes. The capturing procedure was repeated on the four following days on exact the same regions, at around the same time-point of day. IL-1α and IL-1RA 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 and IL-1RA, captured in solution. In graph A, the apparent average concentrations of IL-1α black bars and IL-1RA (white bars) have been plotted for the three different body regions. In graph B, the apparent average concentrations of IL-1α black bars and IL-1RA (white bars) have been plotted for the three different body regions for each of the ten persons (1–10 in graphs) analysed. Y-axis: Apparent concentration of IL-1α and IL-1RA on skin in ng/ml. X-axis: Individual participants labeled 1–10. Error bars in graph A represent the standard deviations from average of combined measurements in the 10 healthy volunteers. Error bars in graph B, represent the standard deviations from the average of measurements from five different days for each of the healthy volunteers.

Mentions: To measure the reproducibility of TAP biomarker measurements from skin, TAP biomarker measurements were performed on three different skin areas from healthy volunteers (N = 10) on five consecutive days. For this, FibroTx TAP containing anti-IL-1α and -IL-1RA capturing antibody micro-arrays were incubated on normal appearing skin on cheek (face), collar bone (neck) and on the inside of the lower arm (lower forearm) of healthy volunteers for 20 minutes. FibroTx TAP capture antibody micro-arrays were collected after incubation on skin and stored at 4°C until further analysis. This procedure was repeated the four following days, at the same positions on the different skin areas, approximately at the same time of day. Captured IL-1α and IL-1RA were visualised using anti-IL-1α and -IL-1RA detection antibodies in spot-ELISA, and quantitatively analyzed, as described. As shown in Figure 6, IL-1α and IL-1RA could be efficiently detected on all three different skin areas using TAP. Notably, whereas IL-1α is found in the neck region and on the inside of the lower arm in higher amounts than IL-1RA, the reverse pattern is observed on facial skin (see Figure 6A). There was little day-to-day variation in amounts of IL-1α and IL-1RA on five consecutive days, with average CV values of 21.1% and 18.4%, respectively (see Figure 6B).Figure 6


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 and IL-1RA from three different skin regions on healthy volunteers on five consecutive days. TAP’s containing capture antibody micro-arrays coated with anti-IL-1α and -IL-1RA capture antibodies were incubated on skin of the inner side of the lower arm (‘Forearm’), cheek (‘Face’) or collar bone (‘Neck’) regions of ten healthy volunteers for 20 minutes. The capturing procedure was repeated on the four following days on exact the same regions, at around the same time-point of day. IL-1α and IL-1RA 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 and IL-1RA, captured in solution. In graph A, the apparent average concentrations of IL-1α black bars and IL-1RA (white bars) have been plotted for the three different body regions. In graph B, the apparent average concentrations of IL-1α black bars and IL-1RA (white bars) have been plotted for the three different body regions for each of the ten persons (1–10 in graphs) analysed. Y-axis: Apparent concentration of IL-1α and IL-1RA on skin in ng/ml. X-axis: Individual participants labeled 1–10. Error bars in graph A represent the standard deviations from average of combined measurements in the 10 healthy volunteers. Error bars in graph B, represent the standard deviations from the average of measurements from five different days for each of the healthy volunteers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig6: Measurements of IL-1α and IL-1RA from three different skin regions on healthy volunteers on five consecutive days. TAP’s containing capture antibody micro-arrays coated with anti-IL-1α and -IL-1RA capture antibodies were incubated on skin of the inner side of the lower arm (‘Forearm’), cheek (‘Face’) or collar bone (‘Neck’) regions of ten healthy volunteers for 20 minutes. The capturing procedure was repeated on the four following days on exact the same regions, at around the same time-point of day. IL-1α and IL-1RA 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 and IL-1RA, captured in solution. In graph A, the apparent average concentrations of IL-1α black bars and IL-1RA (white bars) have been plotted for the three different body regions. In graph B, the apparent average concentrations of IL-1α black bars and IL-1RA (white bars) have been plotted for the three different body regions for each of the ten persons (1–10 in graphs) analysed. Y-axis: Apparent concentration of IL-1α and IL-1RA on skin in ng/ml. X-axis: Individual participants labeled 1–10. Error bars in graph A represent the standard deviations from average of combined measurements in the 10 healthy volunteers. Error bars in graph B, represent the standard deviations from the average of measurements from five different days for each of the healthy volunteers.
Mentions: To measure the reproducibility of TAP biomarker measurements from skin, TAP biomarker measurements were performed on three different skin areas from healthy volunteers (N = 10) on five consecutive days. For this, FibroTx TAP containing anti-IL-1α and -IL-1RA capturing antibody micro-arrays were incubated on normal appearing skin on cheek (face), collar bone (neck) and on the inside of the lower arm (lower forearm) of healthy volunteers for 20 minutes. FibroTx TAP capture antibody micro-arrays were collected after incubation on skin and stored at 4°C until further analysis. This procedure was repeated the four following days, at the same positions on the different skin areas, approximately at the same time of day. Captured IL-1α and IL-1RA were visualised using anti-IL-1α and -IL-1RA detection antibodies in spot-ELISA, and quantitatively analyzed, as described. As shown in Figure 6, IL-1α and IL-1RA could be efficiently detected on all three different skin areas using TAP. Notably, whereas IL-1α is found in the neck region and on the inside of the lower arm in higher amounts than IL-1RA, the reverse pattern is observed on facial skin (see Figure 6A). There was little day-to-day variation in amounts of IL-1α and IL-1RA on five consecutive days, with average CV values of 21.1% and 18.4%, respectively (see Figure 6B).Figure 6

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