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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

Effects of different nitrocellulose materials on the detection of printed human IgG. Panel A: Various amounts of human IgG (1.2, 0.6, 0.3, 0.15 and 0.075 ng /spot) were printed on Whatman Protran BA-85 (0.1, 0.2 or 0.45 μm porosity) or Amersham Hybond-C (0.45 μm porosity) membrane using PBS + 20% glycerol as printing buffer. Printed IgG was visualised in spot-ELISA and signals quantified by determining the pixel intensities of digitized spots. Each line on graph represents the analyses results of IgG printed on a specific nitrocellulose (see Panel A for details). Each data point consists of measurements of five spots on five different strips (N = 25 per data point). X-axis: human IgG amount per spot. Y-axis: Staining intensity defined as the mean pixel intensity measured on a 0–255 grey scale. Panel B: R2 and CV% (Coefficient of variation) range for each tested nitrocellulose membrane type.
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Fig3: Effects of different nitrocellulose materials on the detection of printed human IgG. Panel A: Various amounts of human IgG (1.2, 0.6, 0.3, 0.15 and 0.075 ng /spot) were printed on Whatman Protran BA-85 (0.1, 0.2 or 0.45 μm porosity) or Amersham Hybond-C (0.45 μm porosity) membrane using PBS + 20% glycerol as printing buffer. Printed IgG was visualised in spot-ELISA and signals quantified by determining the pixel intensities of digitized spots. Each line on graph represents the analyses results of IgG printed on a specific nitrocellulose (see Panel A for details). Each data point consists of measurements of five spots on five different strips (N = 25 per data point). X-axis: human IgG amount per spot. Y-axis: Staining intensity defined as the mean pixel intensity measured on a 0–255 grey scale. Panel B: R2 and CV% (Coefficient of variation) range for each tested nitrocellulose membrane type.

Mentions: Accurate TAP biomarker measurements require micro-arrays that stably retain capturing antibodies during analyses and that display minimal variation between individual spots of printed capture antibodies. Therefore, different printing solutions were tested for the generation of stable TAP micro-arrays with minimal variation between printed spots. For this, nitrocellulose strips of Whatman Protran BA 85 nitrocellulose (0.45 μm porosity) were printed with five different amounts (1.2, 0.6, 0.3, 0.15 and 0.075 ng/spot) of human IgG, in five fold, dissolved in PBS formulations containing either 10% or 20% glycerol, that were supplemented or not with various concentrations of either ethanol, Triton-X100 or Tween-20 (see Figure 2 for details). Printed IgG was visualised by spot-ELISA, using HRP-conjugated secondary antibodies specific for the printed IgG. Printed IgG was quantitatively analyzed by determining the pixel intensities of digitised images of the visualised IgG and differences in spot intensities of the spots were measured for each amount of IgG printed with the different printing buffers tested. Regression analyses revealed very strong correlations between spot intensities and amounts of printed proteins for all but one of printing buffers tested (R2 ≥ 0.98 for all buffers tested) (see Figure 2). Also, only minor variation between individual spots was observed for all printing buffers tested. CV values did not exceed 10% for any but two of the combinations of printing buffers and IgG amounts tested (see Figure 2). Nonetheless, substantial differences in signal strengths were observed between printing buffers used and how well dispensed capture antibodies were retained on the membrane during the assay. Micro-arrays printed with IgG dissolved in PBS + 20% glycerol without any further additives yielded the highest signal strength observed for the different printing buffers and was therefore chosen for further TAP micro-array development. In a similar fashion, different nitrocellulose membranes were tested, to identify the optimal printing material for the TAP capture antibody micro-arrays. Quantitative analyses of different amounts of IgG printed either on Whatman Protran BA 85 nitrocellulose, with porosities of either 0.1, 0.2 or 0.45 μm, or Amersham Hybond C-Extra, with 0.45 μm porosity, revealed only minor differences in amounts of printed IgG in different spots during analyses (see Figure 3). Very strong correlations were observed between spot intensities and amounts of printed proteins for all materials tested (R2 > 0.99). Nonetheless, micro-arrays printed on Whatman Protran BA 85 nitrocellulose with 0.45 μm porosity yielded the lowest CV values (3.3-5.3%) for the different IgG amounts tested, indicating the least variability between individually printed spots, and therefore this material was chosen for further TAP development.Figure 2


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)

Effects of different nitrocellulose materials on the detection of printed human IgG. Panel A: Various amounts of human IgG (1.2, 0.6, 0.3, 0.15 and 0.075 ng /spot) were printed on Whatman Protran BA-85 (0.1, 0.2 or 0.45 μm porosity) or Amersham Hybond-C (0.45 μm porosity) membrane using PBS + 20% glycerol as printing buffer. Printed IgG was visualised in spot-ELISA and signals quantified by determining the pixel intensities of digitized spots. Each line on graph represents the analyses results of IgG printed on a specific nitrocellulose (see Panel A for details). Each data point consists of measurements of five spots on five different strips (N = 25 per data point). X-axis: human IgG amount per spot. Y-axis: Staining intensity defined as the mean pixel intensity measured on a 0–255 grey scale. Panel B: R2 and CV% (Coefficient of variation) range for each tested nitrocellulose membrane type.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Effects of different nitrocellulose materials on the detection of printed human IgG. Panel A: Various amounts of human IgG (1.2, 0.6, 0.3, 0.15 and 0.075 ng /spot) were printed on Whatman Protran BA-85 (0.1, 0.2 or 0.45 μm porosity) or Amersham Hybond-C (0.45 μm porosity) membrane using PBS + 20% glycerol as printing buffer. Printed IgG was visualised in spot-ELISA and signals quantified by determining the pixel intensities of digitized spots. Each line on graph represents the analyses results of IgG printed on a specific nitrocellulose (see Panel A for details). Each data point consists of measurements of five spots on five different strips (N = 25 per data point). X-axis: human IgG amount per spot. Y-axis: Staining intensity defined as the mean pixel intensity measured on a 0–255 grey scale. Panel B: R2 and CV% (Coefficient of variation) range for each tested nitrocellulose membrane type.
Mentions: Accurate TAP biomarker measurements require micro-arrays that stably retain capturing antibodies during analyses and that display minimal variation between individual spots of printed capture antibodies. Therefore, different printing solutions were tested for the generation of stable TAP micro-arrays with minimal variation between printed spots. For this, nitrocellulose strips of Whatman Protran BA 85 nitrocellulose (0.45 μm porosity) were printed with five different amounts (1.2, 0.6, 0.3, 0.15 and 0.075 ng/spot) of human IgG, in five fold, dissolved in PBS formulations containing either 10% or 20% glycerol, that were supplemented or not with various concentrations of either ethanol, Triton-X100 or Tween-20 (see Figure 2 for details). Printed IgG was visualised by spot-ELISA, using HRP-conjugated secondary antibodies specific for the printed IgG. Printed IgG was quantitatively analyzed by determining the pixel intensities of digitised images of the visualised IgG and differences in spot intensities of the spots were measured for each amount of IgG printed with the different printing buffers tested. Regression analyses revealed very strong correlations between spot intensities and amounts of printed proteins for all but one of printing buffers tested (R2 ≥ 0.98 for all buffers tested) (see Figure 2). Also, only minor variation between individual spots was observed for all printing buffers tested. CV values did not exceed 10% for any but two of the combinations of printing buffers and IgG amounts tested (see Figure 2). Nonetheless, substantial differences in signal strengths were observed between printing buffers used and how well dispensed capture antibodies were retained on the membrane during the assay. Micro-arrays printed with IgG dissolved in PBS + 20% glycerol without any further additives yielded the highest signal strength observed for the different printing buffers and was therefore chosen for further TAP micro-array development. In a similar fashion, different nitrocellulose membranes were tested, to identify the optimal printing material for the TAP capture antibody micro-arrays. Quantitative analyses of different amounts of IgG printed either on Whatman Protran BA 85 nitrocellulose, with porosities of either 0.1, 0.2 or 0.45 μm, or Amersham Hybond C-Extra, with 0.45 μm porosity, revealed only minor differences in amounts of printed IgG in different spots during analyses (see Figure 3). Very strong correlations were observed between spot intensities and amounts of printed proteins for all materials tested (R2 > 0.99). Nonetheless, micro-arrays printed on Whatman Protran BA 85 nitrocellulose with 0.45 μm porosity yielded the lowest CV values (3.3-5.3%) for the different IgG amounts tested, indicating the least variability between individually printed spots, and therefore this material was chosen for further TAP development.Figure 2

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