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

The human skin is the largest organ of the integumentary system, consisting of multiple layers of ectodermal tissue that protects the underlying muscles, ligaments, bones and internal organs of the body. Cells that reside in the skin, such as keratinocytes, fibroblasts, melanocytes, Merkel cells and Langerhans cells, or immune cells that migrate in and out of the skin, are highly adaptive to the constantly changing outer and inner milieus of the body to prevent e.g. physical damage, temperature changes, dehydration and pathogen invasion [1]. To ensure the integrity of skin, skin cells secrete large amounts of structural, effector and regulatory molecules, including scleroproteins (e.g. collagen type I and IV, keratin) [2–6], cytokines (e.g. IL-1α, IL-1β, IL-1RA, TNF-α and IFN-γ) [7–12], chemokines (e.g. CCL-1) [13, 14], angiogenesis regulators (e.g. VEGF, thrombospondin-1 and -2) [15–17] and anti-microbial peptides (e.g. β-Defensin-1 (hBD-1) and -2 (hBD-2)) [17, 18].

The integrity of skin is constantly threatened by external factors (e.g. mechanical disruption, chemical influences, extensive sun exposure, bacterial infections), but also from ‘within’, for instance due to hormonal changes causing skin ageing or the impact of chronic inflammation (e.g. psoriasis, atopic dermatitis and systemic lupus erythematosus (SLE)). Skin condition is often monitored visually. In dermatology, for instance, clinical scores for psoriasis (Psoriasis Area Severity Index score (PASI) score) and atopic dermatitis (Scoring Atopic Dermatitis (SCORAD) score) are largely based on visual assessments of clinical parameters, such as skin redness, thickness, swelling, crustiness and scaling [19–21]. Alternatively, the condition of skin may be monitored by ultrasound, e.g. to determine the thickness and density of skin layers (epidermis, sub-epidermal low-echogenic band (SLEB) and dermis) [22, 23]. Despite the obvious value of these methods for monitoring skin conditions at any given time, they have little predictive value for how the condition of skin evolves over time, or whether skin will respond to treatment.

Methods that may fulfill such needs are biomarker assessments. Biomarkers are defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention [24]. Biomarkers have been classified as: 1) Type 0 markers, which correlate longitudinally with the natural history of the disease, 2) Type I markers, which capture the effects of an intervention in accordance with the mechanism of action of the drug, and 3) Type II markers, which are surrogate endpoints for changes that predict clinical benefits [25].

Biomarkers are of particular interest due to their predictive value. For instance, presence of antibodies reactive with cyclic-citrulinated peptide (CCP) in the blood is a fairly accurate predictor of development of rheumatoid arthritis, preceding clinical signs with months to even years. Several studies have identified Type 0 biomarkers for pathological skin conditions such as psoriasis and atopic dermatitis, including biomarkers for keratinocyte activity (e.g. presence of K16) and inflammatory response (e.g. up-regulation of IL-8 and TNF-α) [26–32]. Type II biomarkers have been identified for various psoriasis and atopic dermatitis therapies, including biomarkers representing Th17 pathway [33–35], monocyte activity (e.g. TNF-α) [31], lymphocyte activity (Granzyme B) and type I interferon pathways [16–24]. Of particular interest is a study by Malaviya et al., showing that the amount of cleaved Caspase-3-positive cells, accurately predicts response to Enbrel, months before response to therapy can be assessed based on clinical symptoms [25].

Biomarkers are usually biological molecules, such as mRNA or proteins, or metabolites, derived from tissues or body fluids (e.g. blood) via invasive methods. Recently, Portugal-Cohen has published a non-invasive method in which a limited number of soluble biomarkers could be assessed in skin-lavage from lesional skin of a limited number of psoriasis patients and renal failure [7, 36, 37]. A clear increased concentration of IL-1α, TNF-α and IL-6 was found in skin lavage from lesion sites of psoriasis patients, in comparison with skin lavage from non-lesional sites, or from skin of healthy individuals [7]. Similarly, tape stripping of skin, in combination with proteomic analyses of ‘captured’ proteins, has shown increased presence of inflammatory cytokines on inflamed or irritated skin [38–40].

Here we describe the step-by-step development of TAP. The aim of this development was to generate a non-invasive diagnostic tool for skin biomarker measurements, with a sensitivity and dynamic range, as well as convenience of use, which allows biomarker measurements from different skin conditions. To minimize variation between measurements, the core of TAP, consisting of an antibody micro-array that allows multiplex analyses of selected biomarkers, was optimized for optimal and reproducible capturing of analytes from skin. Using IL-1α, IL-1RA, CXCL-1/2 and hBD-1 as model biomarkers, protocols have been developed for qualitative and quantitative analyses of biomarkers from skin using spot-ELISA directly on TAP.

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 skin. Using skin-lavage to extract biomarkers from the surface of skin, Portugal-Cohen et al. have shown clear differences in expression levels of skin hydrophilic biomarkers, including cytokines (IL-1α, TNF-α and IL-6) and antioxidants (uric acid, total antioxidant scavenging capacity), between lesional and non-lesional skin in patients with atopic dermatitis and psoriasis [7, 36]. Similarly, Portugal-Cohen et al. found significant elevations in the expression of IL-10, TNF-α and uric acid on skin of hemodialysis patients with chronic renal failure, in comparison with healthy individuals, suggesting that skin biomarker analyses may be useful for monitoring patients with a variety of diseases with dysfunctional skin manifestations, as well [37]. Another method involves analyses of analytes from dead skin cells obtained from skin by tape stripping methods, e.g. using D-Squame tape. Using tape-stripping in combination with mass spectrometry analyses, Emson et al. have recently shown that they could detect marked quantitative differences in keratinocyte turn-over between lesional and non-lesional skin on psoriasis vulgaris patients [44].

TAP was designed with two goals in mind. The first goal was to develop a highly versatile and sensitive tool for accurate multi-analyte biomarker measurements from the surface of skin. The second goal was to ensure that TAP was easy to use with a minimal burden for patients. To achieve the first goal, the core of TAP was designed as a capture antibody micro-array. The capture antibody micro-arrays allow multi-analyte capturing of biomarkers directly from skin, using minimal amount of fluid between TAP and skin, ensuring much higher concentrations of biomarkers during biomarker capturing than skin lavage-based methods, or during the analyses of individual biomarkers from tape-stripping. In our benchmark studies we found that TAP was far more sensitive than antibody based methods using skin lavage techniques in our assays, which in part could be attributed to differences in the amount of fluid used for the biomarker capturing process. However, we also observed that applying capture antibody micro-arrays in situ, i.e. during skin lavage on skin, was more sensitive than capturing biomarkers from the same skin lavage in vitro. This indicates that capturing of biomarkers directly from skin surface may have other advantages, as well. At present time it is unclear what causes this effect, although one may speculate that analytes captured directly from skin may somehow be protected from degradation by capturing antibodies, which is not the case when analytes become dissolved during skin-lavage.

To provide TAP with the highest sensitivity, we optimized the printing process of the capture micro-arrays. Printing buffers can highly impact the printing of proteins on nitrocellulose, as well as the specifications of nitrocellulose itself [45, 46]. We found that PBS supplemented with 20% glycerol yielded the highest sensitivity. Since the same amounts of IgG were used for optimisation of printing, we conclude that the benefit of PBS + 20% glycerol is that it yields the most stable interaction between IgG and nitrocellulose during the biomarker binding process. Although we did not find any remarkable differences between different nitrocellulose materials used, from different suppliers, and with different porosities, we observed that Whatmann Protran BA85 (0.45 μm porosity) yielded the least variability between printed spots, and hence this material was chosen for TAP production.

In our assays to find the optimal conditions for skin biomarker measurements, using IL-1α, IL-1RA, CXCL-1/2 and hBD-1 as model analytes, we found that the amount of capture antibody printed on the capture antibody micro-array was the most important factor that determined both sensitivity and dynamic range of measuring analytes in vitro. The higher the amount of capture antibodies used, the highest the signal strength, sensitivity and dynamic range of measurements achieved in our experiments. The combination of amount of detection antibody used in our biomarker analyses, and concentration of amplification reagents used, impacted predominantly the curve fit of the concentration range, and thus the accuracy of measurements that can be achieved. Optimal settings, which changed somewhat between analytes measured, yielded R² values of over 0.99, thus indicating that highly accurate biomarker measurements with TAP can be achieved.

To achieve the second goal, convenience of use, we designed TAP as an adhesive bandage. The bandage allows easy fixture of the TAP capture antibody micro-array to skin. This, and the fact that TAP uses a sponge layer as an expandable fluid reservoir that ensures close contact between the micro-array and skin, makes TAP insensitive to gravity issues e.g. such as observed using skin-lavage techniques. In addition, the relatively small size of the TAP micro-array allows measurements of small skin lesions, e.g. as observed in certain forms of psoriasis or bacterial infections, e.g. from tick bites. The methods used for TAP biomarker analyses have been based in large on well-established techniques. In essence, TAP biomarker analyses are based on sandwich ELISA, which is a standard and cost-effective technique for qualitative and quantitative protein analyses established in laboratories all over the world. The design of the micro-array allows for easy adaptation to different sets of capture and detection antibodies used, and thus specific TAP’s for specific purposes can be designed with relative ease. Using TAP, we could detect IL-1α, IL-1RA and hBD-1 from normal skin of healthy volunteers, but not CXCL-1/2 due to its low expression on normal skin. Measuring IL-1α and IL-1RA on the skin of healthy volunteers on five consecutive days, we could show that TAP biomarker levels fluctuated with a CV of around 20%. Since the CV values of TAP measurements under ideal conditions are around 5%, we conclude that there is a low day-to-day variation in biomarker presence on skin. From these measurements it became apparent that the ratio between IL-1a and IL-1RA is different on skin in the lower inner arm and neck regions, in comparison with facial skin, which is very much in alignment with tape-stripping studies performed by Hirao et al. showing that IL-RA is much higher expressed the stratum corneum of sun-exposed skin areas (e.g. the face) than in areas (e.g. inner forearm) not exposed to sun-light [47]. These biomarkers were chosen because they have been described as regulated proteins in different skin conditions, such as atopic dermatitis and psoriasis. Currently, studies are ongoing with TAP to study the expression of various biomarkers in different skin conditions, including psoriasis, and we can see clear differences between skin affected and skin not affected by disease (manuscript in preparation). Using TAP on skin, no adverse effects, such as redness or itching were observed, and thus we can conclude that TAP is a highly convenient and versatile new diagnostic tool for non-invasive biomarker measurements from skin that is safe to use.

With TAP, we could capture and measure a selection of regulatory proteins expressed in the skin, IL-1α, IL-1RA and hBD-1, from the surface of normal skin of healthy volunteers, thus presenting TAP as a practical and highly sensitive skin-diagnostic tool for measuring protein-based biomarkers from skin with minimal effort for operators, as well as with minimal burden for persons being analyzed. 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.