Protein microarray for the analysis of human melanoma biomarkers

The clinical course of malignant melanoma is notoriously variable. Current staging criteria allow assignment to risk categories but do not permit accurate assessment of prognosis. The simultaneous detection of biomarkers reflecting disease stage and likelihood of progression is therefore urgently needed for treatment decisions.Herein we describe the development of a protein microarray platform for the parallel analysis of five melanoma serum biomarker proteins: S100B, VEGF-A, CRP, IL-6 and IL-10. The platform utilizes ARChip Epoxy as a solid support for the covalent immobilization of antibodies using contact printing. Tests are based on sandwich immunoassays with streptavidin–biotin chemistry and fluorescence detection. Using CRP as a model marker optimization of the system in respect to printing buffer, antigen probe concentration (1 mg/mL), assay binding buffer and incubation time (120 min) is outlined. High reproducibility (CV < 20%), weak cross-reactivity (<0.5% for IL-10) and low limit of detection (1.88 × 10−4 mg/L) were achieved. Fit of a four-parameter logistic quantification model to standard curves of the five on-chip assays shows excellent coefficients of determination (R2 ≥ 0.966). Achieved sensitivities are perfectly comparable to classical ELISA kits and obtained working ranges allow discrimination between normal and literature-reported elevated serum levels of melanoma markers.