Antibody-based microarray is a novel technology with great potential within high-throughput proteomics. The process of designing high-performing antibody (protein) microarrays has, however, turned out to be a challenging process. In this study, we have developed further our human recombinant single-chain variable-fragment (scFv) antibody microarray methodology by addressing two crucial technological issues, choice of sample labeling-tag and solid support. We examined the performance of a range of dyes in a one- or two-color approach on a selection of solid supports providing different surface and coupling chemistries, and surface structures. The set-ups were evaluated in terms of sensitivity, specificity, and selectivity. The results showed that a one-color approach, based on NHS-biotin (or ULS-biotin) labeling, on black polymer Maxisorb slides (or Nexterion slide H) was the superior approach for targeting low-abundant (pg/mL) analytes in nonfractionated, complex proteomes, such as human serum or crude cell supernatants. Notably, microarrays displaying adequate spot morphologies, high S/Ns, minimized nonspecific binding, and most importantly a high selectivity, specificity, and sensitivity (>or=fM range) were obtained. Taken together, we have designed the first generation of a high-performing recombinant scFv antibody microarray technology platform on black polymer Maxisorb slides for sensitive profiling of low-abundant analytes in nonfractionated biotinylated complex proteomes.

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein enriched on tumor cells and normal epithelia. It is mainly known for its ability to induce matrix metalloproteinase production in fibroblasts following epithelial-stromal interaction. We sought to examine whether EMMPRIN has a broader role promoting fibroblast-to-myofibroblast differentiation. Because alpha-smooth muscle actin (alphaSMA) is considered a marker of this differentiation process, we analyzed the effect of EMMPRIN on its expression in corneal and skin fibroblasts by Western blots, immunocytochemistry, and a functional assay of collagen lattice contraction. Increasing EMMPRIN expression by cDNA transfection or by treatment with exogenously added recombinant EMMPRIN resulted in an up-regulation of alphaSMA expression. EMMPRIN also increased the contractile properties of the treated fibroblasts as demonstrated by the immunohistochemical appearance of stress fibers and by the accelerated contraction of fibroblast-embedded collagen lattices. Blocking EMMPRIN expression by small interfering RNA inhibited alphaSMA and collagen gel contraction induced not only by EMMPRIN but also by transforming growth factor-beta, a major mediator of myofibroblast differentiation that also regulated EMMPRIN expression. These findings, combined with the fact that EMMPRIN and alphaSMA colocalized to the same cells in the stroma of pathological corneas, expand on the mechanism by which EMMPRIN remodels extracellular matrix during wound healing and cancer.

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