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.

Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.

Antibody microarray is a rapidly emerging, powerful approach with great promise within high-throughput proteomics. However, before a truly proteome-wide analysis can be performed, the antibody array format needs to be miniaturized even further in order to enable ultradense arrays to be fabricated. To this end, we have designed and generated proof-of-concept for the first generation of an atto-vial based recombinant antibody array platform. Briefly, we have designed a novel nanostructured substrate using electron beam lithography. Vials, ranging in volume/size from 6 (200 nm in diameter) to 4000 aL (5 microm in diameter), were fabricated. Human recombinant single-chain Fv antibody fragments, microarray adopted by design, were used as probes. The set-up was interfaced with planar wave-guide technology for evanescant field fluorescence detection. The results showed that protein analytes could be specifically detected in the subzeptomole range for pure systems, using vials down to 57 aL. Further, low-abundant (pg/mL) protein analytes could be detected in directly labeled complex proteomes, such as human whole serum, using 157 aL-vials. Taken together, these results outline the potential of the atto-vial array set-up for miniaturized affinity proteomics-based approaches.

Generating proteomic maps of membrane proteins, common targets for therapeutic interventions and disease diagnostics, has turned out to be a major challenge. Antibody-based microarrays are among the novel rapidly evolving proteomic technologies that may enable global proteome analysis to be performed. Here, we have designed the first generation of a scaleable human recombinant scFv antibody microarray technology platform for cell surface membrane proteomics as well as glycomics targeting intact cells. The results showed that rapid and multiplexed profiling of the cell surface proteome (and glycome) could be performed in a highly specific and sensitive manner and that differential expression patterns due to external stimuli could be monitored.