Evaluation of Solid Supports for Slide- and Well-Based Recombinant Antibody Microarrays.

Antibody microarrays have emerged as an important tool within proteomics, enabling multiplexed protein expression profiling in both health and disease. The design and performance of antibody microarrays and how they are processed are dependent on several factors, of which the interplay between the antibodies and the solid surfaces plays a central role. In this study, we have taken on the first comprehensive view and evaluated the overall impact of solid surfaces on the recombinant antibody microarray design. The results clearly demonstrated the importance of the surface-antibody interaction and showed the effect of the solid supports on the printing process, the array format of planar arrays (slide- and well-based), the assay performance (spot features, reproducibility, specificity and sensitivity) and assay processing (degree of automation). In the end, two high-end recombinant antibody microarray technology platforms were designed, based on slide-based (black polymer) and well-based (clear polymer) arrays, paving the way for future large-scale protein expression profiling efforts.

Technical Advances of the Recombinant Antibody Microarray Technology Platform for Clinical Immunoproteomics.

In the quest for deciphering disease-associated biomarkers, high-performing tools for multiplexed protein expression profiling of crude clinical samples will be crucial. Affinity proteomics, mainly represented by antibody-based microarrays, have during recent years been established as a proteomic tool providing unique opportunities for parallelized protein expression profiling. But despite the progress, several main technical features and assay procedures remains to be (fully) resolved. Among these issues, the handling of protein microarray data, i.e. the biostatistics parts, is one of the key features to solve. In this study, we have therefore further optimized, validated, and standardized our in-house designed recombinant antibody microarray technology platform. To this end, we addressed the main remaining technical issues (e.g. antibody quality, array production, sample labelling, and selected assay conditions) and most importantly key biostatistics subjects (e.g. array data pre-processing and biomarker panel condensation). This represents one of the first antibody array studies in which these key biostatistics subjects have been studied in detail. Here, we thus present the next generation of the recombinant antibody microarray technology platform designed for clinical immunoproteomics.

Generation of miniaturized planar ecombinant antibody arrays using a microcantilever-based printer.

Miniaturized (Ø 10 µm), multiplexed (>5-plex), and high-density (>100 000 spots cm(-2)) antibody arrays will play a key role in generating protein expression profiles in health and disease. However, producing such antibody arrays is challenging, and it is the type and range of available spotters which set the stage. This pilot study explored the use of a novel microspotting tool, Bioplume(TM)-consisting of an array of micromachined silicon cantilevers with integrated microfluidic channels-to produce miniaturized, multiplexed, and high-density planar recombinant antibody arrays for protein expression profiling which targets crude, directly labelled serum. The results demonstrated that 16-plex recombinant antibody arrays could be produced-based on miniaturized spot features (78.5 um(2), Ø 10 µm) at a 7-125-times increased spot density (250 000 spots cm(-2)), interfaced with a fluorescent-based read-out. This prototype platform was found to display adequate reproducibility (spot-to-spot) and an assay sensitivity in the pM range. The feasibility of the array platform for serum protein profiling was outlined.

Multiplexing of miniaturized planar antibody arrays for serum protein profiling--a biomarker discovery in SLE nephritis.

In the quest to decipher disease-associated biomarkers, miniaturized and multiplexed antibody arrays may play a central role in generating protein expression profiles, or protein maps, of crude serum samples. In this conceptual study, we explored a novel, 4-times larger pen design, enabling us to, in a unique manner, simultaneously print 48 different reagents (antibodies) as individual 78.5 µm(2) (10 µm in diameter) sized spots at a density of 38,000 spots cm(-2) using dip-pen nanolithography technology. The antibody array set-up was interfaced with a high-resolution fluorescent-based scanner for sensitive sensing. The performance and applicability of this novel 48-plex recombinant antibody array platform design was demonstrated in a first clinical application targeting SLE nephritis, a severe chronic autoimmune connective tissue disorder, as the model disease. To this end, crude, directly biotinylated serum samples were targeted. The results showed that the miniaturized and multiplexed array platform displayed adequate performance, and that SLE-associated serum biomarker panels reflecting the disease process could be deciphered, outlining the use of miniaturized antibody arrays for disease proteomics and biomarker discovery.

Protein expression profiling of formalin-fixed paraffin-embedded tissue using recombinant antibody microarrays.

Proteomics, the large-scale analysis of proteins, is a rapidly evolving field with an increasing number of key clinical applications, such as diagnosis, prognosis, and classification. In order to generate complete protein expression profiles, or protein atlases, any crude sample format must be addressable in a rapid, multiplex, and sensitive manner. A common and clinically central sample format, formalin-fixed, paraffin-embedded (FFPE) tissue material, holds great potential as a source for disease-associated biomarker signatures. However, despite major efforts, extraction and subsequent profiling of proteins from FFPE tissue has proven to be challenging. In this proof-of-concept study, we have demonstrated for the first time that proteins could be extracted, labeled, and subsequently profiled in a multiplex, sensitive, and reproducible manner using recombinant scFv antibody microarrays. Thus, we have added FFPE samples to the list of sample formats available for high-throughput analysis by affinity proteomics, paving the way for the next generation of biomarker-driven discovery projects.

Design of recombinant antibody microarrays for membrane protein profiling of cell lysates and tissue extracts.

Generating global protein expression profiles, including also membrane proteins, will be crucial for our understanding of biological processes in health and disease. In this study, we have expanded our antibody microarray technology platform and designed the first human recombinant antibody microarray for membrane proteins targeting crude cell lysates and tissue extracts. We have optimized all key technological parameters and successfully developed a setup for extracting, labeling and analyzing non-fractionated membrane proteomes under non-denaturing conditions. Finally, the platform was also extended and shown to be compatible with simultaneous profiling of both membrane proteins and water-soluble proteins.

Tissue proteome profiling of preeclamptic placenta using recombinant antibody microarrays.

PURPOSE: preeclampsia (PE) is a severe, multi-system pregnancy disorder of yet unknown cause, missing means of treatment, and our fundamental understanding of the disease is still impaired. The purpose of this discovery study was to define candidate placenta tissue protein biomarker signatures to further decipher the molecular features of PE. EXPERIMENTAL DESIGN: we used recombinant antibody microarrays for multiplexed protein expression profiling of preeclamptic placenta tissue (n=25) versus normal placenta (n=11) targeting mainly immunoregulatory water-soluble proteins and membrane proteins. Furthermore, the three known subgroups of PE were profiled, including women with early onset preeclampsia and late onset preeclampsia, as well as women with PE and bilateral notching and intrauterine growth restrictions. RESULTS: the data showed that the first generation of candidate PE-associated placenta tissue protein signatures were delineated, indicating that PE (receiver operating characteristics (ROC) AUC value of 0.83) and the subgroups thereof (ROC AUC values ≤ 0.91) could be distinguished. Notably, the data implied that all subgroups, but preeclampsia with bilateral notching and IUGR, could be further classified into novel subsets (ROC AUC values of 1.0) displaying different inflammatory signatures. CONCLUSIONS AND CLINICAL RELEVANCE: we have taken one step further toward de-convoluting the complex features of PE at the molecular level using affinity proteomics.