Proteomic Data Analysis for Differential Profiling of the Autoimmune Diseases SLE, RA, SS, and ANCA-Associated Vasculitis.

Early and correct diagnosis of inflammatory rheumatic diseases (IRD) poses a clinical challenge due to the multifaceted nature of symptoms, which also may change over time. The aim of this study was to perform protein expression profiling of four systemic IRDs, systemic lupus erythematosus (SLE), ANCA-associated systemic vasculitis (SV), rheumatoid arthritis (RA), and Sjögren's syndrome (SS), and healthy controls to identify candidate biomarker signatures for differential classification. A total of 316 serum samples collected from patients with SLE, RA, SS, or SV and from healthy controls were analyzed using 394-plex recombinant antibody microarrays.

Serum Biomarker Signature-Based Liquid Biopsy for Diagnosis of Early-Stage Pancreatic Cancer.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with a 5-year survival of < 10% because of diffuse symptoms leading to late-stage diagnosis. That survival could increase significantly if localized tumors could be detected early. Therefore, we used multiparametric analysis of blood samples to obtain a novel biomarker signature of early-stage PDAC.

Tumor tissue protein signatures reflect histological grade of breast cancer.

Histological grade is one of the most commonly used prognostic factors for patients diagnosed with breast cancer. However, conventional grading has proven technically challenging, and up to 60% of the tumors are classified as histological grade 2, which represents a heterogeneous cohort less informative for clinical decision making. In an attempt to study and extend the molecular puzzle of histologically graded breast cancer, we have in this pilot project searched for additional protein biomarkers in a new space of the proteome.

Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics.

Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance.

Interfacing antibody-based microarrays and digital holography enables label-free detection for loss of cell volume.

Background: We introduce the combination of digital holographic microscopy (DHM) and antibody microarrays as a powerful tool to measure morphological changes in specifically antibody-captured cells. The aim of the study was to develop DHM for analysis of cell death of etoposide-treated suspension cells.

Results/methodology: We demonstrate that the cell number, mean area, thickness and volume were noninvasively measured by using DHM.

Antibody-Based Proteomics.

Antibody-based proteomic approaches play an important role in high-throughput, multiplexed protein expression profiling in health and disease. These antibody-based technologies will provide (miniaturized) set-ups capable of the simultaneously profiling of numerous proteins in a specific, sensitive, and rapid manner, targeting high- as well as low-abundant proteins, even in crude proteomes such as serum. The generated protein expression patterns, or proteomic snapshots, can then be transformed into proteomic maps, or detailed molecular fingerprints, revealing the composition of the target (sample) proteome at a molecular level.

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.

Generation and analyses of human synthetic antibody libraries and their application for protein microarrays.

Antibody-based proteomics offers distinct advantages in the analysis of complex samples for discovery and validation of biomarkers associated with disease. However, its large-scale implementation requires tools and technologies that allow development of suitable antibody or antibody fragments in a high-throughput manner. To address this we designed and constructed two human synthetic antibody fragment (scFv) libraries denoted HelL-11 and HelL-13.

Plasma protein profiling in a stage defined pancreatic cancer cohort - Implications for early diagnosis.

Pancreatic ductal adenocarcinoma (PDAC) is a disease where detection preceding clinical symptoms significantly increases the life expectancy of patients. In this study, a recombinant antibody microarray platform was used to analyze 213 Chinese plasma samples from PDAC patients and normal control (NC) individuals. The cohort was stratified according to disease stage, i.

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.

Advancing the global proteome survey platform by using an oriented single chain antibody fragment immobilization approach.

Increasing the understanding of a proteome and how its protein composition is affected by for example different diseases, such as cancer, has the potential to improve strategies for early diagnosis and therapeutics. The Global Proteome Survey or GPS is a method that combines mass spectrometry and affinity enrichment with the use of antibodies. The technology enables profiling of complex proteomes in a species independent manner.

A Multicenter Trial Defining a Serum Protein Signature Associated with Pancreatic Ductal Adenocarcinoma.

Background. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with rapid tumor progression and poor prognosis. This study was motivated by the lack of sensitive and specific PDAC biomarkers and aimed to identify a diagnostic, serum protein signature for PDAC.

Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.

Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g.

AFFIRM--a multiplexed immunoaffinity platform that combines recombinant antibody fragments and LC-SRM analysis.

Targeted measurements of low abundance proteins in complex mixtures are in high demand in many areas, not the least in clinical applications measuring biomarkers. We here present the novel platform AFFIRM (AFFInity sRM) that utilizes the power of antibody fragments (scFv) to efficiently enrich for target proteins from a complex background and the exquisite specificity of SRM-MS based detection. To demonstrate the ability of AFFIRM, three target proteins of interest were measured in a serum background in single-plexed and multiplexed experiments in a concentration range of 5-1000 ng/mL.

Identification of plasma protein profiles associated with risk groups of prostate cancer patients.

Purpose: Early detection of prostate cancer (PC) using prostate-specific antigen (PSA) in blood reduces PC-death among unscreened men. However, due to modest specificity of PSA at commonly used cut-offs, there are urgent needs for additional biomarkers contributing enhanced risk classification among men with modestly elevated PSA.

Experimental Design: Recombinant antibody microarrays were applied for protein expression profiling of 80 plasma samples from routine PSA-measurements, a priori divided into four risk groups, based on levels of total and %free PSA.

Molecular design of recombinant scFv antibodies for site-specific photocoupling to β-cyclodextrin in solution and onto solid support.

The ability to design and tailor-make antibodies to meet the biophysical demands required by the vast range of current and future antibody-based applications within biotechnology and biomedicine will be essential. In this proof-of-concept study, we have for the first time tailored human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the dock'n'flash technology. In more detail, we have successfully explored the possibility to expand the genetic code of E.

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.

Miniaturization of multiplexed planar recombinant antibody arrays for serum protein profiling.

Background: Antibody-based microarrays are a developing tool for high-throughput proteomics in health and disease. However, in order to enable global proteome profiling, novel miniaturized high-density antibody array formats must be developed.

Results: In this proof-of-concept study, we have designed a miniaturized planar recombinant (single-chain Fragment variable).

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.

Identification of B-cell lymphoma subsets by plasma protein profiling using recombinant antibody microarrays.

B-cell lymphoma (BCL) heterogeneity represents a key issue, often making the classification and clinical management of these patients challenging. In this pilot study, we outlined the first resolved view of BCL disease heterogeneity on the protein level by deciphering disease-associated plasma biomarkers, specific for chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma, using recombinant antibody microarrays targeting mainly immunoregulatory proteins. The results showed the BCLs to be heterogeneous, and revealed potential novel subgroups of each BCL.

Antibody array generation and use.

Affinity proteomics, represented by antibody arrays, is a multiplex technology for high-throughput protein expression profiling of crude proteomes in a highly specific, sensitive, and miniaturized manner. The antibodies are individually deposited in an ordered pattern, an array, onto a solid support. Next, the sample is added, and any specifically bound proteins are detected and quantified using mainly fluorescence as the mode of detection.

Recombinant antibody microarray for profiling the serum proteome of SLE.

Systemic lupus erythematosus (SLE) is a severe autoimmune connective tissue disease. Our current knowledge about the serum proteome, or serum biomarker panels, reflecting disease and disease status is still very limited. Affinity proteomics, represented by recombinant antibody arrays, is a novel, multiplex technology for high-throughput protein expression profiling of crude serum proteomes in a highly specific, sensitive, and miniaturized manner.

Photopatterning of self assembled monolayers on oxide surfaces for the selective attachment of biomolecules.

The immobilization of functional biomolecules to surfaces is a critical process for the development of biosensors for disease diagnostics. In this work we report the patterned attachment of single chain fragment variable (scFv) antibodies to the surface of metal oxides by the photodeprotection of self-assembled monolayers, using near-UV light. The photodeprotection step alters the functionality at the surface; revealing amino groups that are utilized to bind biomolecules in the exposed regions of the substrate only.