In the era towards precision medicine, we here present the individual specific autoantibody signatures of 193 healthy individuals. The self-reactive IgG signatures are stable over time in a way that each individual profile is recognized in longitudinal sampling. The IgG autoantibody reactivity towards an antigen array comprising 335 protein fragments, representing 204 human proteins with potential relevance to autoimmune disorders, was measured in longitudinal plasma samples from 193 healthy individuals. This analysis resulted in unique autoantibody barcodes for each individual that were maintained over one year's time. The reactivity profiles, or signatures, are person specific in regards to the number of reactivities and antigen specificity. Two independent data sets were consistent in that each healthy individual displayed reactivity towards 0-16 antigens, with a median of six. Subsequently, four selected individuals were profiled on in-house produced high-density protein arrays containing 23,000 protein fragments representing 14,000 unique protein coding genes. Based on a unique, broad and deep longitudinal profiling of autoantibody reactivities, our results demonstrate a unique autoreactive profile in each analyzed healthy individual. The need and interest for broad-ranged and high-resolution molecular profiling of healthy individuals is rising. We have here generated and assessed an initial perspective on the global distribution of the self-reactive IgG repertoire in healthy individuals, by investigating 193 well-characterized healthy individuals.

Complement components and their cascade of reactions are important defense mechanisms within both innate and adaptive immunity. Many complement deficient patients still remain undiagnosed because of a lack of high throughput screening tools. Aiming towards neonatal proteome screening for immunodeficiencies, we used a multiplex profiling approach with antibody bead arrays to measure 9 complement proteins in serum and dried blood spots. Several complement components have been described as heat sensitive, thus their heat-dependent detectability was investigated. Using sera from 16 patients with complement deficiencies and 23 controls, we confirmed that the proteins C1q, C2, C3, C6, C9 and factor H were positively affected by heating, thus the identification of deficient patients was improved when preheating samples. Measurements of C7, C8 and factor I were negatively affected by heating and non-heated samples should be used in analysis of these components. In addition, a proof of concept study demonstrated the feasibility of labeling eluates from dried blood spots to perform a subsequent correct classification of C2-deficiencies. Our study demonstrates the potential of using multiplexed single binder assays for screening of complement components that open possibilities to expand such analysis to other forms of deficiencies.

This thesis is about protein profiling in blood-derived samples using suspension bead ar- rays built with protein affinity reagents, and the evaluation of binding characteristics and potential disease relation of such profiles.

A central aim of the presented work was to discover and verify disease associated protein profiles in blood-derived samples such as serum or plasma. This was based on immobiliz- ing antigens or antibodies on color-coded beads for a multiplexed analysis. This concept generally allow for a dual multiplexing because hundreds of samples can be screened for hundreds of proteins in a miniaturized and parallelized fashion. At first, protein antigens were used to study humoral immune responses in cattle suffering from a mycoplasma infec- tion (Paper I). Here, the most immunogenic of the applied antigens were identified based on reactivity profiles from the infected cattle, and were combined into an antigen cocktail to serve as a diagnostic assay in a standard ELISA set-up. Next, antibodies and their em- ployment in assays with directly labeled human samples was initiated. This procedure was applied in a study of kidney disorders where screening of plasma resulted in the discovery of a biomarker candidate, fibulin-1 (Paper II). In parallel to the disease related applica- tions, systematic evaluations of the protein profiles were conducted. Protein profiles from 2,300 antibodies were classified on the bases of binding properties in relation to sample heating and stringent washing (Paper III). With a particular focus on heat dependent de- tectability, a method was developed to visualize those proteins that were captured to the beads in an immunoassay by using Western blotting (Paper IV). In conclusion, this thesis presents examples of the possibilities of comparative plasma profiling enabled by protein bead arrays. 

The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries.

Antibody microarrays offer new opportunities for exploring the proteome and to identify biomarker candidates in human serum and plasma. Here, we have investigated the effect of heat and detergents on an antibody-based suspension bead array (SBA) assay using polyclonal antibodies and biotinylated plasma samples. With protein profiles from more than 2300 antibodies generated in 384-plex antibody SBAs, three major classes of heat and detergent susceptibility could be described. The results show that washing of the beads with SDS (rather than Tween) after target binding lowered intensity levels of basically all profiles and that about 50% of the profiles appeared to be lowered to a similar extent by heating of the sample. About 33% of the profiles appeared to be insensitive to heat treatment while another 17% showed a positive influence of heat to yield elevated profiles. The results suggest that the classification of antibodies is driven by the molecular properties of the antibody-antigen interaction and can generally not be predicted based on protein class or Western blot data. The experimental scheme presented here can be used to systematically categorize antibodies and thereby combine antibodies with similar properties into targeted arrays for analysis of plasma and serum.

There is a need for reliable and sensitive biomarkers for renal impairments to detect early signs of kidney toxicity and to monitor progression of disease. Here, antibody suspension bead arrays were applied to profile plasma samples from patients with four types of kidney disorders: glomerulonephritis, diabetic nephropathy, obstructive uropathy, and analgesic abuse. In total, 200 clinical renal-associated cases and control plasma samples from different cohorts were profiled. Parallel plasma protein profiles were obtained using biotinylated and nonfractionated samples and a selected set of 94 proteins targeted by 129 antigen-purified polyclonal antibodies. Out of the analyzed target proteins, human fibulin-1 was detected at significantly higher levels in the glomerulonephritis patient group compared to the controls and with elevated levels in patient samples for all other renal disorders investigated. Two polyclonal antibodies and one monoclonal antibody directed toward separate, nonoverlapping epitopes showed the same trend in the discovery cohorts. A technical verification using Western blot analysis of selected patient plasma confirmed the trends toward higher abundance of the target protein in disease samples. Furthermore, a verification study was carried out in the context of glomerulonephritis using an independent case and control cohort, and this confirmed the results from the discovery cohort, suggesting that plasma levels of fibulin-1 could serve as a potential indicator to monitor kidney malfunction or kidney damage.

Current approaches within affinity-based proteomics are driven both by the accessibility and availability of antigens and capture reagents, and by suitable multiplexed technologies onto which these are implemented. By combining planar microarrays and other multiparallel systems with sets of reagents, possibilities to discover new and unpredicted protein disease associations, either via directed hypothesis-driven or via undirected hypothesis-generating target selection, can be created. In the following stages, the discoveries made during these screening phases have to be verified for potential clinical relevance based on both technical and biological aspects. The use of affinity tools throughout discovery and verification has the potential to streamline the introduction of new markers, as transition into clinically required assay formats appears straightforward. In this article, we summarize some of the current building blocks within array-and affinity-based proteomic profiling with a focus on body fluids, by giving a perspective on how current and upcoming developments in this bioscience could enable a path of pursuit for biomarker discovery.

The advent of affinity-based proteomics technologies for global protein profiling provides the prospect of finding new molecular biomarkers for common, multifactorial disorders. The molecular phenotypes obtained from studies on such platforms are driven by multiple sources, including genetic, environmental, and experimental. In characterizing the contribution of different sources of variation to the measured phenotypes, the aim is to facilitate the design and interpretation of future biomedical studies employing exploratory and multiplexed technologies. Thus, biometrical genetic modelling of twin or other family data can be used to decompose the variation underlying a phenotype into biological and experimental components. RESULTS: Using antibody suspension bead arrays and antibodies from the Human Protein Atlas, we study unfractionated serum from a longitudinal study on 154 twins. In this study, we provide a detailed description of how the variation in a molecular phenotype in terms of protein profile can be decomposed into familial i.e. genetic and common environmental; individual environmental, short-term biological and experimental components. The results show that across 69 antibodies analyzed in the study, the median proportion of the total variation explained by familial sources is 12% (IQR 1-22%), and the median proportion of the total variation attributable to experimental sources is 63% (IQR 53-72%). CONCLUSION: The variability analysis of antibody arrays highlights the importance to consider variability components and their relative contributions when designing and evaluating studies for biomarker discover with exploratory, high-throughput and multiplexed methods.

Novel analytical methods for a next generation of diagnostic devices combine attributes from sensitive, accurate, fast, simple and multiplexed analysis methods. Here, we describe a possible contribution to these by the application of a lateral flow microarray where a panel of recombinant protein antigens was used to differentiate bovine serum samples in the context of the lung disease contagious bovine pleuropneumonia (CBPP). Lateral flow arrays were produced by attaching nitrocellulose onto microscopic slides and spotting of the recombinant proteins onto the membranes. The developed assay included evaluations of substrate matrix and detection reagents to allow for short assay times and convenient read-out options, and to yield a total assay time from sample application to data acquisition of less than ten minutes. It was found that healthy and disease-affected animals could be discriminated (AUC = 97%), and we suggest that the use of an antigen panel in combination with the lateral flow device offers an emerging analytical tool towards a simplified but accurate on-site diagnosis.