Variance decomposition of protein profiles from antibody arrays using a longitudinal twin model

<p>Abstract</p> <p>Background</p> <p>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 components. 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.</p> <p>Results</p> <p>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%).</p> <p>Conclusion</p> <p>The variability analysis of antibody arrays highlights the importance to consider variability components and their relative contributions when designing and evaluating studies for biomarker discoveries with exploratory, high-throughput and multiplexed methods.</p

Individual and stable autoantibody repertoires in healthy individuals

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.Peer reviewe

Newborn screening for presymptomatic diagnosis of complement and phagocyte deficiencies

The clinical outcomes of primary immunodeficiencies (PIDs) are greatly improved by accurate diagnosis early in life. However, it is not common to consider PIDs before the manifestation of severe clinical symptoms. Including PIDs in the nation-wide newborn screening programs will potentially improve survival and provide better disease management and preventive care in PID patients. This calls for the detection of disease biomarkers in blood and the use of dried blood spot samples, which is a part of routine newborn screening programs worldwide. Here, we developed a newborn screening method based on multiplex protein profiling for parallel diagnosis of 22 innate immunodeficiencies affecting the complement system and respiratory burst function in phagocytosis. The proposed method uses a small fraction of eluted blood from dried blood spots and is applicable for population-scale performance. The diagnosis method is validated through a retrospective screening of immunodeficient patient samples. This diagnostic approach can pave the way for an earlier, more comprehensive and accurate diagnosis of complement and phagocytic disorders, which ultimately lead to a healthy and active life for the PID patientsThis work was supported by the Swedish Research Council (VR) and grants provided by the Stockholm County Council (ALF)

Integration of molecular profiles in a longitudinal wellness profiling cohort

An important aspect of precision medicine is to probe the stability in molecular profiles among healthy individuals over time. Here, we sample a longitudinal wellness cohort with 100 healthy individuals and analyze blood molecular profiles including proteomics, transcriptomics, lipidomics, metabolomics, autoantibodies and immune cell profiling, complemented with gut microbiota composition and routine clinical chemistry. Overall, our results show high variation between individuals across different molecular readouts, while the intra-individual baseline variation is low. The analyses show that each individual has a unique and stable plasma protein profile throughout the study period and that many individuals also show distinct profiles with regards to the other omics datasets, with strong underlying connections between the blood proteome and the clinical chemistry parameters. In conclusion, the results support an individual-based definition of health and show that comprehensive omics profiling in a longitudinal manner is a path forward for precision medicine

Bead based protein profiling in blood

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. QC 20130208</p

Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp. mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay▿ †

A recombinant antigen cocktail enzyme-linked immunosorbent assay (ELISA) for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one-third of the surface proteome proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for humoral immune responses toward 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with P values of <10−6. Only minor cross-reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a fivefold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origins. No false-positive results and only two false-negative results were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offers a powerful combination to drive and further improve serological assays toward reliable, simple, and cost-effective diagnosis of CBPP

Heat differentiated complement factor profiling.

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