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Humoral Immunity Profiling of Subjects with Myalgic Encephalomyelitis Using a Random Peptide Microarray Differentiates Cases from Controls with High Specificity and Sensitivity

29 November 2016
– Authors: Sahajpreet Singh, Phillip Stafford, Karen A. Schlauch, Richard R. Tillett, Martin Gollery, Stephen Albert Johnston, Svetlana F. Khaiboullina, Kenny L. De Meirleir, Shanti Rawat, Tatjana Mijatovic, Krishnamurthy Subramanian, András Palotás, and Vincent C. Lombardi

Myalgic encephalomyelitis (ME) is a complex, heterogeneous illness of unknown etiology. The search for biomarkers that can delineate cases from controls is one of the most active areas of ME research; however, little progress has been made in achieving this goal. In contrast to identifying biomarkers that are directly involved in the pathological process, an immunosignature identifies antibodies raised to proteins expressed during, and potentially involved in, the pathological process.
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Detection of Stage I-IV Ovarian Cancer from a Drop of Serum

– Poster Session, NCCN 20th Annual Conference, March 2015
– Authors: Tarasow T, Haddad M, Legutki JB, Melville S, Solberg O, Sykes K

Ovarian cancer remains the most lethal gynecological disease and the second most common cause of female cancer deaths in western countries. This fact is largely due to most ovarian cancer cases being discovered and diagnosed at late stages. There are currently no recognized and reliable diagnostic tools to effectively screen for ovarian cancer, even in high-risk populations where the probability of a positive diagnosis would be highest. Our laboratories have developed a new concept for disease detection and cancer diagnostics based on immunosignatures. The immunosignature (IS) technology uses arrays of hundreds of thousands of unique peptides designed to broadly survey an individual’s antibody binding repertoire from a drop of blood, serum, or plasma…
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Peptide based diagnostics: Are random-sequence peptides more useful than tiling proteome sequences?

– Journal of Immunological Methods, 2014
– Authors: Navalkar KA, Johnston SA, Stafford P

Diagnostics using peptide ligands have been available for decades. However, their adoption in diagnostics has been limited, not because of poor sensitivity but in many cases due to diminished specificity. Numerous reports suggest that protein-based rather than peptide-based disease detection is more specific. We examined two different approaches to peptide-based diagnostics using Coccidioides (aka Valley Fever) as the disease model. Although the pathogen was discovered more than a century ago, a highly sensitive diagnostic remains unavailable. We present a case study where two different approaches to diagnosing Valley Fever were used…
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Epitope identification from fixed-complexity random-sequence peptide microarrays

– Molecular & Cellular Proteomics, 2014
– Authors: Richer J, Johnston SA, and Stafford P

Antibodies play an important role in modern science and medicine. They are essential in many biological assays, and have emerged as an important class of therapeutics. Unfortunately, current methods for mapping antibody epitopes require costly synthesis or enrichment steps, and no low cost universal platform exists. In order to address this, we tested a random sequence peptide microarray consisting of over 330,000 unique peptides sequences sampling 83% of all possible tetramers and 27% of pentamers.
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The immunosignature of canine lymphoma: characterization and diagnostic application

– BMC Cancer, 2014
– Authors: Johnston SA, Thamm DH, and Legutki JB

Cancer diagnosis in both dogs and humans is complicated by the lack of a non-invasive diagnostic test. To meet this clinical need, we apply the recently developed immunosignature assay to spontaneous canine lymphoma as clinical proof-of-concept. Here we evaluate the immunosignature as a diagnostic for spontaneous canine lymphoma at both at initial diagnosis and evaluating the disease free interval following treatment.
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Scalable High-Density Peptide Arrays for Comprehensive Health Monitoring

– Nature Communications, 2014
– Authors: Legutki JB, Zhao ZG, Greving M, Woodbury N, Johnston SA, Stafford P.

There is an increasing awareness that health care must move from post-symptomatic treatment to presymptomatic intervention. An ideal system would allow regular inexpensive monitoring of health status using circulating antibodies to report on health fluctuations. Recently, we demonstrated that peptide microarrays can do this through antibody signatures (immunosignatures)…
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Immunosignature System for Diagnosis of Cancer

– PNAS, 2014
– Authors: Stafford P, Cichacz Z, Woodbury N, Johnston SA

Over much of the world, healthcare systems are facing an unprecedented challenge to meet the medical needs of an aging population while controlling costs. The early detection and treatment of diseases that are prevalent in older people is likely to be a key aspect of economically efficient, high-quality healthcare. In the case of cancer, the resection of a stage I or stage II tumor is often effectively a cure. An ideal diagnostic would allow early detection of disease on a single platform that could be used for any disease. Here, we demonstrate that the immunosignature diagnosis platform could potentially meet the universal platform requirement. Ongoing work will address the early detection requirement separately.
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Diagnosis and Early Detection of CNS-SLE in MRL/lpr Mice Using Peptide Microarrays

– BMC Immunology, 2014
– Authors: Williams S, Stafford P, Hoffman SA.

These results indicate that immunosignaturing could predict and diagnose lupus and its CNS manifestations. It can also be used to characterize pathogenic autoantibodies, which may help to better understand the underlying mechanisms of CNS-Lupus.
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Application of Immunosignatures to Diagnosis of Valley Fever

– Clinical and Vaccine Immunology, 2014
– Authors: Navalkar KA, Johnston SA, Woodbury N, Galgiani J, Magee DM, Chicacz Z, Stafford P.

Valley fever (VF) is difficult to diagnose, partly because the symptoms of VF are confounded with those of other community-acquired pneumonias. Confirmatory diagnostics detect IgM and IgG antibodies against coccidioidal antigens via immunodiffusion (ID). The false-negative rate can be as high as 50% to 70%, with 5% of symptomatic patients never showing detectable antibody levels. In this study, we tested whether the immunosignature diagnostic can resolve VF false negatives.
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Immunosignatures can predict vaccine efficacy

– PNAS, 2013
– Authors: Legutki JB, Johnston SA.

Vaccines have been the most important medical intervention developed, yet vaccines for many diseases are still needed. Despite its success, the process to develop a vaccine remains empirical, resting on measuring the number of vaccinees that incur or do not incur an infection. Here we test in the mouse flu model whether the “immunosignature” diagnostic technology could be applied to predict vaccine efficacy. Immunosignatures are produced by profiling the antibody repertoire of an individual on a chip arrayed with nonnatural sequence peptides. It is attractive in that it is a simple but comprehensive measure of the complexity of the humoral response. We found that immunosignatures are a promising approach to predicting whether a vaccine will confer protection.
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Immunosignaturing: a critical review

– Cell Press, 2012
– Authors: Sykes K., Legutki JB, Stratford P.

Health is a complex interaction between metabolism, physiology, and immunity. Although it is difficult to define quantitatively, the activity of the humoral immune system provides a reasonable proxy for changes in health. Immunosignaturing is a microarray-based technology that quantitates the dynamics of circulating antibodies. Recent advancements in the field warrant a review of the technology. Here, we provide an introduction to the technique, evaluate the current progress, contrast similar technologies, and suggest applications that immunosignaturing could facilitate.
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Feasibility of an early Alzheimer’s disease immunosignature diagnostic test

– Journal of Neuroimmunology, 2012
– Authors: Restrepo L, Stafford P, Johnston SA.

A practical diagnostic test is needed for early Alzheimer’s disease (AD) detection. Immunosignaturing, a technology that employs antibody binding to a random-sequence peptide microarray, generates profiles that distinguish transgenic mice engineered with familial AD mutations (APPswe/PSEN1-dE9) from non-transgenic littermates. It can also detect an AD-like signature in humans. Here, we assess the changes in the immunosignature at different time points of the disease in mice and humans. We also evaluate the accuracy of the late-stage signature as a test to discriminate between young mice with familial AD mutations from non-transgenic littermates.
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Autoreactive antibodies raised by self derived de novo peptides can identify unrelated antigens on protein microarrays

– Experimental and Molecular Pathology, 2012.
– Authors: Kroening K, Johnston SA, Legutki JB.

is usually presumed that the autoantibody was elicited by the protein bound on the array. However, our studies using human protein and random peptide arrays indicate that antibody specificity may not be as high as commonly thought. Therefore we have tested the assumption of the source of autoantibodies.
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Evaluation of Biological Sample Preparation for Immunosignature-Based Diagnostics

– Clinical and Vaccine Immunology, 2012
– Authors: Chase BA, Johnston SA and Legutki JB.

Immunosignatures derived from dried blood were capable of distinguishing naïve mice from those infected with influenza virus. Saliva was applied to the arrays, and the IgA immunosignature correlated strongly with that from dried blood. Finally, we demonstrate that dried blood retains immunosignature information even when exposed to high temperature. This work expands the potential diagnostic uses for immunosignatures. These features suggest that different forms of archival samples can be used for diagnosis development and that in prospective studies samples can be easily procured.
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Immunosiganturing Can Detect Products from Molecular Markers in Brain Cancer

– PLoS ONE, 2012.
– Authors: Hughes A, Cichacz Z, Scheck A, Coons SW, Johnston SA, Stafford P.

Immunosignaturing shows promise as a general approach to diagnosis. It has been shown to detect immunological signs of infection early during the course of disease and to distinguish Alzheimer’s disease from healthy controls. Here we test whether immunosignatures correspond to clinical classifications of disease using samples from people with brain tumors. Blood samples from patients undergoing craniotomies for therapeutically naïve brain tumors with diagnoses of astrocytoma (23 samples), Glioblastoma multiforme (22 samples), mixed oligodendroglioma/astrocytoma (16 samples), oligodendroglioma (18 samples), and 34 otherwise healthy controls were tested by immunosignature.
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Comparative Study of Classification Algorithms for Immunosignaturing Data

– BMC Bioinformatics. 2012.
– Authors: Kukreja M, Johnston SA, Stafford P.

We characterized several classification algorithms to analyze immunosignaturing data. We selected several datasets that range from easy to difficult to classify, from simple monoclonal binding to complex binding patterns in asthma patients. We then classified the biological samples using 17 different classification algorithms. Using a wide variety of assessment criteria, we found ‘Naïve Bayes’ far more useful than other widely used methods due to its simplicity, robustness, speed and accuracy.
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Physical Characterization of the ‘Immunosignaturing Effect

– Molecular and Cellular Proteomics. 2012.
– Authors: Stafford P, Halperin R, Legutki JB, Magee DM, Galgiani J, Johnston SA

Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimer’s disease display a characteristic and reproducible “immunosignature” on a microarray of 10,000 random sequence peptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works.
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Immunosignaturing Microarrays Distinguish Antibody Profiles of Related Pancreatic Diseases

– Journal of Proteomics and Bioinformatics, 2012
– Authors: Kukreja M, Johnston SA, Stafford P.

Immunosignaturing is a technology that allows the humoral immune response to be observed through the binding of antibodies to random sequence peptides. Profiles of the antibody repertoire produced during infection or during long-term chronic disease have proven to be informative for disease classification. An important unanswered question relative to this technology is whether different diseases that target the same organ and result in similar early phenotypes have similar or distinguishable immunosignatures.
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GuiTope: An Application for Mapping Random-Sequence Peptides to Protein Sequences

– BMC Bioinformatics, 2012
– Authors: Halperin RF, Stafford P, Emery JS, Navalkar KA, Johnston SA

Random-sequence peptide libraries are a commonly used tool to identify novel ligands for binding antibodies, other proteins, and small molecules. It is often of interest to compare the selected peptide sequences to the natural protein binding partners to infer the exact binding site or the importance of particular residues. The ability to search a set of sequences for similarity to a set of peptides may sometimes enable the prediction of an antibody epitope or a novel binding partner. We have developed a software application designed specifically for this task.
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Microarray Technology Displays the Complexities of the Humoral Immune Response

– Expert Reviews in Molecular Diagnostics, 2011
– Authors: Stafford P, Johnston SA.

For years, scientists have appreciated the simple elegance with which antibodies combine an active defense against infection and a passive mechanism to store information about a pathogen. Antibodies usually appear as soon as immunogen is present; this is of particular importance when looking for hallmarks of a chronic disease such as Alzheimer’s, arthritis or cancer.
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Exploring antibody recognition of sequence space through random-sequence microarrays

– Molecular and Cellular Proteomics, 2011
– Authors: Halperin RF, Stafford P, Johnston SA.

A universal platform for efficiently mapping antibody epitopes would be of great use for many applications, ranging from antibody therapeutic development to vaccine design. Here we tested the feasibility of using a random peptide microarray to map antibody epitopes.
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Application of immunosignatures to the assessment of Alzheimer’s disease

– Annals of Neurology, 2011
– Authors: Restrepo L, Stafford P, Magee DM, Johnston SA.

Accurate assessment of Alzheimer’s disease (AD), both pre-symptomatically and at different disease stages, will become increasingly important with the expanding elderly population. There are a number of indications that the immune system is engaged in AD. Here we explore the ability of an antibody-profiling technology to characterize AD and screen for peptides that may be used for a simple diagnostic test.
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Statistical Methods for Analyzing Immunosignatures

– BMC Bioinformatics, 2011
– Authors: Brown P, Stafford P, Johnston SA, Dinu Valentin

Based on this research, we lay out an analytic framework illustrating how immunosignatures may be useful as a general method for screening and presymptomatic screening of disease as well as antibody discovery.
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A general method for characterization of humoral immunity induced by a vaccine or infection

– Vaccine, 2010
– Authors: Legutki JB, Magee DM, Stafford P, Johnston SA.

A universal system to diagnose disease, characterize infection or evaluate the response to a vaccine would be useful. Towards this end we introduce a machine-readable platform that we term “Immunosignaturing”. Taken together the data suggests that immunosignaturing on a random peptide array can serve as a universal platform to assess antibody status in ways that cannot be replicated by conventional immunological assays.
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Epitope mapping from random peptide arrays: a step towards a general approach to identify an unknown pathogen

– Arizona State University. 2007

Effective treatment of infectious diseases could almost always be facilitated by quickly and accurately identifying the causative pathogen, but for many pathogens, no such diagnostic test is currently available. Immunosignaturing is a novel technique that involves arrays of random peptides to assay the antibody response. Each antibody that we’ve tested binds to wholly unique set of these random peptides, resulting in a diagnostic pattern. If these random peptide sequences could be used to predict the true antibody epitope, the immunosignaturing approach could be extended to predicting an unknown pathogen, and eliminate the need for a database of pathogen specific patterns.
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