The “Reverse Capture” Autoantibody Microarray (original) (raw)
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PROTEOMICS, 2006
Diagnosing cancers based on serum profiling is a particularly attractive concept. However, the technical challenges to analysis of the serum proteome arise from the dynamic range of protein amounts. Cancer sera contain antibodies that react with a unique group of autologous cellular antigens, which affords a dramatic amplification of signal in the form of antibodies relative to the amount of the corresponding antigens. The serum autoantibody repertoire from cancer patients might, therefore, be exploited for antigen-antibody profiling. To date, studies of antigen-antibody reactivity using microarrays have relied on recombinant proteins or synthetic peptides as arrayed features. However, recombinant proteins and/or synthetic peptides may fail to accurately detect autoantibody binding due to the lack of proper PTMs. Here we describe the development and use of a "reverse capture" autoantibody microarray. Our "reverse capture" autoantibody microarray is based on the dual-antibody sandwich immunoassay platform of ELISA, which allows the antigens to be immobilized in their native configuration. As "proof-of-principle", we demonstrate its use for antigen-autoantibody profiling with sera from patients with prostate cancer and benign prostate hyperplasia.
Journal of Proteomics, 2012
Autoantibodies represent an attractive biomarker for diagnostic assays principally due to the stability of immunoglobulin in patient serum facilitating measurement with conventional assays. Immune responses to tumorigenesis may facilitate detection of ovarian cancer in the early stages of the disease with identification of a panel of tumour specific autoantibodies. Despite the reporting of many tumour associated autoantibodies using arrays of tumour antigens, this has not led to the advance in diagnostic capability as rapidly as was initially expected. Here we examine the potential diagnostic utility of candidate autoantibody biomarkers identified via screening of serum samples on a high content human protein array from a unique cohort of early stage and late stage ovarian cancer patients. We analyse the performance of autoantibodies to the tumour suppressor protein p53 and the novel autoantigens alpha adducin and endosulfine alpha identified in our array screen. Each antigen has different performance characteristics using conventional ELISA format and Western blot immunoassay. The high attrition rate of promising autoantigens identified by array screening can in part be explained by the presentation of the epitope of the antigen in the subsequent method of validation and this study provides directions on maximising the potential of candidate biomarkers. This article is part of a Special Issue entitled: Translational Proteomics.
Protein arrays as tools for serum autoantibody marker discovery in cancer
Journal of Proteomics, 2009
Protein array technology has begun to play a significant role in the study of protein-protein interactions and in the identification of antigenic targets of serum autoantibodies in a variety of autoimmune disorders. More recently, this technology has been applied to the identification of autoantibody signatures in cancer. The identification of tumour-associated antigens (TAAs) recognised by the patient's immune response represents an exciting approach to identify novel diagnostic cancer biomarkers and may contribute towards a better understanding of the molecular mechanisms involved. Circulating autoantibodies have not only been used to identify TAAs as diagnostic/prognostic markers and potential therapeutic targets, they also represent excellent biomarkers for the early detection of tumours and potential markers for monitoring the efficacy of treatment. Protein array technology offers the ability to screen the humoral immune response in cancer against thousands of proteins in a high throughput technique, thus readily identifying new panels of TAAs. Such an approach should not only aid in improved diagnostics, but has already contributed to the identification of complex autoantibody signatures that may represent disease subgroups, early diagnostics and facilitated the analysis of vaccine trials.
Journal of Immunological Methods, 2013
Easily measurable biomarkers are urgently required to detect early stages of cancer progression. Autoantibodies (aAbs), as a component of the humoral immune response against tumor cells, have such potential of diagnostic markers since they are circulating and stable proteins, produced rapidly and easily amenable to in vitro dosage. The identification of aAbs is based on the characterization of tumor-associated antigens (TAA) against which they are directed. Here, we propose a new method for an unbiased identification of TAA and thereby of aAbs as cancer biomarkers. This method that we called sequential immunoaffinity depletion-differential in gel electrophoresis (SID-DIGE) is based on the immunodepletion of tumor cell lysates with IgG from control and tumor-bearing mice and direct matching of the flow throughs of these immunoaffinity separations on the same 2D format. This strategy reduces the complexity of the samples to be analyzed and maximizes the interest of assessing hundreds of proteins simultaneously. SID-DIGE has also the potential, contrary to existing serological proteome analysis (SERPA) techniques, to detect immunogenic proteins with conformational epitopes, including those resulting from post-translational modifications. Using a model of human colorectal tumors in mice for the proof of principle, we showed that SID-DIGE outperforms the conventional SERPA technique, with the identification of 7 common TAA (validating our approach) and 18 additional aAbs proving the potential of this new method. In particular, the identification of aAbs directed against key enzymes supporting glycolysis gives credential to the role of hypoxia as a major determinant of the tumor proteome and thus as a source of immunogenicity. Overall, the developed methodology allowed efficient screening of sera for the identification of aAbs as potential biomarkers.
Autoantigen microarrays for multiplex characterization of autoantibody responses
Nature Medicine, 2002
We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.
International Journal of Cancer, 2014
The cancer-testis antigens are a group of unrelated proteins aberrantly expressed in various cancers in adult somatic tissues. This aberrant expression can trigger spontaneous immune responses, a phenomenon exploited for the development of disease markers and therapeutic vaccines. However, expression levels often vary amongst patients presenting the same cancer type, and these antigens are therefore unlikely to be individually viable as diagnostic or prognostic markers. Nevertheless, patterns of antigen expression may provide correlates of specific cancer types and disease progression. Herein, we describe the development of a novel, readily customizable cancer-testis antigen microarray platform together with robust bioinformatics tools, with which to quantify anti-cancer testis antigen autoantibody profiles in patient sera. By exploiting the high affinity between autoantibodies and tumor antigens, we achieved linearity of response and an autoantibody quantitation limit in the pg/mL range-equating to a million-fold serum dilution. By using oriented attachment of folded, recombinant antigens and a polyethylene glycol microarray surface coating, we attained minimal non-specific antibody binding. Unlike other proteomics methods, which typically use lower affinity interactions between monoclonal antibodies and tumor antigens for detection, the high sensitivity and specificity realized using our autoantibody-based approach may facilitate the development of better cancer biomarkers, as well as potentially enabling pre-symptomatic diagnosis. We illustrated the usage of our platform by monitoring the response of a melanoma patient cohort to an experimental therapeutic NY-ESO-1-based cancer vaccine; inter alia, we found evidence of determinant spreading in individual patients, as well as differential CT antigen expression and epitope usage.V C 2014 UICC The cancer-testis (CT) antigen family are a group of > 90 structurally and functionally unrelated proteins usually only expressed in the germ cells in the adult testis or ovary and in the trophoblast of the placenta, 1 but which are aberrantly expressed in various cancers in adult somatic tissues as a result of disrupted gene regulation. As the testis is an immune-privileged site, aberrant expression of these proteins in somatic tissues typically triggers a spontaneous immune response to the relevant CT antigen. CT antigens have therefore been exploited as therapeutic tumor vaccines,
A contra capture protein array platform for studying post-translationally modified auto-antigenomes
Molecular & Cellular Proteomics, 2016
Aberrant modifications of proteins occur during disease development and elicit disease-specific antibody responses. We have developed a protein array platform that enables the modification of many proteins in parallel and assesses their immunogenicity without the need to express, purify, and modify proteins individually. We used anticitrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) as a model modification and profiled antibody responses to ϳ190 citrullinated proteins in 20 RA patients. We observed unique antibody reactivity patterns in both clinical anticyclic citrullinated peptide assay positive (CCP؉) and CCP-RA patients. At individual antigen levels, we detected antibodies against known citrullinated autoantigens and discovered and validated five novel antibodies against specific citrullinated antigens (osteopontin (SPP1), flap endonuclease (FEN1), insulin like growth factor binding protein 6 (IGFBP6), insulin like growth factor I (IGF1) and stanniocalcin-2 (STC2)) in RA patients. We also demonstrated the utility of our innovative array platform in the identification of immune-dominant epitope(s) for citrullinated antigens. We believe our platform will promote the study of post-translationally modified antigens at a breadth that has not been achieved before, by both identifying novel autoantigens and investigating their roles in disease development. The developed platforms can potentially be used to study many autoimmune disease-relevant modifications and their immunogenicity.
Tumour auto-antibody screening: performance of protein microarrays using SEREX derived antigens
BMC Cancer, 2010
Background The simplicity and potential of minimal invasive testing using serum from patients make auto-antibody based biomarkers a very promising tool for use in diagnostics of cancer and auto-immune disease. Although several methods exist for elucidating candidate-protein markers, immobilizing these onto membranes and generating so called macroarrays is of limited use for marker validation. Especially when several hundred samples have to be analysed, microarrays could serve as a good alternative since processing macro membranes is cumbersome and reproducibility of results is moderate. Methods Candidate markers identified by SEREX (serological identification of antigens by recombinant expression cloning) screenings of brain and lung tumour were used for macroarray and microarray production. For microarray production recombinant proteins were expressed in E. coli by autoinduction and purified His-tag (histidine-tagged) proteins were then used for the production of protein microarray...
Journal of Proteome Research, 2008
High-throughput studies to determine differential immune (humoral) response to diseases are becoming of increasing interest because the information they provide can help in early diagnosis as well as monitoring of therapeutics. Protein microarrays are a high-throughput and convenient technology that can be applied to the study of the humoral response. Proteins can be arrayed on slides and then probed with serum from different classes of patients to observe differences that may exist among autoantibodies that reflect differences in disease states. However, such studies may be difficult to interpret due to the weak overall signal response of such protein microarrays. We propose that this weak signal response is due to the physical positioning of the disease proteins that renders them sterically hindered from binding partners in the serum. In this study, we hypothesize that reducing the complexity and size of the disease proteins by chemical digestion using cyanogen bromide (CNBr) may enhance the overall signal from the humoral response and facilitate visualization of disease-specific responses in various classes of serum. A modified protein microarray methodology using CNBr digestion is presented here. The new workflow was applied to a set of 10 serum samples from healthy subjects, 10 from patients with chronic pancreatitis and 10 from patients diagnosed with pancreatic cancer and the results were compared to results obtained in the absence of CNBr digestion. CNBr digestion allowed the identification of 10 additional autoantibodies that responded to serum, 5 of which were unique to pancreatitis and cancer sera. This new methodology may increase the sensitivity of microarray studies measuring autoantibodies in serum.