Autoantibody microarrays for biomarker discovery

Identification of autoantigens and the detection of autoantibody reactivity are useful in biomarker discovery and for explaining the role of important biochemical pathways in disease. Despite all of their potential advantages, the main challenge to working with autoantibodies is their sensitivity. Nevertheless, proteomics may hold the key to overcoming this limitation by providing the means to multiplex. Clearly, the ability to detect multiple autoantigens using a platform such as a high-density antigen microarray would improve sensitivity and specificity of detection for autoantibody profiling. The aims of this review are to: briefly describe the current status of antigen–autoantibody microarrays; provide examples of their use in biomarker discoveries; address current limitations; and provide examples and strategies to facilitate their implementation in the clinical setting.     

Protein arrays for autoantibody profiling and fine-specificity mapping

Protein arrays provide a powerful approach to study autoimmune disease. Autoimmune responses activate B cells to produce autoantibodies that recognize self-molecules termed autoantigens, many of which are proteins or protein complexes. Protein arrays enable profiling of the specificity of autoantibody responses against panels of peptides and proteins representing known autoantigens as well as candidate autoantigens. In addition to identifying autoantigens and mapping immunodominant epitopes, proteomic analysis of autoantibody responses will further enable diagnosis, prognosis, and tailoring of antigen-specific tolerizing therapy.     

Autoantibody biomarker opens a new gateway for cancer diagnosis

The list of cancer markers of current interest has grown considerably, but none of the markers used in clinical work is a true tumor marker. These cancer biomarkers are based on the determination of tumor antigens. Here, we report a single method of autoantibody enzyme immunoassay (EIA) screens for a spectrum of serum tumor markers. A comparison of the autoantibody-based EIA to conventional antigen EIA kits, using receiver operating characteristic (ROC) plots, showed that the autoantibody EIA can significantly enhance the sensitivity and specificity of tumor markers. The detection of serum autoantibodies for a spectrum of serum tumor markers, as demonstrated here, suggests that most, if not all, serum cancer biomarkers produce autoantibodies. A unique autoantibody biomarker screening method, as presented here, might therefore facilitate achieving the accurate and early diagnosis of cancer.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

The diagnosis of rheumatoid arthritis (RA) is primarily based on clinical symptoms, so it is often difficult to diagnose RA in very early stages of the disease. A disease-specific autoantibody that could be used as a serological marker would therefore be very useful. Most autoimmune diseases are characterized by a polyclonal B-cell response targeting multiple autoantigens. These immune responses are often not specific for a single disease. In this review, the most important autoantibody/autoantigen systems associated with RA are described and their utility as a diagnostic and prognostic tool, including their specificity, sensitivity and practical application, is discussed. We conclude that, at present, the antibody response directed to citrullinated antigens has the most valuable diagnostic and prognostic potential for RA.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

The diagnosis of rheumatoid arthritis (RA) is primarily based on clinical symptoms, so it is often difficult to diagnose RA in very early stages of the disease. A disease-specific autoantibody that could be used as a serological marker would therefore be very useful. Most autoimmune diseases are characterized by a polyclonal B-cell response targeting multiple autoantigens. These immune responses are often not specific for a single disease. In this review, the most important autoantibody/autoantigen systems associated with RA are described and their utility as a diagnostic and prognostic tool, including their specificity, sensitivity and practical application, is discussed. We conclude that, at present, the antibody response directed to citrullinated antigens has the most valuable diagnostic and prognostic potential for RA.     

Autoantigen microarrays for multiplex characterization of autoantibody responses

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.     

Epitope presentation is an important determinant of the utility of antigens identified from protein arrays in the development of autoantibody diagnostic assays

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.     

Fragmentation of Golgi complex and Golgi autoantigens during apoptosis and necrosis

Anti-Golgi complex autoantibodies are found primarily in patients with Sjögren's syndrome and systemic lupus erythematosus, although they are not restricted to these diseases. Several Golgi autoantigens have been identified that represent a small family of proteins. Common features of all Golgi autoantigens appear to be their distinct structural organization of multiple α-helical coiled-coil rods in the central domains flanked by non-coiled-coil N-termini and C-termini, and their localization to the cytoplasmic face of Golgi cisternae. Many autoantigens in systemic autoimmune diseases have distinct cleavage products in apoptosis or necrosis and this has raised the possibility that cell death may play a role in the generation of potentially immunostimulatory forms of autoantigens. In the present study, we examined changes in the Golgi complex and associated autoantigens during apoptosis and necrosis. Immunofluorescence analysis showed that the Golgi complex was altered and developed distinctive characteristics during apoptosis and necrosis. In addition, immunoblotting analysis showed the generation of antigenic fragments of each Golgi autoantigen, suggesting that they may play a role in sustaining autoantibody production. Further studies are needed to determine whether the differences observed in the Golgi complex during apoptosis or necrosis may account for the production of anti-Golgi complex autoantibodies.     

Autoantibody profiling for the study and treatment of autoimmune disease

Proteomics technologies enable profiling of autoantibody responses using biological fluids derived from patients with autoimmune disease. They provide a powerful tool to characterize autoreactive B-cell responses in diseases including rheumatoid arthritis, multiple sclerosis, autoimmune diabetes, and systemic lupus erythematosus. Autoantibody profiling may serve purposes including classification of individual patients and subsets of patients based on their 'autoantibody fingerprint', examination of epitope spreading and antibody isotype usage, discovery and characterization of candidate autoantigens, and tailoring antigen-specific therapy. In the coming decades, proteomics technologies will broaden our understanding of the underlying mechanisms of and will further our ability to diagnose, prognosticate and treat autoimmune disease.     

Serum Autoantibodies in Chronic Prostate Inflammation in Prostate Cancer Patients

Background

Chronic inflammation is frequently observed on histological analysis of malignant and non-malignant prostate specimens. It is a suspected supporting factor for prostate diseases and their progression and a main cause of false positive PSA tests in cancer screening. We hypothesized that inflammation induces autoantibodies, which may be useful biomarkers. We aimed to identify and validate prostate inflammation associated serum autoantibodies in prostate cancer patients and evaluate the expression of corresponding autoantigens.

Methods

Radical prostatectomy specimens of prostate cancer patients (N = 70) were classified into high and low inflammation groups according to the amount of tissue infiltrating lymphocytes. The corresponding pre-surgery blood serum samples were scrutinized for autoantibodies using a low-density protein array. Selected autoantigens were identified in prostate tissue and their expression pattern analyzed by immunohistochemistry and qPCR. The identified autoantibody profile was cross-checked in an independent sample set (N = 63) using the Luminex-bead protein array technology.

Results

Protein array screening identified 165 autoantibodies differentially abundant in the serum of high compared to low inflammation patients. The expression pattern of three corresponding antigens were established in benign and cancer tissue by immunohistochemistry and qPCR: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Of these, SPAST was significantly increased in prostate tissue with high inflammation. All three autoantigens were differentially expressed in primary and/or castration resistant prostate tumors when analyzed in an inflammation-independent tissue microarray. Cross-validation of the inflammation autoantibody profile on an independent sample set using a Luminex-bead protein array, retrieved 51 of the significantly discriminating autoantibodies. Three autoantibodies were significantly upregulated in both screens, MUT, RAB11B and CSRP2 (p>0.05), two, SPOP and ZNF671, close to statistical significance (p = 0.051 and 0.076).

Conclusions

We provide evidence of an inflammation-specific autoantibody profile and confirm the expression of corresponding autoantigens in prostate tissue. This supports evaluation of autoantibodies as non-invasive markers for prostate inflammation.     

Identification and functional validation of novel autoantigens in equine uveitis

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers.     

Proteomics and autoantibody

Autoimmune response is diverse. This diversity is thought not to take place at the beginning of the autoimmune process but to occur as the disease evolves. It is mainly the consequence of the so-called epitope-spreading phenomenon and of the cross-reactivity of antibodies. Analysing autoantibody repertoire constitutes a powerful means to understand physiopathological processes at work in various diseases, mainly autoimmune diseases. In particular this analysis opens the way to precisely identify autoantigens and their changes in various pathological situations, and allows providing new biological markers in chronic inflammatory diseases. New methodologies have recently emerged for the analysis of the autoantibody repertoire in a given individual. They propose diagnostic approaches no more related upon few markers but founded upon analysis of global changes of the antibody repertoire. They belong to methodologies called target-oriented proteomics. Their common feature is to isolate autoantigens by means of affinity chromatography based upon antibody/antigen reactions. Autoantibodies to be studied interact with a protein substratum susceptible to include autoantibody targets. These interactions take place on solid macro- or microsurfaces, i.e. membrane filters or chips. Several strategies can be used for locating the specific autoantibody/autoantigen complexes and for identifying behind autoantigens. In this paper three approaches, namely, the recombinant protein chips, the SELDI techniques and the 2-D gel electrophoresis linked to mass spectrometry are described and compared.     

Extrapancreatic Autoantibody Profiles in Type I Diabetes

Type I diabetes (T1D) is an autoimmune disease characterized by destruction of insulin-producing β-cells in the pancreas. Although several islet cell autoantigens are known, the breadth and spectrum of autoantibody targets has not been fully explored. Here the luciferase immunoprecipitation systems (LIPS) antibody profiling technology was used to study islet and other organ-specific autoantibody responses in parallel. Examination of an initial cohort of 93 controls and 50 T1D subjects revealed that 16% of the diabetic subjects showed anti-gastric ATPase autoantibodies which did not correlate with autoantibodies against GAD65, IA2, or IA2-β. A more detailed study of a second cohort with 18 potential autoantibody targets revealed marked heterogeneity in autoantibody responses against islet cell autoantigens including two polymorphic variants of ZnT8. A subset of T1D subjects exhibited autoantibodies against several organ-specific targets including gastric ATPase (11%), thyroid peroxidase (14%), and anti-IgA autoantibodies against tissue transglutaminase (12%). Although a few T1D subjects showed autoantibodies against a lung-associated protein KCNRG (6%) and S100-β (8%), no statistically significant autoantibodies were detected against several cytokines. Analysis of the overall autoantibody profiles using a heatmap revealed two major subgroups of approximately similar numbers, consisting of T1D subjects with and without organ-specific autoantibodies. Within the organ-specific subgroup, there was minimal overlap among anti-gastric ATPase, anti-thyroid peroxidase, and anti-transglutaminase seropositivity, and these autoantibodies did not correlate with islet cell autoantibodies. Examination of a third cohort, comprising prospectively collected longitudinal samples from high-risk individuals, revealed that anti-gastric ATPase autoantibodies were present in several individuals prior to detection of islet autoantibodies and before clinical onset of T1D. Taken together, these results suggest that autoantibody portraits derived from islet and organ-specific targets will likely be useful for enhancing the clinical management of T1D.     

Two of Them Do It Better: Novel Serum Biomarkers Improve Autoimmune Hepatitis Diagnosis

Background

Autoimmune hepatitis (AIH) is a chronic liver disease of unknown aetiology and characterized by continuing hepatocellular inflammation and necrosis. Autoantibodies represent accessible markers to measure the adaptive immune responses in the clinical investigation. Protein microarrays have become an important tool to discriminate the disease state from control groups, even though there is no agreed-upon standard to analyze the results.

Results

In the present study 15 sera of patients with AIH and 78 healthy donors (HD) have been tested against 1626 proteins by an in house-developed array. Using a Partial Least Squares Discriminant Analysis (PLS-DA) the resulting data interpretation led to the identification of both new and previously identified proteins. Two new proteins AHPA9419 and Chondroadherin precursor (UNQ9419 and CHAD, respectively), and previously identified candidates as well, have been confirmed in a validation phase by DELFIA assay using a new cohort of AIH patients. A receiver operating characteristic analysis was used for the evaluation of biomarker candidates. The sensitivity of each autoantigen in AIH ranged from 65 to 88%; moreover, when the combination of the two new autoantigens was analyzed, the sensitivity increased to 95%.

Conclusions

Our findings demonstrate that the detection of autoantibodies against the two autoantigens could improve the performance in discriminating AIH patients from control classes and in combination with previously identified autoantigens and they could be used in diagnostic/prognostic markers.     

BMI-1 autoantibody as a new potential biomarker for cervical carcinoma

BMI-1 is overexpressed in a variety of cancers, which can elicit an immune response leading to the induction of autoantibodies. However, BMI-1 autoantibody as a biomarker has seldom been studied with the exception of nasopharyngeal carcinoma. Whether BMI-1 autoantibodies can be used as a biomarker for cervical carcinoma is unclear. In this study,BMI-1 proteins were isolated by screening of a T7 phage cDNA library from mixed cervical carcinoma tissues. We analyzed BMI-1 autoantibody levels in serum samples from 67 patients with cervical carcinoma and 65 controls using ELISA and immunoblot. BMI-1 mRNA or protein levels were over-expressed in cervical carcinoma cell lines. Immunoblot results exhibited increased BMI-1 autoantibody levels in patient sera compared to normal sera. Additionally, the results for antibody affinity assay showed that there was no difference between cervical polyps and normal sera of BMI-1 autoantibody levels, but it was significantly greater in patient sera than that in normal controls (patient 0.827±0.043 and normal 0.445±0.023; P<0.001). What''s more, the levels of BMI-1 autoantibody increased significantly at stage I (0.672±0.019) compared to normal sera (P<0.001), and levels of BMI-1 autoantibodies were increased gradually during the tumor progression (stage I 0.672±0.019; stage II 0.775 ±0.019; stage III 0.890 ±0.027; stage IV 1.043±0.041), which were significantly correlated with disease progression of cervical carcer (P<0.001). Statistical analyses using logistic regression and receiver operating characteristics (ROC) curves indicated that the BMI-1 autoantibody level can be used as a biomarker for cervical carcinoma (sensitivity 0.78 and specificity 0.76; AUC = 0.922). In conclusion, measuring BMI-1 autoantibody levels of patients with cervical cancer could have clinical prognostic value as well as a non-tissue specific biomarker for neoplasms expressing BMI-1.     

Genomic and proteomic biomarkers for cancer: A multitude of opportunities

Biomarkers are molecular indicators of a biological status, and as biochemical species can be assayed to evaluate the presence of cancer and therapeutic interventions. Through a variety of mechanisms cancer cells provide the biomarker material for their own detection. Biomarkers may be detectable in the blood, other body fluids, or tissues. The expectation is that the level of an informative biomarker is related to the specific type of disease present in the body. Biomarkers have potential both as diagnostic indicators and monitors of the effectiveness of clinical interventions. Biomarkers are also able to stratify cancer patients to the most appropriate treatment. Effective biomarkers for the early detection of cancer should provide a patient with a better outcome which in turn will translate into more efficient delivery of healthcare. Technologies for the early detection of cancer have resulted in reductions in disease-associated mortalities from cancers that are otherwise deadly if allowed to progress. Such screening technologies have proven that early detection will decrease the morbidity and mortality from cancer. An emerging theme in biomarker research is the expectation that panels of biomarker analytes rather than single markers will be needed to have sufficient sensitivity and specificity for the presymptomatic detection of cancer. Biomarkers may provide prognostic information of disease enabling interventions using targeted therapeutic agents as well as course-corrections in cancer treatment. Novel genomic, proteomic and metabolomic technologies are being used to discover and validate tumor biomarkers individually and in panels.     

Early detection of colon cancer: new tests on the horizon

This year, the American Cancer Society reported that the rate of decline in both the incidence and mortality of colorectal cancer has increased over the last two decades. This success is felt to be attributable to the early detection and treatment of colonic adenomas and early-stage colorectal cancers. However, the current recommended 'menu of options' for screening is limited by poor patient acceptance, low sensitivity, and both high cost and poor accessibility for application to a large general screening population (colonoscopy). Computerized tomography and magnetic resonance colonography offer an alternative method for the identification of polyps and early lesions in certain patients, but have cost, access, and acceptance limitations that are similar to those of colonoscopy; thus, they present similar barriers to their use in broad population screening. These limitations provide a strong rationale for the development of early colorectal cancer detection biomarkers that are simple to use and are cost effective. A successful biomarker or biomarker panel, coupled with the colonoscopic follow-up of only those patients with positive results, would reduce the burden and morbidity associated with the screening of colonoscopy. This would most likely result in enhanced adherence to colorectal screening, as well as a dramatic reduction in the incidence and mortality rates of colorectal cancer. In this paper, we review recent advances in the discovery of potential colorectal cancer biomarkers. Their applicability to clinical population screening will require large prospective validation.     

Paraneoplastic antigen Ma2 autoantibodies as specific blood biomarkers for detection of early recurrence of small intestine neuroendocrine tumors

Background

Small intestine neuroendocrine tumors (SI-NETs) belong to a rare group of cancers. Most patients have developed metastatic disease at the time of diagnosis, for which there is currently no cure. The delay in diagnosis is a major issue in the clinical management of the patients and new markers are urgently needed. We have previously identified paraneoplastic antigen Ma2 (PNMA2) as a novel SI-NET tissue biomarker. Therefore, we evaluated whether Ma2 autoantibodies detection in the blood stream is useful for the clinical diagnosis and recurrence of SI-NETs.

Methodology/Principal Findings

A novel indirect ELISA was set up to detect Ma2 autoantibodies in blood samples of patients with SI-NET at different stages of disease. The analysis was extended to include typical and atypical lung carcinoids (TLC and ALC), to evaluate whether Ma2 autoantibodies in the blood stream become a general biomarker for NETs. In total, 124 blood samples of SI-NET patients at different stages of disease were included in the study. The novel Ma2 autoantibody ELISA showed high sensitivity, specificity and accuracy with ROC curve analysis underlying an area between 0.734 and 0.816. Ma2 autoantibodies in the blood from SI-NET patients were verified by western blot and sequential immunoprecipitation. Serum antibodies of patients stain Ma2 in the tumor tissue and neurons. We observed that SI-NET patients expressing Ma2 autoantibody levels below the cutoff had a longer progression and recurrence-free survival compared to those with higher titer. We also detected higher levels of Ma2 autoantibodies in blood samples from TLC and ALC patients than from healthy controls, as previously shown in small cell lung carcinoma samples.

Conclusion

Here we show that high Ma2 autoantibody titer in the blood of SI-NET patients is a sensitive and specific biomarker, superior to chromogranin A (CgA) for the risk of recurrence after radical operation of these tumors.     

Profiling humoral autoimmune repertoire of dilated cardiomyopathy (DCM) patients and development of a disease-associated protein chip

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.