High-density Protein Microarrays Research Articles

Functional Analysis of Autoantibody Signatures in Rheumatoid Arthritis.

For the identification of antigenic protein biomarkers for rheumatoid arthritis (RA), we conducted IgG profiling on high density protein microarrays. Plasma IgG of 96 human samples (healthy controls, osteoarthritis, seropositive and seronegative RA, n = 24 each) and time-series plasma of a pristane-induced arthritis (PIA) rat model (n = 24 total) were probed on AIT’s 16k protein microarray. To investigate the analogy of underlying disease pathways, differential reactivity analysis was conducted. A total of n = 602 differentially reactive antigens (DIRAGs) at a significance cutoff of p < 0.05 were identified between seropositive and seronegative RA for the human samples. Correlation with the clinical disease activity index revealed an inverse correlation of antibodies against self-proteins found in pathways relevant for antigen presentation and immune regulation. The PIA model showed n = 1291 significant DIRAGs within acute disease. Significant DIRAGs for (I) seropositive, (II) seronegative and (III) PIA were subjected to the Reactome pathway browser which also revealed pathways relevant for antigen presentation and immune regulation; of these, seven overlapping pathways had high significance. We therefore conclude that the PIA model reflects the biological similarities of the disease pathogenesis. Our data show that protein array analysis can elucidate biological differences and pathways relevant in disease as well be a useful additional layer of omics information.

Panproteome Analysis of the Human Antibody Response to Bacterial Vaccines and Challenge.

High-density protein microarray is an established technology for characterizing host antibody profiles against entire pathogen proteomes. As one of the highest throughput technologies for antigen discovery, proteome microarrays are a translational research tool for identification of vaccine candidates and biomarkers of susceptibility or protection from microbial challenge. The application has been expanded in recent years due to increased availability of bacterial genomic sequences for a broader range of species and strain diversity. Panproteome microarrays now allow for fine characterization of antibody specificity and cross-reactivity that may be relevant to vaccine design and biomarker discovery, as well as a fuller understanding of factors underlying themes of bacterial evolution and host-pathogen interactions. In this chapter, we present a workflow for design of panproteome microarrays and demonstrate statistical analysis of panproteomic human antibody responses to bacterial vaccination and challenge. Focus is particularly drawn to the bioinformatics and statistical tools and providing nontrivial, real examples that may help foster hypotheses and rational design of panproteomic studies.

Identification of Tumor Antigens in Ovarian Cancers Using Local and Circulating Tumor-Specific Antibodies.

Ovarian cancers include several disease subtypes and patients often present with advanced metastatic disease and a poor prognosis. New biomarkers for early diagnosis and targeted therapy are, therefore, urgently required. This study uses antibodies produced locally in tumor-draining lymph nodes (ASC probes) of individual ovarian cancer patients to screen two separate protein microarray platforms and identify cognate tumor antigens. The resulting antigen profiles were unique for each individual cancer patient and were used to generate a 50-antigen custom microarray. Serum from a separate cohort of ovarian cancer patients encompassing four disease subtypes was screened on the custom array and we identified 28.8% of all ovarian cancers, with a higher sensitivity for mucinous (50.0%) and serous (40.0%) subtypes. Combining local and circulating antibodies with high-density protein microarrays can identify novel, patient-specific tumor-associated antigens that may have diagnostic, prognostic or therapeutic uses in ovarian cancer.

Novel autoantibodies identified in ACPA-negative rheumatoid arthritis

ObjectivesLack of effective biomarkers in anti-citrullinated protein antibody (ACPA)-negative rheumatoid arthritis (RA) impedes early diagnosis and treatment. This study aimed to identify novel autoantibodies in RA and verify their diagnostic...

Analysis of Protein-Protein Interactions by Protein Microarrays.

The analysis of the proteome and the interactome would be useful for a better understanding of the pathophysiology of several disorders, allowing the identification of potential specific markers for early diagnosis and prognosis, as well as potential targets of intervention. Among different proteomic approaches, high-density protein microarrays have become an interesting tool for the screening of protein-protein interactions and the interactome definition of disease-associated dysregulated proteins. This information might contribute to the identification of altered signaling pathways and protein functions involved in the pathogenesis of a disease. Remarkably, protein microarrays have been already satisfactorily employed for the study of protein-protein interactions in cancer, allergy, or neurodegenerative diseases. Here, we describe the utilization of recombinant protein microarrays for the identification of protein-protein interactions to help in the definition of disease-specific dysregulated interactomes.

A Systematic Analysis Workflow for High-Density Customized Protein Microarrays in Biomarker Screening.

High-density protein microarrays constitute a promising high-throughput platform for the characterization of protein expression patterns, biomarker discovery, and validation. Different types of protein microarrays have been described according to several features (such as content, format, and detection system) presenting advantages and disadvantages which are relevant for the specific application and purposes. Therefore, an experimental design is key for any screening based on protein microarrays assays; in fact, the data analysis strategy is directly related to the experimental design, type of protein microarray and consequently the final outcome, the data and results interpretation, is also directly linked. Here, it is proposed a systematic workflow for biomarker discovery based on tailor-made protein microarrays platforms which obtain comprehensively info for the functional protein characterization in high-throughput format.

Autoantibody biomarkers for the detection of serous ovarian cancer

ObjectiveThe purpose of this study was to identify a panel of novel serum tumor antigen-associated autoantibody (TAAb) biomarkers for the diagnosis of high-grade serous ovarian cancer. MethodsTo detect TAAb we probed high-density programmable protein microarrays (NAPPA) containing 10,247 antigens with sera from patients with serous ovarian cancer (n=30 cases/30 healthy controls) and measured bound IgG. We identified 735 promising tumor antigens and evaluated these with an independent set of serous ovarian cancer sera (n=30 cases/30 benign disease controls/30 healthy controls). Thirty-nine potential tumor autoantigens were identified and evaluated using an orthogonal programmable ELISA platform against a total of 153 sera samples (n=63 cases/30 benign disease controls/60 healthy controls). Sensitivities at 95% specificity were calculated and a classifier for the detection of high-grade serous ovarian cancer was constructed. ResultsWe identified 11-TAAbs (ICAM3, CTAG2, p53, STYXL1, PVR, POMC, NUDT11, TRIM39, UHMK1, KSR1, and NXF3) that distinguished high-grade serous ovarian cancer cases from healthy controls with a combined 45% sensitivity at 98% specificity. ConclusionThese are potential circulating biomarkers for the detection of serous ovarian cancer, and warrant confirmation in larger clinical cohorts.

Protein Microarrays: Flexible Tools for Scientific Innovation.

Protein microarrays have emerged as a powerful tool for the scientific community, and their greatest advantage lies in the fact that thousands of reactions can be performed in a parallel and unbiased manner. The first high-density protein microarray, dubbed the "yeast proteome array," consisted of approximately 5800 full-length yeast proteins and was initially used to identify protein-lipid interactions. Further assays were subsequently developed to allow measurement of protein-DNA, protein-RNA, and protein-protein interactions, as well as four well-known posttranslational modifications: phosphorylation, acetylation, ubiquitylation, and SUMOylation. In this introduction, we describe the advent of high-density protein microarrays, as well as current methods for assessing a wide variety of protein interactions and posttranslational modifications.

Abstract 1706: Evaluation of LGR5 protein expression in colon cancer stem cells

Abstract Cancer stem cells are a subpopulation of cancer cells responsible for cancer initiation, development and metastasis. A number of studies demonstrated that Leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5) can drive cancer development through triggering canonical Wnt signaling and its downstream gene expression. LGR5 is an important biomarker specifically expressed on colon cancer stem cells. In this study, we have successfully developed an LGR5 antibody with high specificity to detect endogenous LGR5 expression in different immunoassay application. By screening multiple anti-LGR5 hybridoma clones, antibody generated by clone UMAB212 was proven to be highly specific on our high density protein microarray chip assay. Here our experimental data demonstrated that clone UMAB212 recognizes not only human LGR5 protein but also mouse LGR5 protein in both western blot and flow cytometry applications. No cross-reactivity was observed with the other two LGR family members, LGR4 and LGR6. Furthermore, this antibody also works great on immunohistochemistry application on FFPE tissue blocks. In summary, UMAB212 is great tool for us to study LGR5 protein in different immunoassay setting and it could also be a potential cancer diagnostic reagent. Citation Format: Wei Fu, Rachel Gonzalez, Kehu Yuan, Caiwei Chen, Haitao Wei, Hsiangmin Lu, Qi Ren, Evelin Logis, Sean Kelly, Wei-Wu He, Donghui Ma. Evaluation of LGR5 protein expression in colon cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1706.

Abstract 438: Development of an autoantibody panel that reflects disease pathogenesis in ovarian cancer

Abstract Introduction The serous subtype of ovarian cancer is responsible for most ovarian cancer deaths. When patients are diagnosed with early stage disease localized to the ovary or pelvis, current therapies can achieve survival rates up to 75-90%. However, approximately 70% of cases are diagnosed at an advanced stage, with a 5-year survival rate of less than 20%. A strategy based on autoantibodies which occur early during tumor development has merit for early cancer detection. We have applied a combined approach of mass spectrometry of circulating antigen-antibody complexes and with high-density protein microarrays to develop ovarian cancer serum markers based on autoantibody reactivity. Materials and methods Serum samples were obtained from women diagnosed with ovarian cancer (cases) and healthy controls undergoing ovarian cancer screening, women undergoing gynecologic surgery for various conditions but with normal ovarian pathology (surgical controls), and women without malignancy but with benign ovarian disease (benign controls). All samples were obtained following Institutional Review Board approval and informed consent. For autoantibody analysis, we used recombinant protein arrays containing 5,005 recombinants arrayed in duplicate and Alexa 647-labeled anti-human IgG were purchased from Invitrogen. Circulating Immunoglobulin (Ig) bound antigen analysis was performed using LC-MS/MS. Results Autoantibodies against 20 targets were identified in ovarian cancer patients compared to healthy, surgical and benign controls by protein array analysis with P&amp;lt;0.01. Ingenuity Pathway Analysis of these 20 antigens, exhibited major nodes of p53, MYC, and ESR1 associated networks reflecting ovarian cancer pathogenesis. Concordant findings were observed with LC-MS/MS analysis of Ig-antigen complexes. Conclusion We have uncovered a set of 20 protein antigens associated with the development of autoantibodies in ovarian cancer that reflect disease pathogenesis and that have potential relevance for early detection. Citation Format: Makoto Kobayashi, Clayton R. Boldt, Wei-Lei Yang, Clemente Aguilar-Bonavides, Hong Wang, Zhen Lu, Robert C. Bast, Samir M. Hanash. Development of an autoantibody panel that reflects disease pathogenesis in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 438.

Application of Flexible Display Technology to Low Cost Multiplexed Point-of-Care Medical Diagnostic Testing

Abstract 62 One of the key roadblocks limiting the transition of high-sensitivity and high-specificity point-of-care technologies from the research laboratory to wide spread use is the availability of a low-cost-high-volume manufacturing technology. This work presents a new interdisciplinary approach combining low cost commercial display manufacturing technology with programmable high density protein microarray printing technology to fabricate disposable point-of-care immunosensors with clinical level sensitivity. Our approach is designed to leverage advances in commercial display technology to reduce pre-functionalized biosensor substrate costs to pennies per cm2, as well as to leverage the display industry’s ability to manufacture an immense number of low cost consumer electronic products annually. For this work, we demonstrate that our new approach can offer diagnostic sensitivity at or below 10 pg/mL, which approaches the lower limit of detection of typical clinical laboratory instrumentation. Our new approach is also designed to overcome the limited analytical sensitivity of existing POC devices (&gt;100x improved sensitivity). It also contains new capability for multiplexed biomarker detection (&gt;10 antigens) in a single low cost POC device through an innovative disposable and scalable architecture, based on flat panel display technology. Here, we demonstrate multiplexed detection of antibodies to the HPV16 proteins E2, E6, and E7, which are circulating biomarkers for cervical as well as head and neck cancers. This detection technology has 100 percent correlation to our current laboratory-based measurement instrumentation. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST: Benjamin A. Katchman Patents, Royalties, Other Intellectual Property: Arizona State University Joseph T. Smith Patents, Royalties, Other Intellectual Property: Arizona State University Jennifer Blain Christen Patents, Royalties, Other Intellectual Property: Arizona State University Karen S. Anderson Stock or Other Ownership: Provista Diagnostics Consulting or Advisory Role: Provista Diagnostics Patents, Royalties, Other Intellectual Property: Arizona State University

Development of a novel multiplex beads-based assay for autoantibody detection for colorectal cancer diagnosis.

Humoral response in cancer patients can be used for early cancer detection. By screening high-density protein microarrays with sera from colorectal cancer (CRC) patients and controls, we identified 16 tumor-associated antigens (TAAs) exhibiting high diagnostic value. This high number of TAAs requires the development of multiplex assays combining different antigens for a faster and more accurate prediction of CRC. Here, we have developed and optimized a bead-based assay using nine selected TAAs and two controls to provide a multiplex test for early CRC diagnosis. We screened a collection of 307 CRC patients' and control sera with the beads assay to identify and validate the best TAA combination for CRC detection. The multiplex bead-based assay exhibited a similar diagnostic performance to detect the humoral response in comparison to multiple ELISA analyses. After multivariate analysis, a panel composed of GTF2B, EDIL3, HCK, PIM1, STK4, and p53, together with gender and age, was identified as the best combination of TAAs for CRC diagnosis, achieving an AUC of 89.7%, with 66% sensitivity at 90.0% fixed specificity. The model was validated using bootstrapping analysis. In summary, we have developed a novel multiplex bead assay that after validation with a larger independent cohort of sera could be utilized in a high-throughput manner for population screening to facilitate the detection of early CRC patients.

Use of a High-Density Protein Microarray to Identify Autoantibodies in Subjects with Type 2 Diabetes Mellitus and an HLA Background Associated with Reduced Insulin Secretion.

New biomarkers for type 2 diabetes mellitus (T2DM) may aid diagnosis, drug development or clinical treatment. Evidence is increasing for the adaptive immune system’s role in T2DM and suggests the presence of unidentified autoantibodies. While high-density protein microarrays have emerged as a useful technology to identify possible novel autoantigens in autoimmune diseases, its application in T2DM has lagged. In Pima Indians, the HLA haplotype (HLA-DRB1*02) is protective against T2DM and, when studied when they have normal glucose tolerance, subjects with this HLA haplotype have higher insulin secretion compared to those without the protective haplotype. Possible autoantibody biomarkers were identified using microarrays containing 9480 proteins in plasma from Pima Indians with T2DM without the protective haplotype (n = 7) compared with those with normal glucose regulation (NGR) with the protective haplotype (n = 11). A subsequent validation phase involving 45 cases and 45 controls, matched by age, sex and specimen storage time, evaluated 77 proteins. Eleven autoantigens had higher antibody signals among T2DM subjects with the lower insulin-secretion HLA background compared with NGR subjects with the higher insulin-secretion HLA background (p<0.05, adjusted for multiple comparisons). PPARG2 and UBE2M had lowest p-values (adjusted p = 0.023) while PPARG2 and RGS17 had highest case-to-control antibody signal ratios (1.7). A multi-protein classifier involving the 11 autoantigens had sensitivity, specificity, and area under the receiver operating characteristics curve of 0.73, 0.80, and 0.83 (95% CI 0.74–0.91, p = 3.4x10-8), respectively. This study identified 11 novel autoantigens which were associated with T2DM and an HLA background associated with reduced insulin secretion. While further studies are needed to distinguish whether these antibodies are associated with insulin secretion via the HLA background, T2DM more broadly, or a combination of the two, this study may aid the search for autoantibody biomarkers by narrowing the list of protein targets.

Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling

High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions.

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis to determine a high confidence set of protein interactions for downstream validation in vivo.

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Abstract 3393: Using protein microarray technology to screen anti-PD-1 monoclonal antibodies for specificity and applications in anatomic pathology

Abstract Programmed death-1 (PD-1) expresses in many tumors in response to inflammation. It is up-regulated in activated T lymphocytes and inhibits T-cell function upon binding to its ligands PD-L1 and PDL2 and serves as a key checkpoint of the immune system. Pembrolizumab is the first anti-PD-1 therapy approved in the United States and received FDA's Breakthrough Therapy designation for advanced melanoma. Thus, to evaluate PD1 protein levels in formalin-fixed paraffin-embedded tissue samples, a high quality monoclonal antibody validated for immunohistochemistry (IHC) is needed. To develop the best monoclonal antibody for PD-1 IHC analysis, 41 monoclonal antibodies were generated. 8 of them work for IHC application on FFPE tissue sections. To identify clones that are mono-specific on target, only two clones passed our high density protein microarray chip tests. Other immunoassays and species cross-reactivity tests were also explored. In summary, two newly generated monoclonal antibodies demonstrated ultra-specificity against PD-1 protein and superior performance for IHC analyses. These two clones could be utilized as companion diagnostic reagents to stratify cancer patients before pembrolizumab prescription. Citation Format: Caiwei Chen, Yanlin Tang, Haitao Wei, Kehu Yuan, Guiyin Wu, Jian Chen, Boyang Chu, Guangli Wang, Youmin Shu, Wei-Wu He, Donghui Ma. Using protein microarray technology to screen anti-PD-1 monoclonal antibodies for specificity and applications in anatomic pathology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3393. doi:10.1158/1538-7445.AM2015-3393

Abstract 561: Development of a highly specific Met antibody for screening Met overexpression in NSCLC

Abstract Approximately 5%-22% of non-small cell lung cancer (NSCLC) patients with secondary resistance to EGFR TKIs had evidence of amplification of the Met oncogene. This dysregulation of Met signaling was implicated in the proliferation and eventual metastasis of NSCLC, and therefore represents an attractive target for anticancer drug development. Currently, there are a number of clinical trials for Met targeted small molecule and monoclonal antibody therapies. Immunohistochemistry (IHC) analysis of the Met protein will be very important for these Met targeted therapies and requires a highly specific antibody to prevent false positives. To develop a highly specific monoclonal antibody against Met for IHC analysis in NSCLC, we generated 97 monoclonal antibodies. IHC evaluation showed that 8 of the antibodies had suitable performance in Met positive NSCLC patient tissues. The 8 Met monoclonal antibodies were further tested on a high density protein microarray chip containing 17,000 human proteins. The microarray analysis showed that clone 3G5 was highly specific as evidenced with it only reacting with Met on the microarray. This new 3G5 Met monoclonal antibody will be a promising candidate as a diagnostic reagent for detecting Met protein in NSCLC with minimal false positives. Citation Format: Caiwei Chen, Hsiangmin Emily Lu, Haitao Wei, Kehu Yuan, Donghui Ma, Wei-Wu He. Development of a highly specific Met antibody for screening Met overexpression in NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 561. doi:10.1158/1538-7445.AM2015-561

High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

Using Protein Microarray Technology to Screen Anti‐PD‐1 Monoclonal Antibodies for Specificity and Applications in Anatomic Pathology

Programmed death‐1 (PD‐1) expresses in many tumors in response to inflammation. It is up‐regulated in activated T lymphocytes and inhibits T‐cell function upon binding to its ligands PD‐L1 and PDL2 and serves as a key checkpoint of the immune system. KEYTRUDA (pembrolizumab) is the first anti‐PD‐1 therapy approved in the United States and received FDA's Breakthrough Therapy designation for advanced melanoma. Thus, to evaluate PD1 protein levels in formalin‐fixed paraffin‐embedded tissue samples, a high quality monoclonal antibody validated for immunohistochemistry (IHC) is needed.To develop the best monoclonal antibody for PD‐1 IHC analysis, 41 monoclonal antibodies were generated. 8 of them work for IHC application on FFPE tissue sections. To identify clones that are mono‐specific on target, only two clones passed our high density protein microarray chip tests. Other immunoassays and species cross‐reactivity tests were also explored.In summary, two newly generated monoclonal antibodies demonstrated ultra‐specificity against PD‐1 protein and superior performance for IHC analyses. These two clones could be utilized as companion diagnostic reagents to stratify cancer patients before KEYTRUDA prescription.This research is partly supported by Chinese 863 research grant number 2014AA020909.