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The antibody array has become a powerful technology in recent years and is widely used to detect the expression levels of various proteins such as cytokines, growth factors, chemokines, and angiogenic factors, some of which are involved in cancer progression. In this chapter, we describe a protein array technology called enhanced protein profiling array, which can simultaneously and quantitatively measure the expression levels of a few proteins in hundreds or thousands of samples, and an example of its use is presented.

Background: Biomarker-based tests for diagnosing TB currently rely on detecting Mycobacterium tuberculosis (Mtb) antigen-specific cellular responses. While this approach can detect Mtb infection, it is not efficient in diagnosing TB, especially for patients who lack aetiological evidence of the disease., Methods: We prospectively enrolled three cohorts for our study for a total of 630 subjects, including 160 individuals to screen protein biomarkers of TB, 368 individuals to establish and test the predictive model and 102 individuals for biomarker validation. Whole blood cultures were stimulated with pooled Mtb-peptides or mitogen, and 640 proteins within the culture supernatant were analysed simultaneously using an antibody-based array. Sixteen candidate biomarkers of TB identified during screening were then developed into a custom multiplexed antibody array for biomarker validation., Results: A two-round screening strategy identified eight-protein biomarkers of TB: I-TAC, I-309, MIG, Granulysin, FAP, MEP1B, Furin and LYVE-1. The sensitivity and specificity of the eight-protein biosignature in diagnosing TB were determined for the training (n=276), test (n=92) and prediction (n=102) cohorts. The training cohort had a 100% specificity (95% CI 98% to 100%) and 100% sensitivity (95% CI 96% to 100%) using a random forest algorithm approach by cross-validation. In the test cohort, the specificity and sensitivity were 83% (95% CI 71% to 91%) and 76% (95% CI 56% to 90%), respectively. In the prediction cohort, the specificity was 84% (95% CI 74% to 92%) and the sensitivity was 75% (95% CI 57% to 89%)., Conclusions: An eight-protein biosignature to diagnose TB in a high-burden TB clinical setting was identified., Competing Interests: Competing interests: HZ, YY and R-PH are employees of RayBiotech Life, a company producing commercial antibody arrays, including the antibody array targeting 640 human proteins that was used in this study. The custom antibody array targeting 16 proteins was also produced by RayBiotech., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Recent research shows bidirectional communication between the normal brain and the peripheral immune system. Glioma is a primary brain tumor characterized by systemic immunosuppression. To better understand gliomagenesis, we evaluated associations between 277 prediagnostic serum cytokines and glioma. We used glioma (n = 487) and matched control (n = 487) specimens from the Janus Serum Bank Cohort in Oslo, Norway. Conditional logistic regression allowed us to identify those cytokines that were individually associated with glioma. Next, we used heat maps to compare case to control Pearson correlation matrices of 12 cytokines modeled in an in silico study of the interaction between the microenvironment and the tumor. We did the same for case-control correlation matrices of lasso-selected cytokines and all 277 cytokines in the data set. Cytokines related to glioma risk (P ≤ .05) more than 10 years before diagnosis are sIL10RB, VEGF, beta-Catenin and CCL22. LIF was associated with decreased glioma risk within five years before glioma diagnosis (odds ratio (OR) = 0.47, 95% confidence interval (CI) = 0.23, 0.94). After adjustment for cytokines above, the previously observed interaction between IL4 and sIL4RA persisted (> 20 years before diagnosis, OR = 1.72, 95% CI = 1.20, 2.47). In addition, during this period, case correlations among 12 cytokines were weaker than were those among controls. This pattern was also observed among 30 lasso- selected cytokines and all 277 cytokines. We identified four cytokines and one interaction term that were independently related to glioma risk. We have documented prediagnostic changes in serum cytokine levels that may reflect the presence of a preclinical tumor.

The development of oncoprotein-targeted anticancer drugs is an invaluable weapon in the war against cancer. However, cancers do not give up without a fight. They may develop multiple mechanisms of drug resistance, including apoptosis inhibition, drug expulsion, and increased proliferation that reduce the effectiveness of the drug. The collective work of researchers has highlighted the role of cytokines in the mechanisms of cancer drug resistance, as well as in cancer cell progression. Furthermore, recent studies have described how specific cytokines secreted by cancer stromal cells confer resistance to chemotherapeutic treatments. In order to gain a better understanding of mechanism of cancer drug resistance and a prediction of treatment outcome, it is imperative that correlations are established between global cytokine profiles and cancer drug resistance. Here we discuss the recent discoveries in this field of research and discuss their implications for the future development of effective anti-cancer medicines., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Allergy is inversely related to glioma risk. To determine whether prediagnostic allergy-related serum proteins are associated with glioma, we conducted a nested case-control study of seven cytokines (IL4, IL13, IL5, IL6, IL10, IFNG, TGFB2), two soluble cytokine receptors (sIL4RA, sIL13RA2) and three allergy-related transcription factors (FOXP3, STAT3, STAT6) using serum specimens from the Janus Serum Bank Cohort in Oslo, Norway. Blood donors subsequently diagnosed with glioma (n = 487) were matched to controls (n = 487) on age and date of blood draw and sex. We first estimated individual effects of the 12 serum proteins and then interactions between IL4 and IL13 and their receptors using conditional logistic regression. We next tested equality of case-control inter-correlations among the 12 serum proteins. We found that TGFB2 is inversely related to glioblastoma (Odds Ratio (OR) = 0.87, 95% Confidence Interval (CI)) = 0.76, 0.98). In addition, ≤ 5 years before diagnosis, we observed associations between IL4 (OR = 0.82, 95% CI = 0.66, 1.01), sIL4RA (OR = 0.80, 95% CI = 0.65, 1.00), their interaction (OR = 1.06, 95% CI = 1.01, 1.12) and glioblastoma. This interaction was apparent > 20 years before diagnosis (IL4-sIL4RA OR = 1.20, 95% CI = 1.05, 1.37). Findings for glioma were similar. Case correlations were different from control correlations stratified on time before diagnosis. Five years or less before diagnosis, correlations among case serum proteins were weaker than were those among controls. Our findings suggest that IL4 and sIL4RA reduce glioma risk long before diagnosis and early gliomagenesis affects circulating immune function proteins.

Until recently, the intrinsically high level of cross-talk between immune cells, the complexity of immune cell development, and the pleiotropic nature of cytokine signaling have hampered progress in understanding the mechanisms of immunosuppression by which tumor cells circumvent native and adaptive immune responses. One technology that has helped to shed light on this complex signaling network is the cytokine antibody array, which facilitates simultaneous screening of dozens to hundreds of secreted signal proteins in complex biological samples. The combined applications of traditional methods of molecular and cell biology with the high-content, high-throughput screening capabilities of cytokine antibody arrays and other multiplexed immunoassays have revealed a complex mechanism that involves multiple cytokine signals contributed not just by tumor cells but by stromal cells and a wide spectrum of immune cell types. This review will summarize the interactions among cancerous and immune cell types, as well as the key cytokine signals that are required for tumors to survive immunoediting in a dormant state or to grow and spread by escaping it. Additionally, it will present examples of how probing secreted cell-cell signal networks in the tumor microenvironment (TME) with cytokine screens have contributed to our current understanding of these processes and discuss the implications of this understanding to antitumor therapies., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Dried blood spot samples (DBSS) on filter paper offer several advantages compared to conventional serum/plasma samples: they do not require any phlebotomy or separation of blood by centrifugation; they are less invasive; they allow sample stability and shipment at room temperature; and they pose a negligible risk of infection with blood-borne viruses, such as HIV, HBV and HCV, to those who handle them. Therefore dried blood spot samples (DBSS) on filter paper can be a quick, convenient and inexpensive means of obtaining blood samples for biomarker discovery, disease screening, diagnosis and treatment monitoring in non-hospitalized, public health settings. In this study, we investigated for the first time the potential application of dried blood spot samples (DBSS) in protein expression profiling using antibody array technology. First, optimal conditions for array assay performance using dried blood spot samples (DBSS) was established, including sample elution buffer, elution time, elution temperature and assay blocking buffer. Second, we analyzed dried blood spot samples (DBSS) using three distinct antibody array platforms, including sandwich-based antibody arrays, quantitative antibody arrays and biotin-label-based antibody arrays. In comparison with paired serum samples, detection of circulating proteins in dried blood spot samples (DBSS) correlated well for both low- and high-abundance proteins on all three antibody array platforms. In conclusion, our study strongly indicates the novel application of multiplex antibody array platforms to analyze dried blood spot samples (DBSS) on filter paper represents a viable, cost-effective method for protein profiling, biomarker discovery and disease screening in a large, population-based survey., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Protein arrays have shown potential applications in cancer research. After several decades of research, it has become evident that many cytokines are central to the development of cancer and its treatment. Cytokine antibody arrays that have been designed to simultaneously detect multiple cytokines are not only available, but show a diversity of applications in the study of many diseases in addition to cancer. This review will focus on the implementation of cytokine antibody arrays in many aspects of cancer research, such as biomarker discovery, molecular mechanisms of cancer development, preclinical studies and the effects of cancer compounds.

The introduction of cytokine antibody arrays has added a new approach for investigators to simultaneously measure multiple cytokine levels in biological samples. Several different platforms have been developed. The ability to measure hundreds of cytokine levels with high specificity and sensitivity within a very limited amount of samples is a powerful tool. Many investigators worldwide have applied this novel technology in their biomedical research, particularly in drug discovery. Undoubtedly, the technology will continue to be improved and the application increased in the next several years.

Cytokines play important roles in many aspects of cell physiology and pathology. The simultaneous determination of multiple cytokine-expression levels is receiving much attention in the research community. Multiple cytokine-expression levels can be simultaneously determined using enzyme-linked immunosorbent assay (ELISA)-based protein array technology. In this approach, target proteins are captured by the arrayed capture antibody and then detected in a sandwich ELISA format using a cocktail of biotinylated detection antibodies. The signals are visualized by either horseradish peroxidase (HRP)-conjugated streptavidin and enhanced chemiluminescence, or cy3-conjugated streptavidin and laser scanner. Several key factors and steps are described, including selection of solid supports, selection of suitable antibodies, determination of specificity and sensitivity of cytokine protein arrays, array design, sample preparation, and detailed experimental procedures for macroarray and microarray formats. An account of the successful development and application of cytokine protein arrays is presented.

Cytokines play important roles in normal cell functions and changes in cytokines have been implicated in many diseases. Recent efforts have focused on developing cytokine antibody arrays. These arrays allow investigators to simultaneously detect multiple cytokines in qualitative and quantitative ways. Cytokine antibody array systems feature high sensitivity, specificity and throughput. This novel technology opens up an expanding spectrum of applications in drug discovery, including target discovery, target validation, screening for lead compounds, compound optimization and clinical trials.

Global analysis of protein expression holds great promise in basic research and patient care. Previously we demonstrated that multiple cytokines could be detected simultaneously using an enzyme-linked immunosorbent assay protein array system with high sensitivity and specificity. In this paper, we described a biotin-labeled-based protein array system to detect multiple cytokines simultaneously from biological samples. In this new approach, proteins from a variety of biological sources are labeled with biotin. The biotin-labeled proteins are then incubated with antibody chips. Targeted proteins are captured by the array antibodies spotted on the antibody chips. The presence of targeted proteins is detected using Cy3- or Cy5-conjugated streptavidin and signals are imaged by laser scanner. The system also can be easily adapted to a two-color binding assay, allowing measurement of the levels of proteins in a test sample with respect to a reference sample at the same chip. To demonstrate its potential applications, we applied this technology to profile human cytokines, chemokines, growth factors, angiogenic factors and proteases in estrogen receptor (ER)+ and ER- cells. These results suggest that biotin-labeled-based antibody chip technology can provide a practical and powerful means of profiling hundreds or thousands of proteins for research and clinical purposes.

Objective: To determine whether peroxisome proliferator-activated receptor (PPAR)-gamma ligands can affect the expression of interleukin-6 (IL-6) and cytokines related to the pathogenesis of endometriosis., Design: In vitro study to determine whether PPARs are expressed in human endometrial cells and determine the effects of various PPAR-gamma ligands on IL-6 and other cytokine expression in these cells., Setting: Academic medical center., Patient(s): Women presenting for infertility workup., Intervention(s): Endometrial cell cultures were treated with PPAR-gamma ligands., Main Outcome Measure(s): Interleukin-6, IL-8, colony stimulating factor-1 (CSF-1) and macrophage chemotactic factor (MCP-1) protein secretion, messenger RNA expression of IL-6, PPAR-alpha, -beta, and -gamma., Result(s): Using a human endometrial cell line (EM42), as well as primary stromal and epithelial endometrial cells, we show the presence of PPAR-alpha, -beta, and -gamma by reverse transcription-polymerase chain reaction (RT-PCR) in these cells. PPAR-gamma ligands stimulated IL-6 secretion and induced enhancement of IL-6 mRNA levels. These ligands also stimulated the secretion of IL-8 and CSF-1., Conclusion(s): PPAR-gamma may play a role in the pathogenesis of endometriosis related to the production of IL-6 and some other cytokines.

Previously we demonstrated that multiple cytokines could be simultaneously detected using an antibody-based protein array system with high sensitivity and specificity from conditioned medium and serum. Here, we created a higher density array system to simultaneously detect 35 cytokines from cell lysates and tissue lysates. This assay combines the advantages of the specificity of enzyme-linked immunosorbent assays (ELISA), sensitivity of enhanced chemiluminescence (ECL), and high-throughput of microspot. In this system, capture antibodies dissolved in methanol were spotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were then incubated with tissue lysates or cell lysates. After removing unbound proteins by extensive washing, the membranes were exposed to horseradish peroxidase (HRP)-conjugated antibody(ies). The signals were visualized with an ECL system. High specificity, sensitivity, and accuracy of this approach were demonstrated. This approach can be used in any general laboratory setting without any sophisticated equipment. It should be feasible to extend this concept to develop a high-throughput protein array system. Combining nitrocellulose membrane-based and PVDF membrane-based approaches, the human cytokine array system can be applied to detect multiple cytokine expression from cell lysate, tissue lysate, serum, plasma, and conditioned medium. Future applications of this new approach include direct protein expression profiling, immunological disease diagnostics, and discovery of new biomarkers.

Previously, we demonstrated that vitamin E supplementation decreases autoantibodies to oxidized lipid-protein complexes (J. Med. Food 1 (2000) 247). Utilizing an in vitro modeling system, we also demonstrated that vitamin E blocks the tumor promotion process in liver epithelial cells (Carcinogenesis 20 (1999) 485 and Mol. Carcinog. 30 (2001) 209). To investigate the molecular mechanisms of vitamin E function, we developed a human cytokine array system that is capable of detecting the expression of 35 cytokines simultaneously. Using this new technology, we analyzed the potential vitamin E-regulated cytokines in vitamin E supplementation individuals. The cytokine arrays showed that expression of several cytokines, particularly monocyte chemoattractant protein-1 (MCP-1), was profoundly reduced in vitamin E supplementation individuals. Moreover, addition of vitamin E to several cultured cells significantly down-regulated the expression of MCP-1. Our results suggested that MCP-1 may be one of the most important targets of antioxidant vitamin E. To the best of our knowledge, this is the first report describing the down-regulation of MCP-1 in vitamin E supplementation in vivo.

HydroGel-coated slide is a porous substrate based on a polymer matrix that provides a three-dimensional hydrophilic environment similar to free solution suitable for biomolecular interactions. This substrate has been used to develop fluorescence-based multiplexed cytokine immunoassays. Forty-three monoclonal antibodies (mAb) of cytokines and chemokines were printed at a volume of 350 pL per spot using a Packard BioChip Arrayer. For each probe, four replicates were printed at a pitch of 500 microm in the layout of a 13 x 16 pattern on a 12 x 12 mm2 HydroGel pad. Cytokines and chemokines that are captured by the arrayed mAbs are detected by using another biotinylated mAb, following by the addition of a Texas Red-conjugated streptavidin. The fluorescent images of arrays were recorded using a Packard ScanArray 5000 confocal slide scanner and quantitated using Packard QuantArray software. Experiments demonstrated that 43 cytokines and chemokines could be simultaneously screened and quantitated in conditioned culture media, cell lysates, and human plasma. Using this chip, we have examined cytokine expression in breast cancer cells and identified the chemokines associated with human cervical cancers.

Asthma is a chronic inflammatory disease of the airways, resulting in bronchial hyperresponsiveness with every allergen exposure. It is now clear that asthma is not a single disease, but rather a multifaceted syndrome that results from a variety of biologic mechanisms. Asthma is further problematic given that the disease consists of many variants, each with its own etiologic and pathophysiologic factors, including different cellular responses and inflammatory phenotypes. These facets make the rapid and accurate diagnosis (not to mention treatments) of asthma extremely difficult. Protein biomarkers can serve as powerful detection tools in both clinical and basic research applications. Recent endeavors from biomedical researchers have developed technical platforms, such as cytokine antibody arrays, that have been employed and used to further the global analysis of asthma biomarker studies. In this review, we discuss potential asthma biomarkers involved in the pathophysiologic process and eventual pathogenesis of asthma, how these biomarkers are being utilized, and how further testing methods might help improve the diagnosis and treatment strain that current asthma patients suffer. [ABSTRACT FROM AUTHOR]