Antibody Array Platforms Research Articles

A Cell Surface-Binding Antibody Atlas Nominates a MUC18-Directed Antibody-Drug Conjugate for Targeting Melanoma.

Discovery of melanoma-targeting antibodies using a proteome-scale array and use of a cutting-edge linker-payload system led to development of a MUC18-targeting antibody-exatecan conjugate with clinical potential for treating major melanoma subtypes.

Systemic Circulating Leptin – Aiding New Dimension of Immune-Related Skin Carcinogenesis and Lipid Metabolism

AIM: The link between the development of melanoma skin cancer and obesity is gaining recently an increased attention. A plethora of immune-related factors sustain a chronic inflammatory state modulated by the metabolic imbalance, the main examples being tumor necrosis factor alpha, interleukin 6, monocyte chemoattractant protein (MCP1), and several adipokines. Among the adipokines leptin (LEP) is steadily increasing interests as it involved in modulating different signaling pathways (e.g., JAK2/STAT3 and MAPK cascade/FOS) and immune cells fate by promoting the switch of memory cells to T helper-1 cell response, increases CD4+CD25- T-cell proliferation, and reduces autophagy during T-cell receptor stimulation. Our work investigates in a pilot study, using protein microarray technology, the LEP circulatory levels in melanoma patients in comparison to other cytokines and chemokines already reported as significantly involved in melanoma initiation and progression. METHODS: Serum from melanoma cases and pool of serum from healthy volunteers were analyzed by an antibody array platform for a 42-cytokine profile. RESULTS: The investigation of cytokines, chemokines, and growth factors has shown that all the proinflammatory circulating molecules are elevated in the melanoma patient’s serum. Inflammatory cytokines were found elevated and were correlated to the more advanced stage of the investigated patient (M2). The highest circulatory level was registered for LEP in both melanoma cases; moreover, in M1 patient, although the LEP level was completely comparable with the one found in M2, its IL-1 serum level was normal. In addition, there are ten analytes that show clear abnormal values and in patient M2 high LEP concentration is correlated with high IL-1, MCP-2, MCP-3, MCSF, and TARC serum concentrations. CONCLUSION: Leptin is pleiotropic adipocytokine involved in various diseases, including skin cancer and melanoma. As it is involved in favoring inflammation and further tumorigenesis, the development of effective leptin-based therapeutic approaches opens new possibilities of adjuvant therapy in skin cancers.

Antibody Arrays Identified Cycle-Dependent Plasma Biomarker Candidates of Peritoneal Endometriosis.

Endometriosis is an estrogen-dependent inflammatory disease affecting women in their reproductive age. Due to non-specific symptoms, women with endometriosis are often misdiagnosed or are accurately diagnosed only after several years. Diagnosis of peritoneal endometriosis is especially challenging and relies only on laparoscopic surgery. To date, different molecules have been proposed as potential non-invasive biomarkers of endometriosis; however, none have been confirmed as clinically useful. Therefore, this study aimed to discover novel plasma biomarker candidates for peritoneal endometriosis using an antibody array platform. This study included patients with endometriosis-like symptoms characterized by the absence (controls) or presence of peritoneal endometriosis (cases) after laparoscopic surgery and histological evaluation. Patients were further divided into secretory and proliferative groups, according to the phase of their menstrual cycle. Their plasma samples were collected and analyzed on an antibody array platform targeting more than 1350 proteins with over 1820 antibodies. In the proliferative group, the analysis revealed three differential proteins between cases and controls: ITB3, ITA2B2, and ACVL-1. In the secretory group, none of the examined proteins reached the log-fold change (logFC) and significance thresholds simultaneously. The potential of the identified differential proteins as plasma biomarker candidates for peritoneal endometriosis should be evaluated on a larger cohort, and their role in endometriosis should be investigated in further studies.

Immune Cell Isolation from Murine Intestine for Antibody Array Analysis.

Gut mucosal immune cells play an essential role in health due to their ability to orchestrate host signaling events in response to exogenous antigens. These antigens may originate from microorganisms including viruses, commensal or pathogenic bacteria, or single-celled eukaryotes, as well as from dietary foodstuff-derived proteins or products. A critical technological capacity to understand host responses to antigens is the ability to efficiently isolate and functionally characterize immune cells from intestinal tissues. Additionally, after characterization, it is of paramount importance to understand the exact functions of these immune cells under different disease states or genetic variables. Here, we outline methods for immune cell isolation from murine small and large intestines with the goal of undertaking a functional analysis of isolated cell types using antibody array platforms.

Identification of the receptor tyrosine kinases (RTKs)-oriented functional targets of miR-206 by an antibody-based protein array

This study demonstrated the feasibility and benefit of an antibody-based experimental approach to identify microRNA functional targets from hundreds of predicted genes using miR-206 as an example. Using a receptor tyrosine kinase (RTK) antibody array, we identified 7 phosphorylated RTKs that were significantly differentially regulated after miR-206-mimic transfection. We then focused on MET, the most varied RTK, and bioinformatically constructed a MET-centred network using computationally predicted miR-206 targets. Within this network, we analyzed two validated targets, PAX3 and SNX2, and one candidate target, EIF4E, may account for the inhibitory effect of miR-206 on MET phosphorylation. Luciferase and Western-blot assays indicated that EIF4E was a direct target of miR-206. This concept may also be applicable for other microRNAs and other antibody array platforms.

Abstract 2495: Quantitative screening of protein biomarkers of hepatocellular carcinoma by antibody array

Abstract Protein biomarkers can be key indicators for evaluating the presence or progression of a human disease or therapeutic response to treatment. We recently carried out a biomarker screening in hepatocellular carcinoma (HCC) using our antibody array platform. Using 25 pairs of HCC and adjacent control tissue samples, the expression levels of 274 proteins including growth factors, inflammation factors, angiogenesis, apoptosis factors and adhesion molecules, were examined using an antibody arrays. A panel of proteins demonstrating significant differences in expression levels between HCC and adjacent control samples was identified by significance analysis of microarrays (SAM) and Wilcoxon signed-rank test, combined with fold-change determination. Additionally, proteomic analysis algorithms were used to capture GO annotations and to construct signaling pathways and protein-protein interaction networks for these proteins. Proteins selected through these comprehensive analyses were designated as potential biomarkers and were well able to distinguish hepatocellular carcinoma tissue samples from paired, adjacent controls with cluster tools. The expression levels of several proteins were further evaluated by enzyme-linked immunosorbent assay (ELISA). Finally, we performed follow-up verification studies on selected proteins (Nidogen-1 and CEACAM-1) for detection of HCC in 164 pairs of serum samples from HCC patients and normal subjects with reverse phase protein array (RPPA). Our work reveals promising protein biomarkers for detection of HCC. Citation Format: Shuhong Luo, Ruochun Huang, Zhongsheng Wang, Yun-Ru Chen, Jinfei Lin, Ruo-Pan Huang. Quantitative screening of protein biomarkers of hepatocellular carcinoma by antibody array. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2495. doi:10.1158/1538-7445.AM2014-2495

Miniaturization of multiplexed planar recombinant antibody arrays for serum protein profiling.

Antibody-based microarrays are a developing tool for high-throughput proteomics in health and disease. However, in order to enable global proteome profiling, novel miniaturized high-density antibody array formats must be developed. In this proof-of-concept study, we have designed a miniaturized planar recombinant (single-chain Fragment variable). antibody array technology platform for multiplexed profiling of non-fractionated, directly labelled serum samples. The size of the individual spot features was reduced 225-times (78.5μm(2)/spot) and the array density was increased 19-times (38,000 spots/cm(2)). These miniaturized, multiplexed arrays were produced, using a desktop nanofabrication system based on dip-pen nanolithography technology, and interfaced with a high-resolution fluorescent-based scanner. The reproducibility, sensitivity, specificity, and applicability of the set-up were demonstrated by profiling a set of well-characterized serum samples. The designed antibody array platform opens up new possibilities for large-scale, multiplex profiling of crude proteomes in a miniaturized fashion.

Protein expression profiling by antibody array analysis with use of dried blood spot samples on filter paper

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.

Extracellular vesicles as prospective carriers of oncogenic protein signatures in adult and paediatric brain tumours

Extracellular vesicles (EVs), including exosomes, act as biological effectors and as carriers of oncogenic signatures in human cancer. The molecular composition and accessibility of EVs in biofluids open unprecedented diagnostic opportunities in malignancies where tumour tissue is difficult to sample, especially in primary and metastatic brain tumours. The ongoing genetic discovery of driver mutations defines the ever increasing numbers of distinct molecular subtypes of brain tumours (orphan diseases), a complexity that may soon be translated into alterations in functional proteins and their oncogenic networks. This may likely be extended to real time changes engendered by the disease progression, tumour heterogeneity, inter-individual variations and therapeutic responses. Meeting these challenges through EV analysis is dependent on technological progress in such areas as generation of mutation- and phospho-specific antibodies, antibody array platforms, nanotechnology, microfluidics, NMR spectroscopy, MS and MRM approaches of quantitative proteomics, which should not be underestimated. Still, vesiculation emerges as a unique process that could be harnessed for the benefit of more individualised patient care.

Sensing the insulin signaling pathway with an antibody array

The development of insulin resistance and type 2 diabetes is determined by various factors, including defects within the insulin signaling pathway. Mediators of insulin resistance operate through activation of various protein kinase C isoforms, IκB kinase β (IKKβ), and/or c-Jun N-terminal kinase, and subsequent inhibition of the proximal insulin signaling pathway via the insulin receptor substrate 1 and Akt. These mechanisms are still largely unresolved because of the complexity of the molecular events. In this study, an expression and activation state profiling of multiple known key signaling biomolecules involved in insulin metabolic and mitogenic signaling pathways was evaluated using a phosphospecific antibody array platform. The results of the arrayed antibodies were verified by the multiplexed bead array assay and conventional Western blot analysis, and confirmed the well-known inhibitory effects of phorbol esters on insulin signaling pathway activation. Of interest, the increase in protein kinase C signaling responses with phorbol esters was associated with activation of the lipid phosphatase PTEN and a 27 kDa HSP. Thus, this insulin signaling antibody array provides a powerful and effective way to investigate the mechanism of insulin resistance and likely assist the development of innovative therapeutic drugs for type 2 diabetes.

Antibody Arrays Identify Potential Diagnostic Markers of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. Effective treatment of HCC patients is hampered by the lack of sensitive and specific diagnostic markers of HCC. Alpha-fetoprotein (AFP), the currently used HCC marker, misses 30%-50% of HCC patients, who therefore remain undiagnosed and untreated. In order to identify novel diagnostic markers that can be used individually or in combination with AFP, we used an antibody array platform to detect the levels of candidate proteins in the plasma of HCC patients (n = 48) and patients with chronic hepatitis B or C viral infections (n = 19) (both of which are the major risk factors of HCC). We identified 7 proteins that significantly differentiate HCC patients from hepatitis patients (p < 0.05) (AFP, CTNNB, CSF1, SELL, IGFBP6, IL6R, and VCAM1). Importantly, we also identified 8 proteins that significantly differentiate HCC patients with 'normal' levels of AFP (< 20 ng/ml) from hepatitis patients (p < 0.05) (IL1RN, IFNG, CDKN1A, RETN, CXCL14, CTNNB, FGF2, and SELL). These markers are potentially important complementary markers to AFP. Using an independent immunoassay method in an independent group of 23 HCC patients and 22 hepatitis patients, we validated that plasma levels of CTNNB were significantly higher in the HCC group (p = 0.020). In conclusion, we used an antibody array platform to identify potential circulating diagnostic markers of HCC, some of which may be valuable when used in combination with AFP. The clinical utility of these newly identified HCC diagnostic markers needs to be systematically evaluated.

Design of atto‐vial based recombinant antibody arrays combined with a planar wave‐guide detection system

Antibody microarray is a rapidly emerging, powerful approach with great promise within high-throughput proteomics. However, before a truly proteome-wide analysis can be performed, the antibody array format needs to be miniaturized even further in order to enable ultradense arrays to be fabricated. To this end, we have designed and generated proof-of-concept for the first generation of an atto-vial based recombinant antibody array platform. Briefly, we have designed a novel nanostructured substrate using electron beam lithography. Vials, ranging in volume/size from 6 (200 nm in diameter) to 4000 aL (5 microm in diameter), were fabricated. Human recombinant single-chain Fv antibody fragments, microarray adopted by design, were used as probes. The set-up was interfaced with planar wave-guide technology for evanescant field fluorescence detection. The results showed that protein analytes could be specifically detected in the subzeptomole range for pure systems, using vials down to 57 aL. Further, low-abundant (pg/mL) protein analytes could be detected in directly labeled complex proteomes, such as human whole serum, using 157 aL-vials. Taken together, these results outline the potential of the atto-vial array set-up for miniaturized affinity proteomics-based approaches.

Applications of antibody array platforms

Antibody arrays are valuable for the parallel analysis of multiple proteins in small sample volumes. The earliest and most widely used application of antibody arrays has been to measure multiple protein abundances, using sandwich assays and label-based assays, for biomarker discovery and biological studies. Modifications to these assays have led to studies profiling specific protein post-translational modifications. Additional novel uses include profiling enzyme activities and protein cell-surface expression. Finally, array-based antibody platforms are being used to assist the development and characterization of antibodies. Continued progress in the technology will surely lead to extensions of these applications and the development of new ways of using the methods.