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Antibody Microarrays (antibody + microarray)

Distribution by Scientific Domains

Life Sciences	50%
Medical Sciences	21%

Selected Abstracts

Clinical application of antibody microarray in chronic kidney disease: How far to go?

PROTEOMICS - CLINICAL APPLICATIONS, Issue 7-8 2008
Lin-Li Lv
Abstract Chronic kidney disease (CKD) that affects about 10% of the adult population has been shown as a worldwide public health problem in recent years. Both basic and clinical investigations have identified complex disease-associated protein networks involved in the pathophysiologic processes of CKD. The traditional single-assay approach and proteomic analysis of those related proteins have given birth to a steadily increasing panel of molecules that may have the potential to serve as biomarkers for CKD. However, both approaches suffered from some shortcomings from a technological point of view. Antibody microarray (AbM) is characterized by high sensitivity, specificity, and quantitative ability for a particular set of known proteins. However, its application in CKD has been very limited so far. The objective of this review, therefore, is to address the potential applications of AbM in studying of CKD. We will briefly discuss the proteins involved in the development of CKD, future directions in which AbM approaches would probably display its potential and also some key issues that need to be considered in application of this novel technique. [source]

A chip-based miniaturized format for protein-expression profiling: The exploitation of comprehensively produced antibodies

ELECTROPHORESIS, Issue 18 2006
Hisashi Koga Dr.
Abstract Numerous antibodies have been developed and validated in recent years, and show promise for use in novel functional protein assays. Such assays would be an alternative to pre-existing comprehensive assays, such as DNA microarrays. Antibody microarrays are thought to represent those functional protein assays. While a variety of attempts have been made to apply DNA microarray technology to antibody microarrays, a fully optimized protocol has not been established. We have been conducting a project to comprehensively produce antibodies against mouse KIAA ("KI" stands for "Kazusa DNA Research Institute" and "AA" are reference characters) proteins. Using our library of antibodies, we established a novel antibody microarray format that utilizes surface plasmon resonance (SPR) technology. A label-free real-time measurement of protein expression in crude cell lysates was achieved by direct readout of the bindings using SPR. Further refinement of the antibody microarray format enabled us to detect a smaller quantity of target proteins in the lysate without the bulk effect. In this review, we first summarize available antibody array formats and then describe the above-mentioned format utilizing updated SPR technology. [source]

Construction of an antibody microarray based on agarose-coated slides

ELECTROPHORESIS, Issue 3 2007
Lin-Li Lv
Abstract The antibody microarray, a high-throughput multiplex immunoassay method, has become a significant tool for quantitative proteomics studies. We describe here the strategies for optimizing the condition of antibody microarray building based on agarose-coated slides. In this study, modified glass slides were robotically printed with capture antibodies against monocyte chemoattractant protein 1 (MCP-1), then dilutions of the cytokine were applied to the arrays, and the protein was detected with biotin-labeled antibody coupled with Cy3-conjugated streptavidin. Thus a protein profiling microarray based on sandwich immunoassay has been established. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, shelf life of the postprinting slides, blocking buffers, and reproducibility of the system. A calibration curve with a correlation coefficient of 0.9995 was established which suggested that the matrix can retain arrayed proteins in near-quantitative fashion. The results revealed high signal uniformity and reproducibility with regard to intra-array (1.3%) and the interarray (8.7%) variation at the capture antibody concentration of 125,µg/mL. Besides, the printed arrays could be stored for at least two months without any apparent change of the performance parameters. [source]

Identification of repertoires of surface antigens on leukemias using an antibody microarray

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2003
Larissa Belov
Abstract We have previously described a microarray of cluster of differentiation (CD) antibodies that enables concurrent determination of more than 60 CD antigens on leukocytes. This procedure does not require protein purification or labeling, or a secondary detection system. Whole cells are captured by a microarray of 10 nL antibody dots immobilized on a nitrocellulose film on a microscope slide. Distinct patterns of cell binding are observed for different leukemias or lymphomas. These haematological malignancies arise from precursor cells of T- or B-lymphocytic, or myeloid lineages of hematopoiesis. The dot patterns obtained from patients are distinct from those of peripheral blood leukocytes from normal subjects. This microarray technology has recently undergone a number of refinements. The microarray now contains more CD antibodies, and a scanner for imaging dot patterns and software for data analysis provide an extensive immunophenotype sufficient for diagnosis of common leukemias. The technology is being evaluated for diagnosis of leukemias with parallel use of conventional diagnostic criteria. [source]

Antibody microarray analysis of serum glycans in esophageal squamous cell carcinoma cases and controls

PROTEOMICS - CLINICAL APPLICATIONS, Issue 8 2009
Changxia Shao
Abstract The objective of this study was to characterize specific serum glycan profile in esophageal squamous cell carcinoma (ESCC) cases and matched controls. A recently developed lectin-based antibody array was applied to detect various cancer-associated glycotopes in sera from 23 cases and 23 controls. Glycan levels were highly expressed in sera of ESCC patients as compared with controls. These included fucosylation level on interleukin (IL) 8; mannose level on haptoglobin; N-acetylglucosamine levels on IL6, mucin (MUC)1, and von Willebrand factor (vWf); sialyl Lewis a (sLea) levels on blood group Lewis X, IL6, IL10, MUC1, and serum amyloid A (SAA); sialyl Tn antigen (sTn) levels on cathepsin D, gelsolin, IL10, and vWf; T antigen levels on IL8, IL10, blood group Lewis X, vitronectin, and vWf (p<0.05). In addition, receiver-operating characteristics (ROC) analysis showed significantly discriminal improvement between cases and controls as measured by area under ROC curve. The highest sum of sensitivity and specificity was 1.52 for carbohydrate antigen 19-9 detection on both MUC1 and SAA, with area under ROC curves of 0.73 and 0.71, respectively. Taken together, this lectin-based antibody microarray allows efficient profiling of variations in specific glycans on proteins in ESCC cases as compared with controls, some of which might be useful for clinical diagnosis, early detection, and/or treatment. [source]

Clinical application of antibody microarray in chronic kidney disease: How far to go?

Monocyte chemotactic protein-1 (MCP-1/CCL2) is associated with prostatic growth dysregulation and benign prostatic hyperplasia

THE PROSTATE, Issue 5 2010
Kazutoshi Fujita
Abstract BACKGROUND Chronic inflammation is commonly observed in benign prostate hyperplasia (BPH), and prostate tissue often contains increased inflammatory infiltrates, including T cells and macrophages. Cytokines are not only key mediators of inflammation but may also play important roles in the initiation and progression of BPH. METHODS In order to determine what cytokines might be involved in prostatic enlargement, expressed prostatic secretions (EPS) from ex vivo prostates were analyzed by human cytokine antibody microarray and ELISA. Prostate epithelial cells (PrEC) and prostate stromal cells (PrSC) were used for ELISA, proliferation, and Western blot assays. RESULTS Monocyte chemotactic protein-1 (MCP-1/CCL2) was one of the most elevated proteins in secretions from large prostate glands. PrSC were found to secrete MCP-1; Western blotting showed that both PrSC and PrEC express the MCP-1 receptor CCR2 which by RT-PCR was the CCR2b isoform. Proliferation assays showed that MCP-1 stimulates the proliferation of PrEC, but not PrSC, and that a specific MCP-1 antagonist (RS102895) suppressed this effect. Conditioned medium from PrSC stimulated the proliferation of PrEC as well, an effect completely inhibited by both RS102895 and a neutralizing anti-MCP-1 monoclonal antibody. The inflammatory cytokines interleukin (IL)-1,, interferon-,, and IL-2 enhanced the secretion of MCP-1 from PrEC and PrSC. In addition, MCP-1 levels in EPS correlated with mRNA levels of the macrophage marker CD68 in the same secretions. CONCLUSIONS The cytokine MCP-1, of apparent prostatic stromal cell origin, may play an important role in prostatic enlargement and BPH, and is a candidate biomarker for these pathologic processes. Prostate 70: 473,481, 2010. © 2009 Wiley-Liss, Inc. [source]

Construction of an antibody microarray based on agarose-coated slides

A chip-based miniaturized format for protein-expression profiling: The exploitation of comprehensively produced antibodies

In Pursuit of Zero: Polymer Brushes that Resist the Adsorption of Proteins

ADVANCED MATERIALS, Issue 23 2009
Angus Hucknall
Abstract Protein resistant or "non-fouling" surfaces are of great interest for a variety of biomedical and biotechnology applications. This article briefly reviews the development of protein resistant surfaces, followed by recent research on a new methodology to fabricate non-fouling surfaces by surface-initiated polymerization. We show that polymer brushes synthesized by surface-initiated polymerization that present short oligo(ethylene glycol) side chains are exceptionally resistant to protein adsorption and cell adhesion. The importance of the protein and cell resistance conferred by these polymer brushes is illustrated by their use as substrates for the fabrication of antibody microarrays that exhibit femtomolar limits of detection in complex fluids such as serum and blood with relaxed requirements for intermediate wash steps. This example highlights the important point that the reduction in background noise afforded by protein-resistant surfaces can greatly simplify the development of ultrasensitive heterogeneous, surface-based clinical and proteomic assays with increased sensitivity and utility. [source]

Upregulation of ,-Catenin Levels in Superior Frontal Cortex of Chronic Alcoholics

ALCOHOLISM, Issue 6 2008
Ali M. Al-Housseini
Background:, Chronic and excessive alcohol misuse results in neuroadaptive changes in the brain. The complex nature of behavioral, psychological, emotional, and neuropathological characteristics associated with alcoholism is likely a reflection of the network of proteins that are affected by alcohol-induced gene expression patterns in specific brain regions. At the molecular level, however, knowledge remains limited regarding alterations in protein expression levels affected by chronic alcohol abuse. Thus, novel techniques that allow a comprehensive assessment of this complexity will enable the simultaneous assessment of changes across a group of proteins in the relevant neural circuitry. Methods:, A proteomics analysis was performed using antibody microarrays to determine differential protein levels in superior frontal cortices between chronic alcoholics and age- and gender-matched control subjects. Seventeen proteins related to the catenin signaling pathway were analyzed, including ,-, ,-, and ,-catenins, their upstream activators cadherin-3 (type I cadherin) and cadherin-5 (type II cadherin), and 5 cytoplasmic regulators c-Src, CK1,, GSK-3,, PP2A-C,, and APC, as well as the nuclear complex partner of ,-catenin CBP and 2 downstream genes Myc and cyclin D1. ILK, G,1, G,1, and G,2, which are activity regulators of GSK-3,, were also analyzed. Results:, Both ,- and ,-catenin showed significantly increased levels, while ,-catenin did not change significantly, in chronic alcoholics. In addition, the level of the ,-catenin downstream gene product Myc was significantly increased. Average levels of the catenin regulators c-Src, CK1,, and APC were also increased in chronic alcoholics, but the changes were not statistically significant. Conclusion:, Chronic and excessive alcohol consumption leads to an upregulation of ,- and ,-catenin levels, which in turn increase downstream gene expressions such as Myc that is controlled by ,-catenin signaling. This study showed that the ,-catenin signal transduction pathway was upregulated by chronic alcohol abuse, and prompts further investigation of mechanisms underlying the upregulation of ,- and ,-catenins in alcoholism, which may have considerable pathogenic and therapeutic relevance. [source]

Kinetics of antigen binding to antibody microspots: Strong limitation by mass transport to the surface

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2006
Wlad Kusnezow Dr.
Abstract It is well documented that diffusion has generally a strong effect on the binding kinetics in the microtiter plate immunoassays. However, a systematic quantitative experimental evaluation of the microspot kinetics is still missing in the literature. Our work aims at filling this important gap of knowledge on the example of antigen binding to antibody microspots. A mathematical model was derived within the framework of two-compartment model and applied to the quantitative analysis of the experimental data obtained for typical antibody microspot assays. A strong mass-transport dependence of the antigen-antibody microspot kinetics was identified to be one of the main restrictions of this new technology. The binding reactions are slowed down in the microspot immunoassays by several orders of magnitude as compared with the corresponding well-stirred bulk reactions. The task to relax the mass-transport limitations should thus be one of the most important issues in designing the antibody microarrays. These limitations notwithstanding, the detection range of more than five orders of magnitude and the high sensitivity in the low femtomolar range were experimentally achieved in our study, demonstrating thus an enormous potential of this highly capable technology. [source]

Versatile protein microarray based on carbohydrate-binding modules

PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2005
Keren Ofir
Abstract Non-DNA microarrays, such as protein, peptide and small molecule microarrays, can potentially revolutionize the high-throughput screening tools currently used in basic and pharmaceutical research. However, fundamental obstacles remain that limit their rapid and widespread implementation as an alternative bioanalytical approach. These include the prerequisite for numerous proteins in active and purified form, ineffectual immobilization strategies and inadequate means for quality control of the considerable numbers of multiple reagents. This study describes a simple yet efficient strategy for the production of non-DNA microarrays, based on the tenacious affinity of a carbohydrate-binding module (CBM) for its three-dimensional substrate, i.e., cellulose. Various microarray formats are described, e.g., conventional and single-chain antibody microarrays and peptide microarrays for serodiagnosis of human immunodeficiency virus patients. CBM-based microarray technology overcomes many of the previous obstacles that have hindered fabrication of non-DNA microarrays and provides a technically simple but effective alternative to conventional microarray technology. [source]

Protein screening in vitreous samples of patients with retinal vein occlusion

ACTA OPHTHALMOLOGICA, Issue 2009
HT AGOSTINI
Purpose The aim of the study was to identify proteins involved in the pathogenesis of retinopathy after retinal vein occlusion. In retinal vein occlusion, proteins penetrate from leaky vessels into the vitreous. Alternatively, retinal cells produce protein factors and release them into the vitreous. Methods Vitreous and plasma samples of patients with retinal vein occlusion or macular pucker / macular hole were analyzed by antibody microarrays and ELISA. Results An antibody based microarray with more than 500 target for screening vitreous samples initially was less enlightening than antibody arrays providing the possibility to quantify up to 30 proteins in an ELISA-like microassay. Standard curves of antibody microarrays are as linear as those of ELISAs. VEGF values were similar to values measured by ELISA. Conclusion In our screen, we found some candidate factors which are currently investigated for their potential of influencing retinopathy after retinal vein occlusion. The use of microarrays to identify protein factors involved in retinal disease in the vitreous will be discussed. [source]