The Experts below are selected from a list of 4584 Experts worldwide ranked by ideXlab platform

Christer Wingren - One of the best experts on this subject based on the ideXlab platform.

  • Evaluation of Solid Supports for Slide- and Well-Based Recombinant Antibody Microarrays.
    Microarrays, 2016
    Co-Authors: Anna Sandström Gerdtsson, Linda Dexlin-mellby, Payam Delfani, Erica Berglund, Carl Arne Krister Borrebaeck, Christer Wingren
    Abstract:

    Antibody Microarrays have emerged as an important tool within proteomics, enabling multiplexed protein expression profiling in both health and disease. The design and performance of Antibody Microarrays and how they are processed are dependent on several factors, of which the interplay between the antibodies and the solid surfaces plays a central role. In this study, we have taken on the first comprehensive view and evaluated the overall impact of solid surfaces on the recombinant Antibody microarray design. The results clearly demonstrated the importance of the surface-Antibody interaction and showed the effect of the solid supports on the printing process, the array format of planar arrays (slide- and well-based), the assay performance (spot features, reproducibility, specificity and sensitivity) and assay processing (degree of automation). In the end, two high-end recombinant Antibody microarray technology platforms were designed, based on slide-based (black polymer) and well-based (clear polymer) arrays, paving the way for future large-scale protein expression profiling efforts.

  • identification of b cell lymphoma subsets by plasma protein profiling using recombinant Antibody Microarrays
    Leukemia Research, 2014
    Co-Authors: Frida Pauly, Carl Arne Krister Borrebaeck, Karin E Smedby, Mats Jerkeman, Henrik Hjalgrim, Mattias Ohlsson, Richard Rosenquist, Christer Wingren
    Abstract:

    B-cell lymphoma (BCL) heterogeneity represents a key issue, often making the classification and clinical management of these patients challenging. In this pilot study, we outlined the first resolved view of BCL disease heterogeneity on the protein level by deciphering disease-associated plasma biomarkers, specific for chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma, using recombinant Antibody Microarrays targeting mainly immunoregulatory proteins. The results showed the BCLs to be heterogeneous, and revealed potential novel subgroups of each BCL. In the case of diffuse large B-cell lymphoma, we also indicated a link between the novel subgroups and survival.

  • protein expression profiling of formalin fixed paraffin embedded tissue using recombinant Antibody Microarrays
    Journal of Proteome Research, 2013
    Co-Authors: Frida Pauly, Carl Arne Krister Borrebaeck, Linda Dexlinmellby, Mats Ohlin, Niclas Olsson, Karin Jirstrom, Michael Dictor, Saskia Schoenmakers, Christer Wingren
    Abstract:

    Proteomics, the large-scale analysis of proteins, is a rapidly evolving field with an increasing number of key clinical applications, such as diagnosis, prognosis, and classification. In order to generate complete protein expression profiles, or protein atlases, any crude sample format must be addressable in a rapid, multiplex, and sensitive manner. A common and clinically central sample format, formalin-fixed, paraffin-embedded (FFPE) tissue material, holds great potential as a source for disease-associated biomarker signatures. However, despite major efforts, extraction and subsequent profiling of proteins from FFPE tissue has proven to be challenging. In this proof-of-concept study, we have demonstrated for the first time that proteins could be extracted, labeled, and subsequently profiled in a multiplex, sensitive, and reproducible manner using recombinant scFv Antibody Microarrays. Thus, we have added FFPE samples to the list of sample formats available for high-throughput analysis by affinity proteomics, paving the way for the next generation of biomarker-driven discovery projects.

  • Serum proteome profiling of pancreatitis using recombinant Antibody Microarrays reveals disease‐associated biomarker signatures
    Proteomics. Clinical applications, 2012
    Co-Authors: Anna Sandstrom, Carl Arne Krister Borrebaeck, Roland Andersson, Ralf Segersvärd, Mattias Löhr, Christer Wingren
    Abstract:

    PURPOSE: Pancreatitis is an inflammatory state of the pancreas, for which high-performing serological biomarkers are lacking. The aim of the present study was to evaluate the use of affinity proteomics for identifying potential markers of disease and stratifying pancreatitis subtypes. EXPERIMENTAL DESIGN: High-content, recombinant Antibody Microarrays were applied for serum protein expression profiling of 113 serum samples from patients with chronic, acute, and autoimmune pancreatitis, as well as healthy controls. The sample groups were compared using supervised classification based on support vector machine analysis. RESULTS: This discovery study showed that pancreatitis subtypes could be discriminated with high accuracy. Using unfiltered data, the individual subtypes, as well as the combined pancreatitis cohort, were distinguished from healthy controls with high AUC values (0.96-1.00). Moreover, characteristic protein patterns and AUC values in the range of 0.69-0.95 were observed for the individual pancreatitis entities when compared to each other, and to all other samples combined. CONCLUSIONS AND CLINICAL RELEVANCE: This study demonstrated the potential of the Antibody microarray approach for stratification of pancreatitis. Distinct candidate multiplex serum biomarker signatures for chronic, acute, and autoimmune pancreatitis were defined, which could enhance our fundamental knowledge of the underlying molecular mechanisms, and potentially lead to improved diagnosis. (Less)

  • Serum proteome profiling of pancreatitis using recombinant Antibody Microarrays reveals disease‐associated biomarker signatures
    Proteomics Clinical Applications, 2012
    Co-Authors: Anna Sandstrom, Carl Arne Krister Borrebaeck, Roland Andersson, Ralf Segersvärd, Mattias Löhr, Christer Wingren
    Abstract:

    PURPOSE: Pancreatitis is an inflammatory state of the pancreas, for which high-performing serological biomarkers are lacking. The aim of the present study was to evaluate the use of affinity proteomics for identifying potential markers of disease and stratifying pancreatitis subtypes. EXPERIMENTAL DESIGN: High-content, recombinant Antibody Microarrays were applied for serum protein expression profiling of 113 serum samples from patients with chronic, acute, and autoimmune pancreatitis, as well as healthy controls. The sample groups were compared using supervised classification based on support vector machine analysis. RESULTS: This discovery study showed that pancreatitis subtypes could be discriminated with high accuracy. Using unfiltered data, the individual subtypes, as well as the combined pancreatitis cohort, were distinguished from healthy controls with high AUC values (0.96-1.00). Moreover, characteristic protein patterns and AUC values in the range of 0.69-0.95 were observed for the individual pancreatitis entities when compared to each other, and to all other samples combined. CONCLUSIONS AND CLINICAL RELEVANCE: This study demonstrated the potential of the Antibody microarray approach for stratification of pancreatitis. Distinct candidate multiplex serum biomarker signatures for chronic, acute, and autoimmune pancreatitis were defined, which could enhance our fundamental knowledge of the underlying molecular mechanisms, and potentially lead to improved diagnosis.

Carl Arne Krister Borrebaeck - One of the best experts on this subject based on the ideXlab platform.

  • Evaluation of Solid Supports for Slide- and Well-Based Recombinant Antibody Microarrays.
    Microarrays, 2016
    Co-Authors: Anna Sandström Gerdtsson, Linda Dexlin-mellby, Payam Delfani, Erica Berglund, Carl Arne Krister Borrebaeck, Christer Wingren
    Abstract:

    Antibody Microarrays have emerged as an important tool within proteomics, enabling multiplexed protein expression profiling in both health and disease. The design and performance of Antibody Microarrays and how they are processed are dependent on several factors, of which the interplay between the antibodies and the solid surfaces plays a central role. In this study, we have taken on the first comprehensive view and evaluated the overall impact of solid surfaces on the recombinant Antibody microarray design. The results clearly demonstrated the importance of the surface-Antibody interaction and showed the effect of the solid supports on the printing process, the array format of planar arrays (slide- and well-based), the assay performance (spot features, reproducibility, specificity and sensitivity) and assay processing (degree of automation). In the end, two high-end recombinant Antibody microarray technology platforms were designed, based on slide-based (black polymer) and well-based (clear polymer) arrays, paving the way for future large-scale protein expression profiling efforts.

  • identification of b cell lymphoma subsets by plasma protein profiling using recombinant Antibody Microarrays
    Leukemia Research, 2014
    Co-Authors: Frida Pauly, Carl Arne Krister Borrebaeck, Karin E Smedby, Mats Jerkeman, Henrik Hjalgrim, Mattias Ohlsson, Richard Rosenquist, Christer Wingren
    Abstract:

    B-cell lymphoma (BCL) heterogeneity represents a key issue, often making the classification and clinical management of these patients challenging. In this pilot study, we outlined the first resolved view of BCL disease heterogeneity on the protein level by deciphering disease-associated plasma biomarkers, specific for chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma, using recombinant Antibody Microarrays targeting mainly immunoregulatory proteins. The results showed the BCLs to be heterogeneous, and revealed potential novel subgroups of each BCL. In the case of diffuse large B-cell lymphoma, we also indicated a link between the novel subgroups and survival.

  • protein expression profiling of formalin fixed paraffin embedded tissue using recombinant Antibody Microarrays
    Journal of Proteome Research, 2013
    Co-Authors: Frida Pauly, Carl Arne Krister Borrebaeck, Linda Dexlinmellby, Mats Ohlin, Niclas Olsson, Karin Jirstrom, Michael Dictor, Saskia Schoenmakers, Christer Wingren
    Abstract:

    Proteomics, the large-scale analysis of proteins, is a rapidly evolving field with an increasing number of key clinical applications, such as diagnosis, prognosis, and classification. In order to generate complete protein expression profiles, or protein atlases, any crude sample format must be addressable in a rapid, multiplex, and sensitive manner. A common and clinically central sample format, formalin-fixed, paraffin-embedded (FFPE) tissue material, holds great potential as a source for disease-associated biomarker signatures. However, despite major efforts, extraction and subsequent profiling of proteins from FFPE tissue has proven to be challenging. In this proof-of-concept study, we have demonstrated for the first time that proteins could be extracted, labeled, and subsequently profiled in a multiplex, sensitive, and reproducible manner using recombinant scFv Antibody Microarrays. Thus, we have added FFPE samples to the list of sample formats available for high-throughput analysis by affinity proteomics, paving the way for the next generation of biomarker-driven discovery projects.

  • Serum proteome profiling of pancreatitis using recombinant Antibody Microarrays reveals disease‐associated biomarker signatures
    Proteomics Clinical Applications, 2012
    Co-Authors: Anna Sandstrom, Carl Arne Krister Borrebaeck, Roland Andersson, Ralf Segersvärd, Mattias Löhr, Christer Wingren
    Abstract:

    PURPOSE: Pancreatitis is an inflammatory state of the pancreas, for which high-performing serological biomarkers are lacking. The aim of the present study was to evaluate the use of affinity proteomics for identifying potential markers of disease and stratifying pancreatitis subtypes. EXPERIMENTAL DESIGN: High-content, recombinant Antibody Microarrays were applied for serum protein expression profiling of 113 serum samples from patients with chronic, acute, and autoimmune pancreatitis, as well as healthy controls. The sample groups were compared using supervised classification based on support vector machine analysis. RESULTS: This discovery study showed that pancreatitis subtypes could be discriminated with high accuracy. Using unfiltered data, the individual subtypes, as well as the combined pancreatitis cohort, were distinguished from healthy controls with high AUC values (0.96-1.00). Moreover, characteristic protein patterns and AUC values in the range of 0.69-0.95 were observed for the individual pancreatitis entities when compared to each other, and to all other samples combined. CONCLUSIONS AND CLINICAL RELEVANCE: This study demonstrated the potential of the Antibody microarray approach for stratification of pancreatitis. Distinct candidate multiplex serum biomarker signatures for chronic, acute, and autoimmune pancreatitis were defined, which could enhance our fundamental knowledge of the underlying molecular mechanisms, and potentially lead to improved diagnosis.

  • Serum proteome profiling of pancreatitis using recombinant Antibody Microarrays reveals disease‐associated biomarker signatures
    Proteomics. Clinical applications, 2012
    Co-Authors: Anna Sandstrom, Carl Arne Krister Borrebaeck, Roland Andersson, Ralf Segersvärd, Mattias Löhr, Christer Wingren
    Abstract:

    PURPOSE: Pancreatitis is an inflammatory state of the pancreas, for which high-performing serological biomarkers are lacking. The aim of the present study was to evaluate the use of affinity proteomics for identifying potential markers of disease and stratifying pancreatitis subtypes. EXPERIMENTAL DESIGN: High-content, recombinant Antibody Microarrays were applied for serum protein expression profiling of 113 serum samples from patients with chronic, acute, and autoimmune pancreatitis, as well as healthy controls. The sample groups were compared using supervised classification based on support vector machine analysis. RESULTS: This discovery study showed that pancreatitis subtypes could be discriminated with high accuracy. Using unfiltered data, the individual subtypes, as well as the combined pancreatitis cohort, were distinguished from healthy controls with high AUC values (0.96-1.00). Moreover, characteristic protein patterns and AUC values in the range of 0.69-0.95 were observed for the individual pancreatitis entities when compared to each other, and to all other samples combined. CONCLUSIONS AND CLINICAL RELEVANCE: This study demonstrated the potential of the Antibody microarray approach for stratification of pancreatitis. Distinct candidate multiplex serum biomarker signatures for chronic, acute, and autoimmune pancreatitis were defined, which could enhance our fundamental knowledge of the underlying molecular mechanisms, and potentially lead to improved diagnosis. (Less)

Jorg D Hoheisel - One of the best experts on this subject based on the ideXlab platform.

  • Antibody Microarrays: the crucial impact of mass transport on assay kinetics and sensitivity.
    Expert Review of Molecular Diagnostics, 2014
    Co-Authors: Wlad Kusnezow, Jorg D Hoheisel, Yana V. Syagailo, Igor Goychuk, David Wild
    Abstract:

    Although they are superficially similar to DNA Microarrays, immunoassay Microarrays represent a daunting technological challenge owing to the much wider diversity of proteins. Yet, as the leading edge of bioscience migrates from genomics to proteomics, the complexity and enormous dynamic range of proteins in a cell necessitate an analytic tool with exceptional specificity and sensitivity. In theory, microspot immunoassays could fulfill this need. However, Antibody Microarrays have had limited success to date, and have often required a highly sensitive detection system and/or sophisticated immobilization approach to be of any use for the profiling of complex specimens. There is a solid body of work on the theory of microspot reaction kinetics, yet much of the published experimental work on protein microarray development pays insufficient attention to the kinetic aspects of this interaction. This review explains that one of the main limitations for the sensitivity of current generation microspot immunoassay...

  • plasma protein analysis of patients with different b cell lymphomas using high content Antibody Microarrays
    Proteomics Clinical Applications, 2013
    Co-Authors: Christoph Schröder, Kurt Fellenberg, Harish Srinivasan, Martin Sill, Jakob Linseisen, Nikolaus Becker, Alexandra Nieters, Jorg D Hoheisel
    Abstract:

    Purpose In this study, plasma samples from a multicentric case-control study on lymphoma were analyzed for the identification of proteins useful for diagnosis. Experimental design The protein content in the plasma of 100 patients suffering from the three most common B-cell lymphomas and 100 control samples was studied with Antibody Microarrays composed of 810 antibodies that target cancer-associated proteins. Sample pools were screened for an identification of marker proteins. Then, the samples were analyzed individually to validate the usability of these markers. Results More than 200 proteins with disease-associated abundance changes were found. The evaluation on individual patients confirmed some molecules as robust informative markers while others were inadequate for this purpose. In addition, the analysis revealed distinct subgroups for each of the three investigated B-cell lymphoma subtypes. With this information, we delineated a classifier that discriminates the different lymphoma entities. Conclusions and clinical relevance Variations in plasma protein abundance permit discrimination between different patient groups. After validation on a larger study cohort, the findings could have diagnostic as well as differential diagnostic potential. Beside this, methodological aspects were critically evaluated, such as the value of sample pooling for the identification of biomarkers that are useful for a diagnosis on individual patients.

  • Robust Protein Profiling with Complex Antibody Microarrays in a Dual-Colour Mode
    Methods of Molecular Biology, 2011
    Co-Authors: Christoph Schröder, Mohamed Ss Alhamdani, Kurt Fellenberg, Andrea S. Bauer, Anette Jacob, Jorg D Hoheisel
    Abstract:

    : Antibody Microarrays are a multiplexing technique for the analyses of hundreds of different analytes in parallel from small sample volumes of few microlitres only. With sensitivities in the picomolar to femtomolar range, they are gaining importance in proteomic analyses. These sensitivities can be obtained for complex protein samples without any pre-fractionation or signal amplification. Also, no expensive or elaborate protein depletion steps are needed. As with custom DNA-Microarrays, the implementation of a dual-colour assay adds to assay robustness and reproducibility and was therefore a focus of our technical implementation. In order to perform Antibody microarray experiments for large sets of samples and analytes in a robust manner, it was essential to optimise the experimental layout, the protein extraction, labelling and incubation as well as data processing steps. Here, we present our current protocol, which is used for the simultaneous analysis of the abundance of more than 800 proteins in plasma, urine, and tissue samples.

  • Secretome profiling with Antibody Microarrays
    Molecular BioSystems, 2011
    Co-Authors: Shakhawan Abdulrahman Mustafa, Jorg D Hoheisel, Mohamed Ss Alhamdani
    Abstract:

    Following the advances in human genome sequencing, attention has shifted in part toward the elucidation of the encoded biological functions. Since proteins are the driving forces behind very many biological activities, large-scale examinations of their expression variations, their functional roles and regulation have moved to the central stage. A significant fraction of the human proteome consists of secreted proteins. Exploring this set of molecules offers unique opportunities for understanding molecular interactions between cells and fosters biomarker discovery that could advance the detection and monitoring of diseases. Antibody Microarrays are among the relatively new proteomic methodologies that may advance the field significantly because of their relative simplicity, robust performance and high sensitivity down to single-molecule detection. In addition, several aspects such as variations in amount, structure and activity can be assayed at a time. Antibody Microarrays are therefore likely to improve the analytical capabilities in proteomics and consequently permit the production of even more informative and reliable data. This review looks at recent applications of this novel platform technology in secretome analysis and reflects on the future.

  • assessment and optimisation of normalisation methods for dual colour Antibody Microarrays
    BMC Bioinformatics, 2010
    Co-Authors: Martin Sill, Christoph Schröder, Jorg D Hoheisel, Axel Benner, Manuela Zucknick
    Abstract:

    Background: Recent advances in Antibody microarray technology have made it possible to measure the expression of hundreds of proteins simultaneously in a competitive dual-colour approach similar to dual-colour gene expression Microarrays. Thus, the established normalisation methods for gene expression Microarrays, e.g. loess regression, can in principle be applied to protein Microarrays. However, the typical assumptions of such normalisation methods might be violated due to a bias in the selection of the proteins to be measured. Due to high costs and limited availability of high quality antibodies, the current arrays usually focus on a high proportion of regulated targets. Housekeeping features could be used to circumvent this problem, but they are typically underrepresented on protein arrays. Therefore, it might be beneficial to select invariant features among the features already represented on available arrays for normalisation by a dedicated selection algorithm. Results: We compare the performance of several normalisation methods that have been established for dualcolour gene expression Microarrays. The focus is on an invariant selection algorithm, for which effective improvements are proposed. In a simulation study the performances of the different normalisation methods are compared with respect to their impact on the ability to correctly detect differentially expressed features. Furthermore, we apply the different normalisation methods to a pancreatic cancer data set to assess the impact on the classification power. Conclusions: The simulation study and the data application demonstrate the superior performance of the improved invariant selection algorithms in comparison to other normalisation methods, especially in situations where the assumptions of the usual global loess normalisation are violated.

Cornelia Steinhauer - One of the best experts on this subject based on the ideXlab platform.

  • improved affinity coupling for Antibody Microarrays engineering of double his 6 tagged single framework recombinant Antibody fragments
    Proteomics, 2006
    Co-Authors: Cornelia Steinhauer, Mingyue He, Michael J Taussig, Christer Wingren, Farid Khan, Carl Arne Krister Borrebaeck
    Abstract:

    Antibody-based microarray is a novel technology with great promise in biomedicine that will provide unique means to perform global proteome analysis. In the process of designing the high-density Antibody Microarrays required, several critical key issues have been identified that remain to be resolved. In particular, there is a great need for specific and selective approaches enabling non-purified probes to be directly purified, orientated and coupled in a generic one-step procedure directly on the chip. In this study, we report on the successful design of affinity-tagged human recombinant single-chain fragment variable Antibody fragments for improved affinity coupling in array applications. By replacing the standard single-histidine (His)(6)-tag with a consecutive double-(His)(6)-tag, the binding to Ni2+-nitrilotriacetic acid-coated substrates was significantly improved. Surface plasmon resonance analysis showed a significantly tighter binding with at least a threefold slower dissociation. The improved binding characteristics thus enabled non-purified probes even in the format of crude expression supernatants to be directly applied thereby eliminating the need for any time-consuming pre-purification step(s) prior to the immobilization. While the double-(HiS)(6)-tag probes were found to be expressed equally well as compared to the single-(His)(6)-tag probes, they displayed better long-term functional on-chip stability. Taken together, the results demonstrate the generic potential of double-(HiS)(6)-tag recombinant antibodies for the facile fabrication of high-density Antibody Microarrays. (Less)

  • improved affinity coupling for Antibody Microarrays engineering of double his 6 tagged single framework recombinant Antibody fragments
    Proteomics, 2006
    Co-Authors: Cornelia Steinhauer, Michael J Taussig, Christer Wingren, Farid Khan, Carl Arne Krister Borrebaeck
    Abstract:

    Antibody-based microarray is a novel technology with great promise in biomedicine that will provide unique means to perform global proteome analysis. In the process of designing the high-density Antibody Microarrays required, several critical key issues have been identified that remain to be resolved. In particular, there is a great need for specific and selective approaches enabling non-purified probes to be directly purified, orientated and coupled in a generic one-step procedure directly on the chip. In this study, we report on the successful design of affinity-tagged human recombinant single-chain fragment variable Antibody fragments for improved affinity coupling in array applications. By replacing the standard single-histidine (His)(6)-tag with a consecutive double-(His)(6)-tag, the binding to Ni(2+)-nitrilotriacetic acid-coated substrates was significantly improved. Surface plasmon resonance analysis showed a significantly tighter binding with at least a threefold slower dissociation. The improved binding characteristics thus enabled non-purified probes even in the format of crude expression supernatants to be directly applied thereby eliminating the need for any time-consuming pre-purification step(s) prior to the immobilization. While the double-(His)(6)-tag probes were found to be expressed equally well as compared to the single-(His)(6)-tag probes, they displayed better long-term functional on-chip stability. Taken together, the results demonstrate the generic potential of double-(His)(6)-tag recombinant antibodies for the facile fabrication of high-density Antibody Microarrays.

  • Biocompatibility of surfaces for Antibody Microarrays: design of macroporous silicon substrates
    Analytical Biochemistry, 2005
    Co-Authors: Cornelia Steinhauer, Carl Arne Krister Borrebaeck, A. Ressine, Thomas Laurell, György Marko-varga, Christer Wingren
    Abstract:

    Major efforts to develop Antibody microarray technology to enable global proteome analysis to be performed in a facile manner are under way. In this process, the design and the properties of the substrate will play crucial roles. In the present study, we have developed novel, highly biocompatible solid supports for Microarrays, using adsorbed recombinant human single-framework Antibody fragments as probes. Several silicon-based supports, including planar silicon, micro- and macroporous silicon, and nitrocellulose-coated variants thereof, were designed and evaluated in a stepwise procedure. The surfaces were scored based on biocompatibility and probe binding capacity as judged by spot morphology, signal intensities, signal to noise ratios, dynamic range, sensitivity, and reproducibility. A set of five commercially available substrates, selected to represent a set of supports providing different surface and coupling chemistries, was used as reference surfaces. The results showed that several well-performing silicon-based supports could be designed; in particular, a nitrocellulose-coated macroporous variant, MAP3-NC7, received the highest scores. In comparison, MAP3-NC7 displayed properties equal to or better than those of the reference substrates. Taken together, designed surfaces based on silicon can undoubtedly meet the requirements of the next generation of solid supports for Antibody Microarrays. (c) 2004 Elsevier Inc. All rights reserved.

  • Biocompatibility of surfaces for Antibody Microarrays: design of macroporous silicon substrates
    Analytical biochemistry, 2005
    Co-Authors: Cornelia Steinhauer, Carl Arne Krister Borrebaeck, A. Ressine, Thomas Laurell, György Marko-varga, Christer Wingren
    Abstract:

    Major efforts to develop Antibody microarray technology to enable global proteome analysis to be performed in a facile manner are under way. In this process, the design and the properties of the substrate will play crucial roles. In the present study, we have developed novel, highly biocompatible solid supports for Microarrays, using adsorbed recombinant human single-framework Antibody fragments as probes. Several silicon-based supports, including planar silicon, micro- and macroporous silicon, and nitrocellulose-coated variants thereof, were designed and evaluated in a stepwise procedure. The surfaces were scored based on biocompatibility and probe binding capacity as judged by spot morphology, signal intensities, signal to noise ratios, dynamic range, sensitivity, and reproducibility. A set of five commercially available substrates, selected to represent a set of supports providing different surface and coupling chemistries, was used as reference surfaces. The results showed that several well-performing silicon-based supports could be designed; in particular, a nitrocellulose-coated macroporous variant, MAP3-NC7, received the highest scores. In comparison, MAP3-NC7 displayed properties equal to or better than those of the reference substrates. Taken together, designed surfaces based on silicon can undoubtedly meet the requirements of the next generation of solid supports for Antibody Microarrays.

  • Biocompatability of surfaces for Antibody Microarrays: Design of macroporous silicon substrates
    2005
    Co-Authors: Cornelia Steinhauer, Carl Arne Krister Borrebaeck, A. Ressine, Thomas Laurell, György Marko-varga, Christer Wingren
    Abstract:

    Antibody microarray is a novel technology with great promise within proteomics. Intense work is under way to evolve this methodology into the high-throughput proteomic research tool needed by the research community. Despite recent advances, there is a growing need for additional highperformance substrates for Antibody Microarrays as well as for protein arrays in general. In this study, we have sucessfully designed novel, highly biocompatible and well-performing silicon-based supports that has the capacity to play a significant role within current and future Antibody and protein microarray applications within the field of proteomics.

Mohamed Ss Alhamdani - One of the best experts on this subject based on the ideXlab platform.

  • Robust Protein Profiling with Complex Antibody Microarrays in a Dual-Colour Mode
    Methods of Molecular Biology, 2011
    Co-Authors: Christoph Schröder, Mohamed Ss Alhamdani, Kurt Fellenberg, Andrea S. Bauer, Anette Jacob, Jorg D Hoheisel
    Abstract:

    : Antibody Microarrays are a multiplexing technique for the analyses of hundreds of different analytes in parallel from small sample volumes of few microlitres only. With sensitivities in the picomolar to femtomolar range, they are gaining importance in proteomic analyses. These sensitivities can be obtained for complex protein samples without any pre-fractionation or signal amplification. Also, no expensive or elaborate protein depletion steps are needed. As with custom DNA-Microarrays, the implementation of a dual-colour assay adds to assay robustness and reproducibility and was therefore a focus of our technical implementation. In order to perform Antibody microarray experiments for large sets of samples and analytes in a robust manner, it was essential to optimise the experimental layout, the protein extraction, labelling and incubation as well as data processing steps. Here, we present our current protocol, which is used for the simultaneous analysis of the abundance of more than 800 proteins in plasma, urine, and tissue samples.

  • Secretome profiling with Antibody Microarrays
    Molecular BioSystems, 2011
    Co-Authors: Shakhawan Abdulrahman Mustafa, Jorg D Hoheisel, Mohamed Ss Alhamdani
    Abstract:

    Following the advances in human genome sequencing, attention has shifted in part toward the elucidation of the encoded biological functions. Since proteins are the driving forces behind very many biological activities, large-scale examinations of their expression variations, their functional roles and regulation have moved to the central stage. A significant fraction of the human proteome consists of secreted proteins. Exploring this set of molecules offers unique opportunities for understanding molecular interactions between cells and fosters biomarker discovery that could advance the detection and monitoring of diseases. Antibody Microarrays are among the relatively new proteomic methodologies that may advance the field significantly because of their relative simplicity, robust performance and high sensitivity down to single-molecule detection. In addition, several aspects such as variations in amount, structure and activity can be assayed at a time. Antibody Microarrays are therefore likely to improve the analytical capabilities in proteomics and consequently permit the production of even more informative and reliable data. This review looks at recent applications of this novel platform technology in secretome analysis and reflects on the future.

  • Analysis conditions for proteomic profiling of mammalian tissue and cell extracts with Antibody Microarrays
    Proteomics, 2010
    Co-Authors: Mohamed Ss Alhamdani, Christoph Schröder, Jorg D Hoheisel
    Abstract:

    Antibody Microarrays are a developing tool for global proteomic profiling. A protocol was established that permits robust analyses of protein extracts from mammalian tissues and cells rather than body fluids. The factors optimized were buffer composition for surface blocking, blocking duration, protein handling and processing, labeling parameters like type of dye, molar ratio of label versus protein, and dye removal, as well as incubation parameters such as duration, temperature, buffer, and sample agitation.

  • single step procedure for the isolation of proteins at near native conditions from mammalian tissue for proteomic analysis on Antibody Microarrays
    Journal of Proteome Research, 2010
    Co-Authors: Mohamed Ss Alhamdani, Christoph Schröder, Andrea S. Bauer, Jens Werner, Nathalia Giese, Jorg D Hoheisel
    Abstract:

    The process of extracting comprehensive proteome representations is a crucial step for many proteomic studies. While Antibody Microarrays are an evolving and promising methodology in proteomics, the issue of protein extraction from tissues for this kind of analysis has never been addressed. Here, we describe a single-step extraction buffer for the isolation of proteins from mammalian tissues under native conditions in an effective and reproducible manner. Protein was extracted from cell lines BxPC-3 and SU.86.86, rat organs (pancreas, liver, heart and lung) and human pancreatic cancer tissues using several buffer systems that contained individual nonionic or zwitterionic detergents in comparison to commercial extraction buffers. Also, detergent combinations were used that included at least one polymeric phenylethylene glycol, a long-chain amidosulfobetaine, cholate and a zwitterionic detergent. Extracts were analyzed for protein quantity and quality. The detergent cocktails exhibited superior extraction capacity. Additionally, they demonstrated a substantially higher recovery of membrane and compartmental proteins as well as much better preservation of protein functionality. Also, they did not interfere with subsequent analysis steps such as labeling. In Western blot and Antibody microarray assays, they outperformed the other buffer systems, indicating that they should also be useful for other types of proteomic studies.

  • Oncoproteomic profiling with Antibody Microarrays
    Genome Medicine, 2009
    Co-Authors: Mohamed Ss Alhamdani, Christoph Schröder, Jorg D Hoheisel
    Abstract:

    The incidence of cancer and its associated mortality are increasing globally, indicating an urgent need to develop even more effective and sensitive sets of biomarkers that could help in early diagnosis and consequent intervention. Given that many cellular processes are carried out by proteins, cancer research has recently shifted toward an exploration of the full proteome for such discovery. Among the advanced methodologies that are being developed for analyzing the proteome, Antibody Microarrays have become a prominent tool for gathering the information required for a better understanding of disease biology, early detection, discrimination of tumors and monitoring of disease progression. Here, we review the technical aspects and challenges in the development and use of Antibody microarray assays and examine recently reported applications in oncoproteomics.