The Experts below are selected from a list of 2631 Experts worldwide ranked by ideXlab platform
Christer Wingren - One of the best experts on this subject based on the ideXlab platform.
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Biocompatability of surfaces for Antibody Microarrays: Design of macroporous silicon substrates
2020Co-Authors: Cornelia Steinhauer, Carl A K Borrebaeck, Anton Ressine, Thomas Laurell, György Marko-varga, Christer WingrenAbstract: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.
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technical advances of the recombinant Antibody Microarray technology platform for clinical immunoproteomics
PLOS ONE, 2016Co-Authors: Payam Delfani, Carl Arne Krister Borrebaeck, Linda Dexlin Mellby, Malin Nordstrom, Andreas Holmer, Mattias Ohlsson, Christer WingrenAbstract:In the quest for deciphering disease-associated biomarkers, high-performing tools for multiplexed protein expression profiling of crude clinical samples will be crucial. Affinity proteomics, mainly represented by Antibody-based Microarrays, have during recent years been established as a proteomic tool providing unique opportunities for parallelized protein expression profiling. But despite the progress, several main technical features and assay procedures remains to be (fully) resolved. Among these issues, the handling of protein Microarray data, i.e. the biostatistics parts, is one of the key features to solve. In this study, we have therefore further optimized, validated, and standardized our in-house designed recombinant Antibody Microarray technology platform. To this end, we addressed the main remaining technical issues (e.g. Antibody quality, array production, sample labelling, and selected assay conditions) and most importantly key biostatistics subjects (e.g. array data pre-processing and biomarker panel condensation). This represents one of the first Antibody array studies in which these key biostatistics subjects have been studied in detail. Here, we thus present the next generation of the recombinant Antibody Microarray technology platform designed for clinical immunoproteomics.
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Evaluation of Solid Supports for Slide- and Well-Based Recombinant Antibody Microarrays.
Microarrays, 2016Co-Authors: Anna Sandström Gerdtsson, Linda Dexlin-mellby, Payam Delfani, Erica Berglund, Carl Arne Krister Borrebaeck, Christer WingrenAbstract: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.
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recombinant Antibody Microarray for profiling the serum proteome of sle
Methods of Molecular Biology, 2014Co-Authors: Carl A K Borrebaeck, Gunnar Sturfelt, Christer WingrenAbstract:Systemic lupus erythematosus (SLE) is a severe autoimmune connective tissue disease. Our current knowledge about the serum proteome, or serum biomarker panels, reflecting disease and disease status is still very limited. Affinity proteomics, represented by recombinant Antibody arrays, is a novel, multiplex technology for high-throughput protein expression profiling of crude serum proteomes in a highly specific, sensitive, and miniaturized manner. The antibodies are deposited one by one in an ordered pattern, an array, onto a solid support. Next, the sample is added, and any specifically bound proteins are detected and quantified. The binding pattern is then converted into a relative protein expression map, or protein map, deciphering the composition of the sample at the molecular level. The methodology provides unique opportunities for delineating serum biomarkers reflecting SLE, thus paving the way for improved diagnosis, classification, and prognosis. (Less)
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Design of recombinant Antibody Microarrays for urinary proteomics
Proteomics Clinical Applications, 2012Co-Authors: Malin Kristensson, Karolina Olsson, Björn Wullt, Joyce Carlson, Gunnar Sturfelt, Carl Arne Krister Borrebaeck, Christer WingrenAbstract:PURPOSE: Urinary proteomics has become a key discipline within clinical proteomics for noninvasive diagnosis and monitoring of disease, and biomarker discovery. In order to decipher complex proteomes, high demands will, however, be placed upon the methodology applied. The purpose of this study was to develop a recombinant Antibody Microarray platform for urinary proteomics. EXPERIMENTAL DESIGN: We adopted our previously in-house developed recombinant Antibody Microarray set-up and redesigned the platform for urinary proteomics. In this process, the key Antibody array assay parameters, such as sample handling, sample labeling protocol, and assay conditions, etc, reflecting the unique properties of urine as sample format, were addressed and reoptimized in a step-by-step procedure. RESULTS: In this proof-of-concept study, we have designed the first generation of a recombinant Antibody Microarray technology platform for urinary proteomics. The results showed that multiplexed, sensitive (pg/mL range), and reproducible urine protein expression profiling could be performed targeting directly labeled, nonfractionated urine. CONCLUSION AND CLINICAL RELEVANCE: We have demonstrated that crude, directly labeled urine samples could be profiled in a rapid, reproducible, sensitive, and multiplexed manner after minimal sample prehandling. These findings could potentially pave the way for enhanced urinary proteomics and understanding of renal physiology with implications in both health and disease. (Less)
Carl Arne Krister Borrebaeck - One of the best experts on this subject based on the ideXlab platform.
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technical advances of the recombinant Antibody Microarray technology platform for clinical immunoproteomics
PLOS ONE, 2016Co-Authors: Payam Delfani, Carl Arne Krister Borrebaeck, Linda Dexlin Mellby, Malin Nordstrom, Andreas Holmer, Mattias Ohlsson, Christer WingrenAbstract:In the quest for deciphering disease-associated biomarkers, high-performing tools for multiplexed protein expression profiling of crude clinical samples will be crucial. Affinity proteomics, mainly represented by Antibody-based Microarrays, have during recent years been established as a proteomic tool providing unique opportunities for parallelized protein expression profiling. But despite the progress, several main technical features and assay procedures remains to be (fully) resolved. Among these issues, the handling of protein Microarray data, i.e. the biostatistics parts, is one of the key features to solve. In this study, we have therefore further optimized, validated, and standardized our in-house designed recombinant Antibody Microarray technology platform. To this end, we addressed the main remaining technical issues (e.g. Antibody quality, array production, sample labelling, and selected assay conditions) and most importantly key biostatistics subjects (e.g. array data pre-processing and biomarker panel condensation). This represents one of the first Antibody array studies in which these key biostatistics subjects have been studied in detail. Here, we thus present the next generation of the recombinant Antibody Microarray technology platform designed for clinical immunoproteomics.
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Evaluation of Solid Supports for Slide- and Well-Based Recombinant Antibody Microarrays.
Microarrays, 2016Co-Authors: Anna Sandström Gerdtsson, Linda Dexlin-mellby, Payam Delfani, Erica Berglund, Carl Arne Krister Borrebaeck, Christer WingrenAbstract: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.
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Design of recombinant Antibody Microarrays for urinary proteomics
Proteomics Clinical Applications, 2012Co-Authors: Malin Kristensson, Karolina Olsson, Björn Wullt, Joyce Carlson, Gunnar Sturfelt, Carl Arne Krister Borrebaeck, Christer WingrenAbstract:PURPOSE: Urinary proteomics has become a key discipline within clinical proteomics for noninvasive diagnosis and monitoring of disease, and biomarker discovery. In order to decipher complex proteomes, high demands will, however, be placed upon the methodology applied. The purpose of this study was to develop a recombinant Antibody Microarray platform for urinary proteomics. EXPERIMENTAL DESIGN: We adopted our previously in-house developed recombinant Antibody Microarray set-up and redesigned the platform for urinary proteomics. In this process, the key Antibody array assay parameters, such as sample handling, sample labeling protocol, and assay conditions, etc, reflecting the unique properties of urine as sample format, were addressed and reoptimized in a step-by-step procedure. RESULTS: In this proof-of-concept study, we have designed the first generation of a recombinant Antibody Microarray technology platform for urinary proteomics. The results showed that multiplexed, sensitive (pg/mL range), and reproducible urine protein expression profiling could be performed targeting directly labeled, nonfractionated urine. CONCLUSION AND CLINICAL RELEVANCE: We have demonstrated that crude, directly labeled urine samples could be profiled in a rapid, reproducible, sensitive, and multiplexed manner after minimal sample prehandling. These findings could potentially pave the way for enhanced urinary proteomics and understanding of renal physiology with implications in both health and disease. (Less)
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Detection of pancreatic cancer using Antibody Microarray-based serum protein profiling
Proteomics, 2008Co-Authors: Johan Ingvarsson, Christer Wingren, Anders Carlsson, Peter Ellmark, Britta Wahren, Gunnel Engstrom, Ulrika Harmenberg, Morten Krogh, Carsten Peterson, Carl Arne Krister BorrebaeckAbstract:The driving force behind oncoproteomics is to identify protein signatures that are associated with a particular malignancy. Here, we have used a recombinant scFv Antibody Microarray in an attempt to classify sera derived from pancreatic adenocarcinoma patients versus healthy subjects. Based on analysis of nonfractionated, directly labeled, whole human serum proteomes we have identified a protein signature based on 19 nonredundant analytes, that discriminates between cancer patients and healthy subjects. Furthermore, a potential protein signature, consisting of 21 protein analytes, could be defined that was shown to be associated with cancer patients having a life expectancy of
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detection of pancreatic cancer using Antibody Microarray based serum protein profiling
Proteomics, 2008Co-Authors: Johan Ingvarsson, Christer Wingren, Anders Carlsson, Peter Ellmark, Britta Wahren, Gunnel Engstrom, Ulrika Harmenberg, Morten Krogh, Carsten Peterson, Carl Arne Krister BorrebaeckAbstract:The driving force behind oncoproteomics is to identify protein signatures that are associated with a particular malignancy. Here, we have used a recombinant scFv Antibody Microarray in an attempt to classify sera derived from pancreatic adenocarcinoma patients versus healthy subjects. Based on analysis of nonfractionated, directly labeled, whole human serum proteomes we have identified a protein signature based on 19 nonredundant analytes, that discriminates between cancer patients and healthy subjects. Furthermore, a potential protein signature, consisting of 21 protein analytes, could be defined that was shown to be associated with cancer patients having a life expectancy of <12 months. Taken together, the data suggest that Antibody Microarray analysis of complex proteomes will be a useful tool to define disease associated protein signatures.
Brian B Haab - One of the best experts on this subject based on the ideXlab platform.
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Antibody Microarray profiling reveals individual and combined serum proteins associated with pancreatic cancer
Cancer Research, 2005Co-Authors: Randal P Orchekowski, Darren Hamelinck, Lin Li, Ewa Gliwa, Matthew W Vanbrocklin, Jorge A Marrero, George Vande F Woude, Ziding Feng, Randall E Brand, Brian B HaabAbstract:We used Antibody Microarrays to probe the associations of multiple serum proteins with pancreatic cancer and to explore the use of combined measurements for sample classification. Serum samples from pancreatic cancer patients (n = 61), patients with benign pancreatic disease (n = 31), and healthy control subjects (n = 50) were probed in replicate experiment sets by two-color, rolling circle amplification on Microarrays containing 92 antibodies and control proteins. The antibodies that had reproducibly different binding levels between the patient classes revealed different types of alterations, reflecting inflammation (high C-reactive protein, α-1-antitrypsin, and serum amyloid A), immune response (high IgA), leakage of cell breakdown products (low plasma gelsolin), and possibly altered vitamin K usage or glucose regulation (high protein-induced vitamin K antagonist-II). The accuracy of the most significant Antibody Microarray measurements was confirmed through immunoblot and antigen dilution experiments. A logistic-regression algorithm distinguished the cancer samples from the healthy control samples with a 90% and 93% sensitivity and a 90% and 94% specificity in duplicate experiment sets. The cancer samples were distinguished from the benign disease samples with a 95% and 92% sensitivity and an 88% and 74% specificity in duplicate experiment sets. The classification accuracies were significantly improved over those achieved using individual antibodies. This study furthered the development of Antibody Microarrays for molecular profiling, provided insights into the nature of serum-protein alterations in pancreatic cancer patients, and showed the potential of combined measurements to improve sample classification accuracy.
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distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon Antibody Microarray analysis
BMC Cancer, 2005Co-Authors: Rork Kuick, Randal P Orchekowski, Brian B Haab, David E Misek, Alissa K Greenberg, Dean E Brenner, Gilbert S Omenn, Samir M HanashAbstract:Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response.
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immunoassay and Antibody Microarray analysis of the hupo plasma proteome project reference specimens systematic variation between sample types and calibration of mass spectrometry data
Proteomics, 2005Co-Authors: Brian B Haab, Bernhard H Geierstanger, George Michailidis, Frank Vitzthum, Sara Forrester, Ryan Okon, Petri Saviranta, Achim Brinker, Martin Sorette, Lorah PerleeAbstract:Four different immunoassay and Antibody Microarray methods performed at four different sites were used to measure the levels of a broad range of proteins (N = 323 assays; 39, 88, 168, and 28 assays at the respective sites; 237 unique analytes) in the human serum and plasma reference specimens distributed by the Plasma Proteome Project (PPP) of the HUPO. The methods provided a means to (1) assess the level of systematic variation in protein abundances associated with blood preparation methods (serum, citrate-anticoagulated-plasma, EDTA-anticoagulated-plasma, or heparin-anticoagulated-plasma) and (2) evaluate the dependence on concentration of MS-based protein identifications from data sets using the HUPO specimens. Some proteins, particularly cytokines, had highly variable concentrations between the different sample preparations, suggesting specific effects of certain anticoagulants on the stability or availability of these proteins. The linkage of Antibody-based measurements from 66 different analytes with the combined MS/MS data from 18 different laboratories showed that protein detection and the quality of MS data increased with analyte concentration. The conclusions from these initial analyses are that the optimal blood preparation method is variable between analytes and that the discovery of blood proteins by MS can be extended to concentrations below the ng/mL range under certain circumstances.more » Continued developments in Antibody-based methods will further advance the scientific goals of the PPP.« less
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Optimized Normalization for Antibody Microarrays and Application to Serum-Protein Profiling
Molecular & Cellular Proteomics, 2005Co-Authors: Darren Hamelinck, Lin Li, Ziding Feng, Heping Zhou, Cornelius Verweij, Deborah A. Dillon, Jose Costa, Brian B HaabAbstract:: The measurements of coordinated patterns of protein abundance using Antibody Microarrays could be used to gain insight into disease biology and to probe the use of combinations of proteins for disease classification. The correct use and interpretation of Antibody Microarray data requires proper normalization of the data, which has not yet been systematically studied. Therefore we undertook a study to determine the optimal normalization of data from Antibody Microarray profiling of proteins in human serum specimens. Forty-three serum samples collected from patients with pancreatic cancer and from control subjects were probed in triplicate on Microarrays containing 48 different antibodies, using a direct labeling, two-color comparative fluorescence detection format. Seven different normalization methods representing major classes of normalization for Antibody Microarray data were compared by their effects on reproducibility, accuracy, and trends in the data set. Normalization with ELISA-determined concentrations of IgM resulted in the most accurate, reproducible, and reliable data. The other normalization methods were deficient in at least one of the criteria. Multiparametric classification of the samples based on the combined measurement of seven of the proteins demonstrated the potential for increased classification accuracy compared with the use of individual measurements. This study establishes reliable normalization for Antibody Microarray data, criteria for assessing normalization performance, and the capability of Antibody Microarrays for serum-protein profiling and multiparametric sample classification.
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Methods and applications of Antibody Microarrays in cancer research
Proteomics, 2003Co-Authors: Brian B HaabAbstract:Antibody Microarrays have great potential for significant value in biological research. Cancer research in particular could benefit from the unique experimental capabilities of this technology. This article examines the current state of Antibody Microarray technological developments and assay formats, along with a review of the demonstrated applications to cancer research. Work is ongoing in the refinement of various aspects of the protocols and the development of robust methods for routine use. Antibody Microarray experimental formats can be broadly categorized into two classes: (1) direct labeling experiments, and (2) dual Antibody sandwich assays. In the direct labeling method, the covalent labeling of all proteins in a complex mixture provides a means for detecting bound proteins after incubation on an Antibody Microarray. If proteins are labeled with a tag, such as biotin, the signal from bound proteins can be amplified. In the sandwich assay, proteins captured on an Antibody Microarray are detected by a cocktail of detection antibodies, each Antibody matched to one of the spotted antibodies. Each format has distinct advantages and disadvantages. Several applications of Antibody arrays to cancer research have been reported, including the analysis of proteins in blood serum, resected frozen tumors, cell lines, and on membranes of blood cells. These demonstrations clearly show the utility of Antibody Microarrays for cancer research and signal the imminent expansion of this platform to many areas of biological research.
Richard I. Christopherson - One of the best experts on this subject based on the ideXlab platform.
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Classification of AML Using a Monoclonal Antibody Microarray
Methods in molecular medicine, 2020Co-Authors: Richard I. Christopherson, Kerryn J. Stoner, Nicole Barber, Larissa Belov, Adrian Woolfson, M. G. Scott, Linda J. Bendall, Stephen P. MulliganAbstract:: A cluster of differentiation (CD) Antibody Microarray called the DotScan Microarray has been developed that enables an extensive immunophenotype to be obtained for a suspension of leukocytes in a single analysis. For a leukemia with a leukemia count of greater than 10 x 10(9)/L, the immunophenotype obtained is essentially that of the leukemic clone. The Antibody Microarray is printed as microscopic (10 nL) dots on a nitrocellulose film on a microscope slide. Cells are captured by the immobilized antibodies and a dot pattern is recorded with an optical array reader giving the immunophenotype of the leukemia. Procedures are being developed that should enable diagnosis of myeloid leukemias by comparison of the dot pattern obtained from an unknown blood sample with a library of consensus patterns for the common leukemias.
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Antibody Microarrays and Multiplexing
Translational Bioinformatics, 2012Co-Authors: Jerry Zhou, Larissa Belov, Nicola J. Armstrong, Richard I. ChristophersonAbstract:This chapter presents a range of statistical methods for Antibody Microarray normalization and data analysis. Commonly used techniques for cluster generation, differential analysis, and classification are covered. The focus is on the implementation of each technique to the technology and its suitability in relation to sample types and experiment design.
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analysis of human liver disease using a cluster of differentiation cd Antibody Microarray
Liver International, 2012Co-Authors: Wassim Rahman, Richard I. Christopherson, Larissa Belov, Pauline Huang, Jeremy S Chrisp, Peter M Stapelberg, F J Warner, Jacob George, David G BowenAbstract:BACKGROUND: A CD Antibody Microarray has been previously developed allowing semi-quantitative identification of greater than 80 CD antigens on circulating leucocytes from peripheral blood samples. This assay, which uses a live cell-capture technique, enables an extensive leucocyte immunophenotype determination in a single analysis and to date this has been used successfully to characterise diseases including human leukaemias and HIV infection. AIMS: To determine CD antigen expression profiles for patients with various liver diseases and to look for preserved disease-specific signatures. METHODS: Three liver disease groups including hepatitis C (HCV) (n = 35), non-alcoholic steatohepatitis (NASH) (n = 21) and alcohol-related liver disease (n = 14) were compared with a normal group (n = 23). Hierarchal Clustering (HCL) and Principal Component Analysis (PCA) of the data revealed distinct binding patterns for patients with and without cirrhosis. RESULTS: Patients with cirrhosis and portal hypertension compared with those without cirrhosis had significantly reduced expression of several markers of T-cell function including CD45, CD8, CD28 and TCR α/β. Disease prediction algorithms based on the expression data were able to discriminate cirrhotics from non-cirrhotics with 71% overall success, which improved to 77% when only patients with HCV were considered. CONCLUSIONS: These results demonstrate disease-specific consensus patterns of expression of CD antigens for patients with chronic liver disease, suggesting that the CD Antibody array is a promising tool in the analysis of human liver disease, and with further refinement may have future research and clinical utility.
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colorectal cancer cell surface protein profiling using an Antibody Microarray and fluorescence multiplexing
Journal of Visualized Experiments, 2011Co-Authors: Jerry Zhou, Larissa Belov, Michael J Solomon, Charles Chan, Stephen Clarke, Richard I. ChristophersonAbstract:The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1. A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs). The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single ‘marker’ has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC Antibody Microarray. The 122-Antibody Microarray consists of a standard 82-Antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the Antibody6. For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC Microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.
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an extended Antibody Microarray for surface profiling metastatic melanoma
Journal of Immunological Methods, 2010Co-Authors: Kimberley L Kaufman, Larissa Belov, Pauline Huang, Swetlana Mactier, Richard A Scolyer, Graham J Mann, Richard I. ChristophersonAbstract:An Antibody Microarray was developed for profiling the surface proteome of melanoma cells, which may facilitate melanoma sub-classification and provide important prognostic information useful in predicting the clinical behavior of the melanoma (e.g., likely sites of metastatic spread), patient outcome and treatment response. Forty-eight antibodies were selected based on their correlation with melanoma development, progression and/or prognosis and printed on nitrocellulose slides. The immobilised antibodies capture live cells expressing corresponding antigens to produce a cell binding dot pattern representing the surface antigen profile (immunophenotype) of the melanoma. Surface antigen signatures were determined for a normal melanocyte and 6 melanoma cell lines and cell suspensions prepared from 10 surgically excised melanoma lymph node metastases. A procedure for obtaining separate surface antigen profiles for melanoma cells and leukocytes from clinical lymph node samples was also developed using anti-CD45 magnetic beads. The capture of live, bead-bound leukocytes on these Antibody Microarrays provides a significant enhancement of this Microarray technology. The Antibody Microarray will be used to profile panels of surgically excised melanoma lymph node metastases (melanoma and leukocyte fractions) to determine whether the immunophenotypes correlate with clinicopathological characteristics, disease progression and clinical outcome.
Johan Ingvarsson - One of the best experts on this subject based on the ideXlab platform.
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Design of recombinant Antibody Microarrays for global proteome analysis
2020Co-Authors: Johan IngvarssonAbstract:Antibody-based Microarrays are among the new, rapidly emerging technologies within the field of proteomics that has the potential to evolve into a key proteomic research tool, providing novel means to perform early diagnostics, identify biomarkers and gain insights into disease biology. The aim of this thesis, based on five original papers, was to develop Antibody Microarrays based on human recombinant scFv Antibody fragments for large-scale comprehensive proteome analysis. In order to accomplish that, several of the key technological Antibody Microarray parameters critical for complex proteome analysis were carefully addressed and optimized (paper I-IV). By successfully optimizing each of the features, a state-of-the-art recombinant Antibody Microarray technology platform was successfully designed. The recombinant Antibody Microarrays could readily be used to perform highly sensitive and multiplexed protein expression analysis of complex samples, such as human plasma. In the final study (paper V), we applied the optimized Microarrays to analyze pancreatic cancer serum samples and compared the protein expression signature to healthy controls. The results showed that a pancreatic cancer associated protein signature could be identified that could discriminate between cancer patients and healthy controls with a 100 % specificity and sensitivity. In addition, we also identified a potential predictor signature, which could be used to identify two cohorts of patients based on the overall survival. These novel findings demonstrated the tremendous potential of Antibody Microarrays in cancer research and clinical applications. (Less)
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Detection of pancreatic cancer using Antibody Microarray-based serum protein profiling
Proteomics, 2008Co-Authors: Johan Ingvarsson, Christer Wingren, Anders Carlsson, Peter Ellmark, Britta Wahren, Gunnel Engstrom, Ulrika Harmenberg, Morten Krogh, Carsten Peterson, Carl Arne Krister BorrebaeckAbstract:The driving force behind oncoproteomics is to identify protein signatures that are associated with a particular malignancy. Here, we have used a recombinant scFv Antibody Microarray in an attempt to classify sera derived from pancreatic adenocarcinoma patients versus healthy subjects. Based on analysis of nonfractionated, directly labeled, whole human serum proteomes we have identified a protein signature based on 19 nonredundant analytes, that discriminates between cancer patients and healthy subjects. Furthermore, a potential protein signature, consisting of 21 protein analytes, could be defined that was shown to be associated with cancer patients having a life expectancy of
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detection of pancreatic cancer using Antibody Microarray based serum protein profiling
Proteomics, 2008Co-Authors: Johan Ingvarsson, Christer Wingren, Anders Carlsson, Peter Ellmark, Britta Wahren, Gunnel Engstrom, Ulrika Harmenberg, Morten Krogh, Carsten Peterson, Carl Arne Krister BorrebaeckAbstract:The driving force behind oncoproteomics is to identify protein signatures that are associated with a particular malignancy. Here, we have used a recombinant scFv Antibody Microarray in an attempt to classify sera derived from pancreatic adenocarcinoma patients versus healthy subjects. Based on analysis of nonfractionated, directly labeled, whole human serum proteomes we have identified a protein signature based on 19 nonredundant analytes, that discriminates between cancer patients and healthy subjects. Furthermore, a potential protein signature, consisting of 21 protein analytes, could be defined that was shown to be associated with cancer patients having a life expectancy of <12 months. Taken together, the data suggest that Antibody Microarray analysis of complex proteomes will be a useful tool to define disease associated protein signatures.
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Design of recombinant Antibody Microarrays for cell surface membrane proteomics
Journal of Proteome Research, 2007Co-Authors: Linda Dexlin, Johan Ingvarsson, Carl A K Borrebaeck, Björn Frendéus, Christer WingrenAbstract:Generating proteomic maps of membrane proteins, common targets for therapeutic interventions and disease diagnostics, has turned out to be a major challenge. Antibody-based Microarrays are among the novel rapidly evolving proteomic technologies that may enable global proteome analysis to be performed. Here, we have designed the first generation of a scaleable human recombinant scFv Antibody Microarray technology platform for cell surface membrane proteomics as well as glycomics targeting intact cells. The results showed that rapid and multiplexed profiling of the cell surface proteome (and glycome) could be performed in a highly specific and sensitive manner and that differential expression patterns due to external stimuli could be monitored.
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design of recombinant Antibody Microarrays for complex proteome analysis choice of sample labeling tag and solid support
Proteomics, 2007Co-Authors: Christer Wingren, Linda Dexlin, Dominika Szul, Johan Ingvarsson, Carl Arne Krister BorrebaeckAbstract:Antibody-based Microarray is a novel technology with great potential within high-throughput proteomics. The process of designing high-performing Antibody (protein) Microarrays has, however, turned out to be a challenging process. In this study, we have developed further our human recombinant single-chain variable-fragment (scFv) Antibody Microarray methodology by addressing two crucial technological issues, choice of sample labeling-tag and solid support. We examined the performance of a range of dyes in a one- or two-color approach on a selection of solid supports providing different surface and coupling chemistries, and surface structures. The set-ups were evaluated in terms of sensitivity specificity, and selectivity. The results showed that a one-color approach, based on NHS-biotin (or ULS-biotin) labeling, on black polymer Maxisorb slides (or Nexterion slide H) was the superior approach for targeting low-abundant (pg/mL) analytes in nonfractionated, complex proteomes, such as human serum or crude cell supernatants. Notably, Microarrays displaying adequate spot morphologies, high S/Ns, minimized nonspecific binding, and most importantly a high selectivity, specificity, and sensitivity (>= fM range) were obtained. Taken together, we have designed the first generation of a high-performing recombinant scFv Antibody Microarray technology platform on black polymer Maxisorb slides for sensitive profiling of low-abundant analytes in nonfractionated biotinylated complex proteomes. (Less)
