| 1. |
- El-Schich, Zahra, et al.
(författare)
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Interfacing antibody-based microarrays and digital holography enables label-free detection for loss of cell volume
- 2015
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Ingår i: Future Science OA. - : Future Science Group. - 2056-5623. ; 1:3
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Tidskriftsartikel (refereegranskat)abstract
- Background: We introduce the combination of digital holographic microscopy (DHM) and antibody microarrays as a powerful tool to measure morphological changes in specifically antibody-captured cells. The aim of the study was to develop DHM for analysis of cell death of etoposide-treated suspension cells. Result/Methodology: We demonstrate that the cell number, mean area, thickness, and volume were non-invasively measured by using DHM. The cell number was stable over time, but the two cell lines showed changes of cell area and cell irregularity after treatment. The cell volume in etoposide-treated cells was decreased, whereas untreated cells showed stable volume. Conclusions: Our results provide proof of concept for using DHM combined with antibody-based microarray technology for detecting morphological changes in captured cells.
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| 2. |
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| 3. |
- Borrebaeck, Carl A K, et al.
(författare)
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Recombinant Antibody Microarray for Profiling the Serum Proteome of SLE.
- 2014
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029. ; 1134, s. 67-78
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
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| 5. |
- Borrebaeck, Carl, et al.
(författare)
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Antibody array generation and use.
- 2014
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 1131, s. 563-571
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Tidskriftsartikel (refereegranskat)abstract
- Affinity proteomics, represented by antibody arrays, is a multiplex technology for high-throughput protein expression profiling of crude proteomes in a highly specific, sensitive, and miniaturized manner. The antibodies are individually deposited in an ordered pattern, an array, onto a solid support. Next, the sample is added, and any specifically bound proteins are detected and quantified using mainly fluorescence as the mode of detection. The binding pattern is then converted into a relative protein expression map, or protein atlas, delineating the composition of the sample at the molecular level. The technology provides unique opportunities for various applications, such as protein expression profiling, biomarker discovery, disease diagnostics, prognostics, evidence-based therapy selection, and disease monitoring. Here, we describe the generation and use of planar antibody arrays for serum protein profiling.
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| 6. |
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| 7. |
- Borrebaeck, Carl, et al.
(författare)
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Design of high-density antibody microarrays for disease proteomics: Key technological issues.
- 2009
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 72:6, s. 928-935
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-based microarray is a novel proteomic technology setting a new standard for molecular profiling of non-fractionated complex proteomes. The first generation of antibody microarrays has already demonstrated its potential for generating detailed protein expression profiles, or protein atlases, of human body fluids in health and disease, paving the way for new discoveries within the field of disease proteomics. The process of designing highly miniaturized, high-density and high-performing antibody microarray set-ups have, however, proven to be challenging. In this mini-review we discuss key technological issues that must be addressed in a cross-disciplinary manner before true global proteome analysis can be performed using antibody microarrays.
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| 8. |
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| 9. |
- Borrebaeck, Carl, et al.
(författare)
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High-throughput proteomics using antibody microarrays: an update
- 2007
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Ingår i: Expert Review of Molecular Diagnostics. - : Informa UK Limited. - 1744-8352 .- 1473-7159. ; 7:5, s. 673-686
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Forskningsöversikt (refereegranskat)abstract
- Antibody-based microarrays are a rapidly emerging technology that has advanced from the first proof-of-concept studies to demanding serum protein profiling applications during recent years, displaying great promise within disease proteomics. Miniaturized micro- and nanoarrays can be fabricated with an almost infinite number of antibodies carrying the desired specificities. While consuming only minute amounts of reagents, multiplexed and ultrasensitive assays can be performed targeting high- as well as low-abundance analytes in complex nonfractionated proteomes. The microarray images generated can then be converted into protein expression profiles or protein atlases, revealing a detailed composition of the sample. The technology will provide unique opportunities for fields such as disease diagnostics, biomarker discovery, patient stratification, predicting disease recurrence and drug target discovery. This review describes an update of high-throughput proteomics, using antibody-based microarrays, focusing on key technological advances and novel applications that have emerged over the last 3 years.
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| 10. |
- Borrebaeck, Carl, et al.
(författare)
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Progress in miniaturisation of proteon arrays - a step closer to high-density nanoarrays
- 2007
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Ingår i: Drug Discovery Today. - : Elsevier BV. - 1878-5832 .- 1359-6446. ; 12:19-20, s. 813-819
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Forskningsöversikt (refereegranskat)abstract
- Protein microarrays is a technology with great promise for high-throughput proteomics. Designing high-performance protein microarrays for global proteome analysis has, however, turned out to be challenging. To this end, major efforts are under way to design novel array formats capable of harboring the tremendous range of probes required to target complex proteomes composed of more than 10 000 analytes. By adopting nanotechnology, the first generation of miniaturized nanoarrays has recently emerged, which opens up new avenues for global proteome analysis and disease proteomics. This review describes the progress and key issues in designing miniaturized protein arrays.
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| 11. |
- Borrebaeck, Carl, et al.
(författare)
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Recombinant antibodies for the generation of antibody arrays.
- 2011
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 785, s. 247-262
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Tidskriftsartikel (refereegranskat)abstract
- Affinity proteomics, mainly represented by antibody microarrays, has in recent years been established as a powerful tool for high-throughput (disease) proteomics. The technology can be used to generate detailed protein expression profiles, or protein maps, of focused set of proteins in crude proteomes and potentially even high-resolution portraits of entire proteomes. The technology provides unique opportunities, for example biomarker discovery, disease diagnostics, patient stratification and monitoring of disease, and taking the next steps toward personalized medicine. However, the process of designing high-performing, high-density antibody micro- and nanoarrays has proven to be challenging, requiring truly cross-disciplinary efforts to be adopted. In this mini-review, we address one of these key technological issues, namely, the choice of probe format, and focus on the use of recombinant antibodies vs. polyclonal and monoclonal antibodies for the generation of antibody arrays.
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| 12. |
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| 13. |
- Brofelth, Mattias, et al.
(författare)
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Multiplex profiling of serum proteins in solution using barcoded antibody fragments and next generation sequencing
- 2020
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Ingår i: Communications Biology. - : NATURE PUBLISHING GROUP. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- The composition of serum proteins is reflecting the current health status and can, with the right tools, be used to detect early signs of disease, such as an emerging cancer. An earlier diagnosis of cancer would greatly increase the chance of an improved outcome for the patients. However, there is still an unmet need for proficient tools to decipher the information in the blood proteome, which calls for further technological development. Here, we present a proof-of-concept study that demonstrates an alternative approach for multiplexed protein profiling of serum samples in solution, using DNA barcoded scFv antibody fragments and next generation sequencing. The outcome shows high accuracy when discriminating samples derived from pancreatic cancer patients and healthy controls and represents a scalable alternative for serum analysis. Brofelth, Ekstrand et al use DNA barcoded scFv antibody fragments and next generation sequencing for multiplex profiling of proteins in serum from pancreatic cancer patients with high accuracy. This approach can potentially be used in high throughput precision diagnosis.
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| 14. |
- Brofelth, Mattias, et al.
(författare)
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Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics
- 2017
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639. ; 1865:8, s. 985-996
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology. In more detail, we have generated the 2nd generation of scFvs carrying the photoreactive UAA p-benzoyl-l-phenylalanine (pBpa). Based on key properties, such as expression levels, activity, and affinity, a preferred choice of site for pBpa, located in the beginning of the C-terminal affinity-tag, was for the first time pin-pointed. Further, the results showed that pBpa mutated antibody could be site-specifically photocoupled to free and surface immobilized β-cyclodextrin (an affinity ligand to pBpa). This paves the way for use of scFv antibodies, engineered for site-specific photochemical-based tagging, functionalization, and orientated surface immobilization, in affinity proteomics.
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| 15. |
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| 16. |
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| 17. |
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| 18. |
- Carlsson, Anders, et al.
(författare)
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Molecular serum portraits in patients with primary breast cancer predict the development of distant metastases.
- 2011
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 108:34, s. 14252-14257
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Tidskriftsartikel (refereegranskat)abstract
- The risk of distant recurrence in breast cancer patients is difficult to assess with current clinical and histopathological parameters, and no validated serum biomarkers currently exist. Using a recently developed recombinant antibody microarray platform containing 135 antibodies against 65 mainly immunoregulatory proteins, we screened 240 sera from 64 patients with primary breast cancer. This unique longitudinal sample material was collected from each patient between 0 and 36 mo after the primary operation. The velocity for each serum protein was determined by comparing the samples collected at the primary operation and then 3-6 mo later. A 21-protein signature was identified, using leave-one-out cross-validation together with a backward elimination strategy in a training cohort. This signature was tested and evaluated subsequently in an independent test cohort (prevalidation). The risk of developing distant recurrence after primary operation could be assessed for each patient, using her molecular portraits. The results from this prevalidation study showed that patients could be classified into high- versus low-risk groups for developing metastatic breast cancer with a receiver operating characteristic area under the curve of 0.85. This risk assessment was not dependent on the type of adjuvant therapy received by the patients. Even more importantly, we demonstrated that this protein signature provided an added value compared with conventional clinical parameters. Consequently, we present here a candidate serum biomarker signature able to classify patients with primary breast cancer according to their risk of developing distant recurrence, with an accuracy outperforming current procedures.
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| 19. |
- Carlsson, Anders, et al.
(författare)
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Plasma proteome profiling reveals biomarker patterns associated with prognosis and therapy selection in glioblastoma multiforme patients
- 2010
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Ingår i: Proteomics Clinical Applications. - : Wiley. - 1862-8354 .- 1862-8346. ; 4:6-7, s. 591-602
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Glioblastoma multiforme (GBM) is a frequent and aggressive type of primary brain tumor with a heterogeneous origin. GBM is highly therapy resistant and carries a dismal prognosis for the patient. The purpose of this discovery study was to define candidate plasma biomarker signatures for improved classification and novel means for selecting patients for refined individualized therapy. Experimental design: Here, we have for the first time investigated the applicability of large-scale recombinant antibody-based microarrays, targeting mainly immunoregulatory analytes, for sensitive and selective plasma protein profiling of GBM patients undergoing immunotherapy with autologous IFN-gamma transfected glioma cells Results: This proof-of-concept study showed that candidate plasma protein signatures associated with GBM were outlined that could be used for GBM classification, monitoring the effects of the immunotherapy as well as for stratifying patients according to the beneficial effect of the adopted immunotherapy Further, central key cytokines that could be utilized for optimization and/or refinement of the immunotherapeutic regime were indicated. Conclusions and clinical relevance: Candidate plasma proteins signatures associated with GBM was outlined, that could be used for GBM classification and for pre-operatively stratifying patients according to the beneficial effect of the adopted immunotherapy.
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| 20. |
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| 21. |
- Carlsson, Anders, et al.
(författare)
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Serum protein profiling of systemic lupus erythematosus and systemic sclerosis using recombinant antibody microarrays.
- 2011
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Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 10
-
Tidskriftsartikel (refereegranskat)abstract
- Systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) are two severe autoimmune connective tissue diseases. The fundamental knowledge about their aetiology is limited and the conditions display complex pathogenesis, multifaceted presentations, and unpredictable courses. Despite significant efforts, the lack of fully validated biomarkers enabling diagnosis, classification, and monitoring of disease activity represents significant unmet clinical needs. In this discovery study, we have for the first time used recombinant antibody microarrays for miniaturized, multiplexed serum protein profiling of SLE and SSc, targeting mainly immunoregulatory proteins. The data showed that several candidate SLE-associated multiplexed serum biomarker signatures were delineated, reflecting disease (diagnosis), disease severity (phenotypic subsets) and disease activity. Selected differentially expressed markers were validated using orthogonal assays and a second, independent patient cohort. Further, biomarker signatures differentiating SLE versus SSc were demonstrated, and the observed differences increased with severity of SLE. In contrast, the data showed that the serum profiles of SSc versus healthy controls were more similar. Hence, we have shown that affinity proteomics could be used to de-convolute crude, non-fractionated serum proteomes, extracting molecular portraits of SLE and SSc, further enhancing our fundamental understanding of these complex autoimmune conditions.
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| 22. |
- Carlsson, Anders, et al.
(författare)
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Serum proteome profiling of metastatic breast cancer using recombinant antibody microarrays.
- 2008
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Ingår i: European Journal of Cancer. - : Elsevier BV. - 1879-0852 .- 0959-8049. ; 44:3, s. 472-480
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Tidskriftsartikel (refereegranskat)abstract
- The driving force behind oncoproteomics is to identify biomarker signatures associated with a particular malignancy. Here, we have for the first time used large-scale recombinant scFv antibody microarrays in an attempt to classify metastatic breast cancer versus healthy controls, based on differential protein expression profiling of whole serum samples. Using this multiplexed and miniaturised assay set-up providing pM range sensitivities, breast cancer could be classified with a specificity and sensitivity of 85% based on 129 serum analytes. However, by adopting a condensed 11 analyte biomarker signature, composed of nine non-redundant serum proteins, we were able to distinguish cancer versus healthy serum proteomes with a 95% sensitivity and specificity, respectively. When a subgroup of patients, not receiving anti-inflammatory drugs, was analysed, a novel eight analyte biomarker signature with a further improved predictive power was indicated. In a longer perspective, antibody microarray analysis could provide a tool for the development of improved diagnostics and intensified biomarker discovery for breast cancer patients.
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| 23. |
- Centlow, Magnus, et al.
(författare)
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Differential gene expression analysis of placentas with increased vascular resistance and pre-eclampsia using whole-genome microarrays.
- 2011
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Ingår i: Journal of Pregnancy. - : Hindawi Limited. - 2090-2727 .- 2090-2735. ; 2011:Mar 8
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Tidskriftsartikel (refereegranskat)abstract
- Pre-eclampsia is a pregnancy complication characterized by hypertension and proteinuria. There are several factors associated with an increased risk of developing pre-eclampsia, one of which is increased uterine artery resistance, referred to as "notching". However, some women do not progress into pre-eclampsia whereas others may have a higher risk of doing so. The placenta, central in pre-eclampsia pathology, may express genes associated with either protection or progression into pre-eclampsia. In order to search for genes associated with protection or progression, whole-genome profiling was performed. Placental tissue from 15 controls, 10 pre-eclamptic, 5 pre-eclampsia with notching, and 5 with notching only were analyzed using microarray and antibody microarrays to study some of the same gene product and functionally related ones. The microarray showed 148 genes to be significantly altered between the four groups. In the preeclamptic group compared to notch only, there was increased expression of genes related to chemotaxis and the NF-kappa B pathway and decreased expression of genes related to antigen processing and presentation, such as human leukocyte antigen B. Our results indicate that progression of pre-eclampsia from notching may involve the development of inflammation. Increased expression of antigen-presenting genes, as seen in the notch-only placenta, may prevent this inflammatory response and, thereby, protect the patient from developing pre-eclampsia.
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| 24. |
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| 25. |
- Czemiel Berndtsson, Justyna, et al.
(författare)
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LTH - en attraktiv arbetsplats för både kvinnor och män?
- 2012
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Ingår i: Ledarutvecklingsprogram för kvinnor och män vid Lunds universitet 2010-2011. - 9789163703522 ; AKKA IV, s. 115-145
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 26. |
- Delfani, Payam, et al.
(författare)
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Technical advances of the recombinant antibody microarray technology platform for clinical immunoproteomics
- 2016
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:7
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Tidskriftsartikel (refereegranskat)abstract
- 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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| 27. |
- Dexlin Mellby, Linda, et al.
(författare)
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Design of recombinant antibody microarrays for cell surface membrane proteomics
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7:1, s. 319-327
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Tidskriftsartikel (refereegranskat)abstract
- 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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| 28. |
- Dexlin Mellby, Linda, et al.
(författare)
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Design of recombinant antibody microarrays for membrane protein profiling of cell lysates and tissue extracts.
- 2011
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Ingår i: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 11, s. 1550-1554
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Tidskriftsartikel (refereegranskat)abstract
- Generating global protein expression profiles, including also membrane proteins, will be crucial for our understanding of biological processes in health and disease. In this study, we have expanded our antibody microarray technology platform and designed the first human recombinant antibody microarray for membrane proteins targeting crude cell lysates and tissue extracts. We have optimized all key technological parameters and successfully developed a setup for extracting, labeling and analyzing non-fractionated membrane proteomes under non-denaturing conditions. Finally, the platform was also extended and shown to be compatible with simultaneous profiling of both membrane proteins and water-soluble proteins.
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| 29. |
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| 30. |
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| 31. |
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| 32. |
- Dexlin Mellby, Linda, et al.
(författare)
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Tissue proteome profiling of preeclamptic placenta using recombinant antibody microarrays
- 2010
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Ingår i: Proteomics Clinical Applications. - : Wiley. - 1862-8354 .- 1862-8346. ; 4:10-11, s. 794-807
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: preeclampsia (PE) is a severe, multi-system pregnancy disorder of yet unknown cause, missing means of treatment, and our fundamental understanding of the disease is still impaired. The purpose of this discovery study was to define candidate placenta tissue protein biomarker signatures to further decipher the molecular features of PE.EXPERIMENTAL DESIGN: we used recombinant antibody microarrays for multiplexed protein expression profiling of preeclamptic placenta tissue (n=25) versus normal placenta (n=11) targeting mainly immunoregulatory water-soluble proteins and membrane proteins. Furthermore, the three known subgroups of PE were profiled, including women with early onset preeclampsia and late onset preeclampsia, as well as women with PE and bilateral notching and intrauterine growth restrictions.RESULTS: the data showed that the first generation of candidate PE-associated placenta tissue protein signatures were delineated, indicating that PE (receiver operating characteristics (ROC) AUC value of 0.83) and the subgroups thereof (ROC AUC values ≤ 0.91) could be distinguished. Notably, the data implied that all subgroups, but preeclampsia with bilateral notching and IUGR, could be further classified into novel subsets (ROC AUC values of 1.0) displaying different inflammatory signatures.CONCLUSIONS AND CLINICAL RELEVANCE: we have taken one step further toward de-convoluting the complex features of PE at the molecular level using affinity proteomics.
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| 33. |
- Duroux, Meg, et al.
(författare)
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Light-induced immobilization of biomolecules as an attractive alternative to micro-droplet dispensing-based arraying technologies
- 2008
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Ingår i: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 7:19, s. 1113-1113
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Tidskriftsartikel (refereegranskat)abstract
- The present work shows how UV light-induced molecular immobilisation (LIMI) of biomolecules onto thiol reactive surfaces can be used to make biosensors, without the need for traditional microdispensing technologies. Using LIMI, arrays of biomolecules can be created with a high degree of reproducibility. This technology can be used to circumvent the need for often expensive nano/microdispensing technologies. The ultimate size of the immobilised spots is defined by the focal area of the UV beam, which for a diffraction-limited beam can be less than 1 m in diameter. LIMI has the added benefit that the immobilised molecules will be spatially oriented and covalently bound to the surface. The activity of the sensor molecules is retained. Antibody sensor arrays made using LIMI demonstrated successful antigen binding. In addition, the pattern of immobilised molecules on the surface is not restricted to conventional array formats. The ultimate consequence of the LIMI is that it is possible to write complex protein patterns using bitmaps at high resolution onto substrates. Thus, LIMI of biomolecules provides a new technological platform for biomolecular immobilisation and the potential for replacing present microdispensing arraying technologies.
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| 34. |
- Ellmark, Peter, et al.
(författare)
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Attovial-based antibody nanoarrays.
- 2009
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Ingår i: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 9:24, s. 5406-5413
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Tidskriftsartikel (refereegranskat)abstract
- Antibody array-based technology is a powerful emerging tool in proteomics, but to enable global proteome analysis, antibody array layouts with even higher density has to be developed. To this end, we have further developed the first generation of a nanoarray platform, based on attoliter sized vials, attovials, which we have characterized and used for the detection of complement factor C1q in human serum samples. Finally, we demonstrated proof-of-concept for individual functionalisation of the attovials with a recombinant antibody.
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| 35. |
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| 36. |
- Ellmark, Peter, et al.
(författare)
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Identification of protein expression signatures associated with Helicobacter pylori infection and gastric adenocarcinoma using recombinant antibody microarrays.
- 2006
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Ingår i: Molecular & cellular proteomics : MCP. - 1535-9476 .- 1535-9484. ; 5:9, s. 1638-46
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Tidskriftsartikel (refereegranskat)abstract
- Antibody microarray based technology is a powerful emerging tool in proteomics, target discovery, and differential analysis. Here, we report the first study where recombinant antibody fragments have been used to construct large scale antibody microarrays, composed of 127 different antibodies against mostly immunoregulatory antigens. The arrays were based on single framework recombinant antibody fragments (SinFabs) designed for high on-chip stability and functionality and were used for the analysis of malignant and normal stomach tissue samples from Helicobacter pylori-positive and -negative patients. Our results demonstrate that distinct tumor- as well as infection-associated protein expression signatures could be identified from these complex tissue proteomes, as well as biomarkers such as IL-9, IL-11, and MCP-4, previously not found in these diseases. In a longer perspective, this study may improve the understanding of H. pylori-induced stomach cancer and lead to development of improved diagnostics.
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| 37. |
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| 38. |
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| 39. |
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| 40. |
- Falk, Ronny, 1970-
(författare)
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Systems enabling antibody-mediated proteomics research
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- As many genome sequencing efforts today are completed, we are now provided with the genetic maps for several organisms, including man. With these maps at hand, the scientific focus is now shifting towards investigations of the functionality of proteins. This task is even more challenging than the genomic field since proteins, in contrast to DNA, do not allow themselves to be specifically probed or amplified by easy and generic methods. However, to achieve knowledge regarding protein function, useful information includes where, when and how much certain proteins are expressed in an organism. Such information can be obtained if protein-specific binding molecules are available as tools. One such class of target specific binders are the antibody molecules, traditionally employed in a broad variety of biotechnical applications, including protein localization studies on both cellular and sub cellular levels.In a first serie of studies, new methodology for recombinant production and purification of antigens for generation of antibodies via immunization routes were investigated. Parallel affinity gene fusion-based expression systems were used for evaluation of different concepts for production of antigen and post-immunization antibody purification. Carefully designed protein antigens from different organisms were produced and used to raise antisera which were affinity purified on their respective antigens to obtain highly specific polyclonal antibodies (monospecific antibodies). One of the constructed expression systems includes an affinity handle, ZSPA-1, previously selected from a combinatorial protein library for its capacity to selectively bind protein A. This allows for convenient, non IgG-dependent, affinity purification of proteins on conventional protein A resins.A strategy where highly target specific antibody preparations could be affinity purified in a more streamlined setup is also presented. By this strategy it was possible to fractionate antibodies showing reactivity to different parts of the antigen into separate fractions. This resulted in affinity purified antibodies showing monospecific but still multi-epitope reactivity. Purified monospecific antibodies were used in different studies including Western blot immunofluorescence and recovery applications. For affinity purification of endogenous target from its native surrounding a selective elution strategy where the recombinant antigen was used to competitively elute the captured target was developed.
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| 41. |
- Gantelius, Jesper, 1980-
(författare)
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Novel diagnostic microarray assay formats towards comprehensive on-site analysis
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent. In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation.
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| 42. |
- Gerdtsson, Anna S, et al.
(författare)
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Evaluation of Solid Supports for Slide- and Well-Based Recombinant Antibody Microarrays
- 2016
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Ingår i: Microarrays. - : MDPI AG. - 2076-3905. ; 5:2
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Tidskriftsartikel (refereegranskat)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.
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| 43. |
- Gerdtsson, Anna Sandström, et al.
(författare)
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Plasma protein profiling in a stage defined pancreatic cancer cohort – Implications for early diagnosis
- 2016
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Ingår i: Molecular Oncology. - : Wiley. - 1574-7891. ; 10:8, s. 1305-1316
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Tidskriftsartikel (refereegranskat)abstract
- Pancreatic ductal adenocarcinoma (PDAC) is a disease where detection preceding clinical symptoms significantly increases the life expectancy of patients. In this study, a recombinant antibody microarray platform was used to analyze 213 Chinese plasma samples from PDAC patients and normal control (NC) individuals. The cohort was stratified according to disease stage, i.e. resectable disease (stage I/II), locally advanced (stage III) and metastatic disease (stage IV). Support vector machine analysis showed that all PDAC stages could be discriminated from controls and that the accuracy increased with disease progression, from stage I to IV. Patients with stage I/II PDAC could be discriminated from NC with high accuracy based on a plasma protein signature, indicating a possibility for early diagnosis and increased detection rate of surgically resectable tumors.
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| 44. |
- Ghatnekar-Nilsson, Sara, et al.
(författare)
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Design of atto-vial based recombinant antibody arrays combined with a planar wave-guide detection system
- 2007
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Ingår i: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 7:4, s. 540-547
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Tidskriftsartikel (refereegranskat)abstract
- Antibody microarray is a rapidly emerging, powerful approach with great promise within high-throughput proteomics. However, before a truly proteome-wide analysis can be performed, the antibody array format needs to be miniaturized even further in order to enable ultradense arrays to be fabricated. To this end, we have designed and generated proof-of-concept for the first generation of an atto-vial based recombinant antibody array platform. Briefly, we have designed a novel nanostructured substrate using electron beam lithography. Vials, ranging in volume/size from 6 (200 nm in diameter) to 4000 aL (5 mu m in diameter), were fabricated. Human recombinant single-chain Fv antibody fragments, microarray adopted by design, were used as probes. The set-up was interfaced with planar wave-guide technology for evanescant field fluorescence detection. The results showed that protein analytes could be specifically detected in the subzeptomole range for pure systems, using vials down to 57 aL. Further, low-abundant (pg/mL) protein analytes could be detected in directly labeled complex proteomes, such as human whole serum, using 157 aL-vials. Taken together, these results outline the potential of the atto-vial array set-up for miniaturized affinity proteomics-based approaches.
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| 45. |
- Gloriam, David E., et al.
(författare)
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A Community Standard Format for the Representation of Protein Affinity Reagents
- 2010
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 9:1, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one online warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site. Molecular & Cellular Proteomics 9: 1-10, 2010.
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| 46. |
- Grannas, Karin, 1983-
(författare)
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Improvements and Applications of in situ Proximity Ligation Assays
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The cells building up the human body is in constant communication with each other. This communication is done through large complex networks of signaling pathways for inter- and intracellular signal transduction. The signaling activity regulates many important processes, for example cell death, proliferation and differentiation. Information within the signaling networks is communicated over the cell membrane, through the cytoplasm and entering the nucleus by protein activities such as protein-protein interactions (PPIs) and post translation modifications (PTMs). The cells adapts to their own environment, responding to multiple stimuli from their surroundings. This in combination with memory of previous responses, difference in cell cycles stages and sometimes altered genetic background generates heterogeneous cell populations in which every cell is slightly different from its neighbor. This calls for methods to study the activity of endogenous proteins in individual cells within a population.In situ proximity ligation assay (in situ PLA) was originally developed to visualize interaction between endogenous proteins in fixed cells and tissue and can also be applied to detect PTMs. This thesis describe the application of in situ PLA to study PPIs in signaling pathways and the work to further develop and improve techniques for proximity dependent detection. In paper I in situ PLA is used to study cross talk between the Hippo and the TGFβ signaling pathways. The study shows the complex formation by the transcription co-factors of the Hippo pathway, Yap and Taz, and the main effectors of the TGFβ pathway Smad2/3. Furthermore the density dependent localization of the interaction is described.Paper II presents a new version of the in situ PLA probes for simultaneous detection of multiple complexes. Visualization of various complexes involving EGFR, Her2 and Her3 is presented as a proof of concept.The efficiency of in situ PLA is limited by several factors, one being the design of PLA probes and oligonucleotide systems. Even upon proximal binding of the probes there is a risk of formation of non-circular ligation products, which cannot be amplified and detected. In Paper III two new PLA probes are presented aiming to reduce the formation of non-circular ligation product and hence increase the detection efficiency of in situ PLA.Paper IV presents a new method for detection of protein complexes and phosphorylation; proxHCR. ProxHCR combines signal amplification by enzyme free hybridization chain reaction (HCR) with the requirement of proximal binding of two affinity probes. As a proof of principle the method is used to detect multiple complexes and protein phosphorylation in fixed cells and tissue.
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| 47. |
- Gunnarsson, Anders, 1981, et al.
(författare)
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Kinetics of Ligand Binding to Membrane Receptors from Equilibrium Fluctuation Analysis of Single Binding Events
- 2011
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 133:38, s. 14852-14855
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Tidskriftsartikel (refereegranskat)abstract
- Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.
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| 48. |
- Gustavsson, Elin, et al.
(författare)
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Surrogate antigens as targets for proteome-wide binder selection
- 2011
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 28:4, s. 302-311
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Tidskriftsartikel (refereegranskat)abstract
- In the last decade, many initiatives have been taken to develop antibodies for proteome-wide studies, as well as characterization and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to conventional antibody generation by immunization and hybridoma technology, since it is an unlimited resource of affinity reagents without batch-to-batch variation and is amendable for high throughput. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens in phage selectionsfor the retrieval of target specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.
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| 49. |
- Hansson, Ulla-Britt, et al.
(författare)
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Antigen-binding sites dominate the surface properties of antibodies
- 1996
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Ingår i: Immunotechnology. - 1380-2933. ; 2:4, s. 276-276
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Tidskriftsartikel (refereegranskat)abstract
- We have found a remarkable relationship between the specificity of antibodies and their chromatographic behavious upon liquid-liquid partition chromatography (LLPC). Well characterized human and murine monoclonal antibodies and Fab/Fv fragments thereof as well as mouse/human chimeric antibodies were employed. While, lgG 1, 2 and 4 antibodies with identical specificities (affinity constants) have identical partition properties, lgG antibodies with different partition properties reacted with different partition epitopes or had different affinities against the same epitope. Hence, the surface properties of the antigen binding sites dominate over all other surfaces of the free antibody molecule. LLPC may also be used to detect conformational changes occuring upon binding of antigen by antibody. Antigen-antibody complexes formed by different lgG antibodies against a large antigen like HSA all had similar surface properties. different from those of both antigen and antibody. In contrast, the surface properties of complexes formed by small antigens haptens are related to those of the lgG antibody. In addition, antigen-antibody complexes were found to have similar surface properties irrespective of the molar ratio of antigen to antibody at which the complexes had been formed.
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| 50. |
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