| 1. |
- Ayoglu, Burcu, et al.
(författare)
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Multiplexed antigen bead arrays for the assessment of antibody selectivity and epitope mapping
- 2018
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Ingår i: Epitope Mapping Protocols. - New York, NY : Humana Press Inc.. ; , s. 239-248
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Bokkapitel (refereegranskat)abstract
- With the increasing number of binding reagents for affinity-based investigations of the human proteome, high-throughput tools for the characterization of the used reagents become essential. For the analysis of binding selectivity, bead-based antigen arrays offer a miniaturized and parallelized assay platform to meet such needs, as they enable two-dimensional multiplexing to analyze up to 384 samples against up to 500 analytes in a single round of analysis. In this chapter, we describe our protocols for the generation of multiplex bead arrays built on immobilized protein fragments, as well as biotinylated peptides. Combined together, these two versions of antigen arrays offer a versatile approach for multiplexed characterization of antibody binding selectivity, off-target interactions, as well as mapping for the amino acids of epitopes involved in antibody binding.
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| 2. |
- Bendes, Annika, et al.
(författare)
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Bead-Based Assays for Validating Proteomic Profiles in Body Fluids
- 2021
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Ingår i: Protein Microarrays for Disease Analysis. - New York, NY : Springer Nature. ; , s. 65-78
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Bokkapitel (refereegranskat)abstract
- Protein biomarkers in biological fluids represent an important resource for improving the clinical management of diseases. Current proteomics technologies are capable of performing high-throughput and multiplex profiling in different types of fluids, often leading to the shortlisting of tens of candidate biomarkers per study. However, before reaching any clinical setting, these discoveries require thorough validation and an assay that would be suitable for routine analyses. In the path from biomarker discovery to validation, the performance of the assay implemented for the intended protein quantification is extremely critical toward achieving reliable and reproducible results. Development of robust sandwich immunoassays for individual candidates is challenging and labor and resource intensive, and multiplies when evaluating a panel of interesting candidates at the same time. Here we describe a versatile pipeline that facilitates the systematic and parallel development of multiple sandwich immunoassays using a bead-based technology.
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| 3. |
- Birgersson, Elin, et al.
(författare)
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Bead-based and multiplexed immunoassays for protein profiling via sequential affinity capture
- 2017
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Ingår i: Serum/Plasma Proteomics. - New York, NY : Humana Press. - 9781493970568 ; , s. 45-54
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Bokkapitel (refereegranskat)abstract
- Antibody microarrays offer high-throughput immunoassays for multiplexed analyses of clinical samples. For such approaches, samples are either labeled in solution to enable a direct readout on the single binder assay format or detected by matched pairs of capture and detection antibodies in dual binder assay format, also known as sandwich assays. Aiming to benefit from the flexibility and capacity offered by single binder assay readout and the specificity and sensitivity of dual binder assays, we developed a multiplexed dual binder procedure that is based on a sequential, rather than combined, antigen binding. The method, entitled dual capture assay (DCA), is composed of an initial antigen capture by antibodies on beads, followed by labeling of captured protein targets on beads, combinatorial elution steps at high and low pH, and a readout using a secondary bead array. Compared to classical single binder assays, the described method demonstrated several advantages such as reduced contribution of off-target binding, lower noise levels, and improved correlation when comparing with clinical reference values. This procedure describes a novel and versatile immunoassay strategy for proteome profiling in body fluids.
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| 4. |
- Iglesias, Maria Jesus, et al.
(författare)
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Affinity Proteomics Assays for Cardiovascular and Atherosclerotic Disease Biomarkers
- 2021
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Ingår i: Protein Microarrays for Disease Analysis. - New York, NY : Springer Nature. ; 2344, s. 163-179
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Bokkapitel (refereegranskat)abstract
- Systematic exploration of the dynamic human plasma proteome enables the discovery of novel protein biomarkers. Using state-of-the-art technologies holds the promise to facilitate a better diagnosis and risk prediction of diseases. Cardiovascular disease (CVD) pathophysiology is characterized for unbalancing of processes such as vascular inflammation, endothelial dysfunction, or lipid profiles among others. Such processes have a direct impact on the dynamic and complex composition of blood and hence the plasma proteome. Therefore, the study of the plasma proteome comprises an excellent exploratory source of biomarker research particularly for CVD. We describe the protocol for performing the discovery of protein biomarker candidates using the suspension bead array technology. The process does not require depletion steps to remove abundant proteins and consumes only a few microliters of sample from the body fluid of interest. The approach is scalable to measure many analytes as well as large numbers of samples. Moreover, we describe a bead-assisted antibody-labeling process that helps to develop quantitative assays for validation purposes and facilitate the translation of the identified candidates into clinical studies.
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| 5. |
- Schwenk, Jochen M, et al.
(författare)
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Assessment of antibody specificity using suspension bead arrays.
- 2011
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Springer. ; , s. 183-189
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Bokkapitel (refereegranskat)abstract
- With the increasing collection of affinity reagents, their validation in terms of functionality and binding specificity becomes a challenge. To match this growing need, miniaturized and parallelized platforms have become available to corroborate the applicability for a broad range of binder scaffolds. Among the -commonly used systems, planar microarrays have been a platform of choice for a long time but alternative systems are emerging, of which one is based on color-coded beads for the creation of arrays in suspension. The latter systems offer to perform a two-dimensional multiplexing by now analyzing up to 384 samples against up to 500 analytes in a single experiment. While the analyte parameter is flexible in terms of its composition, an extended screening can be facilitated without the need to set up a microarray production facility.
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| 6. |
- Sjöberg, Ronald, et al.
(författare)
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High-density antigen microarrays for the assessment of antibody selectivity and off-target binding
- 2018
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Ingår i: Epitope Mapping Protocols. - New York, NY : Humana Press Inc.. - 9781493978397 ; , s. 231-238
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Bokkapitel (refereegranskat)abstract
- With the increasing availability of collections of antibodies, their evaluation in terms of binding selectivity becomes an important but challenging task. Planar antigen microarrays are very suitable tools to address this task and provide a powerful proteomics platform for the characterization of the binding selectivity of antibodies toward thousands of antigens in parallel. In this chapter, we describe our in-house developed procedures for the generation of high-density planar antigen microarrays with over 21,000 features. We also provide the details of the assay protocol, which we routinely use for the assessment of binding selectivity of the polyclonal antibodies generated within the Human Protein Atlas.
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