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Circumventing the requirement of binding saturation for receptor quantification using interaction kinetic extrapolation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Biomedical Radiation Sciences.
2011 (English)In: Nuclear medicine communications, ISSN 0143-3636, E-ISSN 1473-5628, Vol. 32, no 9, 863-867 p.Article in journal (Refereed) Published
Abstract [en]

Quantification of the number of receptors per cell (NRPC) is important when assessing whether a tumor surface biomarker is suitable for medical imaging. One common method for NPRC quantification is to use a binding saturation assay, which is time consuming and requires large amounts of reagents. The aim of this study was to evaluate an alternative method based on kinetic extrapolation (KEX) and compare it with the classical manual saturation technique with regard to accuracy as well as time and reagent consumption. Epidermal growth factor receptor (EGFR) and HER2 receptor surface expression were quantified on five tumor cell lines using three (125)I-labeled and (131)I-labeled ligands (cetuximab and EGF for EGFR, trastuzumab for HER2 receptor) for both techniques. The KEX method involved interaction measurements in the LigandTracer, followed by KEX through computerized real-time interaction analysis to correct for nonsaturation on cells. Variability and NRPC estimates of the EGFR and HER2 receptor levels using the KEX method were comparable with the results from the classical saturation technique. However, the ligand consumption for the KEX method was 26-46% of the classical saturation technique. Furthermore, the KEX method reduced the workload radically. From the observations described in this study, we believe that the KEX method enables fast, credible, and easy NRPC quantification with a reduction in reagent consumption.

Place, publisher, year, edition, pages
2011. Vol. 32, no 9, 863-867 p.
Keyword [en]
kinetics, ligand-receptor interaction, receptor quantification
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-158879DOI: 10.1097/MNM.0b013e3283483e1cISI: 000294345300015OAI: oai:DiVA.org:uu-158879DiVA: diva2:442031
Available from: 2011-09-20 Created: 2011-09-19 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions: Resolving How the Epidermal Growth Factor Binds to Its Receptor
Open this publication in new window or tab >>Novel Methods for Analysis of Heterogeneous Protein-Cell Interactions: Resolving How the Epidermal Growth Factor Binds to Its Receptor
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cells are complex biological units with advanced signalling systems, a dynamic capacity to adapt to its environment, and the ability to divide and grow. In fact, they are of such high level of complexity that it has deemed extremely difficult or even impossible to completely understand cells as complete units. The search for comprehending the cell has instead been divided into small, relatively isolated research fields, in which simplified models are used to explain cell biology. The result produced through these reductionistic investigations is integral for our current description of biology. However, there comes a time when it is possible to go beyond such simplifications and investigate cell biology at a higher level of complexity. That time is now.

This thesis describes the development of mathematical tools to investigate intricate biological systems, with focus on heterogeneous protein interactions. By the use of simulations, real-time measurements and kinetic fits, standard assays for specificity measurements and receptor quantification were scrutinized in order to find optimal experimental settings and reduce labour time as well as reagent cost. A novel analysis platform, called Interaction Map, was characterized and applied on several types of interactions. Interaction Map decomposes a time-resolved binding curve and presents information on the kinetics and magnitude of each interaction that contributed to the curve. This provides a greater understanding of parallel interactions involved in the same biological system, such as a cell. The heterogeneity of the epidermal growth factor receptor (EGFR) system was investigated with Interaction Map applied on data from the instrument LigandTracer, together with complementing manual assays. By further introducing disturbances to the system, such as tyrosine kinase inhibitors and variation in temperature, information was obtained about dimerization, internalization and degradation rates.

In the long term, analysis of binding kinetics and combinations of parallel interactions can improve the understanding of complex biomolecular mechanisms in cells and may explain some of the differences observed between cell lines, medical treatments and groups of patients.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 854
Keyword
Heterogeneity, Kinetics, EGFR, HER2, LigandTracer, Interaction Map, Internalization, Specificity
National Category
Medical Biotechnology Cell and Molecular Biology
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-183872 (URN)978-91-554-8570-2 (ISBN)
Public defence
2013-02-15, Rudbeck Hall, Rudbeck Laboratory, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-01-24 Created: 2012-11-05 Last updated: 2013-02-11Bibliographically approved

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