Uppsala, Sweden
Uppsala, Sweden

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Patent
Ridgeview Instruments AB | Date: 2017-07-12

This invention relates to a solid support suitable for improving the detection of how different species interact with each other. The solid support has multiple defined areas divided by low dividers, and optionally independent containers divided by high dividers, each container having at least two defined areas. The solid support is typically used in measurements where the total amount of liquid is temporarily reduced during measurement. The solid support extends the possibilities of the measurement system.


Grant
Agency: European Commission | Branch: H2020 | Program: MSCA-ITN-ETN | Phase: MSCA-ITN-2015-ETN | Award Amount: 3.84M | Year: 2016

The development of effective novel drugs - especially for rare and neglected diseases - is one of the biggest challenges of the upcoming decades, as illustrated by the recent Ebola outbreak. Moreover, European innovation in new drug registrations is dramatically falling behind compared to the US and Asia. The principal aim of the AEGIS ITN is to implement the first comprehensive, intersectoral cross-disciplinary and structured curriculum for doctoral students in the European Research Area by establishing a unique training platform for the next generation of European researchers in early drug discovery. A significant added value is provided through networking with key European pharmaceutical companies. A key research aim of AEGIS is improving the efficiency and success of early stage drug development by combining innovative methods and techniques to tackle difficult but promising targets (i.e. protein-protein interactions), as potentially valuable drug targets are often neglected due to the high risk associated with their validation. The consortium joins leading academic and industry researchers in an open innovation environment for innovative drug development in Europe. It is supported by several additional partners and stakeholders in the field. Integrated training of the fellows takes place at the host institute and by secondments, research schools and individual training within the AEGIS network. The scientific training includes complementary skills, management, intellectual property rights, fund raising, communication and career planning. AEGIS will improve the availability of a highly skilled workforce for European industries and research, greatly enhance the employability and the career perspectives of young researchers for academia as well as for industry, and will be the seed of a sustainable development in innovative drug discovery, in particular for rare and neglected diseases.


Orlova A.,Uppsala University | Hofstrom C.,KTH Royal Institute of Technology | Strand J.,Uppsala University | Varasteh Z.,Uppsala University | And 5 more authors.
European Journal of Nuclear Medicine and Molecular Imaging | Year: 2013

Purpose: Radionuclide imaging of insulin-like growth factor type 1 receptor (IGF-1R) expression in tumours might be used for selection of patients who would benefit from IGF-1R-targeted therapy. We have previously shown the feasibility of IGF-1R imaging using the Affibody molecule 111In-DOTA- His6-ZIGF1R:4551. The use of 99mTc instead of 111In should improve sensitivity and resolution of imaging, and reduce the dose burden to patients. We hypothesized that inclusion of a HEHEHE tag instead of a His6 tag in ZIGF1R:4551 would permit its convenient purification using IMAC, enable labelling with [99mTc(CO) 3]+, and improve its biodistribution. Methods: Z IGF1R:4551 was expressed with a HEHEHE tag in the N terminus. The resulting (HE)3-ZIGF1R:4551 construct was labelled with [99mTc(CO)3]+. Targeting of IGF-1R-expressing cells using [99mTc(CO)3]+-(HE) 3-ZIGF1R:4551 was evaluated in vitro and in vivo. Results: (HE)3-ZIGF1R:4551 was stably labelled with 99mTc with preserved specific binding to IGF-1R-expressing DU-145 prostate cancer cells in vitro. In mice, [99mTc(CO)3] +-(HE)3-ZIGF1R:4551 accumulated in IGF-1R-expressing organs (pancreas, stomach, lung and salivary gland). [ 99mTc(CO)3]+-(HE)3-Z IGF1R:4551 demonstrated 3.6-fold lower accumulation in the liver and spleen than 111In-DOTA-ZIGF1R:4551. In NMRI nu/nu mice with DU-145 prostate cancer xenografts, the tumour uptake was 1.32 ± 0.11 %ID/g and the tumour-to-blood ratio was 4.4 ± 0.3 at 8 h after injection. The xenografts were visualized using a gamma camera 6 h after injection. Conclusion: 99mTc(CO)3]+-(HE) 3-ZIGF1R:4551 is a promising candidate for visualization of IGF-1R expression in malignant tumours. © 2012 Springer-Verlag Berlin Heidelberg.


McGrath T.F.,Queen's University of Belfast | Andersson K.,Ridgeview Instruments AB | Andersson K.,Uppsala University | Campbell K.,Queen's University of Belfast | And 2 more authors.
Biosensors and Bioelectronics | Year: 2013

A prototype fluorescent based biosensor has been developed for the antibody based detection of food related contaminants. Its performance was characterised and showed a typical antibody binding signal of 200-2000. mV, a short term noise of 9.1. mV, and baseline slope of -0.016. mV/s over 4. h. Bulk signal detection repeatability (n=23) and reproducibility (n=3) were less than 2.4%CV. The biosensor detection unit was evaluated using two food related model systems proving its ability to monitor both binding using commercial products and inhibition through the development of an assay. This assay development potential was evaluated by observing the biosensor's performance whilst appraising several labelled antibody and glass slide configurations. The molecular interaction between biotin and an anti-biotin antibody was shown to be inhibited by 41% due to the presence of biotin in a sample. A food toxin (domoic acid) calibration curve was produced, with %CVs ranging from 2.7 to 7.8%, and a midpoint of approximately 17. ng/ml with further optimisation possible. The ultimate aim of this study was to demonstrate the working principles of this innovative biosensor as a potential portable tool with the opportunity of interchangeable assays. The biosensor design is applicable for the requirements of routine food contaminant analysis, with respect to performance, functionality and cost. © 2012 Elsevier B.V.


Bjorkelund H.,Uppsala University | Bjorkelund H.,Ridgeview Instruments AB | Gedda L.,Uppsala University | Andersson K.,Uppsala University | Andersson K.,Ridgeview Instruments AB
Journal of Molecular Recognition | Year: 2011

The competition measurement using simultaneous incubation of labeled and unlabeled Ligand is a common method to assess the specificity of a biomolecular interaction. In this paper we show that invalid assumptions about the interactions may lead to improper experimental setups which in turn can result in inaccurate conclusions about the specificity. To improve understanding of competition measurements, simulations in MATLAB as well as real-time interaction analysis using LigandTracer have been performed. We show that use of a concentration of unlabeled Ligand of at least 10 × KD is necessary for assay accuracy. Increasing the incubation time to assure equilibrium, adding a pre-incubation phase, and a general understanding of the reversibility of an interaction may also improve the reliability of the measurement and the conclusions drawn about specificity. These findings may lower the risk of false negative results as well as reducing the amount of reagent needed. Copyright © 2010 John Wiley & Sons, Ltd.


Tolmachev V.,Uppsala University | Orlova A.,Uppsala University | Andersson K.,Ridgeview Instruments AB
Methods in Molecular Biology | Year: 2014

The use of radionuclide labels allows to study the pharmacokinetics of monoclonal antibodies, to control the specificity of their targeting and to monitor the response to an antibody treatment with high accuracy. Selection of label depends on the processing of an antibody after binding to an antigen, and on the character of information to be derived from the study (distribution of antibody in the extracellular space, target occupancy or determination of sites of metabolism). This chapter provides protocols for labelling of antibodies with iodine-125 (suitable also for other radioisotopes of iodine) and with indium-111. Since radiolabelling might damage and reduce the immunoreactive fraction and/or affinity of an antibody, the methods for assessment of these characteristics of an antibody are provided for control. © 2014 Springer Science+Business Media, LLC.


Borzecka K.,Nencki Institute of Experimental Biology | Plociennikowska A.,Nencki Institute of Experimental Biology | Bjorkelund H.,Ridgeview Instruments AB | Bjorkelund H.,Uppsala University | And 2 more authors.
Mediators of Inflammation | Year: 2013

Activation of macrophages with lipopolysaccharide (LPS) involves a sequential engagement of serum LPS-binding protein (LBP), plasma membrane CD14, and TLR4/MD-2 signaling complex. We analyzed participation of CD14 in TNF-α production stimulated with 1-1000 ng/mL of smooth or rough LPS (sLPS or rLPS) and in sLPS binding to RAW264 and J744 cells. CD14 was indispensable for TNF-α generation induced by a low concentration, 1 ng/mL, of sLPS and rLPS. At higher doses of both LPS forms (100-1000 ng/mL), TNF-α release required CD14 to much lower extent. Among the two forms of LPS, rLPS-induced TNF-α production was less CD14-dependent and could proceed in the absence of serum as an LBP source. On the other hand, the involvement of CD14 was crucial for the binding of 1000 ng/mL of sLPS judging from an inhibitory effect of the anti-CD14 antibody. The binding of sLPS was also strongly inhibited by dextran sulfate, a competitive ligand of scavenger receptors (SR). In the presence of dextran sulfate, sLPS-induced production of TNF-α was upregulated about 1.6-fold. The data indicate that CD14 together with SR participates in the binding of high doses of sLPS. However, CD14 contribution to TNF-α production induced by high concentrations of sLPS and rLPS can be limited. © 2013 Kinga Borzȩcka et al.


Bjorkelund H.,Ridgeview Instruments AB | Bjorkelund H.,Uppsala University | Gedda L.,Uppsala University | Andersson K.,Ridgeview Instruments AB | Andersson K.,Uppsala University
PLoS ONE | Year: 2011

The interaction of the epidermal growth factor (EGF) with its receptor (EGFR) is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of 125I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracerH. Highly repeatable and precise measurements show that the overall apparent affinity of the 125I-EGF - EGFR interaction is greatly dependent on cell line at normal culturing conditions, ranging from K D≈200 pM on SKBR3 cells to K D≈8 nM on A431 cells. The 125I-EGF - EGFR binding curves (irrespective of cell line) have strong signs of multiple simultaneous interactions. Furthermore, for the cell lines A431 and SKOV3, gefitinib treatment increases the 125I-EGF - EGFR affinity, in particular when the cells are starved. The 125I-EGF - EGFR interaction on cell line U343 is sensitive to starvation while as on SKBR3 it is insensitive to gefitinib and starvation. The intriguing pattern of the binding characteristics proves that the cellular context is important when deciphering how EGF interacts with EGFR. From a general perspective, care is advisable when generalizing ligand-receptor interaction results across multiple cell-lines. © 2011 Björkelund et al.


Stenberg J.,Uppsala University | Stenberg J.,Ridgeview Instruments AB | Spiegelberg D.,Uppsala University | Karlsson H.,Uppsala University | Nestor M.,Uppsala University
Nuclear Medicine and Biology | Year: 2014

Medical imaging by use of immunotargeting generally relies on a labeled molecule binding to a specific target on the cell surface. It is important to utilize both cell-based and time-resolved binding assays in order to understand the properties of such molecular interactions in a relevant setting.In this report we describe the detailed characterization of the interaction properties for AbD15179, a promising CD44v6-targeting antibody fragment for radio-immunotargeting. Influence of labeling and cell-line model on the protein interaction kinetics was assessed using three different labeling approaches (111In, 125I and FITC) on three different squamous carcinoma cell lines. Interactions were measured using time-resolved assays on living cells, and further analyzed with Interaction Map®.Results demonstrated a general biphasic appearance of a high- and a low-affinity binding event in all cases. The relative contribution from these two interactions differed between conjugates. For 125I-Fab, the population of low-affinity binders could be significantly increased by extending the chloramine T exposure during labeling, whereas the 111In-labeling predominantly resulted in a high-affinity interaction. Interactions were also shown to be cell line dependent, with e.g. SCC-25 cells generally mediating a faster dissociation of conjugates compared to the other cell lines.In conclusion, we report both cell line dependent and labeling associated variations in interaction kinetics for AbD15179 binding to CD44v6. This has implications for cell-based kinetic assays and applications based on labeled conjugates in general, as well as in a clinical setting, where each individual tumor may create different kinetic profiles for the same conjugate. © 2014 Elsevier Inc.


Patent
Ridgeview Instruments AB | Date: 2011-03-16

A method of analysis or diagnosis of solid objects is based on real-time detection of how predefined probes interact with structures present on or in the solid object combined with the calculation of how the recorded binding curves of said probes are distributed in terms of interaction properties. The interaction properties are input to a classification algorithm which automatically determines statues of the solid object. The method is particularly advantageous for solid biological objects like tissue slices combined with antibody probes, said antibody recognizing receptors known to be over-expressed in disease states on said tissue slice.

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