Determining a receptor-ligand interaction useful in e.g. drug screening involves repetitive application of reversibly binding fluorescent ligands with fast association-dissociation kinetics
NOVELTY - Determining a receptor-ligand interaction involves repeatedly contacting a cell comprising a receptor, with a ligand, of the receptor in which the ligand has a fluorescence labeling group and binds reversibly to the receptor under conditions in which the ligand associates with the receptor and then dissociates; and then repeatedly monitoring the fluorescence of the cell. USE - In a screening procedure for the identification and/or characterization of pharmaceutical drug candidate molecules and for determining a receptor-ligand interaction to allow sensitive measurements on single cells carried out in a microfluidic structure or on a chip (claimed) and can be used to characterize single or multiple cells by determining the presence of receptors or other proteins on the surface of the cells e.g. native cells, multiple cells like tissue sections, genetically modified cells or with cells immobilized on a solid surface, or in a matrix like a gel, or with cells in a liquid medium. ADVANTAGE - The method enables pharmacological investigations to be performed on single cells expressing the natural amount of receptor, which usually is very difficult due to photobleaching. The invention performs fluorescence binding assays, the reversible sequential binding assay that solves problems associated with previous measurements of ligand-receptor interactions by applying repetitively-completely reversible and specific fluorescently labeled ligands with fast association-dissociation kinetics. The new approach overcomes problems related to photobleaching so that cells expressing low amounts of receptors down to the single-molecule level can be investigated. The high sensitivity of the method allows the detection of little abundant proteins, thus enabling characterization of native cells. It can be applied even in the case of strong photobleaching opening the possibility to investigate single cells with low receptor concentrations comparable to native conditions in tissue cells. Furthermore, the influence of stimuli, such as ligands, can be rapidly and repetitively investigated using our reversible binding assay. A key advantage of the measurement approach is that pharmacological investigations can be performed on single cells expressing the natural amount of receptor, which usually is very difficult due to photobleaching. Using repetitive measurements, binding parameters can be extracted and investigations can be performed even when photobleaching becomes extremely strong as it is the case at lowest expression levels. Ultimately, single-molecule sensitivity can be achieved as illustrated on the nAChR. This opens the possibility to perform pharmacological investigations on tissue cells expressing minimal amounts of membrane receptors. By performing repetitive measurements, tenth of image sequences can be acquired to collect extensive statistics within a single experiment. High-throughput screening of potential drugs can be performed on a single cell with a rate only depending on the association-dissociation cycle time, consuming only minimal amount of testing components. One can expect that the use of ligands with faster kinetics could reduce this time down to 5 minutes. In addition, the use of more complex microstructures enables complete binding assays on a single cell or increasing statistics by parallel approaches using only some hundred nano liters of cell suspension. This way, screening more than 250 ligands per day using one single cell extracted from an animal tissue or obtained from a body fluid is a realistic goal.
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Record created on 2010-03-17, modified on 2016-08-08