Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Patent
1998-10-01
2000-10-31
Russel, Jeffrey E.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
422239, 436166, 5303913, 530395, 530402, 536 111, 536 221, C07K 113, G01N 33533, G01N 33535, G01N 33543
Patent
active
061400904
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for the labeling of molecules and a device for performing the method.
The modification of proteins, polycarboxylic acids and other biopolymers by labeling with a reporter molecule which may be luminescent, in particular, is of great importance to many fields of applications, such as immunology, cellular biology or the examination of molecular interactions such as those occurring, for example, between ligands and receptors, or proteins and nucleic acids. The use of fluorescence-labeled molecules is not limited to the performance of assays. Fluorescence-labeling does not only gain importance to the extent to which radioactive assay methods become fewer. The labeled substances are employed in fluorescence-immunological assays (e.g., ELISA) or in various fluorescence techniques, such as fluorescence microscopy, fluorescence polarization and fluorescence life measurements, or fluorescence correlation spectroscopy.
Methods for the fluorescence-labeling of biopolymers have been known. Thus, it is possible to couple reactive dyes, such as the sulfonyl chloride derivative of sulforhodamine 101 (Texas Red.RTM.), covalently to amino groups of the protein by directly adding them to a protein solution. However, one disadvantage is the rapid hydrolysis of the reactive dye in aqueous solution at room temperature since the dye molecules are prematurely inactivated before reacting as desired with the molecules to be labeled. Thus, such coupling reactions are preferably performed at 4.degree. C. (Molecular Probes MP 353 Dec. 21, 1993). The coupling times are between one and four hours depending on the reactive dye chosen and the protein concentration (Titus et al., Journal of Immunological Methods, 50, 193-204, 1992). Rinderknecht (Experentia 16, 430, 1960) describes another fluorescence-labeling procedure using Celite adsorbed fluorescent dyes which are added to the solution containing the substances to be labeled. By providing the fluorescent dyes on such an increased surface area, coupling times of 30 minutes can be achieved. After the reaction, the Celite material is separated off by centrifugation.
In particular, the above mentioned methods have a drawback in that the performance of the methods requires a person skilled in dealing with hydrolysis-sensitive substances and an equipment for preventing health hazards, such as an exhaust hood. In addition, it has been found disadvantageous that the reproducibility of the methods is highly dependent on accidental circumstances, such as the skill of those performing the reaction.
The object of the invention has been to provide an industrially applicable method for the labeling, especially fluorescence-labeling, of molecules which can be reproducibly performed by unskilled persons or automatically.
The object of the invention is achieved by a method for the labeling of molecules by reacting the molecules in a reaction chamber in which a solid phase (matrix) loaded with a reactive component is provided, and after the coupling has taken place, the labeled molecules leave the reaction chamber through a porous means; and a device having a cavity, in the lumen of which at least one porous means and a matrix loaded with a reactive component are provide, for performing the method according to the invention.
The method according to the invention is characterized in that the molecules to be labeled enter a reaction chamber in which a matrix loaded with a reactive component is provided, and after the coupling has taken place, the fluorescence-labeled molecules leave the reaction chamber through a porous means.
The labeling reaction is preferably a fluorescence-labeling reaction. The method according to the invention is also advantageously suitable for the labeling with various other reagents or substances which are detectable as such or contain a detectable portion. Thus, according to the invention, the labeling may be done with haptens which are susceptible to immunological detection. Similarly, the method according to the invention may also be
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Bauer Gunter
Kreuzer Oliver
Meyer-Almes Franz-Josef
Schierholz Bernd
Werner Irmgard
EVOTEC BioSystems AG
Russel Jeffrey E.
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