DE19818485B4 - Reaction vessels coated with a cis-diol-specific affinity medium, as well as processes for their preparation - Google Patents

Abstract

Reaction vessel whose Surface coated is with a cis-diol-specific Affinity medium.

Description

The The present invention relates to reaction vessels whose surface with a cis-diol-specific affinity medium is coated, as well Process for their preparation and test kits containing such Reaction vessels.

In the field of medical diagnostics, there is a general desire to provide cost-effective, easy-to-use, precise and rapid detection methods and corresponding test kits for the most diverse substances to be detected. An important field in diagnostics is the detection of catecholamines, such as epinephrine, norepinephrine or dopamine, in samples of body fluids. A first important step in these detection methods is the extraction of the catecholamines from the respective sample. Conventionally, substances which serve as affinity ligands which recognize the cis-diol structures of the catecholamines, eg immobilized boric acid or boric acid derivatives, are used for this extraction, which effects substantial purification and concurrent concentration (see, for example, Speek et al., Clinica Chimica Acta 128 (FIG. 1983), 103-113 or Gelijkens and De Leenheer, Journal of Chromatography 183 (1980), 78-82) or alumina (see eg Cooper et al., Journal of Neuroscience Methods 22 (1987), 31-39 or Refshauge et al ., Life Sciences 14 (1974), 311-322). Further affinity ligands and corresponding analytes are in DE 43 10 964 A1 (Page 2, Table 1) compiled by way of example. Usually, for extraction, the appropriate sample is mixed with an affinity medium in suspension, which medium may also be in a chromatography column or other vessel. In EP 0 288 425 B1 For example, a column filled with an immunoglobulin G-specific affinity medium is described. In DE 44 20 732 A1 a device for the separation of nucleic acids is described, wherein an immobilization of nucleic acids to an adsorbent contained in the reaction space, such as silicate or glass particles, or a biospecific immobilization of nucleic acids to biospecific adsorbents, eg affinity materials can be used. In the DE 44 20 732 A1 cited document EP 03 89 063 A2 describes a method of isolating nucleic acids that includes a biospecific affinity medium contained in a reaction space. This in DE 195 12 361 A1 claimed method for purifying biological material, provides in one step the biological material as bound to a compressible porous matrix form. Columnar accumulations of granular affinity material are described here. DE 195 12 369 A1 discloses a columnar device for isolating nucleic acids filled with a nucleic acid binding material. After washing the affinity medium, the substances bound to it are separated again (see, for example, Mazzeo and Krull, Biochromatography 4 (1989), 124-130 and Hageman and Kuehn, Methods in Biology 11: Practical Protein Chromatography, Ed Kennedy and Fowell, The Humana Press Inc. (1992), 45-71). These processes, which include several centrifugation, pipetting or washing steps, represent a significant source of error in the precise determination of the concentration of the substance to be detected and are also time-consuming.

Of the present application is therefore the object of funds to disposal to provide the extraction of substances to be detected Simplify samples.

These The object is achieved by the Provision of the embodiments specified in the patent claims solved. Thus, the present invention relates to a reaction vessel whose surface coated with a cis-diol specific affinity medium.

Such reaction vessels now allow the extraction step in the detection of substances with cis-diol structure, such as polyphenols (eg catecholamines and flavonoids), 1,2-hydroxy acids, 1,2-hydroxylamines, enzymes (eg serine proteinases) and others Proteins (eg, albumin), nucleic acids (eg, nucleosides, nucleotides, and RNAs), carbohydrates (eg, glucose), and other compounds containing carbohydrate structures can be carried out more quickly and accurately. Using the reaction vessels according to the invention eliminates all centrifugation steps in the extraction step and the pipetting steps are reduced. The separation of the liquid from the solid phase takes place in this case by decanting and knocking the reaction vessels on an absorbent pad. The US 5,565,622 A describes a method for extracting components from a liquid sample, wherein the inner surface of a hollow needle is coated with a stationary phase which is not an affinity medium or a cis-diol specific affinity medium.

The term "reaction vessel" in the context of the present invention includes any type of vessels conventionally used in diagnostics that are suitable for receiving a sample, in particular in liquid form. These include, for example, any type of centrifuge tubes, macro or microtiter plates, multi-dishes, Cell or tissue culture plates or dishes, tubes, bottles or pans. The volume of these vessels does not matter. It is preferably in a range from 5 μl to 100 ml, particularly preferably in a range from 500 μl to 5 ml and in particular in a range from 1.5 ml to 2.5 ml. The reaction vessels should preferably be made of plastic. Preferably, they consist entirely or partially of polystyrene.

Of the Expression "whose surface coated "means that at least the majority that surface, which comes in contact with the sample to be examined, with the cis-diol-specific affinity medium is coated. "Coated" means that this affinity medium the surface of the reaction vessel and not in contact with the sample to be examined solves.

Under the term "cis-diol-specific Affinity Media "will be part of the Present invention understood a material consisting of a carrier material (Matrix), which has chemical structures (ligands), which can specifically bind substances with a cis-diol structure. The support material is preferably a material as it is for the preparation of chromatography separation media is used.

In a preferred embodiment is the matrix of cis-diol-specific Affinity medium polyacrylamide.

In a particularly preferred embodiment is the cis-diol-specific affinity medium a boronate affinity gel. Such a gel is commercially available, e.g. under the name affi-gel 601 (BIO-RAD, catalog numbers 153-6101 and 153-6102).

• (a) Inkontaktbringen des cis-Diol-spezifischen Affinitätsmediums mit der inneren Oberfläche des zu beschichtenden Reaktionsgefäßes;
• (b) Erwärmen des Reaktionsgefäßes; und
• (c) Abkühlen des Reaktionsgefäßes.

Furthermore, the present invention relates to a method for producing a reaction vessel according to the invention comprising the following steps:

• (a) contacting the cis-diol specific affinity medium with the inner surface of the reaction vessel to be coated;
• (b) heating the reaction vessel; and
• (c) cooling the reaction vessel.

The In contact with the reaction vessel according to step (a) of the method according to the invention is preferably such that a reasonable amount of the cis-diol specific affinity medium brought into the reaction vessel will and there for a longer one Period remains. "Appropriate Quantity "means while an amount that is suitable, the surface of the reaction vessel for desired Extent too cover.

The Contacting after step (a) of the process can be both such take place that the cis-diol-specific Affinity medium as such is brought directly into contact with the reaction vessel. Alternatively, the affinity medium but also with other solids, e.g. other affinity media, Stabilizers, fillers and preservatives, or with gases, e.g. Air, nitrogen or argon, and contact the reaction vessel to be brought. Preferably, the cis-diol specific affinity medium becomes before contacting the reaction vessel with a liquid mixed and the mixture of affinity medium and liquid is introduced into the reaction vessel. The mixing is preferably carried out such that the cis-diol-specific Affinity medium under constant stir to a liquid is given. The resulting mixture is then preferably for a longer time, especially for approx. 30 minutes, further stirred, to the affinity medium well slurried is and an optically homogeneous, finely divided mixture of the affinity medium in the liquid is present. Liquids, which can be used in this context are, for example, aqueous solutions (e.g. Buffer), organic solvents (e.g., hydrocarbons, alcohols, ethers), acids and alkalis. At the liquid it is preferably ultrapure water.

Becomes the affinity medium with a liquid mixed, after step (a), a drying step is performed, the guaranteed that in that Mixture of existing liquid largely, preferably to a residual moisture content of less than 1%, and most preferably completely, is removed. Especially the drying of the introduced into the reaction vessel is preferred Mixture of cis-diol-specific affinity gel and a liquid performed in such a way that this with the mixture provided reaction vessel over a longer period, preferably for 5 minutes up to 5 hours, stored at a temperature between 4 ° C and 30 ° C.

The height of Temperature and the duration of the heating according to step (b) of the method according to the invention depends, if necessary, on the material of the coating to be coated Reaction vessel as well according to the type of matrix of the affinity medium. The parameters can however adjusted by the skilled person by suitable experiments without problems become.

A "heating" in the context of the present invention preferably means heating to a temperature of at least 50 ° C., preferably of at least 60 ° C., and particularly preferably of at least 70 ° C. Further, heating preferably means heating to not more than 100 ° C, preferably not more than 90 ° C. Heating in the range of 70 ° C to 90 ° C is particularly preferred. The duration of the heating is preferably at least 30 minutes, preferably at least 60 minutes and particularly preferably at least 120 minutes. The duration preferably does not exceed 360 minutes, preferably not 300 minutes and more preferably not 240 minutes. Most preferably, the heating takes place for 1 to 5 hours.

The cooling down of the coated reaction vessel according to step (c) the method according to the invention is preferably done by standing at room temperature.

in the In connection with the method applies to the reaction vessel and the cis-diol-specific affinity medium the same as explained above.

In a preferred embodiment this is for coating by means of the method according to the invention cis-diol-specific affinity medium used a boronate affinity gel.

In a further preferred embodiment is the matrix of the cis-diol-specific affinity medium polyacrylamide.

The The following example serves the closer one explanation the invention.

example

Preparation of boronate affinity medium coat macrotiter plates

On the precision balance 28.8 g Affi Gel 601 (Bio-Rad) are weighed out. The gel is under stir added to 1200 ml ultrapure water. Stirring with the aid of a magnetic stirrer is for 30 minutes continued at room temperature. The homogeneous mixture is used for the coating from macrotiter plates (NUNC).

to Coating of these plates is a pipette (Eppendorf) used. 250 μl of the gel / water mixture are added to each well of the macroplate Pipette. The Combitip should always be before and between the pipetting runs reared up and ejected become. The gel should rise evenly after pipetting distribute the bottom of each well.

The Macrotiter plates with the introduced gel / water mixture are for 48 Hours under the hood by evaporating the water at room temperature dried.

The dried macrotiter plates are placed in a drying oven (VTR 5050, Heraeus Instruments) and stacked up within 2 hours a temperature of 80 ° C heated. Maintaining this temperature of 80 ° C, heating for another 2 hours continued. Subsequently The macrotiter plates are removed from the oven and through Standing for Cooled for 2 hours at room temperature.

Claims (9)

Reaction vessel whose surface coated with a cis-diol specific affinity medium. Reaction vessel according to claim 1, wherein the cis-diol specific affinity medium is a boronate affinity gel. Reaction vessel according to claim 1 or 2, wherein the matrix of the affinity medium is polyacrylamide. Reaction vessel after a the claims 1 to 3, containing a macro- or microtiter plate, multi-shell, cell or tissue culture plate or cup, tube, bottle or pan is. Process for the preparation of a reaction vessel, the surface coated with a cis-diol specific affinity medium comprising following steps: (a) contacting the cis-diol specific affinity medium with the inner surface the reaction vessel to be coated; (b) heating the Reaction vessel on a temperature of at least 50 ° C for one Duration of at least 30 minutes; and (c) cooling the Reaction vessel on Room temperature. The method of claim 5 further comprising Step (a) a drying step. A method according to claim 5 or 6, wherein the reaction vessel is polystyrene is. A method according to any one of claims 5 to 7, wherein the cis-diol specific affinity medium a boronate affinity gel is. Method according to one of claims 5 to 8, wherein the matrix the affinity medium Polyacrylamide is.