DE19620636A1 - Appts. for affinity processes allows multiple simultaneous analysis - Google Patents

Abstract

Appts. for performing biochemical and chemical affinity processes comprises a tubular support on the inside wall which is an immobilised affinity sorbent.

Description

The invention relates to a device for performing Chemical or biochemical affinity process ren and a method for operating such gene device. One uses in affinity procedures the specific affinity of a reaction partner, on the surface of a solid phase (carrier) is fixed (immobilized) in order from a substance selectively add the specific component tie. The other substances can then easily e.g. B. can be separated by washing. To the ver Widest affinity methods include immunoassay and affinity chromatography.

In affinity chromatography after Ent remove the unbound substances from the valuable substance deliberately replaced (eluted) and ideally as pure  fabric won. Usually has one at the same time Concentration took place.

The immunoassay is a technique for the detection of Substances (e.g. proteins, bacteria, viruses, toxins etc.) based on an antigen-antibody reaction. Antibodies are proteins that selectively correspond to them antigen (substances that cause an immune response trigger) bind.

For immunoassays and affinity chromatography There are a number of different applications Chen carrier materials (solid phases). While in the Affinity chromatography inorganic and organic Carrier materials such as agarose or silica gel in the form of spheres or irregularly shaped particles as Column materials are used at Immuno assays the microtiter plate made of polystyrene as solid Most widespread phase. In some cases com also immunoassays based on membranes (e.g. made of nylon or nitrocellulose), glass plates, porous glass or silica gel particles as well as latex balls Chen for use.

A disadvantage of the prior art is the fact that the mostly planar arrangement of the carrier in particular especially in immunoassays not an effective one - in particular automatic - operation of the devices allowed and that the relationship between active Surface and sample volume is too low.

In affinity chromatography are columnar Devices used with porous supports substances (column materials), for example in Spherical shape, are filled. Such devices  have the disadvantage, however, that the diffusion paths due to the inner surface of the carrier substances are very long. This usually only makes one Part of the actual surface to be used binding of the resource is used.

Another disadvantage of packed columns is that high pressure drop in the bed. Therefore, the Operate such devices powerful pump pen, which requires high investment and operating costs cause. Due to the flow in the packed pillar is also a wide dwell given time distribution. The one available With packed columns, the surface is not geometrical precisely defined. Deviations in the enrichment This means that performance can also be achieved with the same form len occur. In addition, it is often highly unspecific to observe adsorption.

The present invention is therefore based on the object based on a device for carrying out chemi shear or biochemical affinity processes create a favorable relationship between Trä surface and sample volume as well as a narrow displacement distribution of time guaranteed, with low pump performance and reproducible character has tika. Another object of the invention is it, a process for effective and automati operable operation of this device put.

This object is achieved by an inventive one Device with the characteristic features of the An Proverb 1 and the procedure for one advantageous operation of the device by the kenn  Drawing features of claim 9. Advantageous Refinements and developments result from the respective subclaims.

According to the invention is as a carrier material for a device for carrying out chemi shear or biochemical affinity methods tubular support with on the support use immobilized affinity sorbent on the inside wall det. Such a device is suitable for for example as a fixed phase for the implementation of Immunoassays or affinity chromatography turns.

The tube shape according to the invention allows an advantage stick operation of the device in the flow process. This facilitates automation of immunoas says.

The tubular support material ensures this also an optimal relationship between the top of the institution area and sample volume. In particular, the Trä can be freely selected, whereby the ver relationship between active surface and required Sample volume can be set advantageously. The use of kapil is particularly advantageous laren carriers, which are extremely favorable due to their relationship between active surface and pro mark the volume. To increase throughput zes can also increase the pipe diameter will.

Since the affinity sorbent according to the invention on the Trä gutter wall is immobilized, no pillars materials needed. For this reason, the Strö  not through the tubular support influenced by column materials and the Druckwi the level is low. The result is a close ver because of the time distribution and the low number of samples required lumina. In addition, the diffusion paths between the Affinity partners, compared to porous columns materials, short and you get a geometrically good defined surface. The latter guarantees a good reproducibility of immunoassys.

Tubular supports according to the invention are preferred of glass. The invention is particularly preferred Carrier made of melted silicon dioxide (fused Si lica). Fused silica is characterized by a very high purity. A commercial advantage Retained, covered capillaries made of fused silica is their high flexibility and mechanical stability.

To improve immobilization of affinity it is extremely advantageous to sorbent the glass or Fused silica according to the invention in an upstream To activate digestion processes. As a result of the up In the final process, the treated surface has a high density of functional groups.

This can result in activation, for example gene that the surfaces of the fused silica hydroxy be lated. This can be done with a good reproducer Availability preferred by a hydrothermal alkali happen. By creating free Hydroxyl groups on the surface can, for example proteins are covalently immobilized. Except that is surprisingly a sharp decrease of nonspecific absorptions.  

In addition to glasses, other materials, for example wise polymers such as polystyrene, as inventive tubular support material can be used. The Carrier wall can be used to prepare for the Immobi pretreatment of an affinity sorbent.

Suitable as an immobilized affinity sorbent for example a component of a biochemical Affinity pair, e.g. B. a partner of the Anti systems gene / antibody, enzyme / substrate, receptor / hormone, Koh lenhydrate / lectin or nucleic acid / complementary nu cleic acid. Other suitable immobilized affini Tat sorbents are complex ligands that are selective bind certain metal ions or host complexes that Can accommodate guest molecules. Immobilization the affinity sorbent can be adsorptive or covalent respectively.

Advantageously, several tubular supports can be arranged in parallel next to each other. One of the like bundle arrangement allows simultaneous Carrying out several affinity procedures. The through rate can be increased in this way. also can be distinguished at the same time, for example perform immunoassays, and so within short different components of a solution certify. Depending on the application are on the wearer walls have the same or different affinity immobilized in sorbent.

The operation of a device according to the invention in Flow method is a particularly advantageous one Embodiment of this invention. So Pipet animal robots, which are used for automatic execution widely used, for example, by immunoassays  are through simple and inexpensive flow systems teme to be replaced.

A flow method according to the invention for operation ben a tubular device with on the inside immobilized affinity sorbent includes next a step of incubating a sample oil solution. Incubation means both the con clock of the flowing as well as the standing medium with the carrier according to the invention. Then can a step of incubating a reagent solution fol The reagent solution contains an immunoassay for example an enzyme-labeled antibody, which in the subsequently added substrate solution catalyzes a specific indicator reaction. The Reaction products of this reaction are then eating, e.g. B. as an indication of the presence of the ana lysing substance in the sample solution, demonstrated (Enzyme linked immunosorbent assay test, ELISA test).

Finally, a regeneration step can take place through which the immobilized component of the Af financial system is renewed. With affinity schoma topographical applications consists of regeneration step in the elution of the bound affinity part ners.

After a regeneration step, a new Ver cycle will be started. Thus, one another subsequently several process cycles are carried out the. This is particularly automatic Way possible. According to at least at least one, preferably after each, of the process a washing step was carried out.  

With the help of several tubes arranged side by side Shaped carriers can have multiple affinity processes Perform ren at the same time.

The invention is based on a selected embodiment illustrated in more detail. It demonstrate:

Fig. 1 is a schematic representation of the surface of a fused silica capillary Oberflä prior to treatment with a pulping process,

Fig. 2 is a schematic representation of the surface of a fused silica capillary after treatment with a digestion process, and

Fig. 3 is a schematic representation of an immobilized on the surface of a fused silica capillary affinity sorbent.

Capillaries made of fused silica, such as are known from gas chromatography, can be reproducibly activated by a closure method so that they can be used as a solid phase in immunoassays or in affinity chromatography. The immunologically active receptor layer is applied to the inner surface of the capillary by covalent bonding. Commercially available fused silica capillaries consist of pure SiO₂ and are pulled out of the melt at 2000 ° C. By covering the capillaries with a jacket made of polyimide, they become flexible. Due to the extreme manufacturing conditions, there are no or only a few free hydroxyl groups on the surface of the capillaries ( FIG. 1). In order to be able to fix (immobilize) proteins (e.g. antibodies) better covalently on the inner surface, the capillaries are first hydroxylated, ie free hydroxyl groups are created on the surface ( FIG. 2). Via these hydroxyl groups, organofunctional groups to which proteins are coupled can be introduced by reaction with organosilanes ( FIG. 3).

For coupling proteins to organofunctional ones Groups on inorganic surfaces are in the Literature various methods have been described which in principle all apply to activated fused silica Capillaries can be transferred.

The digestion process should be optimized so that on the one hand a sufficient density of hydroxyl group pen on the inner surface of the capillary will create, on the other hand the flexibility and maintain the mechanical stability of the capillaries remains. A suitable digestion ver driving described in more detail.

Read through a hydrothermal alkaline digestion fused silica capillaries are reproducible hy doxylation without losing flexibility or mecha loss of stability. A piece of fused silica Capillary becomes 75% with a 0.01 molar potassium hydroxide solution filled and at both ends with one Acetylene welding torch melted down. The capillary is annealed at 200 ° C for 10 hours, at room temperature cooled and 5 minutes in a flow with distilled water rinsed water. The hydroxylated capillary can be filled with water and stored indefinitely the.  

The following is a possible procedure wrote to put a protein on the surface of the hy droxylated fused silica capillary covalently to immo bilize.

The hydroxylated capillary is with a 5% aqueous 3-2- (aminoethylamino) propyltrimethoxysilane solution (12 hours at 85 ° C) silanized and on finally rinsed with water in the flow. After Ak Activation with a 2.5% glutardialdehyde solution (1 hour at room temperature) the with PBS (phos phat buffered saline) rinsed capillary for example wise for the immunological detection of T-2 toxin with a solution of T-2 toxin-BSA conjugate in PBS (200 µg / ml) filled and 12 hours at room temperature ditched. Then the capillary must still Treated with a blocking protein for 30 minutes to remove any remaining free binding sites saturate.

The following is a procedure for operating a such prepared capillary for the flow injection tion analysis (flow method) detailed will. The principle is based on an indirectly compe titive ELISA test for the detection of T-2 toxin.

The sample solution to be analyzed is anti-T-2 Toxin antibodies and anti-mouse IgG urease conjugate to move. After 20 minutes, the solution is applied for 5 Minutes in contact with the capillary. Ent if the sample does not hold any toxin, the complex binds out anti-T-2 toxin antibody and anti-mouse IgG urease Conjugate to the surface of the capillary in the mobilized toxin molecules. Contains the sample Toxin, the binding sites of the anti-T-2 toxin  Saturated antibodies with free toxin. The complex from anti-T-2 toxin antibodies and anti-mouse IgG-ure ase conjugate can now no longer on the capillary connect surface and will be in the subsequent wash step (duration 1.5 minutes). Subsequently the substrate solution is incubated for 20 min. The substrate solution contains sodium chloride, potassium chloride, urea and acid-reacting BSA (bovine serum albumin), so that a pH is reproducible Sets a value between 5.4 and 5.6. Contains the pro be no toxin, cleaves it to the capillary surface bound urease the urea NH₃ and CO₂ where increases due to the pH of the substrate solution. This An increase in the pH value can serve as an indication that that toxin is absent and e.g. B. by an Ar ray of two ion-sensitive field effect transistors be detected. After another wash step incubation of the regeneration solution follows preparation of the capillary for the next analysis cycle lus. Such an analysis cycle lasts including Approximately 40 times the regeneration of the receptor layer Minutes.

Claims (13)

1. Apparatus for carrying out chemical or biochemical affinity processes consisting of carrier and immobilized Affinitätssor bens, characterized in that the carrier is tubular and the affinity sorbent is immobilized on the inner wall of the carrier. 2. Device according to claim 1, characterized draws, that the carrier is designed as a capillary. 3. Device according to one of the preceding An sayings, characterized, that the carrier is made of glass. 4. The device according to claim 3, characterized draws, that the carrier made of melted quartz glass (Fused silica). 5. Device according to one of the preceding An sayings, characterized, that the carrier ak in a digestion process is activated and after activation free Hy has droxyl groups.   6. Device according to one of the preceding An sayings, characterized, that the immobilized affinity sorbent is a Component of the systems antigen / antibody, Enzyme / substrate, receptor / hormone, carbohydrate / Lectin, nucleic acid / complementary nucleic acid, Metal ion complex ligand or host molecule guest is molecular complex. 7. Device according to one of the preceding An sayings, characterized, that at least two beams in parallel next to each other which are arranged and on the carrier inner walls either different or at least partly the same affinity sorbents immobili are based. 8. Device according to one of the preceding An sayings, characterized, that the device is a device for through conducting an immunoassay or an affini chromatography is. 9. Method for operating a device for Implementation of chemical or biochemical Af financial process, characterized, that a tubular support with on the support inner wall of immobilized affinity sorbent in the Flow process is operated. 10. The method according to claim 9, characterized in net, that the flow process is a step of Incubation of a sample solution as well as at least one of the steps of incubating a reagent solution  solution and / or incubation of a substrate solution and / or incubation of a regeneration solution includes. 11. The method according to claim 10, characterized in net, that following at least one of the procedures steps followed by a washing step. 12. The method according to any one of claims 9 to 11, characterized, that several process cycles in automated Be done in a row. 13. The method according to any one of claims 9 to 12, characterized, that with the help of several arranged side by side ter tubular support, either under different or at least partially the same Af have sorbents, several affini procedures are carried out simultaneously.