DD286671A5 - GEFAESSKOMBINATION FOR DETECTION OF BIOMOLECULES - Google Patents

Abstract

This invention relates to a vascular combination for the detection of biomolecules. The invention likewise provides a method for detection by means of the vessel combinations according to the invention. Areas of application of the invention are molecular biology, immunology, diagnostics and microbiology. The Gefaeszkombination consists of one or more analysis vessels, which are composed of one or more small reaction chambers (1) of 1 to 20 ml volume and a large incubation chamber (2) of 50 ml to 100 ml volume, the reaction chambers (1) as depressions the bottom and / or the side walls of the incubation chamber (2) are formed. The detection method consists in the reaction chamber, a probe molecule, for. As an antigen or an antibody to fix on the surface, anschlieszend to give the biomolecule to be analyzed in solution in the reaction chamber and incubation chamber, perform the specific reaction and finally the biomolecule to be analyzed, z. B. by an enzyme reaction to detect in the reaction chamber. With the solution according to the invention, high-sensitivity molecular biology detection methods can be carried out with the smallest amounts of detection reagent at large sample volumes to be analyzed. In addition, a parallel determination of different biomolecules from the same sample to be analyzed is possible {vascular combination; Detection method, molecular biological; Reaction chamber, small; Incubation chamber, large; depressions; Sondenmolekuel; Antibody; Antigen; Enzyme reaction}

Description

For this 2 pages drawings

Field of application of the invention

This invention relates to a vascular combination for performing post-immune reactions for biomolecules and methods practicable by means of this vascular combination. The vascular combination, their individual analysis vessels and the detection methods can be applied in molecular biology, immunology, diagnostics as well as in microbiology.

Characteristic of the known state of the art

Microtiter plate assays are widely used analytical techniques in molecular biology, see e.g. B.J.D. Buxbaum and Y. Dudni, Anal. Biochem. 169, 209-215 (1988) or product catalog of Flow-Laboratories 1989, pp. 94-95. Both antibodies and antigens are usually determined using the ELISA technique, see, for. B. B. E. Engval! and P. Pcrlmann, Immunochemistry 8, 871-874 (1971). These microtiter plates have chamber volumes of 0.1 to 1 ml, which are used for all Nachwoisschritte with the same volume. This requires a high consumption of detection ragozia and an unfavorable geometry of the reaction surface, which has an adverse effect on the background during the Nachwois reaction. In the so-called ultramicroliter test plates (polystyrene, PVC), a chamber with a volume of approximately 20 μΙ is used for all detection steps (see, for example, EP 203,443), while the SIA technique in Tropfon has a volume of approx Glass plates without actual reaction chambers. Although this leads to an improvement in the geometry of the reaction surface of the detection and thus the background of the detection reaction, but has the disadvantage that the volume of the analyzed probo is very small, waJ oino reducing the sensitivity (detection pg sought component / ml Tostflüssigkoit) Folgo has.

The disadvantage of bekannton tachnese solutions is that only one continuous reaction chamber is used, which does not allow the carrying out of negative controls or a parallol examination of several PiO molecules to be analyzed from an available probo to be analyzed (see, for example, US Pat EP 206,561 and EP 246,760).

ZIoI the invention

The aim of the invention is ee to construct a Gofäßkombination in which molecular biology detection methods of highest sensitivity with smallest amounts of the detection reagent can be carried out at large sample volumes to be analyzed. In addition, a parallel determination of different biomolecules from the same sample to be analyzed should be possible.

Explanation of the essence of the invention

According Eifindungsgemäb a vessel combination of one or meieren analysis vessels is provided, which are composed of one or more small reaction chambers of 1 to 20μΙ volume (1) and a large incubation chamber of 50 .mu.Ι to 100 ml volume (2), woboi dio reaction chambers (1 ) are formed as depressions of the bottom and / or the side walls of the incubation chamber (2) (FIGS. 1 and 2).

The inventive construction of the vessel combination of analysis vessels with different sized chambers, which are interconnected, decisive advantages are achieved over conventional methods and analysis vessels, which consist in that biomolecules can be detected from large volumes to be analyzed in very small reaction volumes and at the same time a parallel determination of different biomolecules from the same sample to be analyzed is made possible. The combination of the large test volume with very small reaction volume makes it possible to minimize the surface used for the detection reaction and thus a favorable signal / background ratio (increase in sensitivity). At the same time, very small amounts of the detection reagent are needed. The use of incubation chambers with several reaction chambers (FIG. 2), the possibility of carrying out control and parallel tests, is particularly advantageous.

The material from which the reaction chambers and the incubation chamber are made may be the same (e.g., polystyrene) or different from each other, e.g. As polystyrene and PVC. It is possible for the reaction chambers, a material such. As nitrocellulose, and for the incubation another material, eg. As PVC, to use. For the chambers primarily synthetic polymers are used, for. Polystyrene, PVC, polyethylene, polypropylene, polyester, polyamide, polyvinylidene fluoride, polytetrafluoroethylene, etc .; however, modified natural polymers may also be used, e.g. As nitrocellulose, cellulose acetate, etc. In addition, the materials may have charges by z. B. are fixed negative and / or positive groups on the surface. PVC, polystyrene, nitrocellulose and polyvinylidene fluoride are preferred for the reaction chambers and the incubation chamber.

The reaction chamber and the incubation chamber can be made of optically transparent or optically impermeable or partially permeable, colored or non-colored material. For the implementation of the detection method of the invention, the nature of the material is not critical, as long as the constructive parameters are met and binding of the probe molecule is made possible. Thus, as a further advantage of the method according to the invention is that in the manufacture of the device according to the invention compliance with tight tolerances for implementing dos Nachwoises with optical methods is not required unbodingt, only in cases is necessary if an optical detection ancestor, eg by color reactions , it is asked for. A preferred constructive solution of the device according to the invention is to form the reaction chamber as a depression of the bottom of the incubation chamber. As shown in the various embodiments of Figure 1, the recess is preferably in the form of a Kegeis (A), a cylinder (B), a truncated cone (C), a spherical segment (H) or nus a combination of two or more geometrical Bodies, for example of cylinder and cone (D) or cylinder and truncated cone, or in the form of rotational bodies, for. B. in the form of a U-shaped body of revolution (G) or two Kogelabschnitten (F), or in the form of annular recesses, for. B. with rectangular cross-section (E), with triangular cross-section (I) or in the form of depressions on the edge with a rectangular cross section (K). The preparation of such forms of polymers is well known and may e.g. B. by pressing, pressing, Spr'tzguß, drilling, milling done. Furthermore, it is possible to coat one of the chambers or a part of one or both of the chambers with material other than that of which the device is made up.The coating can also be carried out with lauungon-containing surface-active agents having surfaces of the reaction chambers is that they have negative charges.

For the detection of biomolecules, more than one reaction vessel is necessary in the Rogol, since the safety of a control sample (negative control) mitgoführt is often analyzed multiple samples odor different Verdünnungon be applied. In the automated implementation of the proof then as many samples are examined simultaneously in parallel, z. 48 or 96 or more. For such applications, it is favorable to connect the devices according to the invention to one another, ie at least two, whereby the connection can be carried out mechanically, by gluing or pre-pressing. Preferably, a vascular combination is provided, which consists of several analysis vessels that are permanently or temporarily intermittently mechanically vorbundon each other. In particular, Gufäßkof.jinationsen be made, consisting of 8 to 100 analysis vessels that are permanently verbundon together. The Vorbindung can z. B. by sticking together, screwing, gluing the analysis of the invention individually or in blocks; but it can also be so proceeded that the Analysenkofäße invention already in blocks of mohr than one, preferably 8 to 100, are produced, wherein the above-mentioned production methods are used. A favorable variant for the formation of the Orfindungsgomäßon Gofäßkombination to carry out multiple determinations is to connect an incubation chamber (2) with a plurality of reaction chambers (1) (Fig. ?.). In this case, an incubation chamber with 1 to 50 reaction chambers can be formed. These reaction chambers can be arranged in many different ways. Bnproven it is favorable if the reaction chambers are arranged symmetrically or in a Rastor. This facilitates an evaluation, in particular automatic evaluation. A bosonders preferred embodiment of this ei flündungsgomäßen vascular combination is to form the Roaktionskammem as Vortiefungen the soil and / or Soitenwändo the incubation chamber. In this case, dio reaction chambers can have a different shape, as it is particularly favorable for the intended application case. FIG. 2 shows some embodiments of an analysis vessel with a plurality of reaction chambers (1) in an incubation chamber. In variant (A), an analysis vessel is shown with two conical reaction vessels arranged in parallel with conical shape. Dio version (8) shows similar reaction chambers (1), which are arranged symmetrically. Finally, in version (C), the analysis vessel is shown with a multiplicity of rcaution chambers (1) arranged symmetrically in raster form.

Dio inventive devices are intended primarily for detection ancestors in medicine, molecular biology, immunology, serology and microbiology, of course, can also be used for other purposes or applications.

The invention likewise relates to a method for the detection of biomolecules by means of the inventive combination of felts. The detection reactions to be performed may have as ziol the detection of a particular antibody or a particular antigen or more thereof. The detection is carried out according to the invention in the following steps:

a) fixing one or more probe molecules or a biomolecule to be analyzed or a mixture of OiomoloV.üleri containing the biomolecule to be analyzed on the oborface of the reaction chamber (s) (1);

b) incubation with the sample to be analyzed which contains one or more biomolecules to be detected or with one or more probe molecules in the analytical gavage (s);

c) Detection of the Analyzing Biomolecules in the Reaction Chamber (s) (1).

The probe molecule is a molecule that specifically recognizes and specifically binds a biomolecule to be analyzed.

The detection of the nalysierenden to a biomolecule occurs after a second specific reaction of the meanwhile, a probe molecule or directly to the surface of the Reakticnskammer (1) bound molecule with a second enzyme labeled probe by means of the corresponding He zymreaktion.

According to a first, preferred embodiment of the detection method according to the invention, steps (a) and (b) are carried out as follows:

a) fixing one or more probe molecules on the surface of the reaction chamber (s) (1);

b) incubation with the sample to be analyzed containing one oiler of several biomolecules to be detected and washing in the analysis vessel (s),

c) Detection of the biomolecule to be analyzed in the reaction chamber.

In carrying out the detection process according to the invention, the essential advantage of the combination according to the invention becomes clear: use is made of the smallest volumes, e.g. 5 or 10μΙ containing either a probe molecule or a biomolecule to be analyzed, and binds this only on the surface of the reaction chamber. The remaining free surface is blocked by means of suitable substances (del. Agents, foreign proteins) against non-specific binding of the biomolecule or sondon molecule to be analyzed. Subsequently, the fixed biomolecule is contacted and reacted with either the analyte biomolecule or a probe molecule in a comparatively large volume of a liquid, which may also be a biological fluid such as blood, serum or urine. As a result of this possibility of using large volumes, concentration and / or purification steps are generally superfluous, since the fixed biomolecule selectively binds its corresponding partner from the large volume.

For example, as a probe molecule, z. As antibodies, antigens, t pseudo-reactive proteins such as protein A or protein G can be used. Detection occurs after a second specific reaction with an enzyme-linked probe. The coupling of the enzyme to the sondo can be done chemically by bifunctional reagents. Particularly favorable for the detection method according to the invention ί ind enzyme-coupled probes which are microbiologically produced as fusion proteins, as described, for. As described in EP 304.934.

The biomolecule to be detected can now also be an antigen or an antibody. As antigens, for example, Hüllprotoino of viruses or their fragments, proteins of human serum or oligopeptides are possible. Antibodies are detected when an antigen specific for them is bound to the surface of the reaction chamber. Beispiclo for antibodies to be detected are z. B. against HIV, against hepatitis B virus, etc., in the detection of an envelope protein, for example of HIV, is bound to the Oberllache the Reaktionskamrrer, z. 8. the gp48 or pg 110. Furthermore, monoclonal antibodies can be bound, ar. dio binds the biomolecule to be analyzed. Subsequently, the detection is performed with a second antibody and a enzyme-coupled probe (second specific reaction). According to a second embodiment of the detection method according to the invention, steps (a) and (b) are carried out as follows:

a) Fixation of a Biomcloküls to be analyzed or a mixture of biomolecules containing the biomolecule to be analyzed! It, on the surface of the roaction chamber (s) (1);

b) Incubation with one or more sondon molecules in the analysis vessel (on).

c) Detection of the biomolecule (s) to be analyzed in the reaction chamber (s).

For detecting an antibody, according to the first embodiment, steps (a) to (c) are to be carried out as follows:

a) fixing an antigen (probe) on the surface of a reaction chamber and optionally blocking and / oror washing steps;

b) incubation of the fiordorton antigen with the sample to be analyzed containing a desired antibody, washing steps, incubation with an enzyme-coupled probe, washing steps;

c) detection of the antibody by an enzyme reaction.

For the detection of an antigen according to the first embodiment, steps (a) to (c) were carried out as follows:

a) fixing an antibody (probe) on the surface of a reaction chamber and optionally blocking and / or washing steps;

b) incubation of the immobilized antibody with the sample to be analyzed containing an antigen, optionally washing steps, incubation with an enzyme-coupled probe which reacts specifically with the antigen but not with dom-fixed antibody. Washes;

c) detection of the antigen by an enzyme reaction.

In particular, in a variant of the detection of an antigen, steps (a) to (c) were carried out as follows:

a) fixing an antigen to be analyzed on the surface of a reaction chamber and optionally blocking and / or washing steps;

b) incubating the fixed antigen with an enzyme-coupled probe and optionally blocking and / or washing stepo;

c) Detection of the antigen by an enzyme reaction.

In the preferred variants and embodiments, the biomolecule to be analyzed is detected by an enzyme reaction. The reaction of the particular enzyme with its substrate, if appropriate in the presence of a cosubstrate, produces a detectable reaction product. This reaction product may be a dye, a fluorescent molecule or a radiolabelled molecule. Dioxygenation of colored reaction products e.g. with peroxidase, glucoionidaso or glucose oxidase is known. According to EP 304,934, the detection by the radioactive labeling of a substrate, for. B. from

Kanamycin by (Y ³² P) ATP. As an enzyme-coupled probe is z. For example, a fusion protein of protein A and neomycin phosphotransferase II (NPT II) is used. Such detection methods using fusion proteins from NPT II and molecular probes are preferred for carrying out the method according to the invention. By this invention, vascular combinations are provided in which molecular biological detection reaction can be carried out in a volume of 1 to 10μΙ. The previously achieved detection limit for the biomolecule to be detected, z. As an antigen such as human serum albumin, is 100 fg, which means over dam known state of the art at least 100 times increase.

AusfÜhrungsbelspiolo example 1

Holes of 4mm diameter and 1.2mm depth are placed in the bottom of the incubation chambers of a polystyrene flat bottom microtiter plate (VEB MLW Polyplast Halberstadt) with a 0.4ml chamber volume. This hole leads to conical reaction chambers of about 6μΙ volume.

Example 2

It is worked with the same starting material as in Example 1 with a cutter of 4 mm diameter and milled to 1mm depth. This work catfish leads to reaction chambers of cylindrical shape with a reaction volume of about 12.5μΙ.

Example 3 The same material as in Example 1 is used. In the bottom of the incubation chambers is a thorn at one Pressed temperature of 120 ° C, so that a conical reaction chamber of about 10μί volume is produced. Example 4 The procedure is as in Example 1, but a PVC material is used. Example 5 Example 1 is repeated. After completion of the analysis vessels in each reaction chamber, a 1% solution of Nitrocellulose introduced into acetone and the solvent evaporated.

Example β

A dilution series of 10ng to 0.1 pg human serum albumin (HSA) including a zero control in each 5μΙ PBS

were filled in the Reaktionsgofäße the combination of the described in Example 1 and the antigen over

Fixed at 4 ⁰ C overnight. After three wiping with PBS, do η the incubation vessel and the coagulation chamber with 100 μΙ 5% Skimmed milk powder emulsion filled in PBS and blocked for 30 minutes at room temperature. The antibody action takes place

by means of an anti-human serum albumin serum (rabbit, dilution 1: 1000.5% skim milk powder in PBS, 0.2%

Tween * 20) in a volume of 100 μΙ overnight at room temperature. After washing 6 times (5 minutes) with PBS, 0.2% Tweon * 20 is treated with a fusion protein comprising protein A and noomycin phosphotransferase II, according to EP 304,934

(1 μΙ / 100μΙ 5% igo skimmed milk powder emulsion in PBS) for one hour at room temperature. Thereafter, it is washed 5 more times with PBS for 5 min. Dio Enzyme reaction is carried out in a volume of 5μΙ enzyme buffer in the crude vessel for 1 hour at 37 * C.

Enzyme puff: 20 mM Tris-HCl, pH 7.2; 125mM NH ₄ Cl, 20mM MgCl ₂ , 10mM DTE, 50mg / ml kanamycin sulfate, 5x 10'cpm | γ- "Ρ |

ATP / ml.

From the enzyme reaction solution, 1 μΙ aliquots are spotted onto phosphocellulose paper, left ointrock-free, with water

washed and autoradiographed. 0.1 μg of human serum albumin can be proven beyond doubt.

Beisptol 7

Into a polystyrene tube of 11 mm internal diameter and 17 mm high, four wells of 4 mm diameter and 1.8 mm depth are placed in the bed, so that they can accommodate 10μΙ (see Figure 2B). In one of the wells 10μΙ PBS with 100pg HSA, in the second 10μΙ PBS with 1pg HSA, in the third 10μΙ PBS with 100pg myoglobin and in the fourth 10μΙ PBS with 5% skimmed milk powder. Thereafter, it is fixed overnight at 4 "C and then proceeded as in Example 1 on.

From each reaction chamber, aliquots are then spotted on phosphor cellulose paper, allowed to dry, washed and then autoradiographed for 30 minutes at room temperature. Only two probes with HSA can be clearly detected.

Claims (17)

1. Vessel combination for the detection of biomolecules, characterized in that it consists of one or more analysis vessels consisting of one or more small reaction chambers of 1 to 20 μΙ volume (1) and a large incubation chamber of 50 μΙ to 100 ml volume (2) are constructed, wherein the reaction chambers (1) as depressions of the bottom and / or the side walls of the incubation chamber (2) are formed. 2. Vessel combination according to claim 1, characterized in that it consists of several analysis vessels which are permanently or temporarily mechanically connected to each other. 3. Vessel combination according to claim 1 or 2, characterized in that it consists of 8 to 100 analysis vessels, which are permanently connected to each other. 4. Vessel combination according to claim 1, characterized in that the material from which the reaction chamber (s) (1) and the material from which the incubation chamber (2) is equal or different from each other. 5. Vessel combination according to claim 1, characterized in that the material from which the reaction chambers (1) are made, is different from each other. 6. Vessel combination according to claim 1,4 and 5, characterized in that the material for producing the reaction chamber (s) and / or the incubation chamber (2) is PVC, polystyrene, nitrocellulose or polyvinylidene fluoride. 7. Vessel combination according to claim 1 and 4 to 6, characterized in that the surface of one or more reaction chambers (1) has negative charges. 8. Vessel combination according to claim 1, characterized in that an incubation chamber (2) with 1 to 50 reaction chambers (1) is formed. 9. Vessel combination according to claim 1 and 8, characterized in that the reaction chambers (1) are arranged symmetrically or grid-like. 10. Vessel combination according to claim 1 and 8, characterized in that the reaction chambers (1) as recesses of the bottom and / or the side walls of the incubation chamber (2) have a different shape. 11. Vessel combination according to claim 1,8 and 10, characterized in that the reaction chambers (1) have the shape of a cone, cylinder, truncated cone, spherical section, combination of cylinder and cone or truncated cone or a U-shaped body of revolution. 12. A method for the detection of biomolecules, characterized in that the detection is carried out in a vascular combination according to claim 1 in the following steps: a) fixing one or more probe molecules or a biomolecule to be analyzed or a mixture of biomolecules containing the biomolecule to be analyzed on the surface of the reaction chamber (s) (1); b) incubation with the sample to be analyzed containing one or more biomolecules to be detected or with one or more probe molecules in the assay vessel (s); c) Detection of the biomolecule (s) to be analyzed in the reaction chamber (s) (1). 13. The method according to claim 12, characterized in that the steps (a) and (b) are carried out as follows: a) fixing one or more probe molecules on the surface of the reaction chamber (s) (1); b) incubation with the sample to be analyzed, containing one or more biomolecules to be detected, and washing in the analysis vessel (s). 14. Verfver according to claim 12, characterized in that the steps (a) and (b) are carried out as follows: a) fixing a biomolecule to be analyzed or a mixture of biomolecules containing the biomolecule to be analyzed on the surface of the reaction chamber (s) (1); b) Incubation with one or more molecules of the probe in the analysis vessel (s) 15. The method according to claim 13, characterized in that the steps (a) to (c) are carried out as follows: a) fixing an antigen (probe) on the surface of a reaction chamber and optionally blocking and / or washing steps; b) incubation of the fixed antigen with the sample to be analyzed containing a desired antibody, washing steps, incubation with an enzyme-coupled probe, washing steps; c) detection of the antibody by an enzyme reaction. 16. The method according to claim 13, characterized in that the steps (a) to (c) are carried out as follows; a) fixing an antibody on the surface of a reaction chamber and optionally blocking and / or washing steps; b) incubation of the immobilized antibody with the sample to be analyzed containing an antigen, optionally washing steps, incubation with an enzyme-coupled probe which reacts specifically with the antigen but not with the fixed antibody, washing steps; c) detection of the antigen by an enzyme reaction. 17. The method according to claim 14, characterized in that the steps (a) to (c) are carried out as follows: a) fixing an antigen to be analyzed on the surface of a reaction chamber and optionally blocking and / or washing steps; b) incubation of the fixed antigen with an enzyme-coupled probe and optionally blocking and / or washing steps; c) detection of the antigen by an enzyme reaction.