DE10200374A1 - Method for the qualitative and quantitative comparative detection of chemical substances - Google Patents

Abstract

Disclosed is a method for qualitative and quantitative comparative detection of chemical substances. At least one type of chemical substances and at least one type of marker/receptor unit is provided. The marker/ receptor units respectively consist of a known marker and a receptor unit joined thereto. The receptor unit can bind a chemical substance. The marker component of the marker/receptor units is a desoxyribo nucleic acid oligomer, a peptide nucleic acid oligomer or a nucleic acid oligomer with a structurally analogous backbone. The chemical substances and the marker/receptor units are brought into contact with each other, resulting in the formation of complexes of at least one marker/receptor unit and a chemical substance. The complexes of the marker/receptor unit and a chemical substance are subsequently separated from the non-bound marker/receptor units and one or more chemical bonds of complexes of a marker/receptor unit and a chemical substance are cleaved in such a way that the marker component is separated from the complex of the marker/receptor unit and the chemical substance. The separated marker components are washed out and detected by an appropriate method. According to the inventive method, at least one type of chemical substance is provided which is of the same type of chemical substance as the chemical substances provided above or which is different from said chemical substances. The same type and amount of marker/receptor units as those previously mentioned are also provided. The latter chemical substances and the latter marker/receptor units are brought into contact with each other, resulting in the formation of complexes of at least one marker/receptor unit and a chemical substance. The non-bound marker/receptor units are separated. Subsequently, one or more chemical bonds of complexes of a marker/receptor unit and a chemical substance are cleaved in such a way that the marker component of the marker/receptor unit is separated. The separated marker components are washed out and detected by means of an appropriate method. Finally, the results of the two analyses of the marker components thus obtained are compared.

Description

Technical field

The present invention relates to a method for qualitative and quantitative comparative detection of chemical substances.

State of the art

The quantitative recording of all proteins of a cell of a certain one Organism at a certain point in time, often as a (cellular) proteome Research or (cellular) proteomics is the key to one functional understanding of the physiological, in the special case pathological State of the cell in question. The "proteomic" information enriches both basic and applied research. It delivers the molecular basis for the state of a cell and offers the possibility of that Change in the expression and modification pattern of the proteins in response for a specific disorder (e.g. an illness or the administration of a certain medication). This is what proteome research does a fundamental meaning for the target search and selection in the biomedical research, i.e. the development of new drugs. The (Cellular or global) proteome can also be used to diagnose Diseases and therapy control can be used. Thus the Proteome research basically the same goals as genome research, namely the correlation of the state of an organism or a cell (the Phenotype) with basic molecular building blocks of this organism the parallel analysis of as many such components as possible (e.g. the mRNA or the Proteins).

The complete set of genetic information is referred to as the genome has a cell. Functional genome research (functional genomics or phenomics) generally includes the expression and behavior of the gene Products. Within the "functional genomics" the areas become cellular Transcriptome research, cellular proteome research, global proteome research Research and metabolome research distinguished. As a cellular transcriptome Research (cellular transcriptomics) is the comparative study of mRNA exposure profiles with the help of e.g. B. called DNA chips. The quantitative isolation and identification of all proteins in a cell is considered cellular Proteome research (cellular proteomics) known. At the global proteome Research (global proteomics) will encode all proteins of the whole Genome analyzed and without specifying a specific cell type or specific growth conditions (physiological state of the cell). The proteome itself is a building block of the metabolome or the metabolome Research (metabolomics) is about understanding the whole Metabolism of an organism.

The proteome is much larger than the genome Information content. While the genome has the pattern of inheritance and the immediate transcription patterns of the genes in the form of the cDNA and mRNA Captured qualitatively and quantitatively, the complexity of the Transcription products, i.e. the proteins, through post-translational Modifications of many proteins (phosphorylation, glycosylation, proteolytic Processing etc.) further. So the same genome leads because of this post-translational variations and through transport mechanisms that transcribed gene products spread over the whole organism different proteomes and thus different physiological (or pathological) conditions.

The proteome also has a target search and selection or Diagnosing diseases against the genome has two main advantages: On the one hand, the correlation between proteome and phenotype is closer others can (at least partially) the proteome in the typical clinical samples such as serum, cerebrospinal fluid or urine. This simple accessible samples do not contain DNA, but proteins, so the samples to be examined for potential proteomic markers of a disease can, but not to genomic markers.

In comparison to genome research, however, the analysis of the proteome is much more difficult to carry out because - in contrast to the Principle of hybridization of DNA, cDNA or mRNA in the genome - for proteins there is no simple, universally applicable and clear detection principle. Both the isolation and separation as well as the actual detection must individually to the biochemical properties (e.g. hydrophobicity, hydrophilicity, Acidophilia, thermophilia) of the respective proteins or protein groups adapted become.

The analysis of the proteome can be divided into the following steps (see F. Lottspeicher, "Proteom Analysis: A Pathway to the Functional Analysis of Proteins", Angewandte Chemie, International Edition, 1999, Vol. 38, 2476-2492): Determination of the start parameters, sample preparation, protein separation, Protein quantification and data analysis. The start parameters should guarantee that the material that is undergoing proteome analysis is reproducible can be manufactured. Sample preparation includes a. Cell cultivation and Cell disruption under denaturing or non-denaturing conditions. There Proteins do not have uniform physical and chemical properties own sample preparation requires an exact protocol to Ensure reproducibility.

The currently most common, high-resolution method of protein separation is the 2D gel electrophoresis in which proteins after (i) charge (by means of isoelectric focusing, IEF gel) and (ii) size (using SDS-PAGE gel Electrophoresis). The problem also lies with this method in the reproducibility, in this case in the reproducibility of the two-dimensional pattern on the gel. External parameters like the protein Transfer from the first, IEF gel dimension to the second, SDS-PAGE gel Dimension, the electrophoresis device, the gel composition, the Electrophoresis time for IEF gels, staining methods, operating personnel etc. can lead to a clear variation of the patterns.

Protein identification and quantification is usually carried out. d. R. through mass spectrometric methods that require a transfer of the gel-separated Proteins from the gel into other environments (e.g. organic matrices in MALDI Mass Spectroscopy, MALDI-MS). This transfer into one too other environment can lead to modifications of the proteins and thus Misinformation or loss of information.

The final necessary data analysis is carried out using appropriate methods of bioinformatics.

In addition to the limited reproducibility, they are very important personnel- and apparatus-intensive sample preparation and analysis (MALDI-MS- Quantification) significant disadvantages of the described procedure for Proteome analysis. In addition, only a low degree of parallelization is achieved, because of the approximately 20,000 to 30,000 expressed proteins of a cell also in the complex 2D gels separated and only a few hundred different proteins can then also be detected. The 2D gel technique is also not automatable and therefore personnel and cost intensive.

Alternatives known from the prior art in the field of Sample preparation and separation are immunoprecipitation and chromatographic separation. In addition to irreversibly bound and therefore for the analysis of lost protein mainly reveals the low separating power of the widespread chromatographic methods a major problem of this Alternatives. In the first chromatographic step, the proteome in a few hundred fractions with different protein compositions separated, which are then carried out in new chromatographic steps, so serial, must be further separated.

To increase the degree of parallelization, various strategies for proposed at least partial, chip-based proteome analysis (cf. Alan Dove: "Proteomics: translating genomics into products? ', Nature Biotechnology, 1999, vol. 17, 232-236). For example, B. "separation chips" with different protein-specific surface chemistry or specific protein Binding partners are structured for the two-dimensional separation of the Proteomes can be used. The fractionated proteins should then be analyzed by mass spectroscopy. In a related strategy suggested isolating the proteins of the isolated proteome first (uniformly) with a To label fluorescent dye, then on an antibody chip (antibody Array) two-dimensionally and finally based on the Quantify fluorescence intensity (e.g. using CCD cameras). To a very specific antibody library is required, the The specificity / selectivity of the antibodies must be extremely high. In addition, the Antibody chip strategy the disadvantages of non-specific fluorescence and one due to the two-dimensional reading process of fluorescence by z. B. CCD cameras in combination with confocal microscope optics are extremely expensive Detection method. In addition, the standardization of the reaction provides Linking the fluorescent dye to the various proteins (qualitative and quantitative) is an unsolved problem.

The proteome analysis thus has the potential to become an extremely helpful one Technology in biomedical research and medical diagnostics too develop. However, this requires the problems described above overcome, namely the insufficient reproducibility, the low Degree of parallelization and the enormous amount of preparative work highly qualified staff and expensive technical equipment.

When examining clinical pictures or in comparative studies it is often not necessary to explicitly add the components of a proteome determine. In most cases, a sample with one is sufficient To compare the reference sample, e.g. B. the proteome of a healthy person with the proteome of a sick patient.

Presentation of the invention

The object of the present invention is therefore to provide a method for qualitative and to provide quantitative comparative detection of chemical substances, which does not have the disadvantages of the prior art.

This object is achieved qualitatively and by the method quantitative comparative detection of chemical substances according to independent claim 1 solved. Further advantageous details, aspects and Embodiments of the present invention result from the dependent Claims, the description and the examples.

In the method according to the invention for qualitative and quantitative comparative detection of chemical substances is at least one kind of chemical substances and at least one type of marker receptor Units are provided, the marker receptor units being each a known marker and one associated with the marker Receptor unit exist and the receptor unit is a chemical substance can bind specifically. Chemical substances and marking Receptor units are brought into contact, creating a Formation of complexes from at least one labeling receptor unit and chemical substance takes place. Then the complexes of labeling Receptor unit and chemical substance from the unbound Labeling receptor units separated and one or more chemical Binding of the complexes of labeling-receptor unit and chemical Substance cleaved so that the labeling component from the complex Marker receptor unit and chemical substance is separated. The separated marking components are washed out and by a suitable method is detected. In the method according to the invention also provided at least one type of chemical substance, wherein are the same types of chemical substances as those already in use provided types of chemical substances or different from them Can act types of chemical substances. In addition, the same type and Amount of marker receptor units that have already been provided provided again. The latest chemical substances and the last marker receptor units provided are in each other Contacted, thereby forming complexes from at least one Labeling receptor unit and chemical substance is made. The unbound label receptor units are separated. Then one or more chemical bonds of the complexes split from marker-receptor unit and chemical substance so that the labeling component of the labeling receptor unit is separated. The separated marking components are washed out and by a suitable method is detected. In conclusion, the results obtained of the two analyzes of the labeling components.

According to a preferred embodiment of the present invention at least one type of chemical substance, at least one type of Receptor units and at least one type of marker receptor units provided, wherein the marker receptor units from one each known marker and each connected to a receptor unit exist and the receptor unit specifically bind a chemical substance can. The chemical substances, the marker receptor units and the Receptor units are in contact with one another in any order brought, whereby a formation of complexes from one or more Labeling receptor unit (s), chemical substance and receptor unit he follows. The receptor units are before or after contacting chemical substances, marker receptor units to a suitable Material bound, whereby the complexes formed from labeling receptor Unit, chemical substance and receptor unit bound to the material are present and thereby a separation of unbound label receptor Units. Then one or more chemical bonds the complexes of labeling-receptor unit bound to the material, chemical substance and receptor unit split so that the labeling Part of the marker receptor unit is separated from the material. The Marking components separated from the material are washed out and detected by a suitable method. In the invention Process also uses at least one type of chemical substance provided, which are the same types of chemical substances as with the types of chemical substances already provided or around of which can be different types of chemical substances. moreover will be the same type and amount of marker receptor units that already exist was provided, and the same type and amount of receptor units that has also already been provided. The last one provided chemical substances, the last provided marking Receptor units and the last receptor units provided are in any order brought into contact with each other, thereby forming a Complexes of marker-receptor unit, chemical substance and Receptor unit takes place. The last provided receptor units will be before or after contacting the last provided chemical Substances, last provided marker receptor units and last provided receptor units bound to a suitable material, whereby the complexes formed from marker-receptor unit, chemical Substance and receptor unit are bound to the material and thus a separation of unbound marker receptor units takes place. Then one or more chemical bonds are attached to the Material-bound complexes of marker-receptor unit, chemical The substance and receptor unit are split so that the labeling component the marker receptor unit is separated from the material. The one from that Separate marking components are washed out and through a suitable method is detected. Finally, the ones obtained Results of the two analyzes of the labeling components were compared.

According to a special embodiment of the method, the use the receptor unit is eliminated if the chemical substance is of a kind that the complex of chemical substance and labeling receptor unit itself can be physically separated. Whole cells, larger parts of cells or Cell compartments meet this requirement.

According to a preferred embodiment of the present invention, a any unspecified library of receptors, in particular protein / peptide binding molecules, oligomers or polymers, too Recognize post-translational modifications, reproducible through a corresponding chemistry known to the person skilled in the art on a support material immobilized. A part of a cellular proteome becomes attached to this modified matrix bound to the receptors by specific interaction. Subsequently becomes a second unspecified library of labeling receptors used to label part of the bound cellular proteome. In In this second receptor library, each individual specific receptor carries one Known or unknown, but clear marking. The marking of the binding receptors can be isolated or copied and in the next step be determined qualitatively or quantitatively. This analysis data becomes the Comparison with an identical procedure (identical carrier immobilized receptors, identical second label receptor Library) on another cellular proteome for proteome studies, e.g. B. comparative protein expression studies used.

The methods according to the present invention become very special preferably used for the detection of changes in cell components. at the chemical substances are therefore preferably proteins and their post-translational modifications, especially a complete one Proteome. But it also contains other biologically relevant molecules such as Nucleic acids, glycolipids of the cell membranes, glycosides of the extracellular Matrix or the cell wall of microorganisms, signal molecules of another kind such as certain types of hormones, cytokines and inhibitors, or metabolites that be formed under special conditions. Furthermore, the procedure used to cause pathogenic changes to the cell components investigate what chemical changes of all kinds arises from a non-natural process or reaction.

Building the complex from receptor to protein from the subject to be examined Proteome and the labeled receptor can in different steps. So it is possible to build the whole complex in Build up solution to attach it to a carrier in the second step immobilize. The structure of the two components receptor and Protein are performed in solution, followed by immobilization of the Dimers on a solid matrix. The final step is on the fixed Phase the entire complex is formed with the marker receptor.

Novel in this method is u. a. that when building a structure from three Components only have a unique marker assignment in the second Receptor library is necessary. The composition of the matrix immobilized receptors and the labeled receptor The library does not have to be known before the experiment. The only condition for comparative qualitative and / or quantitative proteome analysis is the constant composition of the marker receptor library and the immobilized receptors.

As the receptor unit, the marker receptor unit can be used are: antibody, cell receptor protein, hormone or cytokine receptor, regulatory protein, DNA or RNA binding protein, enzyme, protein other nature or peptide, a genetically engineered equivalent of this Proteins or parts thereof, DNA, RNA or other nucleic acid products, a genetically engineered equivalent of these nucleic acid products or Parts thereof, an oligoglycoside or its derivatives such as glycoproteins and Glycolipids and genetically engineered equivalents of these derivatives of Oligoglycosides, a lectin, a hormone, cytokine, or other biological Signal molecule, as well as their analogs and antagonists, an inhibitor of Proteins and their analogues, genetically engineered equivalents of these Molecules, a hapten or other modification of proteins or Nucleic acid derivatives, a peptide or nucleic acid aptamer.

Receptor units or labeled receptors can be one wide range of specifically binding molecules such as. B. antibodies, Lectins, receptor proteins, aptamers, inhibitors, peptides, RNA, DNA, Oligonucleotides, PNA, LNA, capture proteins, sugar or haptens. The Receptor units can be bound to the support by covalent bonds z. B. via an amide bond, or via a hapten z. B. biotin, Digoxigenin.

The labeled receptor library can e.g. B. from genetically engineered monoclonal antibodies. The Labels can be bound to the antibody by covalent bonds z. B. via an amide bond, or via a hapten such. B. biotin, Digoxigenin or via a crosslinker, which is a chemical or enzymatic has a cleavable bond between the antibody and the label. Not covalent bonds between antibodies and labels can be created with chaotropic salts or detergents.

According to the invention, nucleic acid oligomers can be used as the label become. Under the term "nucleic acid oligomers" each type of Oligonucleotide, DNA, or RNA understood. Peptides or are also conceivable mass-specific markers as markers of the receptors. The antibody Bio libraries can also be generated using phage display methods.

Genetically generated antibodies can also be used as immobilization receptors a phage display library can be immobilized on the solid matrix. This first antibody pool can e.g. B. by reductive cleavage of a chemical Binding can be obtained when the phage crosses a disulfide bridge Have antibodies and a virus envelope. About the resulting free thiol group the antibodies can be placed on a solid pre-activated with maleimide Carrier material are tied. Then on the affinity material the cellular proteome or a part thereof, with a particular Protein pattern depending on the immobilized antibody set on the column is bound. A second set is used to complete the sandwich structure antibody pool produced via phage display. You can all phages are used. After washing out the not The immobilized phage RNA / DNA is isolated and bound by means of Methods known to the person skilled in the art worked up to obtain the obtained Evaluate oligonucleotides qualitatively and quantitatively with DNA arrays. phages Display methods are an ideal option using genetically engineered methods Antibody libraries to generate receptor units whose Binding specificities are not known before the procedure.

Methods known to the person skilled in the art are u. a. Amplification of the phages RNA / DNA using PCR, digestion of the viral genome with restriction enzymes and / or specific linear amplification of one corresponding to the label Part of the viral genome / antibody gene by RNA polymerases (e.g. T7 RNA Polymerase) or DNA polymerases (e.g. primer extension with Kleenow Fragment, Tli DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase).

According to a preferred embodiment, a first pool of antibodies immobilized on a polymeric support. The connection of the antibodies can via a hapten (e.g. biotin, digoxigenin) or in the simplest case by one chemical crosslinkers. Then on the affinity material cellular proteome or a part thereof, where a specific Protein pattern depending on the immobilized antibody set on the column is bound. A second is used to complete the immuno-sandwich Pool of antibodies labeled with specific oligonucleotides to which Given an affinity column. After washing out the free not in immune complexes Bound antibodies become the bonds between the immobilized Antibodies and the oligonucleotides chemically cleaved. The received unlabeled oligonucleotides are evaluated quantitatively using DNA arrays, which gives you a specific protein profile depending on the selection of the two Receives antibody pools. This procedure enables spatial separation of selection process and detection process so that both are independent can be carried out from each other.

Alternatively, the entire sandwich-immune complex can be built up in solution and be immobilized on a polymeric support in the second step.

It is through a predefined selection of antibodies for the two pools possible a predefined protein profile and its change over time follow. In toxicological and drug effectiveness studies, e.g. B. Changes in the signal cascade and regulation mechanisms, activation of receptor proteins can be specifically examined. With cytological Characterization of tumor tissue, it is possible to mark the tumor and the to determine tumor-triggering factors.

The following pairs of receptors and chemical substances have the required binding affinity and can therefore be used in the context of the present invention:

According to a preferred embodiment of the present invention, the Connection of the receptor units to a suitable material before Bringing the receptor units into contact with the chemical substances and the marker receptor units.

In addition, one or more washing steps are preferably carried out. In particular, washing steps are carried out after the complexes are formed Receptor unit, chemical substance and marker receptor unit and the binding of the complexes to the material. Through this Washing steps are all non-specifically bound material and all Unbound marker receptor units removed.

Preferred as the receptor unit is an antigen, an antibody, a genetically engineered antibody, an F _ab fragment, in particular a genetically modified F _ab fragment, a protein, in particular a DNA or RNA binding protein, a genetically engineered equivalent of one Protein or parts thereof, a peptide, an enzyme, an enzyme antagonist, a hormone, a hormone receptor, a hapten, an oligoglycoside or a lectin.

It is also preferred to use marker-receptor units which, as the receptor unit, comprise an antigen, an antibody, a genetically engineered antibody, an F _ab fragment, in particular a genetically modified F _ab fragment, a protein, in particular a DNA or RNA-binding protein, a genetically engineered equivalent of a protein or parts thereof, a peptide, an enzyme, an enzyme antagonist, a hormone, a hormone receptor, a hapten, an oligoglycoside or a lectin.

The marking component is preferably a Nucleic acid oligomer, it is particularly preferred a deoxyribonucleic acid oligomer, a ribonucleic acid oligomer Peptide nucleic acid oligomer or around a nucleic acid oligomer with structural analogous backbone, the nucleic acid oligomer component being different Nucleic acid oligomer with complementary base sequence, sequence-specific can bind with the formation of base pairs.

The receptor unit of a nucleic acid oligomer receptor unit is preferred to one of the phosphoric acid units, to one of the thiophosphate units one of the phosphoric acid amide units, to one of the sugar units, especially to a sugar-hydroxy group, to a carbonyl, carboxyl, Amide or amine group, or to one of the bases of the nucleic acid oligomer Partially linked, especially to a terminal 3 'or 5' unit.

The receptor unit of a nucleic acid oligomer receptor unit is particularly preferably linked to the nucleic acid oligomer component via a group which can be cleaved by induction, in particular via a -CHOH-CHOH group, a -CO-O-CH ₂ -CH ₂ -O- CO group, an -SS group, an - N = N group or via psoralen.

According to a particularly preferred embodiment of the present invention, the receptor unit of a nucleic acid oligomer receptor unit and the nucleic acid oligomer component of such a unit are each modified with a chemical substance, the two chemical substances represent specific binding partners to one another and the receptor unit is over the specific binding of the two chemical substances to the nucleic acid oligomer component. As the two chemical substances, two specific binding partners are particularly preferably selected from the group consisting of biotin and monovalent avidin, biotin and monovaltent streptavidin, biotin and avidin, biotin and streptavidin, chitin and chitin-binding protein, antigen and antibody, antigen and F _ab fragment of the antibody used. If an antigen and an antibody are used, two specific binding partners are very particularly preferably selected from the group consisting of biotin and anti-biotin antibody, FITC and anti-FITC antibody, digoxigenin and anti-digoxigenin antibody, DNP and anti -DNP antibody, rhodamine and anti-rhodamine antibody, His-Tag and anti-His-Tag antibody, HA-Tag and anti-HA-Tag antibody, Flag-Tag and anti-Flag-Tag antibody, GST -Tag and anti-GST tag antibodies used.

The receptor unit of a nucleic acid oligomer receptor unit can preferably also be attached to a branched or unbranched part of the molecule of any composition and chain length and the branched or unbranched part of the molecule can alternatively be attached to one of the phosphoric acid units, to one of the thiophosphate units, to one of the Phosphoric acid amide units, to one of the sugar units, in particular to a sugar hydroxy group, to a carbonyl, carboxyl, amide or amine group, or to one of the bases of the nucleic acid oligomer component, in particular to one terminal 3 'or 5' unit. The receptor unit can particularly preferably be connected to a branched or unbranched part of the molecule which contains at least one group which can be cleaved by induction and which is located in the part of the molecule which connects the receptor unit and the nucleic acid oligomer component, the receptor unit in particular to one branched or unbranched part of the molecule is attached, the at least one -CHOH-CHOH group, a -CO-O-CH ₂ -CH ₂ -O-CO group, an -SS group, an -N = N group or psoralen contains.

It is also preferred that the branched or unbranched molecular part in the part of the molecule connecting the receptor unit and the nucleic acid oligomer component contains a bond between two chemical substances, the two chemical substances being specific binding partners and the binding of the two chemical substances of the mutually specific bond of the two chemical substances. In particular, two specific binding partners are selected as the two chemical substances from the group consisting of biotin and monovalent avidin, biotin and monovalent streptavidin, biotin and avidin, biotin and streptavidin, chitin and chitin-binding protein, antigen and antibody, antigen and F _ab Fragment of the antibody is preferably used. If an antigen and an antibody are used, two specific binding partners are very particularly preferably selected from the group consisting of biotin and anti-biotin antibody, FITC and anti-FITC antibody, digoxigenin and anti-digoxigenin antibody, DNP and anti -DNP antibody, rhodamine and anti-rhodamine antibody, His-Tag and anti-His-Tag antibody, HA-Tag and anti-HA-Tag antibody, Flag-Tag and anti-Flag-Tag antibody, GST -Tag and anti-GST- Tag antibodies used.

The method according to the invention is very particularly preferably used for the detection of proteins. The chemical substances are therefore preferably proteins, very particularly preferably a complete proteome. The one or more chemical bonds between the label and the marker receptor unit are cleaved, preferably by adding an oxidizing agent, by adding a reducing agent, by adding an acid suitable for cleaving an amide bond, or by irradiating light, particularly preferably by A reducing or oxidizing agent suitable for cleaving an -SS group, for cleaving a -CHOH-CHOH group or for cleaving a -CO-O-CH ₂ -CH ₂ -O-CO group is added, in particular by adding NaIO ₄ , by adding H ₂ N-OH, by adding DTT or by adding β-mercaptoethanol. These bonds can also be cleaved enzymatically.

The detection of the nucleic acid oligomer components is preferably carried out via the Hybridization to complementary nucleic acid oligomers, the Detection particularly preferably amperometric, cyclic voltametric, impedance spectroscopic or potentiometric.

The particular advantages of the method according to the invention are in particular in the simultaneous detection of several types of nucleic acid oligomer Ingredients. This allows a variety of chemical substances and in particular a large number of proteins can be detected in parallel.

The present invention also includes a kit for performing one of the above described method. The kit includes an effective amount of at least one Type of nucleic acid oligomer receptor units described above. Prefers the kit additionally comprises an effective amount of at least one kind of the above described receptor units. The kit particularly preferably comprises additionally comprises a chromatography column and very particularly preferably the kit additionally contains a dot pre-filled with suitable probe oligonucleotide types Plot membrane for the detection of the nucleic acid oligomer components of the Nucleic acid receptor units.

Ways of Carrying Out the Invention example 1 Binding of the nucleic acid oligomer receptor units to Material immobilized proteins

It is worked in dim light or with red light. 0.1 μg of the desired nucleic acid oligomer-labeled F _ab fragments in PBS in the smallest possible volume (or diluted in a column volume) are placed on the column or membrane on the columns or membranes washed with PBS or PBST. Allow the nucleic acid-labeled F _ab - fragments for 1 hour at room temperature and another 16 hours at 4 ° C to the immobilized on the material proteins bind, and washed with PBST and phosphate wash buffer (50 mM potassium phosphate pH 7.5, 500 mM NaCl ). The remaining non-bound nucleic acid oligomer-labeled F _ab fragments are removed by the washing steps, and only the nucleic acid oligomer-protein binding units that have been bound to the target remain on the column material or the membrane. The nucleic acid oligomer unit can be isolated and further analyzed using one of the methods described in Examples (3-5) below.

Example 2 Immobilization of soluble proteins (targets) on anti-fluorescein Antibody modified material

It is worked in dim light or with red light. The reaction tubes are rinsed with PBST and PBS before use. 5 µl of a soluble native cytoplasmic protein fractions as a target fraction in PBS (cytoplasmic proteins, soluble fraction from core and membrane fractions) are placed on a Biogel PD6 column, which was previously coated with carbonate buffer (50 mM NaHCO ₃ / Na ₂ CO ₃ , pH 9.5, 150 mM NaCl) was equilibrated. It is eluted with carbonate buffer and the protein fractions are collected (cleaning step and buffer change). 0.2 mg FITC (20 mg / ml in DMSO) is added to the collected and pooled protein fractions. The mixture is left to react for 30 min at room temperature. The reaction solution is then placed on a Biogel PD6 column which was previously equilibrated with PBS, it is eluted with PBS and the protein fractions are collected (purification step). 0.02 µg of the desired nucleic acid oligomer-labeled F _ab fragments are added to the combined protein fractions. The nucleic acid oligomer-labeled F _ab fragments are then allowed to bind to the antigens for 1 hour at room temperature and for a further 16 hours at 4 ° C. The entire sample (diluted in approximately one column volume) is placed on an affinity chromatography column which is coated with anti-FITC antibodies. The FITC-labeled antigens are allowed to bind for about 1 hour at room temperature (and possibly for a further 12 hours at 4 ° C), washed with PBST, then with phosphate wash buffer (50 mM, pH 7.2, 500 mM NaCl) and isolates the nucleic acid oligomer unit according to a method described in the following examples (3-5). The remaining free nucleic acid oligomer-labeled F _ab fragments are removed from the column by the washing steps, and only complexes of these nucleic acid oligomer F _ab fragments with the target remain on the column, so that the nucleic acid oligomer units isolated from the column correspond to the amount Target match.

Example 3 Separation of the nucleic acid oligomer from the receptor unit

• a) Denaturation with guanidinium hydrochloride: To the isolated in the eluate, in Pellet-bound, membrane-bound or column-bound Nucleic acid oligomers attached to the receptor unit (and possibly also are bound to the target or in the various immune complexes) 20 mM sodium phosphate buffer pH 7.2, 6 M guanidinium / HCl, 10 mM DTT, 2% TritonX 100 preheated to 90 ° C (approx. One column volume / approx. 3 times the volume with other isolates), elutes in the case of column-bound complexes after 2-3 min with another 3 column volumes of the same buffer and collects in the case of column-bound immune complexes the eluate. The eluate will be short at 15,000 × g centrifuged, 5 µg poly-dT (33) to support the supernatant Precipitation is given, the sample is then filled with half a volume 3 M potassium acetate pH 5.0 and four times the volume of ethanol (-20 ° C) and the DNA precipitated at 15,000 x g at 4 ° C for 40 min. The pellet will separated, washed with 70% ethanol (-20 ° C) and 100% ethanol (-20 ° C) and air dried. Alternatively, add 0.5 volume after adding the carrier DNA Ethanol and 0.5 volume of 3 M potassium acetate pH 5.0 added and on a 'spin column 'with an activated glass fiber membrane. After cooling the Solution (approx. 10 min) is centrifuged, then with the same buffer and then washed with ethanol. The nucleic acid oligomers are finally eluted from the glass fiber membrane with 50 µl TE buffer (kits from Qiagen, Promega, et al. a.). For amplification, the pellet is placed in the PCR buffer (see Example 4) and for direct detection without amplification in the hybridization buffer (cf. Example 5) added.
• b) Splitting the linker after reduction: To split the RS-SR 'unit there one to the membrane-bound or isolated in the eluate, bound in the pellet or columnar nucleic acid oligomer receptor units, optionally are bound to the target or in the various immune complexes, 10 mM DTT in PBS (one column volume or 1-3 volumes of the sample), leaves for 15 min act, elute with 1-2 column volumes of the same buffer and collect in the case of column-bound complexes, the eluate. The eluate will be briefly at 15,000 x g centrifuged. 5 µg of poly-dT (33) are added to the supernatant Support of DNA precipitation given, the sample is then with half a volume of 3 M potassium acetate pH 5.0, and four times the volume Ethanol (-20 ° C) and the DNA at 15,000 × g at 4 ° C for 40 min precipitated. The pellet is separated off with 70% ethanol (-20 ° C) and 100% Washed ethanol (-20 ° C) and air dried. For amplification, this is Pellet in the PCR buffer (see Example 4) and for direct detection without Amplification in the hybridization buffer (see Example 5) added. alternative the nucleic acid oligomers can also be described as described under (i) Glass fiber membranes are cleaned. This method has like that in (iii) and (iv) methods described the advantage that the DNA is separated from the protein and fewer proteins in the eluate are also washed out.
• c) Cleavage of the linker after oxidation: To cleave the RC (OH) -C (OH) R 'unit (in the linker between nucleic acid oligomer and receptor unit), 10 mM NaIO ₄ in PBS (is added to the isolated free or column-bound complexes Column volume or the same volume of the elution solution), takes effect for 15 min, elutes with 1-2 column volumes of PBS and collects the eluate in the case of the column-bound complexes. 5 μg of poly-dT (33) are added to the eluate to support the DNA precipitation and it is isolated as described under (i) via ethanol precipitation or via glass fiber membrane.
• d) Photolytic cleavage of the linker: For cleaving the RN = NR 'unit irradiate the isolated free or suspended in a beaker column-bound complexes in PBS with stirring for 10 min with the light of a 500 W xenon lamp with 330 to 350 nm interference filter. The Column material is filtered, briefly washed with PBS buffer and the filtrate collected. The combined filtrates are supported with 5 µg poly-dT (33) the DNA precipitation and, as described under (i), over ethanol Precipitation or isolated over glass fiber membrane.

Example 4 Amplification and subsequent labeling of the nucleic acid oligomer

• a) Exponentielle Amplifikation: Nach Abtrennung der DNA aus den Komplexen aus Nukleinsäureoligomer-Rezeptor-Einheit, chemischer Substanz und Rezeptor- Einheit (vgl. Beispiel 3) löst man das DNA-enthaltende Pellet in 10 mM Tris/HCl pH 8.0. Ein Aliquot davon (1/20 - 1/100) wird zur Amplifikation eingesetzt. Damit die Amplifizierungsrate exakt ermittelt werden kann wird vor der Amplifikation eine definierte Menge (10 fg, 1 pg, 50 pg, 1 ng) von 4 Kontroll-Nukleinsäureoligomeren (mit eigenen spezifischen Kodierungsregionen und ansonsten identischen Aufbau wie die zur Markierung verwendeten Nukleinsäureoligomere) zum Isolat der Nukleinsäureoligomere zugegeben. Man gibt die beiden universalen Primer P1 und P2 (ein Primer bevorzugt mit einem Detektions-Marker) und die Nukleotid- Bausteine dCTP, dGTP, dATP und dTTP zu und führt z. B. eine Standard-PCR mit 11 bis 18 Zyklen durch um eine in etwa 1000fache bis 100.000fache Amplifikation zu erreichen. Bei Verwendung von unterschiedlichen Primer-Gruppen, die jede für sich universal nur für eine bestimmte Gruppe von Nukleinsäureoligomer-Rezeptor- Einheiten ist, werden für jedes Primer-Paar entsprechend 4 spezifische Kontroll- Nukleinsäureoligomere in definierter Menge vor der PCR zugegeben und die PCR optional in separaten PCR-Reaktionen amplifiziert. Die Lösung wird nach erfolgter PCR-Synthese mit 5 µg Poly-dT(33) zur Unterstützung der Präzipitation und mit 4 Volumen 6 M Guanidiniumchlorid versetzt, um die Polymerase zu zerstören. Zu dieser Lösung wird anschließend ein halbes Volumen 3 M Kaliumacetat pH 5.0, das vierfache Volumen Ethanol (-20°C) gegeben und die DNA bei 15.000 × g bei 4°C über 40 min präzipitiert. Das Pellet wird mit 70% Ethanol (-20°C) und 100% EtOH (-20°C) gewaschen und an der Luft getrocknet. Zur direkten Detektion wird das Pellet im Hybridisierungspuffer gelöst (vgl. Beispiel 5), zur nachträglichen Modifikation mit Detektions-Marker führt man anschließend eine lineare Amplifikation mit gleichzeitigem Einbau eines Detektions-Markers (siehe Beispiel 4ii) oder eine nachträgliche chemische Modifikation mit Detektions-Marker (siehe Beispiel 4iii) durch. Für die nachträgliche Modifikation mit einem Detektions- Marker wird nach der PCR ein zusätzlicher Chromatographieschritt eingeführt, um die nicht umgesetzten Primer von den amplifizierten Nukleinsäureoligomeren abzutrennen. Dazu wird der gesamte Ansatz auf eine Sephacryl S100-Säule aufgetragen und die erste Fraktion (ca. 25 kDa), die die Amplifikationsprodukte enthält, gesammelt. Die zweite Fraktion (ca. 4 kDa), die die Primer enthält, wird verworfen.
• b) Lineare Amplifikation: Unter sonst gleicher Vorgehensweise wie unter Beispiel 4i) erhält man bei Weglassen des Primers P1 (Zusatz von Primer P2, der einen Detektions-Marker wie z. B. Rhodamin trägt) bei Durchlaufen von 10 bis 40 Synthesezyklen (analog zur PCR) eine 10fache bis 40fache Amplifikation als ss- DNA, wobei die komplementäre Sequenz des Nukleinsäureoligomers als ss-DNA erhalten wird. Setzt man die Amplifikationsprodukte aus Beispiel 4i) ein, kann wahlweise auch mit Primer 1 (ebenfalls mit Detektions-Marker) spezifisch das Nukleinsäureoligomer als ss-DNA amplifiziert werden. Vorteil dieses Amplifikationsschrittes ist, dass die zu detektierenden Nukleinsäureoligomere bereits als ss-DNA vorliegen.
• c) Neben der beschriebenen Methode kann auch durch Zugabe von Nukleotiden, die einen Detektions-Marker tragen, über PCR die DNA oder über in- vitro Transskription die RNA mit einem Detektionsmarker versehen werden, daneben kann der Detektions-Marker an das Nukleinsäureoligomer nachträglich angebunden werden.

If the nucleic acid oligomers bound to the receptor units are DNA or RNA which have primer-binding regions which are identical for all the receptor-bound nucleic acid oligomers used within a qualitative and / or quantitative target analysis (proteome analysis etc.) the nucleic acid oligomers present in the test solution, which were separated using one of the methods (i) to (v) in Example 3, are amplified by PCR. With identical primers and identical or almost identical length of all nucleic acid oligomers and the selection criteria for the individual sequences, amplification via PCR is possible without changing the representation of individual nucleic acid oligomers or the composition of the nucleic acid oligomer mixture.

• a) Exponential amplification: After separation of the DNA from the complexes of nucleic acid oligomer-receptor unit, chemical substance and receptor unit (cf. Example 3), the DNA-containing pellet is dissolved in 10 mM Tris / HCl pH 8.0. An aliquot thereof (1/20 - 1/100) is used for amplification. So that the amplification rate can be determined exactly, a defined amount (10 fg, 1 pg, 50 pg, 1 ng) of 4 control nucleic acid oligomers (with their own specific coding regions and otherwise identical structure to the nucleic acid oligomers used for labeling) becomes isolate before amplification of the nucleic acid oligomers added. The two universal primers P1 and P2 (a primer preferably with a detection marker) and the nucleotide building blocks dCTP, dGTP, dATP and dTTP are added and z. B. a standard PCR with 11 to 18 cycles by in order to achieve an approximately 1000-fold to 100,000-fold amplification. When using different primer groups, each of which is universal only for a certain group of nucleic acid oligomer receptor units, 4 specific control nucleic acid oligomers are added in a defined amount for each primer pair before the PCR and the PCR optionally in separate PCR reactions amplified. After the PCR synthesis, 5 μg of poly-dT (33) to support the precipitation and 4 volumes of 6 M guanidinium chloride are added to destroy the polymerase. Half a volume of 3 M potassium acetate pH 5.0, four times the volume of ethanol (-20 ° C.) are then added to this solution and the DNA is precipitated at 15,000 × g at 4 ° C. for 40 min. The pellet is washed with 70% ethanol (-20 ° C) and 100% EtOH (-20 ° C) and air dried. For direct detection, the pellet is dissolved in the hybridization buffer (see Example 5), for subsequent modification with a detection marker, linear amplification is then carried out with simultaneous incorporation of a detection marker (see Example 4ii) or a subsequent chemical modification with a detection marker (see example 4iii). For the subsequent modification with a detection marker, an additional chromatography step is introduced after the PCR in order to separate the unreacted primers from the amplified nucleic acid oligomers. For this purpose, the entire batch is applied to a Sephacryl S100 column and the first fraction (approx. 25 kDa), which contains the amplification products, is collected. The second fraction (approx. 4 kDa), which contains the primers, is discarded.
• b) Linear amplification: otherwise using the same procedure as in example 4i), if the primer P1 is omitted (addition of primer P2, which bears a detection marker such as rhodamine), 10 to 40 synthesis cycles (analogous to PCR) a 10-fold to 40-fold amplification as SS-DNA, the complementary sequence of the nucleic acid oligomer being obtained as SS-DNA. If the amplification products from Example 4i) are used, the nucleic acid oligomer can also be specifically amplified as ss-DNA using primer 1 (likewise with a detection marker). The advantage of this amplification step is that the nucleic acid oligomers to be detected are already present as ss-DNA.
• c) In addition to the described method, the detection marker can also be provided with the addition of nucleotides carrying a detection marker, the DNA via PCR or the RNA via in vitro transcription, and the detection marker can also be subsequently attached to the nucleic acid oligomer ,

Example 5 (Parallel) detection of the nucleic acid oligomers via fluorescence

• 1. Parallel detection with dot-plot technique on modified nylon membrane (Hybond N +) via fluorescence detection: The test sites carry probe oligonucleotides covalently attached to a support material, which are complementary to the nucleic acid oligomers to be detected. The probe oligonucleotides are dropped onto the modified nylon membrane in any but known pattern. The membrane is then baked at 80 ° C. for 2 hours, as a result of which the probe oligonucleotides are firmly bound. Alternatively, the probe oligonucleotide 3 'or 5' terminally carry a linker which has a reactive or activatable group via which the probe oligonucleotide is bound in a defined manner to a suitable support surface (e.g. an NH ₂ group on the probe oligonucleotide and an isothiocyanate group on the support).
• 2. Free binding sites on the membrane are incubated at 45 ° C for 30 min with hybridization buffer 1 (7% SDS, 500 mM Na phosphate buffer pH 7.5, 5 mM EDTA, 0.1 mg / ml ssDNA from an organism with as little as possible phylogenetic kinship, e.g. B. pBR322 from E. coli, 0.5 mg / ml acetylated BSA) saturated (pre-hybridization). The "dots" (test sites with Probe oligonucleotide) are isolated from the hybridization solution containing the Contains nucleic acid oligomers or their amplification products overlaid. Double-stranded DNA nucleic acid oligomers are previously at 5 ° C for 5 minutes denatured and briefly chilled on ice before going to the hybridization solution be added. The probe oligonucleotides and Hybridize the nucleic acid oligomers of the solution at approx. 40 ° C. for approx. 3 to 16 h. After removal of the non-hybridized components (non-stringent washing with 2 × SSC, 0.1% SDS, 1 mM EDTA for 15-60 min at 40 ° C and then stringent washing with 0.4 × SSC, 0.1% SDS, 0.1 mM EDTA, for 5 min at 50 ° C) the corresponding ss- are determined based on the fluorescence pattern Nucleic acid oligomers detected from the target analysis (qualitative analysis) and / or recorded quantitatively.
• 3. Parallel detection on DNA chips via fluorescence detection: As a chip can z. B. a custom made Affymetrix GeneChip® (cf. Wodicka et al. 1997, Nature Biotechnology, 15, 1359ff) can be used. The test sites enter on Carrier material covalently linked probe oligonucleotides that are complementary to the nucleic acid oligomers to be detected. The one for target analysis contain used nucleic acid oligomers (or the amplification products) according to the Affymetrix protocol, a biotin as the primary detection marker.
• 4. 10 µg of the nucleic acid oligomers are used as SS nucleic acid oligomers in 250 µl (per chip used) hybridization buffer 3 (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% TWEEN 20, pH 6.5-6.7) together with 0.1 mg / ml herring Sperm ss-DNA and 0.5 mg / ml acetylated BSA added and on the Brought chip. Then you leave the complementary to each other Hybridize nucleic acid oligomers for 16 h at 43 ° C. After done Hybridization is the chip used to remove the non-hybridized components washed (non-stringent washing with 900 mM NaCl / 60 mM Na phosphate pH 7.6, 6 mM EDTA, 0.05% Triton X100 for 1 h at 40 ° C and then stringent washing with 75 mM NaCl / 5 mM Na phosphate pH 7.6, 0.5 mM EDTA, 0.05% Triton X100 for 15 min at 40 ° C). The connection of the detection marker Streptavidin-modified phycoerithrin is z. B. with convocal fluorescence microscope and coupled CCD camera read. Based on the pattern of fluorescence, certain Nucleic acid oligomers detected (qualitative analysis) or also quantitatively are recorded (quantitative analysis).

Example 6 Exemplary cellular proteome analysis

• a) Protein digestion: tissue cells (> 100 cells), e.g. B. the cellular material Biopsy, - after adding RIPA buffer (150 mM NaCl 1% NP-40, 0.5% Deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, 10 µl per mg cell tissue, however, at least 1 µl) is digested in the microtip sonifier. Much of the cellular proteome of the cells becomes partial as in Example 6ii) denaturing conditions in solution (separation of the remaining insoluble protein fraction (ECM / cytoskeleton) at 800 × g / 4 ° C / 15 min and Residues of cell debris from the separated supernatant at 50,000 × g / 4 ° C / 30 min). The RIPA-buffered supernatant contains the partially denatured soluble ones cytoplasmic proteins and most of the membrane proteins. The in Pellet remaining proteins are discarded.
• b) Construction of the receptor-nucleic acid oligomer complexes: z. B. biotinylated F _ab fragments of the antibodies are used (in general, the selection of the receptor units depends on the question of the proteome analysis and the receptor units are selected accordingly). A synthetic ss-DNA of the sequence is used as the nucleic acid oligomer
  
  
  with the primer binding region P1 (= GACGAGACGTCATGG), the base region P'2 (= CTCACCAGAGCTTCG) complementary to the primer P2, the coding sequence K16 specific for a receptor unit (or a specific receptor unit group), see below, and a 3-terminal TTT-hexyl-NH ₂ unit and a 5'-terminal rhodamine-TTT unit, which were covalently bound in the solid phase synthesis. Nucleic acid oligomer 1 for binding receptor unit 1 has e.g. B. the coding sequence 5'-GTTCCAAGCATGGTTC-3 ', positions 1, 2, 9 and 10 of the coding sequence being independent, positions 3, 5 and 7 depending on position 1 by the permutation rule G → A → C → T → G For a G at position 1 it follows that position 3 is an A, position 5 is a C and position 7 is a T. Similarly, due to the same permutation rule, positions 4, 6 and 8 depend on the independent occupation of position 2, positions 11, 13 and 15 depend on independent position 9 and positions 12, 14 and 16 depend on independent position 10; This simple permutation rule results in 4 ⁴ = 256 different coding sequences due to the 4 freely selectable positions, which take into account that (a) the GC content of the coding sequence (50%) is retained and (b) the individual coding sequences differ by at least 4 Differentiate bases. If you also want to ensure that (c) no more than two of the bases of a base pair (T and / or A or G and / or C) are adjacent, only position 8 can no longer be freely selected within this occupation rule, so it is conditionally dependent , so that there are only 3 occupancy options for position 8 and thus only 4 ³ × 3 = 192 different coding sequences remain from the original 4 ⁴ = 256, which definitely meet the conditions (a) to (c) (of the 16 occupancy options for bases 7 and 8 there are 8 options, since 2 bases of a base pair are adjacent, which then only allow 2 choices for position 9, while for the other 8 options that do not have 2 bases of a base pair adjacent, all 4 options remain remain so that for Base 9 there are only 3 options instead of 4; for a longer coding sequence from any multiple of a Ber Seq nence, i.e. from n × 8 bases, there are analogously (4 ^{2 (n-1)} × 3 ^n-1 ) coding sequences which meet the conditions (a) to (c) or 4 ²ⁿ coding sequences which meet the conditions (a) and (b) are sufficient).
• c) In order to bind the F _ab fragments, all the nucleic acid oligomer components necessary for the different antibodies with different coding sequences (any of the maximum of 192 nucleic acid oligomers generated above) are coupled to the linker dithio-bis-propionic acid sulfosuccinimidyl ester (one separate batch of nucleic acid oligomer per antibody: 1 nMol of the nucleic acid oligomer is taken up with 50 nMol dithio-bis-propionic acid sulfosuccinimidyl ester in approx. 100 µl 100 mM NaHCO ₃ / Na ₂ CO ₃ , pH 9, it is left overnight in the dark at room temperature, isolated and the converted DNA is separated in carbonate buffer). The nucleic acid oligomer linker units of a reaction are then reacted with each type of F _ab fragment and isolated and the number of DNA units per F _ab fragment z. B. via the ratio of the rhodamine fluorescence at 575 nm (proportional to the DNA) to the absorption at 285 nm (proportional to the F _ab fragments).
• d) Target-receptor-unit coupling: The F _ab fragments of all batches dissolved in PBS and labeled with the nucleic acid oligomers are combined (approx. 10 ml) and 10 μl thereof are added to 10 μl solution of the partially denatured proteins in RIPA buffer (corresponding to 1 mg of cell material). The proteins are allowed to couple to the F _ab fragments for about 3 h at room temperature and for about 24 h at 4 ° C. Add 1 µg of a cocktail of polyclonal antibodies in PBS that are specific for the same targets (proteins) as the F _ab fragments (and optionally additional anti-Ig antibodies that are specific for the F _c regions of these antibodies) and has the antibodies coupled to the proteins for a further approx. 3 h at room temperature and for approx. 12 h at 4 ° C.
• e) separation of the complexes from nucleic acid-labeled receptor, target and Target-specific antibody: A protein G-coated agarose column is used equilibrated with PBST, pH 7.5, the solution with the complexes of nucleic acid labeled receptor, target and target-specific antibody for Immobilization of the immune complexes was applied to the column and 2 h at Incubated at room temperature so that the immune complexes can bind. After that the column is eluted with PBS (the eluate is discarded or used later) Control kept) and washed.
• f) Separation, amplification and quantification of the DNA from the carrier-immobilized antibody-target-receptor-DNA-immune complexes: To cleave the -SS unit in the linker between DNA and F _ab fragment, a column volume of 30 mM DTT in PBS is added. The column is left to stand for about 15 minutes, eluted with PBS, and the DNA in the eluate is isolated and purified. The DNA obtained is then amplified exponentially as described under Examples 4i) and iii) using PCR using primer 2 and rhodamine-modified primer P1, the amplified DNA is melted (heating for 5 min at 100 ° C., then cooled on ice), placed on a dot plot membrane provided with probe oligonucleotides complementary to the original coding sequences, hybridized and washed (as described in Example 5i). The fluorescence of the spots is then determined quantitatively (cf. Example 5i).

Claims (33)

1. A method for qualitatively and quantitatively comparative detection of chemical substances comprising the steps a) at least one type of chemical substance is provided, b) at least one type of marker-receptor unit is provided, the marker-receptor units each consisting of a known marker and a respective receptor unit connected to the marker, and the receptor unit can specifically bind a chemical substance, c) the chemical substances provided in step a) and the marker receptor units provided in step b) are brought into contact with one another, whereby complexes are formed from at least one marker receptor unit and chemical substance, d) the complexes of the marker-receptor unit and chemical substance are separated from the unbound marker-receptor units, e) one or more chemical bonds of the complexes of marker-receptor unit and chemical substance are cleaved in such a way that the marking constituent is separated from the complex of marker-receptor unit and chemical substance, f) the marking constituents separated in step e) are washed out and detected by a suitable method, g) at least one type of chemical substance is provided, which can be the same types of chemical substances as in step a) or different types of chemical substances, h) the same type and amount of labeling receptor units as in step b) is provided, i) the chemical substances provided in step g) and the marker receptor units provided in step h) are brought into contact with one another, as a result of which complexes are formed from at least one marker receptor unit and chemical substance, j) the complexes of the marker-receptor unit and chemical substance are separated from the unbound marker-receptor units, k) one or more chemical bonds of the complexes of the marker-receptor unit and chemical substance are cleaved in such a way that the marking constituent is separated from the complex of the marker-receptor unit and chemical substance, l) the marking constituents separated off in step k) are washed out and detected by a suitable method, m) the results obtained in step f) and in step l) are compared with one another. 2. The method of claim 1, wherein after step b) the step a) at least one type of receptor unit is provided, is carried out as step c) the step a) the chemical substances provided in step a), the marker receptor units provided in step b) and the receptor units provided in step bb) are brought into contact with one another in any order, thereby forming complexes from at least one marker -Receptor unit, chemical substance and receptor unit takes place, is carried out as step d) the step a) the receptor units are bound to a suitable material before or after the contacting according to step c), as a result of which the complexes formed from the marker-receptor unit, chemical substance and receptor unit are bound to the material and thereby separate unbound Marker receptor units are carried out, is carried out as step e) the step a) one or more chemical bonds of the complexes of marker-receptor unit, chemical substance and receptor unit bound to the material are cleaved so that the marker component is separated from the material, is carried out as step f) the step a) the marking components separated from the material in step e) are washed out and detected by a suitable method, is carried out after step h) the step a) the same type and amount of receptor units as in step bb) is provided, is carried out as step i) the step a) the chemical substances provided in step g), the marker receptor units provided in step h) and the receptor units provided in step hh) are brought into contact with one another in any order, thereby forming complexes from at least one marker -Receptor unit, chemical substance and receptor unit takes place, is carried out as step j) the step a) the receptor units are bound to a suitable material before or after the contacting according to step i), as a result of which the complexes formed from the marker-receptor unit, chemical substance and receptor unit are bound to the material and thereby separate unbound Marker receptor units are carried out, is carried out as step k) the step a) one or more chemical bonds of the complexes of the marker-receptor unit, chemical substance and receptor unit which are formed in step i) and are bound to the material are cleaved so that the marker component of the marker-receptor unit is separated from the material is separated is carried out and as step l) the step a) the marking components separated from the material in step k) are washed out and detected by a suitable method, is carried out. 3. The method according to claim 2, wherein the connection of the in step bb) provided receptor units to a suitable material in step d) before contacting the receptor units with those in step a) provided chemical substances and the in step b) provided marker receptor units. 4. The method according to claim 2 or 3, wherein the connection of the in step hh) provided receptor units to a suitable material in step j) before contacting the receptor units with those in step g) provided chemical substances and the in step h) provided marker receptor units. 5. The method according to any one of the preceding claims, wherein an additional or several washing steps are carried out, in particular Washing steps are carried out according to steps e) and / or k). 6. The method according to any one of the preceding claims, wherein in step bb) and in step hh) as an receptor unit an antigen, an antibody, a genetically engineered antibody, an F _ab fragment, in particular a genetically modified F _ab fragment Protein, in particular a DNA or RNA binding protein, a peptide, an allergen, an enzyme, an enzyme antagonist, a hormone, a hormone receptor, a hapten, an oligoglycoside or a lectin is provided. 7. The method according to any one of the preceding claims, wherein in step b) and in step h) marker receptor units are provided which as the receptor unit, an antigen, an antibody, a genetically engineered antibody, an F _ab fragment, in particular a genetically modified F _ab fragment, a protein, in particular a DNA or RNA binding protein, a peptide, an allergen, an enzyme, an enzyme antagonist, a hormone, a hormone receptor, a hapten, an oligoglycoside or a lectin , 8. The method according to any one of the preceding claims, wherein marking Receptor units are provided, the labeling component of which Deoxyribonucleic acid oligomer, a ribonucleic acid oligomer Peptide nucleic acid oligomer, a structural nucleic acid oligomer analog backbone, a peptide or a mass-specific label. 9. The method according to claim 8, wherein the receptor unit is one Nucleic acid oligomer receptor unit to one of the phosphoric acid Units, to one of the thiophosphate units, to one of the Phosphoric acid amide units, to one of the sugar units, in particular to a sugar-hydroxy group, to a carbonyl, carboxyl, amide or Amine group, or to one of the bases of the nucleic acid oligomer Component is connected, in particular to a terminal 3'- or 5'- Unit. 10. The method according to any one of the preceding claims, wherein the receptor Unit of a marker-receptor unit via an induction cleavable group is attached to the marker component. 11. The method according to claim 10, wherein the receptor unit via a -CHOH-CHOH group, a -CO-O-CH ₂ -CH ₂ -O-CO group, an -SS group, a -N = N Group or connected via psoralen. 12. The method according to any one of claims 1 to 10, wherein the receptor unit a marker receptor unit and the marker component of one such unit are each modified with a chemical substance that specific chemical binding partners for each other represent and the receptor unit via the mutually specific binding of the two chemical substances on the marking component is connected. 13. The method according to claim 12, wherein as the two chemical substances two specific binding partners selected from the group consisting of biotin and monovalent avidin, biotin and monovaltent streptavidin, biotin and avidin, biotin and streptavidin, chitin and chitin-binding protein, antigen and Antibody, antigen and F _ab fragment of the antibody are used. 14. The method according to claim 13, wherein two as antigen and antibody specific binding partners selected from the group consisting of Biotin and anti-biotin antibodies, FITC and anti-FITC antibodies, digoxigenin and anti-digoxigenin antibody, DNP and anti-DNP antibody, rhodamine and anti-rhodamine antibodies, His-Tag and anti-His-Tag antibodies, HA- Tag and anti-HA-Tag antibodies, Flag-Tag and anti-Flag-Tag antibodies, GST-Tag and anti-GST-Tag antibodies can be used. 15. The method according to claim 8 or 9, wherein the receptor unit Nucleic acid oligomer receptor unit to a branched or unbranched molecular part of any composition and chain length is attached and the branched or unbranched part of the molecule alternatively to one of the phosphoric acid units, to one of the thiophosphate units, to one of the phosphoric acid amide units, to one of the sugar units, especially to a sugar-hydroxy group, to a carbonyl, carboxyl, Amide or amine group, or to one of the bases of the Nucleic acid oligomer component is bound, in particular to one terminal 3 'or 5' unit. 16. The method of claim 15, wherein the receptor unit to one branched or unbranched part of the molecule is bound, at least contains a group which can be split by induction and which contains the receptor Unit and the part of the molecule connecting the nucleic acid oligomer component located. 17. The method according to claim 16, wherein the receptor unit is attached to a branched or unbranched part of the molecule, the at least one -CHOH-CHOH group, a -CO-O-CH ₂ -CH ₂ -O-CO group -S- S group, a -N = N group or psoralen contains. 18. The method of claim 16, wherein the branched or unbranched Molecule part in which the receptor unit and the nucleic acid oligomer Constituent part of the molecule binds two chemical bonds Contains substances, the two chemical substances to each other represent specific binding partners and the binding of the two chemical substances of the specific binding of the two chemical substances. 19. The method according to claim 18, wherein as the two chemical substances two specific binding partners selected from the group consisting of biotin and monovalent avidin, biotin and monovalent streptavidin, biotin and avidin, biotin and streptavidin, chitin and chitin-binding protein, antigen and Antibody, antigen and F _ab fragment of the antibody are used. 20. The method according to claim 19, wherein two as antigen and antibody specific binding partners selected from the group consisting of Biotin and anti-biotin antibodies, FITC and anti-FITC antibodies, DNP and anti-DNP antibodies, digoxigenin and anti-digoxigenin antibodies, Rhodamine and anti-rhodamine antibodies, His-Tag and anti-His-Tag Antibodies, HA-Tag and anti-HA-Tag-Antibodies, Flag-Tag and anti-Flag-Tag Antibodies, GST-Tag and anti-GST-Tag antibodies can be used. 21. The method according to any one of the preceding claims, wherein it is the chemical provided in step a) and / or in step g) Substances are proteins. 22. The method according to claim 21, wherein it is in step a) and / or in Step g) chemical substances provided is a proteome. 23. The method according to any one of the preceding claims, wherein the cleavage one or more chemical bonds of the complexes Labeling receptor unit, chemical substance and receptor unit in Step e) and / or in step k) by adding an oxidizing agent Add a reducing agent, by adding one to cleave one Suitable acid amide bond or by irradiation of light. 24. The method according to claim 23, wherein the cleavage by adding a to cleave an -SS group, to cleave a -CHOH-CHOH group or to cleave a -CO-O-CH ₂ -CH ₂ -O-CO group Suitable reducing or oxidizing agent is carried out, in particular by adding NaIO ₄ , by adding H ₂ N-OH, by adding DTT or by adding β-mercaptoethanol. 25. The method according to any one of the preceding claims, wherein the Connection of receptor unit and label within one Label receptor unit consists of a covalent bond. 26. The method according to any one of claims 1 to 24, wherein the receptor unit a marker-receptor unit on the surface of a virus or a cell is attached and the marker in the virus or cell corresponds to existing DNA or RNA. 27. The method according to any one of the preceding claims, wherein the detection of the nucleic acid oligomer components via the hybridization Complementary nucleic acid oligomers takes place. 28. The method according to claim 27, wherein the detection is amperometric, cyclic voltametric, impedance spectroscopic or potentiometric. 29. The method according to any one of claims 27 or 28, wherein several types of Nucleic acid oligomer components can be detected simultaneously. 30. Kit for performing a procedure according to the previous ones Claims comprising an effective amount of at least one type of Labeling receptor units according to claims 1 to 29. 31. The kit of claim 30, wherein the kit additionally contains an effective amount at least one type of receptor unit according to claims 2 to 29 includes. 32. Kit according to one of claims 30 or 31, wherein the kit additionally one Chromatography column includes. 33. Kit according to one of claims 30 to 32, wherein the kit additionally has a suitable probe oligonucleotide types pre-assigned dot plot membrane for Detection of the nucleic acid oligomer components of the nucleic acid oligomer Includes receptor units.