WO2002016635A2 - Procede pour le marquage de substances chimiques - Google Patents

Procede pour le marquage de substances chimiques Download PDF

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Publication number
WO2002016635A2
WO2002016635A2 PCT/DE2001/003009 DE0103009W WO0216635A2 WO 2002016635 A2 WO2002016635 A2 WO 2002016635A2 DE 0103009 W DE0103009 W DE 0103009W WO 0216635 A2 WO0216635 A2 WO 0216635A2
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nucleic acid
acid oligomer
receptor
units
complexes
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PCT/DE2001/003009
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German (de)
English (en)
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WO2002016635A3 (fr
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Gerhard Hartwich
Michael Bandilla
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Friz Biochem Gmbh
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Priority to EP01971604A priority Critical patent/EP1334208A2/fr
Publication of WO2002016635A2 publication Critical patent/WO2002016635A2/fr
Publication of WO2002016635A3 publication Critical patent/WO2002016635A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present invention relates to a method for labeling and detecting chemical substances.
  • proteome research The quantitative acquisition of all proteins of a cell of a certain organism at a certain point in time, often referred to as (cellular) proteome research or (cellular) proteomics, is the key to a functional understanding of the physiological, in a special case pathological state of the cell in question.
  • the "proteomic" information enriches both basic and applied research. It provides the molecular basis for the condition of a cell and offers the possibility of recording the change in the expression and modification pattern of the proteins in response to a specific disorder (e.g. a disease or the administration of a certain drug).
  • Proteome research is therefore of fundamental importance for target search and selection in biomedical research, i.e. the development of new drugs.
  • the (cellular or global) proteome can also be used to diagnose diseases and control therapy.
  • proteome research therefore pursues the same goals as genome research, namely the correlation of the state of an organism or a cell (of the phenotype) with basic molecular building blocks of this organism through the parallel analysis of as many such building blocks as possible (e.g. the mRNA or the proteins).
  • the proteome has a much larger amount of information than the genome. While the genome captures the inheritance pattern and the direct transcription pattern of the genes in the form of cDNA and mRNA qualitatively and quantitatively, the complexity of the transcription products, i.e. the proteins, increases through post-translational modifications of many proteins (phosphorylation, Glycosylation, proteolytic processing, etc.). The same genome therefore leads to different proteomes and thus different physiological (or pathological) conditions due to these post-translational variations and transport mechanisms that distribute transcribed gene products over the entire organism.
  • the proteome also has two main advantages with regard to target search and selection or the diagnosis of diseases compared to the genome: on the one hand, the correlation between proteome and phenotype is closer, and on the other hand, the proteome can (at least partially) be typical clinical samples such as serum, cerebrospinal fluid or urine can be examined. These easily accessible samples do not contain DNA, but do contain proteins, so that the samples can be examined for potential proteomic markers of a disease, but not for genomic markers.
  • proteome 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 - there is no simple, universally applicable and unambiguous detection principle for proteins.
  • the isolation and separation as well as the actual detection must be individually adapted to the biochemical properties (e.g. hydrophobicity, hydrophilicity, acidophilia, thermophilia) of the respective proteins or protein groups.
  • the analysis of the proteome can be divided into the following steps (cf. F. Lott Acid, "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 starting parameters are intended to guarantee that the material that is subjected to a proteome analysis can be produced reproducibly.
  • Sample preparation includes cell cultivation and cell disruption under denaturing or non-denaturing conditions. Since proteins do not have uniform physical and chemical properties, sample preparation requires an exact protocol to ensure reproducibility.
  • the currently most common, high-resolution method of protein separation is 2D gel electrophoresis, in which proteins are separated according to (i) charge (using isoelectric focusing, IEF gel) and (ii) size (using SDS-PAGE gel electrophoresis) become.
  • the problem with this method also lies in the reproducibility, in this case in the reproducibility of the two-dimensional patterns on the gel. External parameters such as the protein transfer from the first, IEF-gel dimension to the second, SDS-PAGE-gel dimension, the electrophoresis device, the gel composition, the electrophoresis duration for IEF gels, staining methods, operating personnel etc. can become clear Variation of the pattern.
  • Protein identification and quantification is usually done. by mass spectrometric methods, which require a transfer of the gel-separated proteins from the gel to other environments (e.g. organic matrices in MALDI mass spectroscopy, MALDI-MS). This transfer to another environment can also lead to modifications of the proteins and thus to misinformation or loss of information.
  • mass spectrometric methods which require a transfer of the gel-separated proteins from the gel to other environments (e.g. organic matrices in MALDI mass spectroscopy, MALDI-MS). This transfer to another environment can also lead to modifications of the proteins and thus to misinformation or loss of information.
  • the antibody chip strategy has the disadvantages of unspecific fluorescence and, owing to the two-dimensional readout method of fluorescence by, for. B. CCD cameras in combination with cofocal microscope optics extremely expensive detection method.
  • the unification of the reaction to bind the fluorescent dye to the various proteins represents an unsolved problem.
  • Proteome analysis thus has the potential to develop into an extremely helpful technique in biomedical research and medical diagnostics.
  • problems described above must be overcome, namely the insufficient reproducibility, the low degree of parallelization and the enormous amount of preparative work, highly qualified personnel and expensive technical equipment. Presentation of the invention
  • the object of the present invention is therefore to provide a method for marking chemical substances which does not have the disadvantages of the prior art.
  • Functional genome research generally includes the expression and behavior of gene products. Within the "functional genomics” the areas of cellular transcriptome research, cellular proteome research, global proteome research and metabolome research are distinguished. As cellular transcriptome research (cellular transcriptomics), the comparative investigation of the mRNA exposure profiles with the help of e.g. B. DNA chips. The quantitative isolation and identification of all proteins in a cell is known as cellular proteomics research. In global proteome research (global proteomics), all encoded proteins of the entire genome are analyzed, without any definition of a specific cell type or specific growth conditions (physiological state of the cell). The proteome itself is a building block of the metabolome or metabolome research (metabolomics), i.e. the understanding of the entire metabolism of an organism.
  • Affinity chromatographic method in which specific graphical bonds between a tag and its binding partner are used to purify a substance. Either the tag or the binding partner on the substance to be purified is modified and the binding partner or the tag is immobilized on the chromatography material.
  • a combination of tag and binding partner are a specific anti-hapten antibody as a binding partner and a hapten as a tag, or Protein A (or Protein G) as a binding partner and a specific IgG antibody as a tag.
  • protein G or protein A has a special position as binding partner immobilized on the chromatography material, since this protein can quantitatively bind all secondary immune complexes, free F a b fragments, which are used in the nucleic acid oligomer receptor units become, but not bound. alkaline enzyme that cleaves phosphate groups from various phosphatase substrates
  • Alkynyl alkyl or alkenyl groups in which one or more of the C-C single or C C double bonds are replaced by C ⁇ C triple bonds.
  • alkyl denotes a saturated hydrocarbon group that is straight-chain or branched (eg ethyl, 2,5-dimethylhexyl or isopropyl etc.).
  • alkyl refers to a group with two available valences for covalent linkage (e.g. - CH 2 CH 2 -, -CH 2 C (CH 3 ) 2 CH 2 CH 2 C (CH 3 ) 2 CH 2 - or -CH 2 CH 2 CH 2 - etc.).
  • Preferred alkyl groups as substituents or side chains R are those of chain length 1-30 (longest continuous chain of atoms covalently bonded to one another).
  • anti-Fab-antibody antibodies b to the constant regions of the F a fragments are directed. This term is always understood to mean a whole set of antibodies which can bind all F a b fragments regardless of the isotype and species.
  • Antigen Specific binding partner of an antibody any substance, compound, biomolecule or molecule or combinations thereof which, due to its primary, secondary or tertiary structures, has a binding site for an antibody which enables specific binding of this antibody; the ability of a substance to act as an antigen is only restricted by the fact that the antigen-antibody interaction must have a certain affinity, so that the antibody can specifically recognize the substance.
  • Another peculiarity of antigens is to stimulate the production of certain antibodies in an organism (i.e. to stimulate an immune response).
  • An antigen can therefore also represent a complex mixture of chemical substances or entire organisms (such as pollen grains, killed bacteria, viruses, etc.).
  • anti-hapten antibody in the context of the present invention, is used for a group of antibodies which are specifically directed against a specific hapten or hapten analogue.
  • Preferred haptens are biotin or those in the form of the non-carrier-bound but with hapten analogs which specifically bind to the corresponding anti-hapten antibodies, such as FITC, digoxigenin, DNP and rhodamine (cf. also the definition of hapten).
  • anti-Ig antibodies anti-Ig antibodies are anti-immunoglobulin antibodies which are specific for individual isotypes, classes or subclasses of antibodies of a particular species.
  • anti-Ig antibodies are understood to mean those anti-Ig antibodies which, after affinity chromatography purification, are directed only against the constant regions on the F c fragment. Isotypic anti-Ig antibodies which are directed against constant regions of the Fab fragments are explicitly distinguished from them as anti-F a antibodies.
  • anti-Ig antibodies are understood to mean a complete set of anti-immunoglobulin antibodies, which includes all antibodies against all classes and subclasses, as long as the class or subclass is not specifically defined (in contrast to, for example, anti- IgG antibodies bearing the abbreviation of the subclass in the name).
  • the anti-Ig antibodies play an important role Role in the formation of secondary immune complexes: They bind to the specific antibodies of the primary immune complexes and initiate the formation of secondary immune complexes, ie an aggregation of the primary immune complexes into large complexes with an undefined composition, these secondary immune complexes via the anti-Ig antibodies can bind to protein A or protein G.
  • Antibody-specific group of proteins also called immunoglobulins, abbreviated as Ig
  • Ig immunoglobulins
  • B cells of the immune system of vertebrates.
  • Antibodies specifically recognize structural features, in particular the secondary or tertiary structure of any chemical substances (or complexes or aggregates of substances) and bind to them, the substances specifically binding to the antibodies being referred to as antigens.
  • Antibodies are made up of two (identical) H chains and two (identical) L chains, which assemble to form a Y-shaped protein with two arms, the antigen-binding domains, formed by the assembly of an L and an H Chain, also called F a t domain, and a stem region, the effector domain, formed by the C-terminal parts of the two H chains, also called F c domain, being combined.
  • the antigen binding domain has a constant region and a so-called variable region, formed from the N-terminal globular domain of an arm, which differs from antibody to antibody, is characteristic of each antibody and bears the specific binding site for a specific antigen.
  • the constant regions of the effector domains contain the binding regions for specific ones Binding partners of different Zeil lines and binding regions for the complement factor C1q, as well as for protein A and protein G.
  • Naturally occurring antibodies are made up of such a unit or from a complex of 2-5 such units and thus have between 2 and 10 identical ones antigen binding sites.
  • Antibodies are divalent or multivalent accordingly.
  • the B cell binding partners expressed on the cell surface of the B cells have the same structure, only with an additional domain that integrates the molecule into the cell membrane.
  • antibodies There are different classes and subclasses of antibodies: eg IgGI, IgG2a, IgG2b, IgG3, IgG4, IgD, IgM, IgA1, IgA2 and IgE in the mouse, which differ in the region of the constant regions.
  • the antibodies also differ in that there are different combinations of the H chain and different L chains (lamda and kappa). Antibodies with the same combination of H chain and L chain are called isotypes.
  • the individual antibodies are also defined according to their origin, ie according to the species from which the antibodies or the B cells for hybridoma cell fusion are obtained.
  • monoclonal antibodies usually come from mice or rats, in rare cases also from humans, and polyclonal antibodies come from mice, rats, hamsters, guinea pigs, rabbits, goats, sheep, cattle, horses, chicken and goose.
  • the specificity of 'polyclonal' antibodies is determined by the complexity of the antigens and can therefore include an indefinable number of epitopes on a number of different substances. In general, neither the Ig class, the subclass, the isotype nor the origin play a role in the specificity and affinity of the antibodies. Therefore, in the present invention in generally dispenses with this information.
  • Biotin Biotin (a cofactor of certain enzymes) can be covalently bound to proteins as a hapten or as a tag.
  • Biotin carboxylase protein that binds biotin as a cofactor - a peptide that contains a carrier protein domain of this protein is used as a tag to achieve in vivo biotinylation in E. coli of the proteins fused with the tag.
  • B-cells Cells of the immune system that are responsible for producing antibodies during an immune response.
  • Carrier A molecule which is a carrier material for another substance, the substance being bound specifically or nonspecifically, covalently or non-covalently, to the carrier.
  • a carrier can be any material regardless of the size and structure to which a substance can bind.
  • macromolecules are used as the carrier material, which, depending on the purpose for which the carrier is used, ideally has various additional properties, such as, for example Property of easily sedimenting or of being specifically or covalently bound by another substance, such as a protein that can be bound by an antibody.
  • carrier is used exclusively to refer to a compound which is capable of binding to a hapten analogue with only one epitope, without the properties of the epitope being changed and the complex of carrier and hapten analog in addition to the unchanged epitope of the hapten analog has at least one further epitope (on the carrier).
  • Chromatography equivalent also to carrier material. material cytoplasmic proteins that are located in the cytoplasm and can be isolated as soluble protein fractions
  • Detection markers chemical substances with any structure and composition, which are bound to the nucleic acid oligomer or its amplification products and via which the nucleic acid oligomer or its amplification products can be detected.
  • Typical detection markers are e.g. B. fluorescent dyes, radioactive isotopes and functional enzyme units.
  • any modification of a molecule that is suitable for simply connecting a detection marker can also be referred to as a detection marker, such as, for. B. a linked biotin to which the actual avidin-modified detection marker can be linked in a simple reaction.
  • Digoxigenin steroid used as a hapten analogue
  • DNAse enzymes break down the DNA
  • DNP 2,4-dinitrophenol is used as a hapten analogue or as a chromophore for labeling
  • Dot plot membrane Membranes that serve as carrier material for DNA or RNA. DNA or RNA samples are applied to the dot-plot membranes and immobilized as individual, mostly punctiform drops in a defined pattern. ds double strand (double strand)
  • ECM extracellular proteins of the extracellular matrix that are isolated as an insoluble matrix
  • Epitope Any structural feature of an antigen that is recognized by an antibody and that represents a binding site for a specific antibody.
  • F a b / F a b fragment After papain digestion, the proteolytic fragment of IgG antibodies, which contains the variable region that specifically recognizes the antigens, is obtained by papain digestion - the F ab fragment contains only one binding site for the antigen (monovalent) - genetically Antibodies produced are also monovalent depending on the design and are then referred to as F ab fragments in the context of this invention.
  • F ab fragment is also understood to mean the binding domains of other Ig classes which have been isolated using other methods.
  • F c F c fragment is the fragment after papain digestion of IgG antibodies, which contain the C-terminal constant domains (constant regions) of an IgG - in these regions are the binding sites for the components C1q of the complement, for protein A and protein G, as well as for the cellular receptor proteins, which are the constant Specifically recognize and bind regions of the antibodies.
  • F c - ⁇ receptor cellular receptor proteins for the F c region of IgG that specifically bind the antibodies as soon as they have formed an antigen-antibody complex
  • Fluorescein activated by an isothiocyanate side group which makes it possible to covalently bind fluorescein to other substances - fluorescein can be used as a simple fluorescence label, on the other hand it can be used as a hapten analogue, i. e. serve as a non-carrier-bound substance that specifically binds to the corresponding anti-hapten antibodies.
  • functional protein Protein in native or partially denatured form which is still able to perform at least one of the functions corresponding to one of the natural functions of the protein in the organism.
  • Gel electrophoresis Method for the electrophoretic separation of macromolecules such as polypeptides, proteins or nucleic acids, using a gel matrix (usually agarose or polyacrylamide).
  • Hapten Substance that elicits an immune response in the animal with antibody formation when bound to an immunogenic carrier eg a protein
  • immunogenic carrier eg a protein
  • immunogenic means that this carrier itself is an (ideally very well recognized) antigen.
  • the antibodies formed in the immune response against the carrier differ from the immune response against the hapten.
  • a substance is referred to as a hapten if it is bound to a carrier and serves as a binding partner for an antibody.
  • the substance which is the hapten in association with the carrier is generally also recognized and bound by the anti-hapten antibody if it is not bound to a carrier or to another carrier.
  • Hapten analogue A substance that, in conjunction with a carrier, represents hapten.
  • a hapten analogue is recognized and bound by the anti-hapten antibody, even if it is not bound to a carrier.
  • 0.1 mg / ml ssDNA from pBR322 plasmid 0.5 mg / ml acetylated BSA or 7% SDS, 500 mM Na phosphate buffer pH 7.5, 5 mM EDTA or
  • Hybridoma cells chimeric cell lines from B cells (predominantly from mouse and hybridoma cells rat) which, as immortalized cell lines, produce monoclonal antibodies with the specificity of the B cells.
  • IgA high affinity immunoglobulins that support the immune defense in the mucous membranes of the organism.
  • igE high-affinity immunoglobulins which are bound by certain cells of the immune system, occur when certain groups of pathogens and parasites are infected, but also form in the formation of allergies or play an important role in allergic reactions.
  • IgG high-affinity immunoglobulins which are formed in the case of an immune response and which can still be detected in the serum for a long time after an infection.
  • IgM low-affinity immunoglobulins which are formed during the development of the not yet fully differentiated B cells and represent the first immune response to an infection.
  • Immunoassay An assay (detection system) that uses the specific binding of the antibody to its antigen to detect the antigen - a distinction is made between different assays depending on the type of labeling and the detection method. Examples include: RIA (radioimmunoassay), RIST (radioimmunosorbent test) and RAST (radioallergosorbent test) for detection via radioactive labeling, EIA (enzyme linked immune assay) and ELISA (enzyme linked immumosorbent assay) for detection via an enzyme (in the Rule with alkaline phosphatase, ß-galactosidase or horseradish peroxidase) and quantification of the implementation of certain substrates by z. B. colorimetry or chemiluminescence, IF (immunofluorescence) when detected via fluorescent dyes, IP (immunoprecipitation) when detected via direct isolation of the precipitate.
  • RIA radioimmunoassay
  • Isoelectric method for electrophoretic separation of focusing (IEF) polypeptides and proteins according to their pl - i.e. depending on the surface charge, the amino acid composition and post-translational modifications of the polypeptides and proteins
  • Nuclear Proteins Proteins that are isolated with the nuclear fraction.
  • Complement-C1q A component of the complement system C1q that bind specifically to the F c region of IgG and IgM antibodies in an antigen-antibody complex.
  • linkers are commercially available as alkyl, alkenyl, alkynyl, hetero-alkyl, hetero-alkenyl or heteroalkynyl chains, the chain being derivatized at two points with (identical or different) reactive groups. These groups form a covalent chemical bond in simple / known chemical reactions with the corresponding reaction partners.
  • the reactive groups can also be photoactivatable, i. H. the reactive groups are only activated by light of certain or any wavelength.
  • Membrane proteins Proteins that are integrated in or attached to the membrane and can be isolated with the membrane fraction - membrane proteins can be made from these Membrane fractions can be extracted using detergents.
  • the two single strands hybridize in such a way that the base A (or C) of one strand forms hydrogen bonds with the base T (or G) of the other strand (in RNA, T is replaced by uracil) , Any other base pairing does not form correct hydrogen bonds, distorts the structure of the double strand and is referred to as "mismatch”.
  • Modified nucleic acid oligomer with attached receptor-nucleic acid oligomer unit The terms "modified nucleic acid oligomer” and “nucleic acid oligomer-receptor unit” are used equivalently.
  • Monoclonal Monoclonal antibodies are each produced from a single hybridoma cell clone, the antibodies produced from a cell clone all having the same defined antigen binding site and belonging to the same isotype. Accordingly, monoclonal antibodies show specificity against only one defined epitope.
  • Nucleic acid at least two covalently linked nucleotides or at least two covalently linked pyrimidine (e.g. cytosine, thymine or uracil) or purine bases (e.g. adenine or guanine).
  • the term nucleic acid refers to any "backbone" of the covalently linked pyrimidine or purine bases, such as. B. on the sugar-phosphate backbone of the DNA or RNA, on a peptide backbone of the PNA or on analog structures (e.g. phosphoramide, thio-phosphate or dithio-phosphate backbone).
  • An essential feature of a nucleic acid in the sense of the present invention is that it can bind complementary nucleic acids in a sequence-specific manner.
  • Nucleic acid oligomer Nucleic acid of unspecified base length (e.g. nucleic acid octamer: a nucleic acid with any backbone in which 8 pyrimidine or purine bases are covalently bound to one another).
  • nucleic acid octamer a nucleic acid with any backbone in which 8 pyrimidine or purine bases are covalently bound to one another.
  • nucleic acid oligomer is used both as a name for a single molecule and as a name for any number of single molecules of a type of nucleic acid oligomer. The respective meaning is clear from the context.
  • Nucleic acid oligomer complex consisting of a receptor unit and a receptor unit.
  • Nucleic acid oligomer, the complex formation being able to be realized by direct or indirect linker-bridged, covalent or non-covalent, specific binding between nucleic acid oligomer and receptor unit and the complex composition corresponding to a defined stoichiometry.
  • Oligo Abbreviation for oligonucleotide Oligo Abbreviation for oligonucleotide.
  • Oligomer equivalent to nucleic acid oligomer Oligomer equivalent to nucleic acid oligomer.
  • Oligonucleotide equivalent to oligomer or nucleic acid oligomer e.g. B. a DNA, PNA or RNA fragment unspecified base length.
  • pBR322 plasmid E.co/. ' Cloning plasmid, which acts as ss-DNA to block non-specific binding sites on the blot membrane is used.
  • PBS Phosphate buffered saline buffer substance with a defined composition - composed in the context of the present invention from 10 mM Na phosphate, 135 mM NaCl, 5 mM KCI, pH 7.4
  • Phosphate wash- 50 mM potassium phosphate pH 7.5, 500 mM NaCI, 1 mM buffer EDTA photo-inducible Photo-inducible means that a certain property is only developed by irradiation with light of certain or any wavelength.
  • Phycoerythrin pigment-protein complex from cyanobacteria which is used as a fluorescence marker.
  • PNA Peptide nucleic acid synthetic DNA or RNA in which the sugar-phosphate unit is replaced by an amino acid.
  • synthetic DNA or RNA synthetic DNA or RNA in which the sugar-phosphate unit is replaced by an amino acid.
  • sugar-phosphate unit is replaced by the -NH- (CH 2 ) 2 -N (COCH 2 - base) -CH 2 CO - Unit hybridizes PNA with DNA.
  • Poly-dT (33) oligomer made up of 33 deoxy-thymidines.
  • polyclonal antibodies The term refers to a set of antibodies with a generally undefined composition, all of which Specifically recognize and bind the same antigen.
  • Polyclonal antibodies are produced by different B cells of an animal's immune system and are obtained from the sera of the immunized animals. So-called anti-sera do not correspond to purified polyclonal antibodies and are equivalent to them. Depending on the complexity of the antigen used for the immunization, polyclonal antibodies have a broad or more limited range of specificity. The specificity of polyclonal antibodies can be restricted to a portion of the epitopes of the antigen by affinity chromatography enrichment and / or purification, so that high specificity and high affinity can also be achieved with polyclonal antibodies.
  • Primary immune complex immune complex is understood to mean a complex of specific antibody and target, the specific antibody not forming a receptor unit, but being used as a tag for immobilizing the target.
  • Primer binding region Region of a nucleic acid that serves either directly as a primer binding region or as a primer binding region on the corresponding complementary strand of the nucleic acid.
  • Protein A Protein G Proteins that bind specifically to antibodies with an intact F c region of different subclasses (predominantly IgG) of immunoglobulins
  • PVDF membranes Polyvinylidene fluoride membranes with a hydrophobic surface, preferred for the immobilization of Proteins is used
  • Receptor unit any substance, every compound, every biomolecule or molecule or combinations thereof, which because of its primary, secondary or tertiary structures has a binding site for another substance (compound, biomolecule or molecule or combinations) that has a specific binding of these others Substance enables.
  • Receptor units are e.g. B. a protein or peptide, or one of the specific binding partners that bind to a protein or peptide, an antibody or an antigen.
  • Receptor protein Protein (or a functional peptide) whose natural function is to specifically bind one or a defined group of cofactors (binding partners) in order to perform its biologically relevant function.
  • a specific binding in the sense of the present invention is when a substance which is at least one molecule is bound by another conjugate, or a limited group of other conjugated substances which are at least one molecule, the primary , Secondary or tertiary structure of conjugated molecules make a significant contribution to this binding.
  • the specific bond thus differs in the sense of the present invention from a covalent chemical connection of two molecules.
  • the specific bond can be established by one or more ionic bonds, by hydrogen bonds, by van der Waals bridges, by ⁇ interaction, by coordination by means of electron pair donation and acceptance, or by encapsulation of matching molecular structures (e.g. Three-dimensionally designed binding pockets and fitted cofactors).
  • SSC 20 x stock solution 3 M NaCI, 300 mM Na citrate pH 7.0, which is used as a 2x and 0.4x solution for washing blots.
  • ß-galactosidase enzyme that hydrolytically cleaves ß-galactoside.
  • Tag any substance, compound, biomolecule or molecule or combination thereof that, due to its primary, secondary or tertiary structure, has a binding site for another substance (compound, biomolecule or molecule or combination) that enables specific binding of this other substance
  • the term “tag” is understood in a restrictive manner so that this substance does not function as a target or as a receptor unit and not as a detection marker.
  • the term is also to be understood in a restrictive manner so that the tag is covalently bound to another substance, in particular to the targets or carrier materials of the present invention, and serves to immobilize the substance associated with the specific tag binding partner.
  • Typical tags are e.g. B.
  • biotin e.g. HA-Tag, His-Tag, Flag-Tag, GST-Tag Z-Tag
  • functional enzymes such as e.g. AP, POD, ⁇ -galactosidase or luciferase.
  • Target receptor-specific or specific chemical substance every substance, every compound, every biomolecule or Molecule or combinations thereof which, due to its primary, secondary or tertiary structures, has a binding site for another substance (compound, biomolecule or molecule or combinations) which enables specific binding of this other substance.
  • Carrier material is a material that generally binds certain chemical substances while other chemical substances are not bound.
  • Carrier materials are e.g. modified or unmodified silica gel, cellulose, agarose material, nitrocellulose membranes, PVDF membranes, (modified or unmodified) nylon membranes.
  • Tris / HCI Tris where the pH is adjusted with HCI - Tris is equated with Tris / HCI
  • TRITC Tetramethyl-Rhodamine B-Isothiocyanate Tetramethyl-Rhodamine B activated by an isothiocyanate side group, which enables tetramethyl-Rhodamine B to be covalently bound to other substances - Tetramethyl-Rhodamine B can serve as a simple fluorescence label, on the other hand it can as a hapten analogue, i. e. serve as a non-carrier-bound substance that specifically binds to the corresponding anti-hapten antibodies.
  • Cell organelles constituents of different fractions of the cell disruption, which with gentle cell fractionation, mostly as intact membrane fractions with specific Combination of membrane proteins and soluble proteins can be obtained.
  • Examples of cell organelles are mitochondria, lysosomes and endosomes, ER vesicles and Golgi vesicles, peroxisomes and the cell nuclei
  • the present invention comprises a method for labeling chemical substances, comprising the steps: providing one or more chemical substances, providing one or more types of nucleic acid oligomer receptor units, the nucleic acid oligomer receptor units each consisting of a nucleic acid oligomer of a known sequence and in each case one of them connected receptor unit and the receptor unit can specifically bind a chemical substance, and bringing the chemical substances provided into contact with the nucleic acid oligomer receptor units provided, as a result of which complexes are formed from the nucleic acid oligomer receptor unit and the associated chemical substance.
  • the present invention also encompasses a method for the detection of chemical substances, comprising the steps of: providing one or more chemical substances, providing one or more types of nucleic acid oligomer receptor units, bringing the chemical substances into contact with the nucleic acid oligomer receptor units, thereby causing formation of complexes of nucleic acid oligomer receptor unit and chemical substance, binding of the complexes of nucleic acid oligomer receptor unit and chemical substance to a suitable chromatography material, whereby separation from the nucleic acid oligomer receptor units remaining in the eluate takes place, cleavage of one or more chemical Binding of the complexes of nucleic acid oligomer receptor unit and chemical substance bound to the chromatography material such that the nucleic acid oligomer component of the nucleic acid oligomer receptor unit is in the form of a nucleic acid oil igomers or in the form of any modified nucleic acid oligomer, which comprises the complete sequence information of the nucleic acid oligomer
  • the present invention also comprises a method for the detection of chemical substances, comprising the steps: providing one or more chemical substances, providing defined amounts of one or more types of nucleic acid oligomer receptor units, bringing the chemical substances into contact with the defined amounts of nucleic acid oligomer receptor Units, whereby a formation of complexes from nucleic acid oligomer receptor unit and chemical substance takes place, binding of the formed complexes from nucleic acid oligomer receptor unit and chemical substance to a suitable chromatography material, whereby a separation from the nucleic acid oligomer receptor units remaining in the eluate is carried out, the nucleic acid oligomer receptor units remaining in the eluate are washed out and the nucleic acid oligomer components of the nucleic acid oligomer receptor units are detected by a suitable method directly as a nucleic acid oligomer rec eptor unit or after cleavage of the nucleic acid oligomer component from the receptor unit as nucleic acid oligomer or
  • the present invention also comprises a method for the detection of chemical substances, comprising the steps: providing one or more chemical substances, binding the chemical substances to a chromatography material, providing one or more types of nucleic acid oligomer receptor units, bringing the nucleic acid oligomer receptor units into contact with the chemical substances bound to the chromatography material, which results in the formation of complexes of nucleic acid oligomer receptor unit and chemical substance and a separation from the nucleic acid oligomer receptor units remaining in the eluate, cleavage of one or more chemical bonds of the complexes bound to the chromatography material Nucleic acid oligomer receptor unit and chemical substance such that the nucleic acid oligomer component of the nucleic acid oligomer receptor unit in the form of a nucleic acid oligomer or in the form of any desired ized nucleic acid oligomer, which comprises the complete sequence information of the nucleic acid oligomer, is separated from the chromatography material, Washing out the nucleic acid
  • the present invention also comprises a method for the detection of chemical substances comprising the steps: providing one or more chemical substances, binding the chemical substances to a chromatography material, providing defined amounts of one or more types of nucleic acid oligomer receptor units, contacting the defined amounts of nucleic acid oligomer -Receptor units with the chemical substances bound to the chromatography material, as a result of which complexes are formed from the nucleic acid oligomer-receptor unit and chemical substance, washing out the nucleic acid oligomer-receptor units remaining in the eluate and detection of the nucleic acid oligomer constituents of the nucleic acid oligomer receptor.
  • nucleic acid oligomer receptor unit by a suitable method directly as a nucleic acid oligomer receptor unit or after cleavage of the nucleic acid oligomer component from the receptor unit as a nucleic acid oligomer or modified rst nucleic acid oligomer, which comprises the complete sequence information of the nucleic acid oligomer.
  • the present invention also comprises a method for the detection of chemical substances insoluble in aqueous media, comprising the steps:
  • nucleic acid oligomer receptor Providing one or more types of nucleic acid oligomer receptor
  • Nucleic acid oligomer receptor units whereby complexes are formed from the nucleic acid oligomer receptor unit and insoluble chemical substance
  • nucleic acid oligomer component of the nucleic acid oligomer receptor unit in the form of a nucleic acid oligomer or in
  • Form of any modified nucleic acid oligomer is separated from the insoluble chemical, separating the from the insoluble chemical Substance-separated nucleic acid oligomer components and detection of the nucleic acid oligomer components by a suitable method.
  • the present invention also comprises a method for the detection of chemical substances insoluble in aqueous media, comprising the steps: providing one or more types of insoluble chemical substances, providing defined amounts of one or more types of nucleic acid oligomer receptor units, contacting the insoluble chemical substances with the Defined amounts of nucleic acid oligomer receptor units, whereby a formation of complexes from nucleic acid oligomer receptor unit and insoluble chemical substance takes place, separation of the complexes from nucleic acid oligomer receptor unit and insoluble chemical substance, detection of the nucleic acid oligomer components of the insoluble chemical Substance-separated nucleic acid oligomer receptor units located in the supernatant by a suitable method.
  • the present invention also includes a method of detecting hapten analogs comprising the steps of: providing one or more hapten analogs, providing an excess relative to the hapten analogs of one or more types of nucleic acid oligomer receptor units, contacting the hapten Analogs with the nucleic acid oligomer receptor units, as a result of which complexes are formed from the nucleic acid oligomer receptor unit and the hapten analog, providing the hapten analogs in a modified form, the modification being in the binding of a carrier molecule to the hapten analogs exists, whereby a certain type of carrier molecule is bound to each type of hapten analogue, which satisfies the condition that there is no significant change in the specific binding capacity of the epitope of the hapten analogues due to the binding of the carrier molecule, contacting the modified Hapten analogues in excess with the g e formed mixture of excess nucleic acid oligomer receptor units and complexes formed from nucleic
  • the present invention further comprises a method for the detection of chemical substances, comprising the steps: providing one or more chemical substances, providing one or more types of nucleic acid oligomer receptor units in excess relative to the chemical substances, contacting the chemical substances with the nucleic acid oligomer Receptor units, whereby a formation of complexes from nucleic acid oligomer-receptor unit and chemical substance takes place, providing the chemical substances in modified form, the modification consisting in the connection of the chemical substances to a chromatography material, bringing the mixture of excess nucleic acid oligomer receptor into contact -Units and complexes formed from nucleic acid oligomer receptor unit and chemical substance with the chemical substances bound to the chromatography material, thereby forming complexes from excess nucleic acid oligomer receptor units and chemical substances bound to the chromatography material, washing out the complexes of nucleic acid oligomer receptor unit and chemical substance remaining in the eluate and detection of the nucleic acid oligomer components of the complexes of
  • the present invention also comprises a method for the detection of chemical substances, comprising the steps: providing one or more types of nucleic acid oligomer receptor units, providing an excess relative to the one or more types of nucleic acid oligomer receptor units of one or more chemical substances, binding of the chemical substances to a chromatography material, bringing the nucleic acid oligomer receptor units into contact with the chemical substances bound to the chromatography material, thereby forming complexes of nucleic acid oligomer Receptor unit and chemical substance takes place, providing one or more types of second chemical substances, the second chemical substances being able to bind specifically to the chemical substances, bringing the one or more types of second chemical substances into contact with the complexes formed from nucleic acid oligomer-receptor.
  • the specific binding of the second chemical substances to the chemical substances means that at least some of the complexes formed from the nucleic acid oligomer receptor unit and the chemical substance into the inventory parts of the nucleic acid oligomer receptor unit and chemical substance are cleaved, whereby the nucleic acid oligomer receptor units are released into the eluate, washing out the released nucleic acid oligomer receptor units and detection of the nucleic acid oligomer components of the nucleic acid oligomer receptor units directly by a suitable method as a nucleic acid oligomer receptor unit or after the nucleic acid oligomer component has been split off from the receptor unit
  • Nucleic acid oligomer or modified nucleic acid oligomer comprising the complete sequence information of the nucleic acid oligomer.
  • the present invention also comprises a kit for carrying out one of the abovementioned methods, a kit for the marking of chemical substances, a kit for the detection of chemical substances, a kit for the detection of proteins or antigens, a kit for the detection of chemical substances which are insoluble in aqueous media , a kit for the detection of antibodies, a kit for the detection of haptens or hapten analogs, a kit for the detection of DNA- and / or RNA-binding proteins, a kit for the detection of the antagonists of enzymes or the antagonists of DNA- and / or RNA-binding proteins, a kit for screening hybridoma cells and a kit for screening hybridoma cells and simultaneous epitope mapping.
  • Each kit according to the invention comprises one effective amount of one or more types of nucleic acid oligomer receptor units.
  • the present invention relates to a method for labeling chemical substances, comprising the steps: providing one or more chemical substances, providing one or more types of nucleic acid oligomer receptor units, the nucleic acid oligomer receptor units each consisting of a nucleic acid oligomer of a known sequence and each have a receptor attached to it and the receptor can specifically bind a chemical substance, and bringing the chemical substances provided into contact with the nucleic acid oligomer receptor units provided, as a result of which complexes are formed from the nucleic acid oligomer receptor unit and the associated chemical substance.
  • the associated nucleic acid oligomer component of the nucleic acid oligomer receptor unit originally bound to this chemical substance can be used as evidence of the presence of the chemical substance instead of the specific chemical substance.
  • the nucleic acid oligomer component of the nucleic acid oligomer receptor unit thus serves as a universal coding molecule for the associated specific chemical substance, as long as a specific base sequence is assigned to a specific nucleic acid oligomer receptor unit and thus to the specific associated chemical substance.
  • this has the advantages (1) that it is generally easier to detect a nucleic acid oligomer than any chemical substance, especially if it is an undefined mixture of chemical substances with different chemical and physical properties (standardization and parallelization of the detection),
  • the coding nucleic acid oligomer can be amplified by suitable methods and thus even the smallest amounts of a chemical substance can be easily detected and
  • the contacting can take place using the highly specific interaction between the receptor and the associated chemical substance, but the subsequent manipulations and the final detection can also take place under conditions that change or destroy the original appearance of the chemical substances, and the stability between the receptor and chemical substance need not be considered as long as the sequence integrity of the nucleic acid oligomer component is preserved.
  • nucleic acid oligomers used in the method according to the invention are modified by chemical binding of a substance-specific receptor.
  • the preferred nucleic acid oligomer is a compound composed of at least two covalently linked nucleotides or of at least two covalently linked pyrimidine (e.g. cytosine, thymine or uracil) or purine bases (e.g. adenine or guanine) a DNA, RNA or PNA fragment is used.
  • nucleic acid oligomer refers to any "backbone" of the covalently linked pyrimidine or purine bases, such as. B.
  • nucleic acid oligomer on the sugar-phosphate backbone of the DNA, cDNA or RNA, on a peptide backbone of the PNA or on analogous backbone structures, such as. B. a thio-phosphate, a dithio-phosphate or a phosphoramide backbone.
  • An essential feature of a nucleic acid oligomer in the sense of the present invention is that it can bind analogous nucleic acid oligomers in a base sequence-specific manner.
  • nucleic acid oligomer the terms “oligonucleotide”, “nucleic acid” or “oligomer” are used.
  • a “receptor-specific substance” is understood to mean any unit (any substance, any compound, any biomolecule or molecule or combinations thereof) which is specific to a conjugated receptor unit (substance, compound, biomolecule or molecule) or combinations thereof).
  • photolabile means that the cleavage of a group, that is to say its property of disintegrating into two or more fragments under certain external circumstances, is only developed by irradiation with light of a certain or any wavelength.
  • a prerequisite for the method according to the invention is the binding of a receptor to a nucleic acid oligomer.
  • a receptor is directly covalently bound to a nucleic acid oligomer through the reaction of the nucleic acid oligomer with the receptor or indirectly non-covalently through the formation of specific interactions between the (modified) nucleic acid oligomer and the (modified) receptor. This binding can be done in four different ways (ad):
  • a reactive group for binding formation on the nucleic acid oligomer As a reactive group for binding formation on the nucleic acid oligomer, a free phosphoric acid, thiophosphoric acid, thiol, carbonyl, sugar C-3-hydroxy,
  • Carboxylic acid or amine group of the oligonucleotide backbone in particular a group at one of the two ends of the oligonucleotide backbone, is used.
  • the free, terminal phosphoric acid, thiophosphoric acid, thiol, carbonyl, sugar, C-3-hydroxy, carboxylic acid or amine groups have an increased reactivity and are therefore easy to carry out typical reactions such as e.g. B.
  • the coupling group (acid, amine, alcohol, thioalcohol or aldehyde function) required for the covalent attachment of the receptor unit is either naturally present on the receptor or is obtained by chemical modification of the receptor.
  • a nucleic acid oligomer-receptor unit is used, the coupling of the reactive groups between nucleic acid oligomer and receptor containing a group that can be cleaved after induction.
  • a group that can be cleaved after induction can be, for example, a disulfide group, which can be split reductively into the two thiol constituents after induction by a reducing agent such as DTT or ⁇ -mercaptoethanol, a -CHOH-CHOH group which can be obtained by adding an oxidizing agent such as e.g. B.
  • Nal0 4 or a -CO-0-CH 2 -CH 2 -0-CO- group which can be split by adding H 2 N-OH or a photolabile group such as.
  • B. the azo group (-N N group), which can be split after induction by irradiation of light of a suitable wavelength.
  • the nucleic acid oligomer is on a covalently attached part of the molecule (spacer) of any composition and chain length (longest continuous chain of atoms bound to each other), in particular the chain length 1 to 50 Oligonucleotide backbone or modified on a base with a reactive group. The modification is preferably carried out at one of the ends of the oligonucleotide backbone or at a terminal base.
  • spacer for example, an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituent can be used as spacer.
  • a nucleic acid oligomer-receptor unit in which the linker / spacer has a group which can be cleaved after induction after coupling of the reactive groups between linker / spacer and nucleic acid oligomer and between linker / spacer and receptor.
  • Suitable groups which can be cleaved by induction are, for example, a disulfide group which can be cleaved reductively, that is to say after induction by a reducing agent such as DTT or ⁇ -mercaptoethanol, into the two thiol constituents, a - CHOH-CHOH group which can be obtained by adding an oxidizing agent such as.
  • B. the azo group (-N N group), which can be split after induction by irradiation of light of a suitable wavelength.
  • a terminal base or a terminal nucleotide or terminal nucleoside is replaced by the receptor, a linker / spacer-modified (see b)) receptor or a linker / spacer with a reactive group (see b)) replaced.
  • the receptor or linker / spacer-modified receptor or linker / spacer can be linked with a reactive group completely or in parts of this unit with subsequent completion of the unit.
  • a nucleic acid oligomer receptor unit is used in which the linker / spacer after coupling the reactive Groups between linker / spacer and nucleic acid oligomer or between linker / spacer and receptor has a group which can be cleaved after induction.
  • the continuous chain between nucleic acid oligomer and receptor contains a group which can be cleaved by induction, for example a disulfide group, which is reductive, ie after induction by a reducing agent such as DTT or ⁇ -mercaptoethanol, can be split into the two thiol components, a - CHOH-CHOH group, which by adding an oxidizing agent such.
  • B Nal0 4 or a -CO-0-CH 2 -CH 2 -0-CO- group which can be cleaved by adding H 2 N-OH or a photolabile group such as.
  • B. the azo group (-N N group), which can be split after induction by irradiation of light of a suitable wavelength.
  • nucleic acid oligomer is indirectly noncovalently bound to the receptor.
  • z. B biotin directly or indirectly via a spacer / linker (cf. b)) bound to a nucleic acid oligomer and secondly avidin, in particular monovalent avidin and / or streptavidin, in particular monovalent streptavidin and / or anti-biotin antibody (or the F ab fragment thereof) to the receptor (directly or indirectly via a spacer / linker (cf. b)) or vice versa.
  • This modified nucleic acid oligomer unit is linked to the correspondingly modified receptor unit by coupling the two units due to the specific interactions between biotin and avidin (or biotin and streptavidin or biotin and anti-biotin antibody or between biotin and the F ab fragment of anti-biotin antibodies).
  • Further examples for the realization of the indirect noncovalent connection between nucleic acid oligomer and receptor generally result from direct or indirect connection (cf.b) of a (second) chemical substance to the nucleic acid oligomer, which represents a specific binding partner for a third chemical substance, this being third chemical substance is the one with which the receptor was modified.
  • Examples of such second chemical substances for binding to the nucleic acid oligomer are a hapten analogue (FITC, DNP, digoxigenin or rhodamine), an antigen, a His tag, a HA tag, a FLAG tag, a GST tag, a Z domain (monovalent functional domain of protein A), a functional protein, a chitin-binding protein, an avidin, is monovalent avidin, a streptavidin or monovalent streptavidin.
  • the matching specific binding partner i.e.
  • the third chemical substance is an antibody (specific binding partner for the hapten or antigen) or F a fragment (specific binding partner for the hapten or antigen), in particular an anti-FITC antibody (specific binding partner for FITC), an anti-DNP antibody (specific binding partner for DNP), an anti-digoxigenin antibody (specific binding partner for digoxigenin) or an anti-Rhodamine antibody, or an anti-His-Tag antibody (specific binding partner for His -Tag), an anti-HA-TAG antibody (specific binding partner for HA-TAG), an anti-Flag-Tag antibody (specific binding partner for FLAG-TAG), an anti-GST-Tag antibody or glutathione (specific Binding partner for GST-Tag), antibody Fc fragment (specific binding partner for the Z domain), biotin (specific binding partner for avidin, monovalent avidin, streptavidin, monovale ntes streptavidin, anti-biotin antibody or the F ab fragment of anti-biotin antibody) or the specific binding partner for the functional protein such as
  • the specific binding partner of the second chemical substance can also be bound to the nucleic acid oligomer, in which case a receptor which has been modified with the second chemical substance is used for the indirect binding formation between nucleic acid oligomer and receptor.
  • This type of connection has the advantage that, on the one hand, the receptor to be bound to the respective nucleic acid oligomer can be linked stoichiometrically in a simple, independent and separate reaction and, on the other hand, that the binding between nucleic acid oligomer and receptor or between several nucleic acid oligomers and receptors - if applicable Nucleic acid oligomer receptor compound, the identical pair of conjugated chemical substances was used - can be cleaved if necessary (e.g. after the complexes of target and nucleic acid receptor unit have been separated) by a specific antagonist acting on the conjugated chemical substances (or several such antagonists when using different conjugated chemical substances, each used in a significant excess if necessary).
  • a preferred embodiment of the present invention is as a receptor, an antigen, an antibody, particularly a monoclonal antibody, an F ab fragment of an antibody, a peptide or protein, a protein of a multiprotein complex, an allergen, an enzyme, an enzyme inhibitor, a hormone, a hormone receptor protein, ds-DNA (ss-DNA, ss-RNA) with a specific sequence for binding a DNA- (RNA-) binding protein, a DNA- or RNA-binding protein, a hapten analogue, an oligoglycoside or used a lectin.
  • an antigen an antibody, particularly a monoclonal antibody, an F ab fragment of an antibody, a peptide or protein, a protein of a multiprotein complex, an allergen, an enzyme, an enzyme inhibitor, a hormone, a hormone receptor protein, ds-DNA (ss-DNA, ss-RNA) with a specific sequence for binding a DNA- (RNA-) binding protein, a DNA- or
  • the receptor unit can be attached to one of the phosphoric acid units, to one of the thiophosphate units, to one of the thiol units, to one of the phosphoric acid amide units, to one of the sugar units, in particular to one Sugar-hydroxyl group, be attached to a carbonyl, carboxyl, amide or amine group, or to one of the bases of the nucleic acid oligomer component, in particular to a terminal 3 'or 5 ' unit.
  • the cleavable groups between the nucleic acid oligomer component and the receptor unit can also be part of a linker between the nucleic acid oligomer component and the receptor unit.
  • nucleic acid oligomer component of the nucleic acid oligomer receptor unit comprises at least any primer binding region
  • nucleic acid oligomer component of the nucleic acid oligomer receptor unit comprises any two primer binding regions.
  • primer segments that are 5 'of the coding sequence have identical sequence (preferably 8-24 bp long) and 3 'of the coding region have a sequence which is complementary to (one) primer (s) (preferably 8-24 bp long).
  • primer segments that are 5 'of the coding sequence have identical sequence (preferably 8-24 bp long) and 3 'of the coding region have a sequence which is complementary to (one) primer (s) (preferably 8-24 bp long).
  • Particularly advantageous effects result if the different types of nucleic acid oligomer receptor units in an analysis comprise identical primer binding regions.
  • This embodiment has the advantage that the nucleic acid oligomer components of the nucleic acid oligomer receptor unit, in particular those in the form of DNA, RNA or PNA, if necessary (for example after the complexes from the target and nucleic acid receptor unit and subsequent separation of the nucleic acid oligomer component) by PCR or other methods (synthesis using other DNA or RNA polymerases) can be amplified to z. B. also to detect small amounts of target and / or to provide the nucleic acid oligomer components with a detection marker.
  • a binding site for the T7, T3 or SP6 RNA polymerase is located 3 'next to the coding region or 3' next to the 3 'primer binding region, thereby resulting in an in vitro transcription of the coding sequence becomes possible.
  • the different types of nucleic acid oligomer receptor units have no mutually complementary nucleic acid oligomers and, in particular, no regions in the coding regions which are more than 75% complementary to the primer binding regions.
  • a nucleic acid oligomer receptor unit is used which, before, during or after the receptor has been linked, in one of the ways a) to d) additionally with a fluorescent dye and / or with a hapten and / or with biotin ( Avidin / streptavidin)), a uniform base sequence for binding a universal primer (when using cDNA or RNA as nucleic acid oligomer) and / or a reactive group (directly or linker / spacer-linked) is modified.
  • a specific bond is formed between a chemical substance and the receptor of the nucleic acid oligomer-receptor unit.
  • the substance specific for the respective receptor, the target is labeled in any substance mixture with the nucleic acid oligomer-receptor unit, whereby the receptor-modified nucleic acid oligomers can be used as pure substances (a modified nucleic acid oligomer) or mixtures (several different modified nucleic acid oligomers).
  • a prerequisite for the method according to the invention is the specific binding of a chemical substance to a receptor unit of the receptor-modified nucleic acid oligomers specific for this substance.
  • a substance-specific receptor with an associated specific chemical substance are:
  • Antigen including hapten
  • Antibodies monoclonal or polyclonal, antigen (including hapten) including F ab fragment
  • ds-DNA ss-DNA, ss-RNA
  • DNA- (RNA-) binding proteins of a specific sequence for binding DNA- (RNA-) binding proteins
  • RNA binding proteins ds-DNA binding proteins ds-DNA (ss-DNA, ds-RNA, ss-RNA) with a specific sequence for binding DNA (RNA) binding proteins ds-DNA (ss-DNA, ds-RNA, ss-RNA) with antagonists DNA- (RNA-) binding specific sequence for binding proteins DNA- (RNA-) binding proteins
  • Multiprotein complex Proteins of a multi-protein complex functional protein or peptide e.g. specific binding partner of a hormone receptor protein
  • a functional peptide e.g. the hormone
  • a functional peptide e.g. the hormone
  • its antagonist specific binding partner of a functional protein or peptide e.g. functional components
  • Protein or one of the hormone receptor protein) functional peptide e.g. hormone or its antagonist
  • antigens as receptors of the nucleic acid oligomer-receptor unit has the particular advantage that natural and artificially produced antibodies against this antigen can be identified. This method can also be used to detect extremely small amounts of naturally occurring antibodies (e.g. in the blood serum). In addition, a mixture with other related antigens can be tested and the positive results (antigen with bound antibody) can be separated quantitatively and in high concentration from the mixture, since the method does not require separation of the positive results in this critical phase. The antibodies can therefore also be bound in secondary immune complexes with anti-Ig antibodies, which significantly improves the quantitative immobilization with high avidity for all antibodies.
  • the conditions for the chromatographic purification can be optimized such that only the affinity of the individual antigens for the corresponding antibodies has to be taken into account. Elution can be done in fractions with increasing stringency in the washing conditions.
  • the use of antibodies as receptors of the nucleic acid oligomer-receptor unit has the particular advantage that the antibodies against the corresponding antigens have an extremely high specificity with high affinity, the spectrum of the antigens is arbitrarily broad and different physiological forms of the antigen can be distinguished. This ensures that the individual antigens can be identified quantitatively in a complex mixture without having to separate or separate them chromatographically.
  • substances which form hapten analogues can also be detected with the aid of the antibody receptors, the specificity with high affinity also allowing the differentiation of closely related (chemically similar) substances in a complex mixture with other substances which are difficult to separate chromatographically.
  • monoclonal antibodies or affinity-purified polyclonal antibodies which are only directed against a specific region or an epitope of the antigen is particularly preferred since these have an increased specificity, so that e.g. B. active and inactive isoforms, alternatively spliced, or proteolytically processed, or modified forms of the otherwise identical antigens can be distinguished.
  • the use of F ab fragments of the antibodies also has the advantage that only one antigen or hapten binds per F ab fragment and that the separation of the positive results (F ab fragment with associated antigen or hapten) is easier when using secondary immune complexes is because, unlike antibodies, F ab fragments do not bind to the affinity chromatography supports.
  • enzymes as receptors of the nucleic acid oligomer-receptor unit has the particular advantage that a known effective antagonist (or inhibitor or a group thereof) can be assigned to the previously unknown site of action (from a selection of any nucleic acid oligomer-encoded enzymes).
  • the enzymes used as receptors can be used for the quantitative detection of inhibitors, antagonists and cofactors that are not immunogenic.
  • enzymes can be indirectly linked to the nucleic acid oligomers via their cofactors. This allows e.g. B. a connection of a cofactor to the nucleic acid oligomer, which is generally not below physiological conditions must be carried out, since the cofactors are generally much more stable than the enzyme complex.
  • the cofactor can e.g. B. be bound directly to the nucleic acid oligomer during the solid phase synthesis and the nucleic acid oligomer receptor unit is subsequently completed by reconstitution (incubation with cofactor-free enzyme).
  • enzyme inhibitors and antagonists as receptors of the nucleic acid oligomer-receptor unit has the particular advantage that it is possible to search for research-relevant or therapeutically relevant inhibitors or antagonists of an enzyme (or a group of enzymes).
  • these enzyme inhibitors and antagonists used as receptors can be used for the quantitative detection of enzymes, the proportion of the active form of enzymes being selectively detected.
  • enzyme inhibitors and antagonists as long as they are simple chemical compounds, can be easily modified with nucleic acid oligomers because they i. a. do not have to be handled under physiological conditions and thus z. B. can be bound directly to the nucleic acid oligomer during solid phase synthesis.
  • ds-DNA ss-DNA, ss-RNA
  • RNA- DNA- binding proteins
  • such receptors can be used to search for previously unknown sequences which specifically bind to a DNA (RNA) binding protein (or a group of DNA (RNA) binding proteins), and from this information a consensus sequence of Binding site can be determined.
  • these nucleic acid receptor units can be used in combination with the associated targets to determine which of the complexes of ds-DNA (ss-DNA, ss-RNA) sequence and DNA- (RNA-) binding proteins is a potential antagonist acts, which not only determines a consensus binding site, but also has these functional sites a functional relevance.
  • RNA- binding proteins can be linked directly to the coding nucleic acid oligomer in solid phase synthesis. Also, these specific sequences for binding DNA- (RNA-) binding proteins do not necessarily have to be separated for detection (after formation and separation of the receptor-target complexes and possibly subsequent dissociation of the target unit).
  • DNA (RNA) binding proteins as receptors of the nucleic acid oligomer receptor unit has the particular advantage that known ds-DNA (ss-DNA, ds-RNA, ss-RNA) can be characterized with a specific sequence for binding in terms of the spectrum which DNA (RNA) binding proteins bind to these sequences. This is an important tool in promoter studies, alternative splicing and the determination of specific m-RNA transport factors. DNA and RNA binding proteins can also be used to qualitatively and quantitatively demonstrate the availability of binding sites that may be blocked due to a regulatory mechanism
  • proteins of a multiprotein complex as receptors of the nucleic acid oligomer-receptor unit has the particular advantage that multiprotein complexes can thus be detected qualitatively and quantitatively. They can also be used as so-called potential components to search for unknown binding partners for a specific protein or multiprotein complex.
  • multiprotein complexes as receptors of the nucleic acid oligomer-receptor unit has the particular advantage that it can be used to qualitatively and quantitatively detect proteins in physiologically active form that bind to a specific multiprotein complex.
  • functional protein or peptide e.g. a hormone receptor protein
  • a known effective binding partner e.g. a hormone
  • an antagonist to this binding partner or inhibitor or a group thereof
  • these proteins or peptides used as receptors for the quantitative detection of binding partners such as. B. hormones, antagonists to this binding partner (or inhibitors or cofactors) can be used, the proportion of the active form of binding partners being detected.
  • binding partners of a functional protein or a functional peptide e.g. a hormone
  • a potential antagonist as receptors of the nucleic acid oligomer-receptor unit
  • these binding partners can be used for the quantitative detection of the associated receptor proteins (e.g. the hormone receptor protein), the active form of the receptor proteins being detected with a free binding site.
  • lectins as receptors of the nucleic acid oligomer-receptor unit has the particular advantage that glycosylation patterns and / or pathological changes in the glycosylation pattern can be detected qualitatively and quantitatively.
  • lectins are particularly stable and can therefore be easily linked (under broad reaction conditions) to the nucleic acid oligomers.
  • oligoglycosides as receptors of the nucleic acid oligomer-receptor unit has the particular advantage that they serve as binding partners for various receptor proteins and so that these can be detected qualitatively and quantitatively, the active receptor proteins being detected.
  • oligoglycosides can be checked to see whether they serve as binding partners for a specific receptor protein.
  • the specific bond formation between one or more types of nucleic acid oligomer receptor units and one or more types of specific binding chemical substances (targets) can take place in any aggregate states of the nucleic acid oligomer receptor units and the targets, the targets in vitro or in vivo can be present.
  • the bond formation in a soluble environment is preferred, whereby one of the two binding partners, in particular the target (s), does not necessarily have to be in dissolved form. It can also be in the form of a precipitate, enclosed in permeable vesicles (cells), bound to / in (cell) membranes or suitable carrier materials or contained in the solution in the form of micelles.
  • the specific bond formation takes place between one or more types of nucleic acid oligomer receptor units and one or more types of specifically binding chemical substances (targets) by immobilizing the targets on a support and incubating the support-immobilized targets with the nucleic acid oligomer receptor units instead, the incubation taking place by adding the dissolved nucleic acid oligomer receptor units.
  • targets specifically binding chemical substances
  • the subsequent separation of the complexes of nucleic acid oligomer receptor unit and chemical substance from the remaining nucleic acid oligomer receptor units can be carried out by specific precipitation, filtration, centrifugation, by chromatographic methods (column chromatography, affinity chromatography, size exclusion (gel filtration), membrane chromatography etc.) or by gel electrophoresis.
  • carrier material or “chromatography material” refers to any stationary two-dimensional or three-dimensional material which, if necessary by appropriate derivatization, is suitable for one or more of the building blocks of the complex of nucleic acid oligomer. Bind receptor unit and chemical substance.
  • the chromatography material can also be bound to a "chromatography surface", the chromatography surface being understood to mean any material or surface that is applied stationary on a surface and that allows one or more of the building blocks of the complex of nucleic acid oligomer-receptor unit and chemical substance in one to bind according to the type of complexes of nucleic acid oligomer-receptor unit and chemical substance ordered grid, that is, in a defined spatially resolved arrangement of the different complexes of nucleic acid oligomer-receptor unit and chemical substance.
  • the chromatography material - if necessary after previous derivatization - is suitable, the receptor-specific substance, the target, or modifications of the receptor-specific substance (target complexes of receptor-specific substance and one or more further chemical substances which are not the nucleic acid oligomer receptor according to the invention Represent unit), as such or one or more of the building blocks of this target complex, while the non-target-occupied nucleic acid oligomer receptor units are not bound (or vice versa).
  • the targets are immobilized for incubating one or more types of targets with one or more types of nucleic acid oligomer receptor units and / or for separating the complexes of nucleic acid oligomer receptor unit and target from the remaining nucleic acid oligomer units.
  • Receptor units by binding to one
  • Binding to ion exchangers The target or the complex of nucleic acid oligomer receptor unit and target is bound to strong ion exchangers which have been optimized for the binding of the targets in such a way that the nucleic acid oligomer receptor units alone (without an associated target) are not bound become.
  • binding to specially modified chromatography material via two specific binding partners the target alone or the target in the complex of nucleic acid oligomer-receptor unit and target is modified with a further (second) chemical substance which represents a specific binding partner for a third chemical substance, this third chemical substance being the one with which the chromatography material has been modified.
  • Such further (second) chemical substances are e.g. B.
  • hapten analogue biotin, FITC, DNP, digoxigenin or rhodamine
  • an antigen a His tag, an HA tag, a flag tag, a GST tag, a Z domain (monovalent functional domain of Protein A), a functional protein, a chitin-binding protein, an avidin, monovalent avidin, a streptavidin or monovalent streptavidin.
  • an antibody specifically binding partner for the hapten analog, or the antigen
  • F a b fragment specifically binding partner for the hapten analog, or antigen
  • an anti- FITC antibody specifically binding partner for FITC
  • an anti-DNP antibody specifically binding partner for DNP
  • an anti-digoxigenin antibody specifically binding partner for digoxigenin
  • an anti-Rhodamine antibody or an anti-His-Tag -Antibody (specific binding partner for His-Tag)
  • an anti-HA-Tag antibody specifically binding partner for HA-Tag
  • an anti-Flag-Tag antibody specifically binding partner for Flag-Tag
  • an anti-GST Tag antibody or glutathione specifically binding partner for GST tag
  • antibody Fc fragment specifically binding partner for the Z domain
  • biotin specifically binding partner for avidin, monovalent avidi n, streptavidin, monovalent streptavidin, anti-biotin antibody or the F ab fragment of anti-biotin antibody
  • the specific binding partner of the second chemical substance can also be bound to the target alone or the target in the complex of nucleic acid oligomer-receptor unit and target, a chromatography material modified with the second chemical substance being used as the chromatography material.
  • the second and third chemical substances for connecting the target to the chromatography material are different from the second and third chemical substances for the indirect linking of Nucleic acid oligomer and receptor (see section "Binding a receptor to a nucleic acid oligomer"
  • a photocross linker which has an induction-cleavable group, such as. B. via a disulfide group, which can be cleaved reductively, ie after induction by a reducing agent (z. B.
  • DTT or ß-mercaptoethanol in the two thiol components, via a -CHOH-CHOH group, which by adding an oxidizing agent such as z. B Nal0 4 or via a -CO-0-CH 2 -CH 2 -0-CO- group, which can be cleaved by adding H 2 N-OH.
  • the chromatography material can also be modified with a photocrosslinker and the unmodified target is bound to the chromatography material before the complex of nucleic acid oligomer-receptor unit and target is formed by incubating the target with the chromatography material and irradiating light of a suitable wavelength.
  • Binding to the chromatography material via chemical binding The target alone is modified with a linker with a reactive group, in particular an activated linker, before formation of the complex of nucleic acid oligomer-receptor unit and target, such as. B. with a 4-azido group coupled by photocrosslinking to the target 4-azidobenzoic acid N-sulfosuccmimidyl ester sodium salt.
  • the connection to the chromatography material is carried out by bringing the modified target into contact with suitable chromatography material, that is to say with the necessary counter group to form a covalent chemical bond with the reactive group of the linker.
  • a linker which has a group which can be split by induction, such as, for. B. via a disulfide group, which can be cleaved reductively, ie after induction by a reducing agent (z. B. DTT or ß-mercaptoethanol) in the two thiol components, via a -CHOH-CHOH group, which by adding an oxidizing agent such as z. B Nal0 4 or via a -CO-0-CH 2 -CH 2 -0-CO- group, which can be cleaved by adding H 2 N-OH or via a photolabile group such as. B.
  • the chromatography material can also be modified with a chemical crosslinker and the unmodified target is bound to the chromatography material by incubating the target with the chromatography material before the complex of nucleic acid oligomer-receptor unit and target is formed.
  • Exist against target-occupied receptors in the complex of nucleic acid oligomer-receptor unit and target other binding partners such as.
  • B. receptor proteins such as F c - ⁇ receptors or compliment C1q, which act selectively against groups of target-occupied receptors, these can also be used to modify the column material.
  • Binding to a chromatography material modified with protein A, protein G or directed against the F c domain exist for the targets or the target in the complex of nucleic acid oligomer-receptor unit and target next to the Receptor-specific antibodies, or specific monoclonal or polyclonal antibodies (e.g. in the case of antigens or haptens as targets), the targets can be formed after the complexes of nucleic acid oligomer The receptor unit and target are incubated with the antibodies before binding these complexes to the chromatography material, as a result of which so-called primary immune complexes are formed and secondary antibodies after the addition of anti-Ig antibodies.
  • These (primary or) secondary immune complexes can be separated from the remaining non-target-occupied nucleic acid oligomer receptor units by means of chromatography material modified with protein A, protein G or with anti-Ig antibody directed against the F c domain.
  • the antibodies can also be brought into contact with the targets simultaneously with the bringing into contact with the nucleic acid oligomer-receptor units with the targets.
  • the chromatography material is saturated with the type or types of targets, e.g. B. analogous to methods a) to d) in the section "separation of the complexes of nucleic acid oligomer receptor unit and target from non-target-occupied nucleic acid oligomer receptor units").
  • targets e.g. B. analogous to methods a) to d) in the section "separation of the complexes of nucleic acid oligomer receptor unit and target from non-target-occupied nucleic acid oligomer receptor units"
  • the complexes of nucleic acid oligomer receptor unit and target are not separated from the remaining nucleic acid oligomer receptor units by binding to a chromatography material.
  • targets insoluble in aqueous media e.g. B. insoluble proteins as targets
  • the complex formation between nucleic acid oligomer receptor unit and target can be done by incubating a suspension of the targets with the dissolved nucleic acid oligomer receptor units.
  • aqueous medium is understood to mean all solutions with the main constituent water.
  • the soluble ones are not occupied Nucleic acid oligomer receptor units separated by centrifugation or filtration (pelleting of the complex, together with remaining insoluble material). Even with targets with a high molecular weight compared to the molecular weight of the nucleic acid oligomer receptor units, the soluble, unoccupied nucleic acid oligomer receptor units can be separated off by centrifugation after complex formation has taken place. In the case of targets which are large in comparison to the nucleic acid oligomer receptor units, the unoccupied nucleic acid oligomer receptor units can be separated off by size exclusion (gel filtration) or filtration.
  • target-filled nucleic acid oligomer-receptor units can be used as secondary immune complexes (cf. Method f) in the section "separation of the complexes of nucleic acid-oligomer-receptor unit and target from non-target-filled nucleic acid oligomer-receptor units") by filtration or centrifugation (Immunoprecipitation) from the remaining components of the test solution.
  • the present invention also comprises a method for the detection of chemical substances, comprising the steps (i) providing one or more chemical substances, (ii) providing one or more types of nucleic acid oligomer receptor units, (iii) contacting the chemical Substances with the nucleic acid oligomer receptor units, whereby complexes of nucleic acid oligomer receptor unit and chemical substance are formed, (iv) binding of the complexes of nucleic acid oligomer receptor unit and chemical substance to a suitable chromatography material, thereby separating them from the into the eluate non-target-occupied nucleic acid oligomer receptor units, (v) cleavage of one or more chemical bonds of the complexes of nucleic acid oligomer receptor unit and chemical substance bound to the chromatography material such that the nucleic acid oligomer component de r nucleic acid oligomer receptor unit in the form of a nucleic acid oligomer or in the form of any modified nucleic acid oligo
  • the soluble, unoccupied nucleic acid oligomer receptor units can be separated off by centrifugation after complex formation or in comparison to the nucleic acid oligomer -Receptor units large targets the unoccupied nucleic acid oligomer receptor units are separated by size exclusion (gel filtration).
  • the chemical substances can be detected by a method with the following steps: (i) providing one or more chemical substances, (ii) providing defined amounts of one or more types of nucleic acid oligomer receptor units, (iii) contacting the chemical substances with the defined amounts of nucleic acid oligomer receptor units, whereby complexes are formed from nucleic acid oligomer receptor unit and chemical substance, (iv) binding of the complexes from nucleic acid oligomer receptor unit and chemical substance to a suitable chromatography material, whereby a separation from the non-target-populated nucleic acid oligomer receptor units which pass into the eluate takes place, and finally (v) detection of the nucleic acid oligomer components of the non-target-populated nucleic acid oligomer receptor units located in the eluate directly as nucleic acid reoligomer-receptor unit or after cleavage of the nucleic acid oligomer component from the receptor unit as
  • Nucleic acid oligomer or modified nucleic acid oligomer comprising the complete sequence information of the nucleic acid oligomer by a suitable method.
  • the soluble, unoccupied nucleic acid oligomer receptor units can be separated off by centrifugation after complex formation has taken place, or in the case of large targets compared to the molecular weight of the nucleic acid oligomer receptor units Targets that are not occupied by nucleic acid oligomer receptor units are separated by size exclusion (gel filtration).
  • the chemical substances can be detected by a method with the following steps: (i) providing one or more chemical substances, (ii) binding the chemical substances to a chromatography material, (iii) providing one or more types of nucleic acid oligomer -Receptor units, (iv) bringing the nucleic acid oligomer receptor units into contact with the chemical substances bound to the chromatography material, as a result of which complexes are formed from the nucleic acid oligomer receptor unit and chemical substance and thus a separation from the non-target which passes into the eluate occupied nucleic acid oligomer receptor units, (v) cleavage of one or more chemical bonds of the complexes of nucleic acid oligomer receptor unit and chemical substance bound to the chromatography material such that the nucleic acid oligomer component of the nucleic acid eoligomer-receptor unit in the form of a nucleic acid oligomer or in the form of any modified nucleic acid oli
  • the chemical substances can be detected by a method with the following steps: (i) providing one or more chemical substances, (ii) binding the chemical substances to a chromatography material, (iii) providing defined amounts of one or more species of nucleic acid oligomer receptor units, (iv) contacting the defined amounts of nucleic acid oligomer receptor units with the chemical substances bound to the chromatography material, as a result of which complexes of nucleic acid oligomer receptor unit and chemical substance take place and thus a separation from the non-target-populated nucleic acid oligomer receptor units passing into the eluate, and finally (v) detection of the nucleic acid oligomer components of the non-target-populated nucleic acid oligomer receptor units present in the eluate directly as nucleic acid oligomer recipe tor unit or after the spin-off Nucleic acid oligomer component from the receptor unit as
  • Nucleic acid oligomer or modified nucleic acid oligomer comprising the complete sequence information of the nucleic acid oligomer by a suitable method.
  • the present invention also relates to a method for the detection of substances insoluble in aqueous media, in particular insoluble proteins, comprising the steps (i) providing one or more types of insoluble substances, in particular insoluble proteins, (ii) providing one or more types nucleic acid oligomer receptor units, contacting the insoluble proteins with the nucleic acid oligomer receptor units, thereby forming complexes of nucleic acid oligomer receptor unit and insoluble protein, (iii) separating the complexes of nucleic acid oligomer receptor unit and insoluble protein of non-target-occupied nucleic acid oligomer receptor units, (iv) cleavage of one or more chemical bonds of the complexes of nucleic acid oligomer receptor unit and insoluble protein in such a way that the nucleic acid oligomer component of the nucleic acid oligomer -Receptor unit in the form of a nucleic acid oligomer or in the form of any modified nucleic acid oli
  • steps (iii) separating the complexes of nucleic acid oligomer receptor unit and insoluble protein from non-target-occupied nucleic acid oligomer receptor units and (v) separating the dissolved nucleic acid oligomer separated from the insoluble protein Components carried out by centrifugation.
  • the unoccupied nucleic acid oligomer receptor units and / or in step (v) the dissolved nucleic acid oligomer components separated from the insoluble protein can be separated by size exclusion (gel filtration).
  • the present invention also relates to a method for the detection of hapten analogs comprising the steps of (i) providing one or more hapten analogs, (ii) providing an excess of one or more types of nucleic acid oligomer receptor units (relative to the hapten analogs provided in step (i)), (iii) contacting the provided hapten analogs with the nucleic acid oligomer receptor units, as a result of which complexes are formed from the nucleic acid oligomer receptor unit and the hapten analog, (iv) Providing an excess of the hapten analogs provided in step (i) in the form of carrier-modified hapten analogs, (v) contacting the modified hapten analogs provided in step (iv) with the mixture of excess nucleic acid oligomer formed in step (iii) -Receptor units and complexes formed from nucleic acid oligomer-receptor unit u nd hapten analog, resulting in complexes from
  • the modification of the modified hapten analogs provided in step (iv) consists in the attachment of a carrier molecule to the hapten analogs in which a specific type of carrier molecule is bound to each type of hapten analog which meets the condition that the binding of the carrier molecule does not result in a significant change in the specific binding capacity of the epitope of the hapten analogs for binding the receptor component of the nucleic acid oligomer-receptor unit.
  • step (vi) in the case of carriers with a high molecular weight compared to the molecular weight of the nucleic acid oligomer receptor units occupied with hapten analogue after complex formation, the complexes of carrier-modified hapten analogue and nucleic acid oligomer receptor unit can be centrifuged from the Complexes with hapten analogue and nucleic acid oligomer receptor unit are separated or, in the case of carriers which are large compared to the nucleic acid oligomer receptor units occupied with hapten analogue, the complexes of carrier-modified hapten analogue and nucleic acid oligomer receptor unit by size exclusion (Gel filtration) are separated from the complexes of hapten analogue and nucleic acid oligomer receptor units.
  • size exclusion Gal filtration
  • the present invention also comprises a method for the detection of chemical substances, comprising the steps (i) providing one or more chemical substances, (ii) providing one or more types of nucleic acid oligomer receptor units, (iii) contacting the chemical Substances with the nucleic acid oligomer receptor units, whereby complexes are formed from the nucleic acid oligomer receptor unit and chemical substance, (iv) binding of the nucleic acid oligomer receptor units to a chromatography material modified with the targets, thereby separating them from the eluate passing target-occupied nucleic acid oligomer receptor units, and finally (v) detection of the nucleic acid oligomer components of the target-containing nucleic acid oligomer receptor units located in the eluate directly as nucleic acid oligomer receptor unit or after cleavage of the nucleic acid oligo mer component of the receptor unit as a nucleic acid oligomer or modified nucleic acid oligomer, which comprises the complete sequence
  • the present invention also comprises a method for the detection of antagonists for binding partners of receptor proteins or enzymes, comprising the steps (i) providing one or more types of binding partners of receptor proteins or enzymes, (ii) providing one or more types of nucleic acid oligomer receptor -Units being the receptor represents a potential antagonist of the binding partners of receptor proteins or enzymes, (iii) providing an excess of chromatography material modified with the targets receptor proteins or enzymes (compared to the nucleic acid oligomer-receptor units), (iv) contacting the one provided in step (ii) or several types of nucleic acid oligomer receptor units with the target-modified chromatography material provided in step (iii), thereby forming complexes of nucleic acid oligomer-receptor unit and target-modified
  • nucleic acid oligomer receptor unit or after cleavage of the nucleic acid oligomer component from the receptor unit as
  • Nucleic acid oligomer or modified nucleic acid oligomer comprising the complete sequence information of the nucleic acid oligomer by a suitable method.
  • an elution step with a known antagonist is additionally carried out after the fractional elution with the binding partners, and the nucleic acid oligomer constituents obtained in this way are separately detected by a suitable method.
  • the step of cleaving one or more chemical bonds of the complexes of nucleic acid oligomer-receptor unit and modified chromatography material is additionally carried out in such a way that the nucleic acid oligomer component of the nucleic acid oligomer receptor unit is in the form of a nucleic acid oligomer or in the form of any modified nucleic acid oligomer separated from the target-modified chromatography material is inserted and the nucleic acid oligomer components thus obtained are separately detected by a suitable method.
  • an ion exchange material is used as the chromatography material, which has been optimized in this way for the binding of the targets that the nucleic acid oligomer receptor units alone (without an associated target) are not bound.
  • methods IA, IB, IIA, and IIB use chromatography material to immobilize the targets in the course of incubating one or more types of targets with one or more types of nucleic acid oligomer receptor units and / or in In the course of the separation of the complexes of nucleic acid oligomer receptor unit and target from the remaining nucleic acid oligomer receptor units according to one of the steps b) to f) in the section "Separation of the complexes of nucleic acid oligomer receptor unit and target from non-target-occupied nucleic acid oligomer -Receptor units "methods described.
  • one or more types of complexes of carrier-modified hapten analogue with one or more types of nucleic acid oligomer receptor units and / or in the course of incubation are used the separation of the complexes of carrier-modified hapten analog and nucleic acid oligomer receptor unit from the remaining complexes of hapten analog and nucleic acid oligomer receptor units according to one of the b) to f) in the section "separation of the complexes of nucleic acid oligomer receptor Unit and target of non-target-occupied nucleic acid oligomer receptor units "methods described, only in this case - in the methods described, the term "target” or grammatical equivalents is to be replaced by the term "carrier-modified target” or grammatical equivalents.
  • the target is applied according to one of the methods described under b) to f) in the section “separation of the complexes of nucleic acid oligomer receptor unit and target from non-target-occupied nucleic acid oligomer receptor units” the chromatography material bound.
  • the complexes of nucleic acid oligomer receptor unit and target can be separated from the remaining nucleic acid oligomer receptor units by specific precipitation, filtration, centrifugation, by chromatographic methods or by gel filtration.
  • the subsequent identification and quantification of the receptor-specific substances bound to the respective receptors of the receptor-modified nucleic acid oligomers is carried out by an indirect method in which the receptor-specific substances (targets) are determined on the basis of the oligonucleotide tag on the receptor, which is clearly for a specific receptor and thus the corresponding one encoded receptor-specific substance.
  • target-populated nucleic acid oligomer receptor units are replaced by non-target-populated nucleic acid oligomer receptor units (process alternatives IA, IB, IIA, IIB, and III) or of nucleic acid oligomer receptor units which are occupied by a carrier-modified target (process alternative IV) or complexes of nucleic acid oligomer receptor unit and target of nucleic acid oligomer receptor units which are bound to targets bound to chromatography material (process alternative V) separated.
  • either the dissolved, separated, non-target-occupied nucleic acid oligomer receptor units are detected on the basis of their nucleic acid oligomer components (see below), these dissolved, separated, non-target-occupied nucleic acid oligomer components Receptor units can be used directly or after separation of the nucleic acid oligomer component from the nucleic acid oligomer receptor unit, or the separated immobilized target-containing nucleic acid oligomer receptor units can be used after separation of the nucleic acid oligomer component from the immobilized complexes.
  • either the detached target-occupied nucleic acid oligomer receptor units are detected on the basis of their nucleic acid oligomer constituents (see below), these detached target-occupied nucleic acid oligomer receptor units detached directly or after the nucleic acid oligomer constituent has been separated from the nucleic acid oligomer -Receptor unit can be used.
  • nucleic acid oligomer components of the separated, non-target-containing nucleic acid oligomer receptor units are based on the difference between the originally used and finally separated, nucleic acid oligomer receptor units quantitatively and qualitatively representative of the specific chemical substance (the target), which was originally contained in the sample solution.
  • the nucleic acid oligomer components of the separated immobilized target occupy nucleic acid oligomer receptor units (method IA, IB, IIA, IIB and III) or the nucleic acid oligomer components of the separated complexes of target and nucleic acid oligomer receptor unit (method IV) or die the nucleic acid oligomer constituents of the separated, dissolved nucleic acid oligomer receptor units (method VI) represent, quantitatively and qualitatively, the specific chemical substance (the target) which was originally contained in the sample solution.
  • This separation of the nucleic acid oligomer constituents which is necessary or alternatively possible in the method, can be carried out in the following way:
  • Receptor units with guadinium hydrochloride components of these complexes are denatured so that these complexes dissociate and the nucleic acid oligomer component as such or in the form of any modified nucleic acid oligomer component which comprises the complete sequence information of the nucleic acid oligomer is separated ,
  • nucleic acid oligomer receptor unit When using nucleic acid oligomer receptor units which were formed according to one of the preferred embodiments of the binding types a) to c) in the section "binding a receptor to a nucleic acid oligomer", the nucleic acid oligomer receptor can Unit can be split into a nucleic acid oligomer and a receptor component by incubation with the necessary induction agent.
  • nucleic acid oligomer receptor unit When using nucleic acid oligomer receptor units which were formed according to one of the embodiments of the binding type d) in the section "binding a receptor to a nucleic acid oligomer", the nucleic acid oligomer receptor unit can be incubated with an antagonist of one of the two conjugated chemical substances, which were used for the indirect, non-covalent binding between receptor and nucleic acid oligomer are cleaved. If necessary, the incubation is carried out with a clear excess of the antagonist.
  • C) Cleavage of the bond between target and carrier Are the target-occupied nucleic acid oligomer receptor units in the method IA or IIA or the complexes of carrier-modified target and nucleic acid oligomer receptor unit according to one of the methods c) or d) in the section "Separation of the complexes of nucleic acid oligomer receptor unit and target from non-target-occupied nucleic acid oligomer receptor units" bound to chromatography material, the bound complex can be incubated with the necessary Induction agents are separated from the chromatography material.
  • the attached complex can be incubated with an antagonist of one of the two conjugated chemical substances that were used for the indirect, non-covalent connection to the chromatography material , are separated from the chromatography material. If necessary, the incubation is carried out with a clear excess of the antagonist.
  • the methods according to the present invention can be used to label or to detect one or more types of antigens and proteins.
  • the use for labeling or for detecting at least one type of native, denatured or partially denatured protein or antigen is particularly preferred.
  • the methods according to the invention are used for labeling or for the detection of antibodies, antigens or hapten analogues.
  • nucleic acid oligomer receptor units are provided which are selected by linking one or more identical or different functional chemical compounds selected from the group consisting of dye, in particular fluorescent dye, redox-active substance , in particular quinones and transition metal complexes, biotin, hapten, avidin, in particular monovalent avidin and streptavidin, in particular monovalent streptavidin, and / or are additionally modified by a radio label, in particular by 35 P, 32 S, 14 C or 3 H.
  • these nucleic acid oligomer receptor units cannot be provided in a modified form, but the modification can only take place during a method according to the invention.
  • the modification can before any step performed after the step of "providing one or more types of nucleic acid oligomer receptor units".
  • nucleic acid oligomer component of the nucleic acid oligomer receptor unit represents a DNA or RNA sequence and comprise at least any primer binding region are very particularly preferred.
  • an amplification of the nucleic acid oligomer components in particular by RNA polymerases or by DNA polymerases such as. B. in the polymerase chain reaction (PCR).
  • Embodiments are very particularly preferred according to which a defined amount of a nucleic acid oligomer is added before the joint amplification of the one or more types of nucleic acid oligomer components, the added nucleic acid oligomer comprising the same or the same primer sections as the nucleic acid oligomer components.
  • a primer is particularly preferably used which bears a detection marker.
  • the prerequisite for the method according to the invention is the identification and quantification of the receptor-specific substances (targets).
  • the detection of the chemical substances is carried out indirectly via the detection of the separated nucleic acid oligomer component (see above), the detection of the nucleic acid oligomer component via hybridization to a complementary nucleic acid oligomer being preferred.
  • the detection of the hybridization events is electrochemical, in particular amperometric, cyclovoltametric, impedance spectroscopic, potentiometric, by optical spectroscopy, in particular absorption or fluorescence measurement, by vibration spectroscopy, in particular IR, Raman, FTIR or FT- Raman spectroscopic, by total reflection methods, in particular attenuated total reflection, by quartz-crystal-micro-balance, by surface plasmon resonance, by chemiluminescence measurement or by radioactivity measurement.
  • the simultaneous detection of different nucleic acid oligomer constituents is particularly preferred by means of a uniform type of hybridization, as described in the previous paragraph, in particular the detection on so-called DNA chips or so-called dot plot membranes.
  • the present invention also includes a kit for labeling chemical substances, a kit for the detection of chemical substances, a kit for the detection of proteins, a kit for the detection of proteins insoluble in aqueous media, a kit for the detection of antibodies, a kit for the detection of antigens , a kit for the detection of hapten analogs, a kit for the detection of DNA and / or RNA binding proteins and a kit for the detection of the antagonists of DNA and / or RNA binding proteins, a kit for screening for antagonists for binding partners of Enzymes and receptor proteins and a kit for screening hybridoma cells with simultaneous epitope mapping.
  • kits according to the invention comprises an effective amount of one or more types of nucleic acid oligomer receptor units as used in the methods according to the present invention.
  • kits which additionally comprises primers are preferred, in particular primers modified with rhodamine or fluorescin.
  • each kit which additionally comprises the nucleotide building blocks dCTP, dGTP, dATP and dTTP is particularly preferred, a set of nucleotides which includes at least one type of fluorescent dye-modified nucleotide being particularly preferred.
  • kits which additionally comprises a chromatography cartridge for separating the target-containing nucleic acid oligomers is also particularly preferred.
  • the kits preferably additionally comprise a chromatography column coated with biotin.
  • a kit which additionally comprises a gel filtration cartridge is also particularly preferred.
  • a kit is very particularly preferred, the receptor unit being one or more of the types of nucleic acid oligomer receptor encompassed by the kit. Units contain an anti-Biotin-F ab fragment, a monovalent avidin or a monovalent streptavidin.
  • Kits comprising an effective amount of one or more types of nucleic acid oligomer receptor units as used in the methods according to the present invention are particularly preferred. Kits which additionally comprise biotinylated peptides, polypeptides or proteins are particularly preferred.
  • kits which contains an anti-biotin Fab fragment, a monovalent avidin or a monovalent streptavidin as the receptor of the one or more of the types of nucleic acid oligomer receptor units included in the kit.
  • a kit which additionally contains one or more types of biotinylated receptors is particularly preferred.
  • kits which contains an anti-digoxigenin Fab fragment as the receptor of the one or more of the types of nucleic acid oligomer receptor units included in the kit.
  • a kit which also contains one or more types of digoxigenin-labeled receptors is particularly preferred.
  • kits that contains an anti-FITC Fab fragment as the receptor of the one or more of the types of nucleic acid oligomer receptor units included in the kit.
  • a kit which additionally contains one or more types of FITC-labeled receptors is particularly preferred.
  • the one or more of the types of nucleic acid oligomer receptor units included in the kits according to the invention are dissolved in a solvent.
  • a phosphate buffer is particularly preferably contained as solvent.
  • L linker
  • P1 primer binding region
  • K coding sequence
  • P'2 primer binding region
  • R receptor unit
  • FIG. 3 flow chart of a variant of the method according to the invention (the
  • Abbreviation X corresponds to that in FIG. 1): Target 1 to 3 are modified in a first step "A” with identical tags, then in a second step “B” with different nucleic acid oligomer receptor units (receptor unit 2 to 4 with receptor-specific Affinity for target 2 to 4) for binding formation, then this mixture is added to a chromatographic column modified with the binding partner specific for the day (e.g.
  • the non-target-containing nucleic acid oligomer receptor unit being the receptor Unit 4 passes into the eluate and is thus separated from the target-occupied nucleic acid oligomer receptor unit receptor units 2 and 3; finally either the one remaining in the eluate Receptor unit 4 or - after step D, cleavage of functional group X (z. B. SS group) and elution of the nucleic acid oligomer components of receptor units 2 and 3 - the nucleic acid oligomer components of receptor units 2 and 3 using their coding sequence, in both detection variants it follows that the test solution contained the targets 2 and 3, but not the target 4, of the targets sought by the DNA receptors 2 to 4.
  • Example 1 Construction of the nucleic acid oligomer for solid phase synthesis, usable for labeling a receptor unit:
  • nucleic acid oligomer consists 5'-terminal of the universal sequence P1 (primer binding region, e.g. 12 bases long), which has the identical sequence as a universal primer, a sequence K, which is linked to a specific one Receptor unit represents the label specific for this receptor unit (the coding sequence), and 3'-terminally from a further universal sequence P'2 (primer binding region 2, for example 12 bases long), which can be used to sequence a universal one Primer 2 is complementary.
  • P1 primer binding region, e.g. 12 bases long
  • sequence K which is linked to a specific one Receptor unit represents the label specific for this receptor unit (the coding sequence)
  • P'2 primer binding region 2, for example 12 bases long
  • Regions P1 and P'2 are used for later amplification of the base sequence of the nucleic acid oligomer by PCR. If a complete target analysis is to be carried out on one and the same chip and the targets are present in extremely different concentrations, it is also possible to use different primer pairs for different groups, the respective primer binding regions P1 and P'2 passing through corresponding sequences, e.g. B. Primer binding region 2-1 and 2-'2 on nucleic acid oligomer receptor units for targets with low representation, and primer binding region 3-1 and 3-'2 on nucleic acid oligomer receptor units for targets with very low representation, so that these groups of nucleic acid oligomers can be selectively amplified.
  • the selection criteria for the construction of different primers is that they all have the same GC content and the same length, but differ from one another or from the complementary sequences in at least 60% of the bases.
  • the length of the specific region K (including adjacent bases of the primer binding regions), which is used for the detection by hybridization with probe nucleic acid oligomers (cf. Example 19), can vary depending on the GC content of the sequence, the length advantageously being so is chosen that the respective melting point T m of the hybridized nucleic acid oligomers (calculated according to Bolton and McCarthy, 1962) for all nucleic acid oligomers of an analysis do not differ by more than 0.5 ° C.
  • sequence of the coding region K is advantageously chosen such that each specific coding region K differs from any other specific coding region by at least 4 bp, the base mismatches being distributed over the whole molecule in a particularly favorable embodiment, none of the coding sequences to another or is complementary to the primer binding regions and the sequences are not palindromic over more than 75% of the coding sequence.
  • the nucleic acid oligomer is also provided directly or via a spacer with a reactive group, such as a covalently linked C6-NH 2 unit (hexyl-NH 2 ), which bridges the direct or via an additional linker bridged connection of a specific receptor unit (or certain group of receptor units) is used. Furthermore, the nucleic acid oligomer is modified with a detection marker such as rhodamine, which is used for the later detection of the nucleic acid oligomer (the choice of the detection marker being adapted to the type of nucleic acid oligomer detection method).
  • a detection marker such as rhodamine
  • Example 2 Linking a linker which can be cleaved by reduction to a synthetic nucleic acid oligomer:
  • the commercially available oligonucleotide from Example 1 is used as the synthetic nucleic acid oligomer, which carries a C6 linker with a functional NH 2 group at the 3 'end and a rhodamine (oligol) at the ⁇ ' end.
  • 50 ⁇ g of the nucleic acid oligomer and 50 ⁇ g dithio-bis- (sulfosuccinimidyl) propionate are dissolved in 100 ⁇ l potassium phosphate buffer (50mM, pH 8) and incubated for 2-4 hours in the dark at room temperature.
  • the incubation solution is placed on a Biogel PD6 column, which was previously equilibrated with MES buffer (10mM pH 6.0). The column is eluted with MES buffer (10mM pH 6.0).
  • the first fluorescent fraction contains the Oligo2 m ⁇ d-
  • the Oligo2 m0 d is cooled to 0 ° C by adding 5M NaCl ( ⁇ A of the elution volume of the Oligo2 m ⁇ d fraction, and ethanol (3 ⁇ A times the elution volume of the Oligo2 m0d fraction , cooled to -20 ° C.) and then centrifuged at 4 ° C. (30 min at 15000 ⁇ g), the precipitate is washed with ethanol at ⁇ 20 ° C. and dried.
  • Example 3 Linking a light-cleavable linker to a synthetic nucleic acid oligomer:
  • Oligonucleotide from Example 1 used which carries a C6 linker with a functional NH 2 group at the 3 'end and a rhodamine at the 5' end.
  • An azo compound is used as the linker, which is caused by the irradiation of light in the wavelength range can be split by 350 nm. Therefore, when connecting this linker to the nucleic acid oligomer, work must be carried out under dim light or red light.
  • 4 nmoles (approx. 50 ⁇ g) of the nucleic acid oligomer and 50 ⁇ g 4,4′-dihydroxyazobenzene-3,3'dicarboxylic acid are dissolved in 50 ⁇ l MES buffer (100 mM, pH 6.0) and combined.
  • the solution After adding 50 ⁇ g EDC and 70 ⁇ g NHS, the solution is incubated for 2 hours at room temperature and then for 12 hours at 4 ° C.
  • the incubation solution is placed on a Biogel PD6 column, which was previously equilibrated with MES buffer (10mM pH 6.0). The column is eluted with MES buffer (10mM pH 6.0).
  • the first fluorescent fraction contains the Oligo3 m ⁇ d-
  • the Oligo3 mod is added by adding 5M NaCl (% of the elution volume of the Oligo3 m ⁇ d fraction, cooled to 0 ° C), and ethanol (3 A times the elution volume of the Oligo3 m ⁇ d fraction, cooled to -20 ° C) and subsequent centrifugation at 4 ° C (30min at 15000 xg). The precipitate is washed with ethanol at -20 ° C. and dried.
  • the commercially available synthetic oligonucleotide from Example 1 is used as the synthetic nucleic acid oligomer, which carries at the 3 'end a C6 linker with a functional NH 2 group at the ⁇ ' end of a rhodamine (oligol).
  • An active ester (for binding to the oligol -NH 2 group) is used as linker, which additionally has a terminal azido group which can be bound to any other substance by irradiation with light in the wavelength range around 350 nm. Therefore, when connecting the ester to the Oligol -NH 2 group, work must be carried out under dim light or red light.
  • nucleic acid oligomer 50 nmol of the nucleic acid oligomer are taken up in 0.7 ml sterile water and mixed with 100 ⁇ l carbonate buffer (1M NaHC0 3 / Na 2 C0 3 , pH 9).
  • the linker 4-azidobenzoic acid N-sulfosuccinimidyl ester sodium salt 5 ⁇ mol is dissolved in 200 ⁇ l carbonate buffer (0.1 M NaHC0 3 / Na 2 CO 3 , pH 9) and added to the oligol solution. You react overnight.
  • the first fluorescent fraction contains the Oligo4 m0 d-
  • the Oligo4 m ⁇ d is cooled to 0 ° C by adding 5M NaCl (Y 4 of the elution volume of the Oligo4 m ⁇ d fraction) and ethanol (3 V times the elution volume of the Oligo4 m0 d fraction , cooled to - 20 ° C.) and subsequent centrifugation at 4 ° C. (30 min at 15000 ⁇ g), the precipitate is washed with ethanol at ⁇ 20 ° C. and dried.
  • Example 5 Linking a nucleic acid oligomer with an antibody as a receptor unit and determining the number of attached nucleic acid oligomers per antibody:
  • nucleic acid oligomer contains a fluorescent label or a photolabile group in the linker, you must work under dim light or red light.
  • nucleic acid oligomer now terminated with an activated ester is isolated and purified as described in Examples 2 and 3, respectively.
  • the nucleic acid oligomer is dissolved in 50 ⁇ l potassium phosphate buffer (50 mM, pH 7.2, 150 mM NaCl) and combined with a solution of 150 ⁇ g of the F ab fragment of a monoclonal antibody in 20 ⁇ l PBS.
  • the reaction solution is allowed to react for 4 hours at room temperature and for a further 14 hours at 4 ° C.
  • the reaction solution is then applied to a Sephacryl S200 column, which was previously equilibrated with PBS. It is eluted with PBS.
  • the first fluorescent fraction contains the nucleic acid oligomer-F a fragment complexes (approx. 60kDa), the second fluorescent fraction contains the nucleic acid oligomers not linked to the F ab fragment (approx. 13kDa).
  • the number of DNA units per F ab fragment is determined by the ratio of the concentration of the nucleic acid oligomer, determined from the rhodamine fluorescence at 575 nm, b to the concentration of F a fragments, ideally determined by immunoprecipitation with a radioactively labeled antigen, or with an enzyme-labeled antigen (RIA, or EIA) or an alternative method as in (iii ) described.
  • RIA enzyme-labeled antigen
  • Oligo4 mod from Example 4 is used as the nucleic acid oligomer.
  • Oligo4 m ⁇ d (0.5mg) is dissolved in 100 ⁇ l potassium phosphate buffer (50mM, pH 7.2, 150mM NaCI) and combined with a solution of 150 ⁇ g of the F ab fragment of a monoclonal antibody in 30 ⁇ l PBS. This solution is irradiated for 30 min with the light of a xenon lamp with a 330 to 350 nm interference filter attached.
  • the reaction mixture is then applied to a Sephacryl S200 column which was previously equilibrated with PBS. It is eluted with PBS.
  • the first fluorescent fraction contains the nucleic acid oligomer-F ab fragment complexes (approx. 60 kDa), the second fluorescent fraction contains those that are not linked to the F ab fragment
  • Nucleic acid oligomers (approx. 13kDa). The efficiency of the marking is determined as under (i).
  • (iii) Possibilities for determining the number of linked nucleic acid oligomers per Fab fragment
  • the amount of attached nucleic acid oligomers can be determined according to one of the known methods such as fluorescence, radioactivity, etc., depending on the detection marker of the nucleic acid oligomer (fluorophore, 35 P / 32 S, hapten etc.). If the nucleic acid oligomer does not have a detection marker, it can be quantified by hybridization with a labeled nucleic acid oligomer with a complementary sequence that bears a detection marker, or by a more detailed analysis of the absorption spectrum between 240nm and 300nm.
  • the amount of the F ab fragments is ideally carried out by immunoprecipitation with a radioactively labeled antigen, alternatively with an enzyme-labeled antigen:
  • a radioactively labeled antigen for this purpose, different defined amounts of the labeled antigen are added to a defined mega of the receptor unit and determines the maximum amount of antigen precipitated (radioactive detection or via enzyme-dependent chemiluminescence).
  • the advantage of this method is that it also proves that the isolated F ab fragments are intact.
  • an ELISA system with anti-F ab antibodies can be set up if the antigen is not available.
  • HeLa cells are mixed in PBS (in 9 times the volume of the cell pellet) with the additives PMSF (1mM) and EDTA (1mM), optionally with further protease inhibitors, and gently digested with a Potter.
  • the core fraction is then centrifuged (pelleted) together with the insoluble protein fraction (ECM / cytoskeleton) at 800 x g for 15 minutes.
  • the membrane fractions are then separated from the supernatant (centrifugation at 25,000 x g for 60 min and at 240,000 x g for 30 min). All manipulations are carried out at 4 ° C.
  • the supernatant contains the soluble cytoplasmic proteins and can be stored at -20 ° C to - 80 ° C.
  • individual membrane fractions with a specific composition can be isolated using sucrose density centrifugation).
  • the pellet of the membrane fractions at 4 ° C can be resuspended in PBS with PMSF (1mM) and further disrupted with ultrasound.
  • the membranes are centrifuged at 240,000 x g for 30 minutes.
  • the supernatant contains soluble proteins from cell organelles and can be stored at -20 ° C to -80 ° C.
  • the pellet contains the membrane proteins and some large protein complexes such as Ribosomes, and can be stored at -20 ° C to -80 ° C.
  • the pellet of core fraction and insoluble protein fraction is ultrasonically resuspended at 4 ° C.
  • the insoluble protein fraction is centrifuged out of the suspension at 800 x g for 15 min. It mainly contains cytoskeletal proteins and parts of the core membrane. The supernatant becomes the main core membrane fraction at 240,000 x g for 30 min. centrifuged. The supernatant contains soluble proteins from the cell nucleus.
  • Example 7 Isolation of proteins from HeLa cells under denaturing conditions:
  • Cells are taken up in PBS with 6M urea, 1% SDS and 1mM PMSF, briefly heated to 100 ° C (2 min) and treated with ultrasound for 3 min. The insoluble constituents are then centrifuged off at 240,000 ⁇ g for 30 min. The supernatant contains the soluble but denatured proteins. The pelleted insoluble constituents are resuspended in PBST using ultrasound and centrifuged again to remove urea and SDS (240,000 ⁇ g, 30 min). The pellet contains the insoluble protein fraction.
  • the advantage of this method is that almost all proteins are solubilized by this method, but the buffer conditions require a buffer change before adding antibodies (or F ab fragments).
  • the entire fraction of the soluble proteins is applied to a Biogel PD6 column, which has been equilibrated with RIPA buffer, eluted with RIPA buffer and the protein fractions collected.
  • the fraction can be bound to nitrocellulose: 90 ⁇ l of RIPA buffer and 40 ⁇ l of methanol are added to 10 ⁇ l of the fraction of soluble proteins, and an aliquot of 50 ⁇ l is bound to nitrocellulose and one PVDF membrane. The membranes are then washed with PBS, then with PBST (PBS with 0.1% Tween20). Finally, the remaining free binding sites are blocked with 1 mg of recombinant ⁇ -galactosidase in PBST and washed again with PBST.
  • Example 8 Immobilization of the Soluble Native Cytoplasmic Proteins from Example 6 on Chromatography Material:
  • each of the soluble native cytoplasmic protein fractions in PBS from Example 6 (from the first digestion - cytoplasmic proteins, soluble fraction from core and membrane fractions) are immobilized on RP18 or PVDF material or on nitrocellulose material (application of the supernatants).
  • the columns are then washed with PBS and then with PBST (PBS with 0.1% Tween20). Finally, the remaining free binding sites are blocked with 1 mg of recombinant ⁇ -galactosidase in PBST and washed again with PBST.
  • each of the desired nucleic acid oligomer-labeled F ab fragments which were produced according to Example 5, are diluted in PBS in the smallest possible volume (or in a column volume) placed on the column or membrane.
  • the nucleic acid oligomer-labeled F ab fragments are allowed to bind to the proteins immobilized on the chromatography material for 1 hour at room temperature and for a further 16 hours at 4 ° C., and washed with PBST and phosphate wash buffer (50 mM potassium phosphate pH 7.5, 500 mM NaCl).
  • 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 ⁇ g of the desired nucleic acid oligomer-labeled F ab fragments, which were produced according to Example 5, are added to the combined protein fractions.
  • the nucleic acid oligomer-labeled F a 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, 500mM NaCI) and isolated the nucleic acid oligomer unit according to a method described in the following examples (17-19).
  • the washing steps remove the remaining free nucleic acid oligomer-labeled F ab fragments from the column, 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 of target correspond.
  • the reaction tubes are rinsed with PBST and PBS before use.
  • 0.01 ⁇ g of the desired nucleic acid oligomer-labeled F ab fragments which were prepared according to Example 5, are added to 5 ⁇ l of the soluble native cytoplasmic protein fractions in PBS.
  • a different epitope than the nucleic acid-labeled Fab fragments depending 12:05 micrograms specific antibodies, and allows for 1 hour at room temperature and another 16 hours at 4 ° C, the nucleic acid-labeled F from - Bind fragments and the antibodies to the antigens.
  • IgG antibodies are preferably used for this.
  • anti-Ig antibodies When using other antibodies such as IgE, IgM, or IgA, additional anti-Ig antibodies are added.
  • the entire sample (diluted in approximately one column volume) is placed on an affinity chromatography column which is coated with protein A, protein G or anti-IgG antibody (specifically only for the F c region).
  • the immune complexes of antibodies, antigens and nucleic acid oligomer-labeled F ab fragments are allowed to bind for about 1 hour at room temperature (and possibly for a further 12 hours at 4 ° C), washed with PBST and then with phosphate wash buffer (50 mM , pH 7.2, 500mM NaCl) and then isolates the nucleic acid olomer units for the subsequent detection by one of the methods described in the following examples (17-19).
  • Example 12 Execution of a Process Variant According to the Invention Using Native Membrane Proteins Under Native Conditions (Intact Membranes)
  • the reaction tubes are rinsed with PBST and PBS before use.
  • the membrane fraction from Example 6 is resuspended in 9 times the volume of PBS. 0.02 ⁇ g of the desired nucleic acid oligomer-labeled F ab fragments, which were produced according to Example 5, are added to 5 ⁇ l of this suspension.
  • the nucleic acid oligomer-labeled F ab fragments are allowed to bind to the membrane antigens for 1 hour at room temperature and for a further 16 hours at 4 ° C.
  • the membranes are then pelleted at 4 ° C. by centrifugation at 25,000 ⁇ g for 60 minutes (or at 240,000 ⁇ g for 30 minutes).
  • the pellet is resuspended in PBS and transferred to a new reaction vessel. Pelleting and resuspending are then repeated three times as "washing steps". The remaining free nucleic acid oligomer-labeled F ab fragments are removed by the washing steps, and only complexes of these F ab fragments remain bound to the target in the pellet of the membrane fraction, so that the nucleic acid oligomer units isolated from the column correspond to the amount of target. After the washing steps, the nucleic acid oligomer units are isolated and analyzed using one of the methods described in the following examples (17-19).
  • Example 13 Execution of a Process Variant According to the Invention Using Insoluble (Native or Denatured) Proteins:
  • the reaction tubes are rinsed with PBST and PBS before use.
  • the pellet fraction from Examples 6 or 7 is resuspended in 9 times the volume of PBS (if necessary using ultrasound treatment).
  • 0.02 ⁇ g of the desired nucleic acid oligomer-labeled F ab fragments, which were produced according to Example 5, are added to 5 ⁇ l of this suspension.
  • the nucleic acid oligomer-labeled F ab fragments are allowed to bind to the membrane antigens for 1 hour at room temperature and for a further 16 hours at 4 ° C.
  • the proteins are then pelleted at 4 ° C. by centrifugation at 25,000 ⁇ g for 60 minutes (or at 240,000 ⁇ g for 30 minutes).
  • the pellet is resuspended in PBS (possibly also PBST) and transferred to a new reaction vessel. Pelleting and resuspending are then repeated three times as "washing steps". The remaining free nucleic acid oligomer-labeled F ab fragments are removed by the washing steps, and only complexes of these F a fragments remain bound to the target in the pellet of the membrane fraction, so that the nucleic acid oligomer units isolated from the column correspond to the amount of target. After the washing steps, the nucleic acid oligomer units are isolated and analyzed using one of the methods described in the following examples (17-19).
  • Example 14 Implementation of a method variant according to the invention for the detection of hapten analogs:
  • reaction tubes are rinsed with PBST and PBS before use.
  • 10 ⁇ l of 10 ⁇ PBS are added to manO ⁇ l of the solution to be examined (e.g. water-based sample from the food sector, which is to be examined for various aflatoxins and dioxins).
  • 0.1 ⁇ g each of the desired nucleic acid oligomer-labeled F ab fragments are added, which were produced as described in Example 5 and are directed against the desired substances (such as aflatoxins and dioxins).
  • the nucleic acid oligomer-labeled F ab fragments are allowed to bind to the chemical substances for 1 hour at room temperature and for a further 16 hours at 4 ° C.
  • nucleic acid oligomer-labeled F ab fragments that are not associated with the chemical substances (here aflatoxins and dioxins) from the test solution
  • 0.1 ⁇ g of a complex of the chemical substance to be sought are then added, covalently linked to, for example, fluorescein-labeled alkaline phosphatase (FITC-AP) and 5 ⁇ g anti-AP antibody, with the chemical substance acting as a hapten analogue and AP as a carrier that can be immobilized.
  • FITC-AP fluorescein-labeled alkaline phosphatase
  • the remaining nucleic acid oligomer-labeled F ab fragments are allowed to bind to the carrier-modified hapten analogs for 1 hour at room temperature and for a further 6 hours at 4 ° C.
  • 0.1 ⁇ g of a complex of DNP-AP and 0.05 ⁇ g of two control nucleic acid oligomers are used same structure with specific coding sequence added, one carrying an anti-DNP-F a b fragment, the second carrying an anti-FITC-F a fragment as a receptor unit, both of which can be immobilized quantitatively, and a specific nucleic acid oligomer that carries an anti-Taq-F ab fragment that can be eluted quantitatively.
  • the first two control nucleic acid oligomers only carry additional primer binding regions P k-1 and P k-2 specific for these control nucleic acid oligomers in order to be able to selectively amplify them.
  • the entire solution is placed on an affinity chromatography column (diluted in a column volume) on which protein G is immobilized.
  • the immune complexes consisting of nucleic acid oligomer-labeled F a fragments, the hapten analogue and the antibodies bound to AP are allowed to bind for 1 hour at room temperature. Then the affinity column is eluted with PBST.
  • the amount of AP is determined via the fluorescein fluorescence or enzyme-dependent chemofluorescence (correlating with AP) and determines the amount of control nucleic acid oligomers via selective PCR with the primers K-1 and K-2. If the background with AP or control nucleic acid oligomers is too high, the affinity chromatographic separation of the complexes of carrier-modified hapten analogue and nucleic acid oligomer receptor unit is repeated.
  • the eluate contains the complexes of nucleic acid oligomer-labeled F ab fragments and sought chemical substances (receptor-target complexes). If necessary, these complexes can be concentrated using affinity chromatography. To this are added to the eluate anti-F a b antibodies labeled nucleic constant regions F ab - recognize fragments, complexes of nucleic acid-labeled F lets go - fragments and the sought chemical substances for 1 hour at room temperature and more Bind to the antibodies for 16 hours at 4 ° C. The solution is then applied to an affinity chromatography column which Protein G has bound, and allows the immune complexes to bind to Protein G for 1 hour at room temperature and for a further 16 hours at 4 ° C. with a closed pump circuit and constant slow flow. The nucleic acid oligomer units can be isolated from this column or directly from the eluate of the first column, as described in Example 17 below.
  • Example 15 Implementation of a variant of the method according to the invention for the detection of antibodies against antigens in serum or other extracellular liquids:
  • the detection of antibodies against certain antigens allows conclusions to be drawn about contact of the organism with these antigens, which provides important diagnostic information for antigens of pathogenic germs.
  • the detection of the antibodies is usually easier than the detection of the antigens that trigger the immune response. It is worked in dim light or with red light.
  • the reaction tubes are rinsed with PBST and PBS before use.
  • nucleic acid oligomer-labeled antigens which were prepared analogously to the method in Example 5
  • about 1 mg of anti-aliquot are added to 3 aliquots of 10 ⁇ l each of the serum of a patient (or another extracellular fluid) to be tested for antibodies human Ig antibodies (which only recognize the F c regions of the antibodies) against IgE (1st aliquot), IgM (2nd aliquot) or IgG (3rd aliquot), and possibly leaves them in the serum or extracellular fluid existing antibodies against these (nucleic acid oligomer-labeled) antigens or (nucleic acid oligomer-labeled) haptens to the
  • Bind nucleic acid oligomer-labeled antigens whereby the secondary immune complexes with the anti-Ig antibodies form in parallel (incubate for 1 hour at room temperature and for a further 16 hours at 4 ° C).
  • the solution is then applied to an affinity chromatography column (approx. In a column volume) that has bound protein G, and the immune complexes of nucleic acid oligomer-labeled antigen and specific antibody or nucleic acid oligomer-labeled hapten and specific antibody are left in for 1 hour Bind room temperature to Protein G for a further 16 hours at 4 ° C.
  • the affinity chromatography column with PBS and phosphate Wash buffer 50 mM, pH 7.2, 500mM NaCl washed.
  • the washing steps remove the remaining free nucleic acid oligomer-labeled antigens, and only the nucleic acid oligomer-labeled antigens that have been bound by the antibodies (targets) specific for the antigens remain on the column, so that the nucleic acid oligomer units isolated from the column of the amount are specific to specific antibodies against these antigens.
  • the nucleic acid oligomer units are isolated and analyzed using one of the methods described in the following examples (17-19).
  • Aliquotl provides information about the development of allergies or an infection with certain cell parasites
  • Aliquot2 provides information about an acute infection, since IgM antibodies are only formed during a short phase of the not fully differentiated B cells become.
  • Aliquot3 provides information about an infection, regardless of when the infection occurred (in particular, latent and insidious infections can be detected).
  • Example 16 Assay to identify allergy-causing reagents via IgE detection:
  • the reaction tubes are rinsed with PBST and PBS before use.
  • 0.05 ⁇ g of nucleic acid oligomer-labeled allergens (which were prepared analogously to the method in Example 5) are added, and the bind any antibodies to these (nucleic acid oligomer-labeled) allergens that may be present in the serum or extracellular fluid (incubate for 1 hour at room temperature and for a further 16 hours at 4 ° C).
  • the solution is then applied to an affinity chromatography column (approx.
  • One column volume which is coated with anti-human IgE antibodies, and leaves the immune complexes of nucleic acid oligomer-labeled allergen and specific antibody of the patient (target) for 1 hour at room temperature and a further 16 hours at 4 ° C. to the anti-human IgE antibodies immobilized on the column tie.
  • the affinity chromatography column is then washed with PBST and phosphate wash buffer (50 mM, pH 7.2, 500 mM NaCl) and the nucleic acid oligomer units are isolated and analyzed for the subsequent detection according to one of the methods described in Examples 17-19 below.
  • the washing steps remove the remaining free nucleic acid oligomer-labeled allergens from the column and only nucleic acid oligomer-labeled allergens that have been bound by specific antibodies remain on the column, so that the nucleic acid oligomer units isolated from the column are identical to the amount of the specific antibodies for these allergens.
  • Example 17 Separation of the nucleic acid oligomer from the receptor unit:
  • Nucleic acid oligomers which are bound to the receptor unit (and optionally also to the target or in the various immune complexes) (Examples 8 - 16), are added with 20mM sodium phosphate buffer pH 7.2, 6M guanidinium / HCl, 10mM DTT, 2% TritonX 100 prewarmed 90 ° C (approx. One column volume / approx. 3 times the volume with other isolates), elutes in the case of the column-bound complexes after 2-3 min with another 3 column volumes of the same buffer and collects the eluate in the case of the column-bound immune complexes.
  • the eluates are centrifuged briefly at 15,000 xg, 5 ⁇ g poly-dT (33) are added to the supernatant to support the precipitation, the sample is then mixed with half a volume of 3M potassium acetate pH 5.0 and four times the volume of ethanol (-20 ° C) and the DNA precipitated at 15,000 xg at 4 ° C for 40min.
  • the pellet is separated and washed with 70% ethanol (-20 ° C) and 100% ethanol (-20 ° C) and air dried.
  • 1/2 volume of ethanol and! Volume 3M potassium acetate pH 5.0 added and applied to a 'spin column' with an activated glass fiber membrane.
  • nucleic acid oligomers are finally eluted from the glass fiber membrane with 50 ⁇ l TE buffer (kits from Qiagen, Promega, etc.). For amplification the pellet is taken up in the PCR buffer (cf. Example 8), for direct detection without amplification in the hybridization buffer (cf. Example 19).
  • the eluates are briefly centrifuged at 15,000 x g. 5 ⁇ g of poly-dT (33) are added to the supernatant to support the DNA precipitation, the sample is then mixed with half a volume of 3M potassium acetate pH 5.0 and four times the volume of ethanol (-20 ° C.) and the DNA at 15,000 ⁇ g 4 ° C precipitated over 40min.
  • the pellet is separated and washed with 70% ethanol (-20 ° C) and 100% ethanol (-20 ° C) and air dried.
  • the pellet is taken up in the PCR buffer (cf. Example 18), for direct detection without amplification in the hybridization buffer (cf. Example 19).
  • the nucleic acid oligomers can also be purified over glass fiber membranes as described under (i). Like the methods described in iii) and iv), this method has the advantage that the DNA is separated from the protein and fewer proteins in the eluate are also washed out.
  • Example 18 Amplification and subsequent labeling of the nucleic acid oligomer:
  • nucleic acid oligomers bound to the receptor unit are DNA or RNA which have primer binding regions (such as, for example, the nucleic acid oligomer constructed in Example 1)
  • primer binding regions such as, for example, the nucleic acid oligomer constructed in Example 1
  • the nucleic acid oligomers used for all receptor-bound within a qualitative and / or quantitative target analysis (proteome analysis etc.) are identical
  • the nucleic acid oligomers present in the test solution which were separated using one of the methods (i) to (v) in Example 17, can be amplified by PCR.
  • Exponential amplification After separating the DNA from the complexes of nucleic acid oligomer-receptor unit and chemical substance (cf. Example 17), 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 (10fg, 1pg, 50pg, 1ng) of 4 control nucleic acid oligomers (with their own specific coding regions and otherwise identical structure as the nucleic acid oligomers used for labeling) is used to isolate the nucleic acid oligomers before amplification 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, for example, a standard PCR with 11 to 18 cycles is carried out by approximately 1000-fold to achieve up to 100,000 times amplification.
  • a standard PCR with 11 to 18 cycles is carried out by approximately 1000-fold to achieve up to 100,000 times amplification.
  • primer groups each of which is universal in itself only for a certain group of nucleic acid oligomer receptor units (as in Example 1, primers 2-1, 2-2, 3-1 and 3-2) are used for each primer pair corresponding to 4 specific control nucleic acid oligomers added in a defined amount before the PCR, and the PCR optionally amplified in separate PCR reactions.
  • the pellet is dissolved in the hybridization buffer (see Example 19), for subsequent modification with a detection marker, linear amplification is then carried out with simultaneous incorporation of a detection marker as described in Example 18 ii) or a subsequent chemical modification with detection Markers as described in Example iii).
  • an additional chromatography step is introduced after the PCR in order to separate the unreacted primers from the amplified nucleic acid oligomers.
  • the entire batch is applied to a Sephacryl S100 column and the first fraction (approx. 25kDa) containing the amplification products is collected, the second fraction (approx. 4kDa) containing the primers is discarded.
  • the amplification step is that the nucleic acid oligomers to be detected are already present as ss-DNA.
  • the DNA can also be provided with a detection marker by adding nucleotides which carry a detection marker, via PCR or the RNA via in vitro transcription, and the detection marker can also be used analogously to the binding instructions of Examples 2-5 are subsequently attached to the nucleic acid oligomer.
  • 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.
  • the probe oligonucleotide 3 'or 5' terminal ly carry a linker which has a reactive or activatable group via which the probe oligonucleotide is bound to a suitable support surface in a defined manner (e.g. an NH 2 group on the probe oligonucleotide and isothiocyanate group on the support).
  • a linker which has a reactive or activatable group via which the probe oligonucleotide is bound to a suitable support surface in a defined manner (e.g. an NH 2 group on the probe oligonucleotide and isothiocyanate group on the support).
  • Free binding sites on the membrane are incubated for 30 min at 45 ° C with hybridization buffer 1 (7% SDS, 500mM Na-phosphate buffer pH 7.5, 5mM EDTA, 0.1 mg / ml ssDNA from an organism with the lowest possible phylogenetic relationship, e.g. pBR322 E.coli, 0.5 mg / ml acetylated BSA) saturated (pre-hybridization).
  • hybridization buffer 1 7 SDS, 500mM Na-phosphate buffer pH 7.5, 5mM EDTA, 0.1 mg / ml ssDNA from an organism with the lowest possible phylogenetic relationship, e.g. pBR322 E.coli, 0.5 mg / ml acetylated BSA) saturated (pre-hybridization).
  • the 'dots' thus formed are overlaid by the hybridization solution which contains the nucleic acid oligomers isolated in Example 17 or their amplification products.
  • Double-stranded DNA nucleic acid oligomers which are obtained in the amplification according to Example 18 i) are previously denatured at 100 ° C. for 5 minutes and briefly chilled on ice before adding to the hybridization solution.
  • the probe oligonucleotides and nucleic acid oligomers of the solution are allowed to hybridize at approximately 40 ° C. for approximately 3 to 16 hours.
  • a chip e.g. a custom made Affymetrix GeneChip® (see Wodicka et al. 1997, Nature Biotechnology, 15, 1359ff) can be used.
  • the test sites carry probe oligonucleotides which are covalently bound to a carrier material and which are complementary to the nucleic acid oligomers to be detected.
  • the nucleic acid oligomers (or the amplification products) used for the target analysis contain a biotin as the primary detection marker.
  • nucleic acid oligomers 10 ⁇ g are used as ss nucleic acid oligomers in 250 ⁇ l (per chip used) hybridization buffer 3 (100mM MES, 1M NaCI, 20mM EDTA, 0.01% TWEEN 20, pH 6.5 - 6.7) together with 0.1 mg / ml Herings sperm ss - DNA and 0.5 mg / ml acetylated BSA taken up and put on the chip.
  • the mutually complementary nucleic acid oligomers are then allowed to hybridize at 43 ° C. for 16 h.
  • the kit contains a protocol for performing the assay (similar to that described in Examples 6-19, but adapted to the components supplied with the kit).
  • the kit contains modified nucleic acid oligomers with different coding regions to which a component of two conjugated chemical substances, optionally via a linker (which can be cleaved by induction), as described under d) in the section "Binding a receptor to a nucleic acid oligomer" (eg an anti-biotin F ab fragment), is bound (Oligo20-anti-Biotin-F ab , production analogous to Example 5).
  • About the component of two conjugated chemical substances can be modified with the corresponding binding partner of the component any receptor unit such.
  • a biotinylated receptor unit - can be carried out by the user according to his preferences.
  • the kit also contains a set of probe oligonucleotides which, depending on the protocol, are identical or complementary to the coding regions of the nucleic acid oligomers and which are optionally, depending on the complexity of the kit (number of different coding and corresponding probe nucleic acid oligomers) in defined patterns are immobilized on a dot blot membrane or on a DNA chip.
  • the kit can contain a set of primers 1 and / or primers 2, it being possible for the primer or one of the primers to additionally carry a detection marker (cf. Examples 1 and 19).
  • kit can be sets of suitably modified receptor units specially put together for specific applications, such as, for example, biotinylated antibodies or biotinylated F ab fragments (kit for detecting antigens) or biotinylated antigens (kit for detecting antibodies) which are used for Carrier-bound separation can also be modified.
  • suitable chromatography materials for the purification of the nucleic acid oligomer receptor units such as, for example, an affinity chromatography column coated with biotin
  • further antibodies which can be used to immobilize and separate the nucleic acid oligomer receptor units occupied by target such as specific antibodies against the same targets but against another epitope (antigen kit), anti-Ig antibodies which are directed against the F c region of the antibodies sought.
  • a unit adapted to the target analysis for separating the antigens bound to the nucleic acid oligomer-receptor complexes (antigen kit) or Antibodies (antibody kit) from one or more of the components: specific or group-specific antibodies for the antigen targets (for the antigen kit), specific anti-Ig antibodies directed against the F c regions (antibody kit) for Modification of the antigen targets (antigen kit, cf. Example 10) and suitably modified or unmodified carrier material, cf. Examples 8-16, optionally in the form of a disposable cartridge or as a 'spin column'.
  • buffers optionally in ready-to-use form (in particular incubation buffer, washing buffer and an elution buffer which contains the induction agent necessary for cleaving the cleavable linker, hybridization buffer and washing buffer for the detection units such as dot blot membranes, etc.).
  • other additives such.
  • the kit components for PCR amplification such.
  • the kit consists of one or more nucleic acid oligomers with attached Antibiotin-F ab fragment.
  • Nucleic acid oligomers are preferably used DNA, RNA or PNA, which are constructed analogously to Example 1, via a linker which can be cleaved after induction
  • nucleic acid oligomers with an identical antibiotin F ab fragment are offered with a protocol for handling and further use (analogous to the instructions in Example 25) and can be used with biotinylated antigens or biotinylated
  • Antibodies can be combined.
  • the kit comprises a regulation for the preparation of biotinylated F ab fragments and the preparation of the beginning of this example Modified nucleic acid oligomers mentioned (for example Oligo20-anti-Biotin-F a b) and at least one of the necessary components such as papain immobilized on agarose, protease buffer, PMSF as protease inhibitor to stop the proteolytic digestion, a gel filtration column for purification, a biotinylation Agent and the corresponding biotinylation buffer for biotinylation, and a further gel filtration column for separating the free biotin, a biotin affinity chromatography column for separating unreacted biotinylated nucleic acid oligomers and a protein G affinity column for separating non-proteolytically cleaved antibodies and free F c - Fragments, whereby the columns can be made available as single spin columns or disposable cartridges, as well as reaction vessels and other consumables.
  • the protocol comprises the steps: a) addition of buffer for proteolytic digestion and incubation with papain (immobilized on agarose) for 4 hours up to 16 hours (depending on the subclass of the antibody used), addition of PMFS and centrifugation to separate the papain, b ) Incubation of the purified antibody after addition of the incubation buffer with the activated biotin molecule, the molar ratio between antibody and biotin molecule should be approx.
  • Example 21 Kit for screening potential antagonists for an enzyme or a receptor protein:
  • the nucleic acid oligomer receptor units are put together by the user using the above kit (example 20) according to his preferences, the substances which are to be investigated as potential antagonists can be provided by the user himself as biotinylated forms.
  • Each substance is incubated in a specific buffer Nucleic acid oligomer with attached anti-biotin F ab fragment added, incubated for 1-4 h, the incubation solution placed on an affinity chromatography column occupied with biotin, incubated for a further 1-4 h to remove the unattached nucleic acid oligomer anti-Biotin F ab fragments separate.
  • reaction tubes are rinsed with PBST and PBS before use.
  • a protein enzyme, receptor protein or functional peptide with the active domain of these proteins
  • a suitable buffer 10mM potassium phosphate pH 8.0, 135mM potassium chloride, 2mM Mg sulfate
  • IgG antibody which is specifically directed against the protein (or peptide) but does not block the active centers.
  • the substances are allowed to bind to the protein or peptide (incubate for 1 hour at room temperature and for a further 16 hours at 4 ° C.), then the substrate or the natural binding partner is added in physiological concentration and the solution is added to an affinity chromatography column (approx. one column volume), which is covered with ProteinG.
  • the immune complexes of nucleic acid oligomer-labeled substance, protein (or peptide) and the specific antibody are allowed to bind to the ProteinG for 1 hour at room temperature and for a further 16 hours at 4 ° C.
  • the affinity chromatography column is then eluted with the buffer including the natural binding partner in physiological concentration (fraction 1), then including the natural binding partner in 50 times higher concentration (fraction 2) and then including a known high-affinity antagonist in high concentration (fraction 3) - the Fractions are collected separately.
  • the individual elution steps first remove unbound nucleic acid oligomer-labeled substances that cannot or only bind very weakly to the protein (fraction 1), then nucleic acid oligomer-labeled substances that act as low-affinity antagonists in the binding site of the natural binding partner (fraction 2) , then high affinity antagonists (fraction 3). Nucleic acid oligomer-labeled substances remaining on the column that remain bound to the protein or peptide (fraction 4). Fraction 4 contains Substances which are high affinity antagonists and therefore do not dissociate from the protein, but also antagonists or inhibitors which have bound to a binding site other than that of the natural binding partner or the known antagonist, or substances which have no effect on the protein or Have bound peptide.
  • the nucleic acid oligomer units of the substances still remaining on the column can be isolated as fraction 4 by one of the methods described in Example 17.
  • the reaction tubes are rinsed with PBST and PBS before use.
  • An oligo20-anti-biotin Fab (or a nucleic acid oligomer modified analogously with a component of two conjugated chemical substances, cf. Example 20) is used as the nucleic acid oligomer.
  • Peptides that are labeled with biotin are used as receptor units. In the simplest case, the biotinylation is achieved by in vivo labeling in E. coli via recombinant expression as a fusion product with a segment of the biotin carboxylase carrier protein (pin point vectors from Promega). The peptides are part of the antigen used in the immunization and carry one or more epitopes of this antigen.
  • the kit in this version allows the user to manufacture and use these receptor units according to individual preferences.
  • a specific biotinylated peptide is added in a molar ratio of approx. 1.5: 1 in PBS.
  • the nucleic acid oligomer is allowed to couple to the biotinylated peptide for 2 hours at room temperature.
  • the sample is then applied to an affinity column which is coated with biotin and allowed the unbound nucleic acid oligomers for 2 hours at room temperature over anti-biotin F ab - fragment bind to the biotin of the affinity column.
  • biotin-anti-biotin F a b linked ready-made nucleic acid labeled peptides are eluted (nucleic acid-labeled receptor unit).
  • 100 ⁇ l of the cell culture supernatant of a hybridoma cell line are then added to each O.O ⁇ g of a nucleic acid oligomer-labeled peptide and this is left for 2 hours at room temperature (and possibly a further 12 hours at 4 ° C.) of monoclonal antibodies Bind hybridoma cell lines.
  • the solution is then applied to an affinity chromatography column (approx. One column volume), which has bound protein G, and waits for approx.
  • the separation of free nucleic acid oligomer-labeled peptides and complexes of these peptides with the target, the monoclonal antibody (immobilized on the column material) is carried out by the elution and the washing steps, so that at the end of the washing steps the nucleic acid oligomer units according to one of those described in Example 17 Methods for amplification and detection can be isolated.
  • the kit contains fluorescence-labeled primers for amplification, depending on the complexity of the kit (number of different specific coding nucleic acid oligomers), dot blot strips or DNA chips, on which a whole set of probe oligonucleotides is bound in a defined pattern.
  • the advantage of this method is that the screening can start earlier and requires less material (especially antigen) than the usual methods.
  • the kit consists of nucleic acid oligomers with different coding regions, to which a specific DNA or RNA sequence (as ss- or ds-DNA or as ss- or ds- RNA) as a receptor unit for DNA-binding (or RNA-binding) biomolecules, in particular DNA- or RNA-binding proteins (e.g. the sequence ds- ⁇ '- CCAGGCCTGG-3 'for the DNA ( Cytosine- ⁇ ) -methyltransferase Haelll, the loxP site for CRE recombinase, promoter sequences that specifically bind various transcription factors and transcription regulators or RNA sequences bind the splice factors and hn-RNP).
  • DNA-binding or RNA-binding proteins
  • DNA or RNA sequences can also serve as receptor units for antagonists of DNA or RNA-binding proteins (e.g. the sequence ds- ⁇ '-ATGCAT-3 'as a binding site for DNA-binding proteins Topoisomerase II, but also as a binding site of an inhibitor for DNA-binding topoisomerase II such as LU-79 ⁇ 3, see Gallego & Reid, Biochemistry 1999, 38, 15104-15115).
  • the kit contains a protocol for handling the kit, as well as optional primers (P1 and / or P2) that are the same for all nucleic acid oligomers, optionally with a detection marker (e.g.
  • a rhodamine or flurescein that is used to cleave the cleavable linker necessary induction agents, suitable or any DNA-binding (or RNA-binding) biomolecules, their antagonists (suitable or any), which can be additionally modified for the carrier-bound separation, and a detection unit which is matched to the nucleic acid oligomers used (eg a ready-to-use dot-plot membrane with the probe oligonucleotides complementary to the nucleic acid oligomers to be detected and immobilized on coding sequence-specific spots, cf. Example 19).
  • the kit can have a unit adapted to the target analysis for separating the DNA-binding (or RNA-binding) biomolecules containing the target and bound to the nucleic acid oligomer-receptor complexes from the constituents specific antibodies for the DNA-binding (or RNA) -binding) biomolecules or their antagonists, reagent for modifying the DNA-binding (or RNA-binding) biomolecules or their antagonists, cf.
  • Example 24 Exemplary cellular proteome analysis:
  • tissue cells > 100 cells
  • the cell material of a biopsy is - after addition of RIPA buffer (1 ⁇ 0mM NaCI 1% NP-40, 0. ⁇ % deoxycholate, 0.1% SDS, ⁇ OmM Tris, pH 8.0, 10 ⁇ l per mg cell tissue, but at least 1 ⁇ l) unlocked in the microtip sonifier.
  • Example 6 ii A large part of the cellular proteome of the cells is dissolved as in Example 6 ii) under partially denaturing conditions (separation of the remaining insoluble protein fraction (ECM / cytoskeleton) at 800 ⁇ g / 4 ° C./1 ⁇ min and residues of the cell debris from the separated supernatant at 60,000 xg / 4 ° C / 30 min).
  • the RIPA-buffered supernatant contains the partially denatured soluble cytoplasmic proteins and a large part of the membrane proteins.
  • the proteins remaining in the pellet are discarded (or suitable receptor-nucleic acid complexes are bound to the insoluble proteins in the pellet and these target-receptor-nucleic acid oligomer complexes are further analyzed in a second step, as described in Example 13).
  • Nucleic acid oligomer 1 for binding receptor unit 1 has e.g. B.
  • positions 1, 2, 9 and 10 of the coding sequence being independent, positions 3, ⁇ and 7 being dependent on position 1 by the permutation rule G ⁇ A ⁇ C ⁇ T ⁇ G , so for a G at position 1 follows that position 3 is an A, position ⁇ is a C and position 7 is a T.
  • positions 4, 6 and 8 depend on the independent occupation of position 2, positions 11, 13 and 1 ⁇ on independent position 9 and positions 12, 14 and 16 on independent position 10;
  • Coding sequence any of the maximum of 192 nucleic acid oligomers generated above
  • linker dithio-bis-propionic acid sulfosuccinimidyl ester one separate batch of nucleic acid oligomer per antibody: 1 nmol of the nucleic acid oligomer is mixed with ⁇ O nmol dithio-bis-propionic acid sulfosuccinimidyl ester in approx. 100 .mu.l 100 mM NaHC0 3 / Na 2 C0 3 , pH 9 added, allowed to stand overnight in the dark at room temperature, isolated and separated the converted DNA in carbonate buffer analogous to the procedure in Example 3).
  • the Nucleic acid oligomer linker units of a reaction are then reacted with each type of F ab fragment and isolated (analogously to example ⁇ ) and the number of DNA units per F ab fragment z. B. determined on the ratio of the rhodamine fluorescence at ⁇ 7 ⁇ nm (proportional to the DNA) to the absorption at 28 ⁇ nm (proportional to the F a b fragments).
  • 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 ⁇ l thereof are added to 10 ⁇ l solution of the partially denatured proteins in RIPA buffer ( corresponding to 1 mg cell material). The proteins are allowed to couple to the F a fragments for about 3 hours at room temperature and for about 24 hours at 4 ° C.
  • a cocktail of polyclonal antibodies is added in PBS which are specific for the same targets (proteins) as the F ab fragments (and optionally further anti-Ig antibodies which 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.
  • the DNA obtained is then used as described in Example 18 i) and iii) with PCR using amplified exponentially from primer 2 and rhodamine-modified primer P1, the amplified DNA melted (heating for ⁇ min to 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 19 i), and then the fluorescence of the Spots determined quantitatively (see Example 19 i).
  • a transplant requires an intensive diagnostic examination of the available tissue in the shortest possible time, since the organs should be transplanted as quickly as possible.
  • the blood groups and other polymorphic tissue markers in particular the components of the MHC (major histocompatibility complex), must be examined in order to be able to assess rejection reactions and to be able to select the suitable patients for the transplantation; at the same time, it is necessary to rule out a risk of infection via the transplanted tissue.
  • a kit comprises a set of complete nucleic acid oligomer-receptor units and a set of nucleic acid oligomer-anti-biotin-F ab units which are available to produce 20 additional receptor units, as required by the user, analogously to Example can .
  • the receptor units used are, in particular, F ab fragments against the components of the major histocompatibility complex (MHC - currently 1219 alleles), against blood group epitopes (to determine the polymorphic tissue markers), and in particular antigens which are typical of certain infectious diseases (to detect the existence of antibodies against these antigens) and antigens such as rheumatoid factors (to detect antibodies that are typical of an autoimmune disease).
  • MHC - currently 1219 alleles against blood group epitopes
  • antigens which are typical of certain infectious diseases to detect the existence of antibodies against these antigens
  • antigens such as rheumatoid factors
  • the detection of these antibodies is only possible via protein analysis.
  • the antibodies are also relatively easy to access because they are distributed throughout the body via the blood circulatory system. It is particularly important to detect infections caused by pathogens that are only a problem in immunodeficiency, infections by viral pathogens that can persist long after the infection has survived (HIV, herpes simplex, hepatitis A / B / C, cytomegalovirus, zoster, retroviruses , Mycoplasmas Among these antibodies are not easy to identify because the symptoms are difficult to classify (adenovirus, influenza, etc.) and infections caused by other pathogens that are often not recorded due to the gradual course of the disease ( Legionella, borelliosis, tuberculosis, syphilis etc.).
  • the kit can contain further components as described in Example 20.
  • the kit can also control nucleic acid oligomers that are built like all nucleic used and each has a specific coding sequence contained (positive binding controls in three different concentrations, for example with anti-human IgG F ab fragment as a receptor unit which is always bound in the secondary immune complexes; positive binding controls for checking the immobilization of different isotypic antibodies from different organisms, each with an intact antibody, the specificity of which is an antigen that is not found in the test solution, different isotypes or immunoglobulin Classes from different organisms such as IgGI, IgG2a, IgG2b, IgG3, IgG4 and IgM from mouse, rabbit polyclonal antibodies are used; one or more negative binding controls to check the successful separation of the free nucleic acid oligomer receptor units, being antibodies against Proteins of phylogenetically unrelated organisms that do not appear in the test solution are selected, for example an anti-Taq-F ab fragment; a
  • the kit allows the following components to be analyzed in one approach: To determine the blood and tissue characteristics, nucleic acid oligomer-labeled F ab fragments of monoclonal antibodies against these tissue markers that are attached to the Bind antigens specifically and use antibodies with specificity against another epitope of these targets. To determine the antibodies, nucleic acid oligomer-labeled antigens of the pathogens are added to the same approach. These antibodies are bound as secondary immune complexes by adding anti-human IgG, anti-human IgM, anti-human IgA and anti-human IgE antibodies, which in turn can be immobilized via protein G affinity chromatography.

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Abstract

L'invention concerne un procédé pour le marquage et la détection de substances chimiques qui se lient spécifiquement à un récepteur. Selon l'invention, la séquence d'un simple brin d'oligomère d'ADN, d'ARN ou de PNA sert de marquage moléculaire non équivoque appliqué au récepteur correspondant. Après séparation des complexes constitués de la substance chimique et de l'unité oligomère d'acide nucléique récepteur, le constituant oligomère d'acide nucléique et non la substance chimique est identifié et quantifié.
PCT/DE2001/003009 2000-08-25 2001-08-04 Procede pour le marquage de substances chimiques WO2002016635A2 (fr)

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WO2003058240A2 (fr) * 2002-01-08 2003-07-17 Friz Biochem Gmbh Procédé de détection comparative qualitative et quantitative de substances chimiques
EP1699934A2 (fr) * 2003-09-02 2006-09-13 Robert L. Lawton Detection et amplification d'analytes solubles
WO2016019929A2 (fr) 2014-08-05 2016-02-11 Ustav Organicke Chemie A Biochemie Akademie Ved Cr, V.V.I Procédé de détection de formes actives d'analyte et de détermination de la capacité de substances à se lier dans des sites actifs d'analyte
CN115436622A (zh) * 2022-09-26 2022-12-06 重庆医科大学国际体外诊断研究院 一种单分子蛋白的检测方法及其试剂盒和应用

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DE10156329A1 (de) * 2001-07-17 2003-02-06 Frieder Breitling Verfahren und Anordnung zum Anbringen von in Transportmittel immobilisierten Substanzen sowie Monomerpartikel
WO2006002382A2 (fr) * 2004-06-24 2006-01-05 The Scripps Research Institute Reseaux de liants clivables

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WO2003058240A2 (fr) * 2002-01-08 2003-07-17 Friz Biochem Gmbh Procédé de détection comparative qualitative et quantitative de substances chimiques
WO2003058240A3 (fr) * 2002-01-08 2004-01-29 Friz Biochem Gmbh Procédé de détection comparative qualitative et quantitative de substances chimiques
EP1699934A2 (fr) * 2003-09-02 2006-09-13 Robert L. Lawton Detection et amplification d'analytes solubles
JP2007503831A (ja) * 2003-09-02 2007-03-01 エル． ロートン、ロバート 可溶性検体の検出および増幅
EP1699934A4 (fr) * 2003-09-02 2008-05-21 Robert L Lawton Detection et amplification d'analytes solubles
EP2204456A1 (fr) * 2003-09-02 2010-07-07 Robert L. Lawton Détection et amplification d'analytes solubles
JP2012010712A (ja) * 2003-09-02 2012-01-19 Restalyst Pte Ltd 可溶性検体の検出および増幅
EP2808399A1 (fr) * 2003-09-02 2014-12-03 Restalyst Pte Ltd Détection et amplification d'analyse soluble
US9181579B2 (en) 2003-09-02 2015-11-10 Restalyst Pte Ltd Soluble analyte detection and amplification
WO2016019929A2 (fr) 2014-08-05 2016-02-11 Ustav Organicke Chemie A Biochemie Akademie Ved Cr, V.V.I Procédé de détection de formes actives d'analyte et de détermination de la capacité de substances à se lier dans des sites actifs d'analyte
WO2016019929A3 (fr) * 2014-08-05 2016-03-31 Ustav Organicke Chemie A Biochemie Akademie Ved Cr, V.V.I Procédé de détection de formes actives d'analyte et de détermination de la capacité de substances à se lier dans des sites actifs d'analyte
JP2017524133A (ja) * 2014-08-05 2017-08-24 ウスタフ オルガニッケ ヘミエ アー ビオヘミエ アカデミエ ヴェド ツェーエル，ヴェー．ヴェー．イー 分析物の活性型の検出および分析物の活性部位に結合する、物質の能力の決定の、方法
EP3264091A1 (fr) * 2014-08-05 2018-01-03 Ustav Organicke Chemie A Biochemie Av Cr, V.v.i. Procédé de détection de formes actives d'analyte
AU2015299447B2 (en) * 2014-08-05 2018-04-26 USTAV ORGANICKE CHEMIE A BIOCHEMIE AKADEMIE VED CR, v.v.i. Method of detection of analyte active forms and determination of the ability of substances to bind into analyte active sites
JP2019068838A (ja) * 2014-08-05 2019-05-09 ウスタフ オルガニッケ ヘミエ アー ビオヘミエ アカデミエ ヴェド ツェーエル，ヴェー．ヴェー．イー 分析物の活性型の検出および分析物の活性部位に結合する、物質の能力の決定の、方法
AU2018202250B2 (en) * 2014-08-05 2019-12-19 USTAV ORGANICKE CHEMIE A BIOCHEMIE AKADEMIE VED CR, v.v.i. Method of detection of analyte active forms and determination of the ability of substances to bind into analyte active sites
US10718772B2 (en) 2014-08-05 2020-07-21 USTAV ORGANICKE CHEMIE A BIOCHEMIE AKADEMIE VED CR, v.v.i. Method of detection of analyte active forms and determination of the ability of substances to bind into analyte active sites
CN115436622A (zh) * 2022-09-26 2022-12-06 重庆医科大学国际体外诊断研究院 一种单分子蛋白的检测方法及其试剂盒和应用
CN115436622B (zh) * 2022-09-26 2024-03-08 重庆医科大学国际体外诊断研究院 一种单分子蛋白的检测方法及其试剂盒和应用

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