DE10325152B4 - Method for the quantitative determination of an unknown amount of a target molecule species - Google Patents

Abstract

Method for the quantitative determination of an unknown amount of a target molecule species with the following process steps: a) a system containing a defined amount of labeled target molecules of the target molecule species and an unknown amount of unlabeled target molecules of the same target molecule species is free with a defined amount of reporter groups specific for the target molecule species Aptamer molecules contacted, whereby by binding the aptamer molecules to the target molecules of the target molecule species aptamer / unlabeled target molecule complexes as well as aptamer / labeled target molecule complexes form, and where a distribution between the amount of non-bound aptamer molecules, aptamer / unlabeled target molecule- Sets complexes and aptamer / labeled target molecule complexes, b) the aptamer / labeled target molecule complexes are separated from unbound aptamer molecules and aptamer / non-labeled target molecule complexes, c1) the amount of unbound molecules Aptamer molecules is determined quantitatively by means of a quantitative amplification method, and / or c2) the aptamer / labeled target molecule complexes are dissociated and the amount of aptamer molecules released from the complexes is determined quantitatively by means of a quantitative amplification method, d) that in step c1) and / or c2 ) obtained values are entered into a standard curve, which is determined by multiple ...

Description

Field of the invention.

The invention relates to a method for the quantitative determination of an unknown amount of a Zielmolekülspezies, wherein target molecules of the Zielmolekülspezies be contacted with a defined amount of Aptamermolekülen which specifically bind Aptamermoleküle to the target molecules, wherein the Aptamermoleküle with the target molecules aptamer / target molecule complexes, where not bound aptamer molecules and / or aptamer molecules bound in aptamer-target molecule complexes can be quantified. The invention further relates to a test system for carrying out such a method.

Background of the invention and prior art.

Aptamers are oligonucleotides that specifically bind to target molecules of a defined target molecule species with high affinity. The selectivity is based on the ability or property of oligonucleotides to build certain three-dimensional structures, which depend on the nucleic acid sequence. A particular nucleic acid sequence may have a three-dimensional structure specific for a defined target molecule. For the search for oligonucleotides which are specific for a defined target molecule, for example, the SE-LEX (Systematic Evolution of Ligands by Exponential Enrichment) method can be applied. Here, a pool of different oligonucleotides is contacted with a defined target molecule and binding oligonucleotides are selected, enriched and amplified. For this purpose, in detail, for example, to the reference WO 91/19813 A1 directed. The oligonucleotides or aptamers thus obtained are suitable, for example, for detecting the associated target molecules in a binding assay.

In general, a binding assay performed using aptamers has the problem of detecting binding events. From the literature US 5,789,163 It is known to couple an aptamer with, for example, biotin, fluorochromes, radioisotopes, steroids or peptides, said groups or atoms functioning as reporter groups in the binding assay. After contacting a defined amount of reporter-group-containing aptamer molecules with the target molecules and separating unbound aptamer molecules, the reporter group can be used to quantify either unbound or bound aptamer molecules, from which ultimately the amount of target molecules can be calculated.

From the literature US 6,506,887 It is known to provide an aptamer with a prosthetic group whose accessibility to solvent molecules changes upon binding of the aptamer molecule to a target molecule. The prosthetic group is a reporter group because this change is used to generate a measurement signal.

From the literature US 6,399,302 For example, a special aptamer construction is known that contains, inter alia, fluorophore groups, reporter groups.

From the literature US 6,306,598 It is known to provide an oligonucleotide with lipid monomers, wherein by conformational changes a color signal when bound to a target molecule is genertert. Even with the extent known measures thus a reporter group is used.

The common feature of coupling a reporter group to an oligonucleotide has several disadvantages. If such a reporter group is selected after selection of the aptamer, i. H. of the binding oligonucleotide, it is by no means certain that the construct modified to this extent still has the same binding properties to the target molecule as the selected aptamer. Alternatively, the selection can already be carried out on the basis of oligonucleotides which already contain the reporter group. Then, although the binding properties are given, the likelihood of successful selection is reduced, since coupling of a reporter group may inhibit binding to a binding oligonucleotide, or at least reduce affinity. In addition, it can not be ensured that the binding is based on the oligonucleotide component and not possibly on the reporter group. In any case, it is also complicated to couple or modify oligonucleotides with reporter groups.

From the literature US 5,843,653 and US 2002/0051974 A1 Methods are known for the quantitative determination of target molecules by means of aptamers. From the literature WO 91/19813 A1 and SD Jayasena et al., Clinical Chemistry 45 (9): 1628-1650 (1999), it is known that aptamers are specific binding partners for biomolecules.

Technical problem of the invention.

The invention is based on the technical problem of specifying a method for the quantitative determination of an unknown amount of target molecules using aptamers, which is simpler in the generation of binding aptamer constructs.

Broad features of the invention and preferred embodiments.

To solve this technical problem, the invention teaches a method according to claim 1.

The invention is based initially on the recognition that when contacting a defined amount of aptamer molecules with an unknown amount of target molecules, a defined thermodynamic state is established between bound aptamer molecules and unbound. Aptamermolekülen. The distribution in the defined thermodynamic state can be used for the quantitative determination. In this case, the chemical potentials need not be known if the equilibrium distributions are determined as a function of the concentration of the target molecules by means of calibration curves obtained by measurements on the basis of various known amounts of target molecules. It is also essential here that the defined thermodynamic state is not necessarily the equilibrium state. Because of the use of calibration curves created under the same conditions, it is also possible to work in arbitrary (defined by the calibration curve conditions) thermodynamic states prior to equilibration. This also allows comparatively fast measurements. The invention is based on the further realization that when determining the distribution it is not necessary to carry out the determination of binding events by means of reporter groups, since the amount of free aptamer molecules, be it the amount of unbound aptamer molecules, or the amount of bound aptamer molecules after dissociation from the aptamer / Target molecule complex, can also be determined by standard methods of nucleic acid analysis. Such a dissociation is easily carried out, for example, by increasing the temperature. The only important factor in determining the amount of bound aptamer molecules is that unbound aptamer molecules are separated from aptamer / target molecule complexes prior to determination.

In the context of the invention, the quantitative determination of the aptamer molecules is carried out by means of a quantitative amplification method, in particular the RT-PCR or PCR ligase chain reaction. It is also possible to carry out the determination of the aptamer molecules by oligonucleotide determination via quantitative evaluation after gel electrophoresis. For RT-PCR, reference is made, by way of example only, to the reference M. Neusser, "Rapid Method of PCR Optimization with Real-Time PCR", in BIOspektrum, 6/2001.

In principle, a separation of unbound aptamer molecules from aptamer / target molecule complexes can be carried out by any desired methods. For example, aptamer / target molecule complexes, labeled and / or unlabeled, can be separated from unbound aptamer molecules by molecular weight selective separation techniques. Here, a molecular sieve or a membrane with an exclusion limit is used whose height above the molecular weight of unbound aptamers, for example above 10 kD, in particular above 30 kD, and below the molecular weight of the aptamer / target molecule complexes, for example below 300 kD, in particular below 100 kD, lies. Examples of suitable molecular weight-selective membranes are dialysis tubing or ultrafiltration membranes. Other separation methods are: particle coupling and sedimentation or magnetic separation (in the case of magnetic particles), exclusion chromatography, "washing" in the case of solid-phase-bound target molecules.

The label of the target molecules may be selected from the following group: A) binding to a solid phase, in particular to an immobile solid phase or to mobile particles, B) binding to an anchor group for immobilization to a solid phase, and C) binding to a selection group for a physical and / or physical chemical separation process. In case A), the target molecules are bound in a column, for example. Unbound aptamer molecules, as well as aptamer / unlabeled target molecule complexes, can then be separated from the aptamer / labeled target molecule complexes by a washing step subsequent to contacting. Alternatively, target molecules can also be bound to particles, the separation of the above groups then being carried out by simple filtration of the particles. Binding to a solid phase can be done, for example, via a chemical coupling or an adsorptive coupling. Both methods are familiar to the expert and therefore need not be explained in more detail. In case B), the target molecules are provided with a group which allows immobilization or binding to particles before or after contact with the aptamer molecules. If the immobilization or binding is carried out after the contacting, all aptamer / labeled target molecule complexes can then be separated off. Examples of suitable anchor groups are elements of the pairs biotin / streptavidin, digoxigenin / anti-digoxigenin antibody. The complementary element of a pair is then coupled to the solid phase. An example of C) is the coupling to magnetic particles and magnetic separation. Another example is the fluorenceenzence label.

The invention further relates to a test system for carrying out a method according to the invention as claimed in claim 5.

The above statements apply analogously.

With the invention, the following advantages are achieved in connection with each other. First, a highly selective method for the determination of almost any target molecules, isolated or in mixtures, is obtained. Second, the method is highly sensitive because even the smallest amounts of aptamers can be detected quantitatively by means of PCR or RT-PCR. It can thus be used even very small amounts of aptamers. By using only small amounts of aptamers can also achieve increased sensitivity. The aptamers used can be very simple in construction and in the selection, since they are not marked. Working with standard curves also allows working in biological mixtures, such as cell culture media, cell disruptions, sera and other body fluids, which have been taken from an organism.

Definitions.

An aptamer according to the invention consists of (not: contains) nucleotides, the number of bases typically being in the range of 10 to 500, in particular 20 to 100. An aptamer can be DNA or RNA, single or double stranded. The nucleotides may be exclusively natural nucleotides. However, individual or several nucleotides may also be chemically modified, as long as this modification is not usable or utilized as a reporter group in the context of the method according to the invention.

As target molecule species are basically any type of molecules in question. This includes any natural organic molecules, including fragments thereof, but also synthetic organic molecules and modified natural organic molecules. It is only essential that specific aptamers are used for the respective target molecule species, where such aptamers can be determined by customary selection methods. Of particular biochemical interest are proteins or peptides, whereby also fragments, for example binding domains are determinable, if the aptamers are specific for this binding domain. Examples of target molecule species are: proteins, peptides, carbohydrates, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, viruses, bacteria, yeasts, metabolic products or components or decay products of viruses or bacteria, fragments of prominent substances, for example containing a binding domain, pharmaceutical active substances , Environmental toxins, nutritional ingredients, dyes and the like.

Solid phases can be any solid surfaces to which target molecules can be attached, covalently or noncovalently. This includes, for example, membranes or columns of materials commonly used in biotechnology or biochemistry, plastics such as polystyrene, nylon, or PTFF, beads such as paramagnetic beads, charged surfaces such as charged paper, metals such as gold or silver, functionalized glass, Langmuir Bottgett films, etc. Typically, the surfaces are functionalized, for example, with amino, carboxyl, thiol, and / or hydroxyl groups.

A defined amount denotes an amount that is known and predetermined. A defined amount can be used, for example, by using a predetermined amount of a solution, of which it is known in which concentration target molecules or Aptamermoleküle present.

Aptamers are reporter group-free if they contain no non-nucleotides or radioisotopes suitable for detection by a physical, physicochemical or chemical process, or if such groups are not used for detection purposes in the process of the invention.

An aptamer specifically binds to a target molecule when aptamer / target molecule complexes can be separated from structurally distinct aptamers.

A quantitative determination is the determination of an absolute or relative amount, for example a concentration, of a substance, wherein the amount exceeds a detection limit by at least a factor of 2, better by a factor of 5. In contrast, the determination of the presence or absence of a substance is not quantitative. Determination.

A contacting is typically carried out by mixing the substances to be contacted in a solution.

Examples:

Non-limiting examples of specific embodiments of the invention are explained in more detail below.

Example 1: Solid phase quantification of erythropoietin by an aptamer.

The following starting materials are used: aptamer as a DNA oligonucleotide with binding specificity to erythropoietin, PCR tube with an epoxy-activated surface, as a coupling buffer basic buffer without primary and secondary amino groups e.g. Na ₂ HCO ₃ (the binding buffer realizes the buffer conditions under which aptamer binding to erythropoietin is possible, preferably corresponds to the buffer under which the oligonucleotide was selected from a combinatorial pool), PCR reaction buffer (Tris buffer with adapted Mg ²⁺ concentration), PCR reagent: Taq polymerase with suitable primers for the amplification of the aptamer oligonucleotides.

• (a) in Abwesenheit ungebundenen Zielmoleküls,
• (b) in Gegenwart unterschiedlicher bekannter Mengen Zielmolekül (Standards) und
• (c) in Gegenwart der unbekannten Probe.

An excess of erythropoietin dissolved in the binding buffer is coupled to the epoxy-activated surface of the PCR tubes. Unbound erythropoietin is washed away. The erythropoietin-specific aptamer of known structure is incubated in binding buffer in the vessel, in parallel under the following conditions:

• (a) in the absence of unbound target molecule,
• (b) in the presence of different known amounts of target molecule (standards) and
• (c) in the presence of the unknown sample.

After the same incubation time in all cases a) to c), unbound aptamers are removed by washing in each case. For adaptation to the following buffer for the PCR is then washed with the PCR buffer. The bound aptamers are amplified by conventional PCR. After each amplification step, a melting curve is created with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). The curves of the fluorescence cycle diagram are evaluated and the characteristic values (CT value or threshold cycle) as a fractional cycle number at which a defined fluorescence threshold is exceeded.

From the values of approach b), a standard curve of the characteristic values of the standard samples is created. The unknown amount of erythropoietin in approach c) is shown by the interpolated standard curve in a concentration vs. Characteristics determined from the measured characteristic values of the samples.

Example 2: Solid-phase Quantification of Interleukin-6 and C-Reactive Protein by Aptamers with Different Melting Temperatures.

The starting materials used are: two different aptamers as DNA oligonucleotide with: i) known end structure, ii) binding specificity to each interleukin-6 or C-reactive protein and iii) with different melting temperature, PCR with epoxy-activated surface, as PCR Reagent Taq polymerase with suitable primers for the amplification of the aptamer oligonucleotides (buffer according to Example 1).

• (a) in Abwesenheit ungebundener Zielmoleküle,
• (b) in Gegenwart unterschiedlicher bekannter Mengen der Zielmoleküle (Standards) und
• (c) in Gegenwart einer unbekannten Probe inkubiert.

An excess of a mixture of the two target molecules in the coupling buffer is coupled to the surface of PCR tubes. Unbound target molecules are washed away. The two target-molecule-specific aptamers are placed in a binding buffer in the vessel

• (a) in the absence of unbound target molecules,
• (b) in the presence of different known amounts of the target molecules (standards) and
• (c) incubated in the presence of an unknown sample.

After an incubation time which is the same for all batches a) to c), unbound aptamers are removed by washing. For adaptation to the following buffer for the PCR is then washed with the PCR buffer. The bound aptamers are amplified by conventional PCR. After each amplification step, a melting curve is created with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). The curves of the fluorescence cycle diagram are evaluated and for each aptamer the characteristic values (CT value or threshold cycle) as a fractional cycle number, at which a defined fluorescence threshold is exceeded, are determined at the aptamer-specific melting temperature. Alternatively, the first derivative of the curve can be determined and the characteristic values (height and / or area) can be recorded. Overlapping surface areas of the peaks are attributed to the respective melting points by algorithms known from chromatography. Alternatively, the first derivative of the curves at different temperatures may be integrated into an area in the three-dimensional space of fluorescence, cycle number, and temperature, and as a measure, the volume of the space under the area in the specific temperature range of the particular aptamer. Overlapping areas of the resulting spaces are attributed to the respective melting points in analogy to the algorithms known from chromatography.

It is created from approach b) for each aptamer a standard curve (target molecule quantity versus characteristic value). The unknown amount of the individual target molecules in the samples (approach c) are shown in a concentration versus concentration graph over the interpolated standard curve. Characteristics determined from the measured characteristic values of the samples.

Example 3: Solid-phase quantification of 2 domain-specific aptamers to determine total hemoglobin and amount of glycylated domain for glycated hemoglobin HbA1c.

Starting materials used are: two different aptamers as DNA oligonucleotides with i) known end structure, ii) binding specificity to different domains of the Target molecule glycated hemoglobin and iii) with different melting temperature, PCR tube with epoxy-activated surface, as a PCR reagent Taq polymerase with suitable primers for the amplification of the aptamer oligonucleotides (buffer according to Example 1).

• (a) in Abwesenheit ungebundener Zielmoleküle,
• (b) in Gegenwart unterschiedlicher bekannter Mengen der betreffenden Zielmoleküldomänen (Standards) und
• (c) in Gegenwart einer unbekannten Probe inkubiert.

An excess of a mixture of the two target molecules in the coupling buffer is coupled to the surface of PCR tubes. Unbound target molecules are washed away. The two target-molecule-specific aptamers are placed in a binding buffer in the vessel

• (a) in the absence of unbound target molecules,
• (b) in the presence of different known amounts of the respective target molecular domains (standards) and
• (c) incubated in the presence of an unknown sample.

After an incubation period, unbound aptamers are removed by washing. For adaptation to the following buffer for the PCR is then washed with the PCR buffer. The bound aptamers are amplified by conventional PCR. After each amplification step, a melting curve is created with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). The curves of the fluorescence cycle diagram are evaluated analogously to Example 2.

A standard curve is created for each aptamer (target molecule quantity versus characteristic value) (approach b). The unknown amount (batch c) of the individual target molecule domains in the samples is shown in a concentration versus concentration graph over the interpolated standard curve. Characteristics determined from the measured characteristic values of the samples.

Example 4: Supernatant quantification of erythropoietin by an aptamer.

The following starting materials were used:
Aptamer as DNA oligonucleotide with binding specificity to erythropoietin, PCR-tube with epoxy-activated surface, as PCR-reagent Taq-polymerase with suitable primers for the amplification of the aptamer-oligonucleotides, buffer according to Example 1.

• (a) in Abwesenheit ungebundenen Zielmoleküls,
• (b) in Gegenwart unterschiedlicher bekannter Mengen Zielmolekül (Standards) und
• (c) in Gegenwart einer unbekannten Probe inkubiert.

An excess of erythropoietin in the binding buffer is coupled to the epoxy-activated surface of the PCR tubes. Unbound target molecule is washed away. A target-specific aptamer of known structure is found in the binding buffer in the vessel

• (a) in the absence of unbound target molecule,
• (b) in the presence of different known amounts of target molecule (standards) and
• (c) incubated in the presence of an unknown sample.

After a defined incubation period, a defined subset of the binding buffer is placed in a PCR approach. The ratio of binding buffer to PCR reaction buffer is such that the amplification is not disturbed. The included aptamers are amplified by conventional PCR. After each amplification step, a melting curve is created with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). The curves of the fluorescence cycle diagram are evaluated and for each aptamer the characteristic values (CT value or threshold cycle) as a fractional cycle number, at which a defined fluorescence threshold is exceeded, are determined at the aptamer-specific melting temperature. It is created by means of approach b) a standard curve of the characteristic values of the standard samples. The unknown amount of the target molecule in the sample (approach c) is shown in a concentration vs. concentration graph over the interpolated standard curve. Characteristics determined from the measured characteristic values of the sample.

Example 5: Solid phase quantification of erythropoietin by an aptamer after carboxy coupling.

Starting materials used are: aptamer as DNA oligonucleotide with binding specificity to erythropoietin, PCR tube with epoxy-activated surface, as coupling buffer an acidic buffer (pH 6) without primary and secondary amino groups, eg. As citrate buffer, PCR reaction buffer as in the preceding examples, as a PCR reagent Taq polymerase with suitable primers for the amplification of the aptamer oligonucleotides.

• (a) in Abwesenheit ungebundenen Zielmoleküls,
• (b) in Gegenwart unterschiedlicher bekannter Mengen Zielmolekül (Standards) und
• (c) in Gegenwart einer unbekannten Probe inkubiert.

An excess of erythropoietin in the coupling buffer is coupled to a carboxy-activated surface of PCR tubes in the presence of EDC. Unbound target molecule is washed away. A target-specific aptamer of known structure is placed in a binding buffer in the vessel

• (a) in the absence of unbound target molecule,
• (b) in the presence of different known amounts of target molecule (standards) and
• (c) incubated in the presence of an unknown sample.

After a defined incubation period, unbound aptamers are removed by washing. For adaptation to the following buffer for the PCR is then washed with the PCR buffer. The bound aptamers become amplified by conventional PCR. After each amplification step, a melting curve is created with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). Evaluation is carried out according to example 4. A standard curve of the characteristic values of the standard samples is prepared (approach b). The unknown amount of the target molecule in the sample (approach c) is shown in a concentration vs. concentration graph over the interpolated standard curve. Characteristics determined from the measured characteristic values of the samples.

Example 6: Solid-phase quantitation of erythropoietin by an RNA aptamer.

Starting materials used are: aptamer as RNA oligonucleotide with binding specificity to erythropoietin, PCR tube with epoxy-activated surface, buffer according to Example 1, as PCR reagent Taq polymerase with suitable primers for the amplification of aptamer oligonucleotides.

• (a) in Abwesenheit ungebundenen Zielmoleküls,
• (b) in Gegenwart unterschiedlicher bekannter Mengen Zielmolekül (Standards) und
• (c) in Gegenwart einer unbekannten Probe inkubiert.

An excess of target molecule in the binding buffer is coupled to an epoxy-activated surface of PCR tubes. Unbound target molecule is washed away. A target-specific aptamer of known structure is placed in a binding buffer in the vessel

• (a) in the absence of unbound target molecule,
• (b) in the presence of different known amounts of target molecule (standards) and
• (c) incubated in the presence of an unknown sample.

After an incubation period, unbound aptamers are removed by washing. For adaptation to the following buffer for the RT-PCR is then washed with the PCR buffer. The bound aptamers are amplified by conventional RT-PCR. After each amplification step, a melting curve is created with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). The evaluation is carried out according to example 5. A standard curve of the characteristic values of the standard samples (approach b) is created. The unknown amount of the target molecule in the sample (approach c) is shown in a concentration vs. concentration graph over the interpolated standard curve. Characteristics determined from the measured characteristic values of the samples.

Example 7: Solid-phase quantification of erythropoietin-specific aptamers after isolation by SELEX.

The starting materials used are: aptamer as DNA oligonucleotide with binding specificity to erythropoietin, PCR tube with epoxy-activated surface, buffer according to Example 6, as PCR reagent Taq polymerase with suitable primers for the amplification of aptamer oligonucleotides

Excess of erythropoietin in the binding buffer is coupled to an epoxy-activated surface of PCR tubes. Unbound target molecule is washed away.

• (a) in Abwesenheit ungebundenen Zielmoleküls und
• (b) in Gegenwart unterschiedlicher bekannter Mengen Zielmoleküls inkubiert.

A selection of aptamers is performed according to the SELEX procedure, by contacting a combinatorial mixture of oligonucleotides with the target molecule, desorbing and amplifying the bound oligonucleotides by PCR. After each round, the presence and binding activity are determined as follows. Be isolated aptamers

• (a) in the absence of unbound target molecule and
• (b) incubated in the presence of different known amounts of target molecule.

After a defined incubation period, unbound aptamers are removed by washing. For adaptation to the following buffer for the PCR is then washed with the PCR buffer. The bound aptamers are amplified by conventional RT-PCR. After each amplification step, a melting curve is prepared with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). The curves of the fluorescence temperature diagram are evaluated and the characteristic values of the melting curve (area and / or associated height) are determined. A curve of the characteristic values of the standard samples is created (approach b). The 50% binding is a measure of the binding affinity of the aptamer mixture to the soluble target molecule

Example 8: Cut-off (Erythropoietin)

The following starting materials are used: aptamer as DNA oligonucleotide with binding specificity to erythropoietin, PCR tube with epoxy-activated surface, buffer according to Example 7, as PCR reagent Taq polymerase with suitable primers for the amplification of aptamer oligonucleotides.

• (a) in Abwesenheit ungebundenen Zielmoleküls,
• (b) in Gegenwart einer bekannten Menge Zielmolekül (Cut-off) und
• (c) in Gegenwart einer unbekannten Probe inkubiert.

An excess of target molecule in the binding buffer is coupled to an epoxy-activated surface of PCR tubes. Unbound target molecule is washed away. A target-specific aptamer of known structure is placed in a binding buffer in the vessel

• (a) in the absence of unbound target molecule,
• (b) in the presence of a known amount of target molecule (cut-off) and
• (c) incubated in the presence of an unknown sample.

After an incubation period, unbound aptamers are removed by washing. For adaptation to the following buffer for the PCR is then washed with the PCR buffer. The bound aptamers are amplified by conventional PCR. After each amplification step, a melting curve is created with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). The curves of the fluorescence cycle diagram are evaluated and for each aptamer the characteristic values (CT value or threshold cycle) as a fractional cycle number, at which a defined fluorescence threshold is exceeded, are determined at the aptamer-specific melting temperature. The characteristic values of the unknown samples are related to the characteristic values of the cut-off.

Example 9: Membrane chip (detection of bound aptamers)

Starting materials used are: aptamer as DNA oligonucleotide with binding specificity to a target molecule, filter membrane with epoxy-activated surface, buffer according to Example 8, as PCR reagent Taq polymerase with suitable primers for the amplification of aptamer oligonucleotides.

A defined amount of target molecule in the binding buffer is coupled to an epoxy-activated surface of PCR tubes. Unbound target molecule is washed away. A sample is applied to one end of the membrane and transported by capillary forces toward the immobilized proteins. On the way, aptamers are released and transported with the sample. The sample with the dissolved aptamers is passed over the immobilized proteins. By adding PCR reaction buffer, the aptamers bound to the immobilized proteins are amplified. After each amplification step, a melting curve is created with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). The curves of the fluorescence cycle diagram are evaluated and for each aptamer the characteristic values (CT value or threshold cycle) as a fractional cycle number, at which a defined fluorescence threshold is exceeded, are determined at the aptamer-specific melting temperature. The unknown amount of the target molecule in the sample is determined by external calibration in a concentration vs. Characteristics determined from the measured characteristic values of the sample.

Example 10 Membrane Chip (Detection of Unbound Aptamers)

As starting materials, those of Example 9 are used. A defined amount of target molecule in the binding buffer is coupled to an epoxy-activated surface of PCR tubes. Unbound target molecule is washed away. A sample is applied to one end of the membrane and transported by capillary forces toward the immobilized proteins. On the way, aptamers are released and transported with the sample. The sample with the dissolved aptamers is passed over the immobilized proteins. By addition of PCR reaction buffer, the amplified in a region behind the immobilized aptamers. After each amplification step, a melting curve is created with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). The curves of the fluorescence cycle diagram are evaluated and for each aptamer the characteristic values (CT value or threshold cycle) as a fractional cycle number, at which eleven fixed fluorescence threshold values are exceeded, are determined at the aptamer-specific melting temperature. The unknown amount of the target molecule in the sample is determined by external calibration in a concentration vs. Characteristics determined from the measured characteristic values of the sample.

Example 11: Magnetic Particle Separation (Particles)

The starting materials are the substances of Example 10, but instead of the filter membrane magnetic particles with epoxy-activated surface.

• (a) in Abwesenheit ungebundenen Zielmoleküls,
• (b) in Gegenwart unterschiedlicher bekannter Mengen Zielmolekül (Standards) und
• (c) in Gegenwart einer unbekannten Probe inkubiert.

An excess of target molecule in the binding buffer is coupled to an epoxy-activated surface of the magnetic particles. Unbound target molecule is washed away. A target-specific aptamer of known structure is placed in a binding buffer in the vessel

• (a) in the absence of unbound target molecule,
• (b) in the presence of different known amounts of target molecule (standards) and
• (c) incubated in the presence of an unknown sample.

After an incubation period, the particles are fixed in the vessel via a magnet, the surrounding buffer is removed and residual unbound aptamers are removed by washing the particles immobilized by the magnet. For adaptation to the following buffer for the PCR is then washed with the PCR buffer. The bound to the particles aptamers are amplified by conventional PCR. After each amplification step, a melting curve is created with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). Evaluation is carried out analogously to Example 10. A standard curve of the characteristic values of the standard samples is created. The unknown amount of the target molecule in the sample is determined by the interpolated standard curve in a Chart Concentration vs. Characteristics determined from the measured characteristic values of the sample.

Example 12: Particle Filtration (Particles)

As starting materials, those of Example 11, but non-magnetic particles with epoxy-activated surface are used. The further processing is carried out according to Example 11, but instead of the magnetic fixation, the particles are separated from the surrounding buffer via a suitable filter and residual unbound aptamers are removed by adding washing buffer and re-filtration. The evaluation is again according to Example 11.

Example 13: Complex Particle Filtration (Ferritin)

The starting materials used are those of Example 12, but no particles or other solid phases are used. Approaches according to Example 12 are carried out. After a defined incubation time, unbound aptamers are removed by filtration through a 30 kD filter and remaining unbound aptamers are removed by renewed addition of washing buffer and filtration. Further processing and evaluation takes place according to example 12.

Example 14: Complex Particles (sAv-Biotin Elimination) (Example Erythropoietin)

Starting materials used are: aptamer as DNA oligonucleotide with binding specificity to erythropoietin, PCR-tube with epoxy-activated surface, buffer according to Example 1, biotinylated erythropoietin, as PCR reagent Taq polymerase with suitable primers for the amplification of the aptamer oligonucleotides.

An excess of streptavidin in the binding buffer is coupled to an epoxy-activated surface of PCR tubes. Unbound streptavidin is washed away. A target-specific aptamer of known structure is incubated in a binding buffer in the vessel together with the biotinylated target molecule in runs a) -c) of the above Examples. The further processing and evaluation takes place according to example 1.

Example 15: FREE (Förster resonance energy transfer)

The starting materials used are those of Example 1, but as PCR reagent Taq polymerase and primer with reporter molecule on the 5 'primer and quencher molecule on the 3' primer.

Further processing and evaluation are carried out according to Example 1, but the bound aptamers are amplified by means of FREI-PCR by addition of suitable PCR reagent (see above). After each amplification step, a melting curve is created with intercalative incorporation of a fluorescent dye into the double strands (real-time PCR). The curves of the fluorescence cycle diagram are evaluated and for each aptamer the characteristic values (CT value or threshold cycle) as a fractional cycle number, at which a defined fluorescence threshold is exceeded, are determined at the aptamer-specific melting temperature. The further evaluation is carried out according to Example 1.

Example 16: Detection via first derivative of the temperature / fluorescence curve

Starting materials are those of Example 1. The further processing and evaluation is carried out according to Example 1, but the curves of the fluorescence temperature diagram are evaluated so that the first derivative of the curve is formed. The parameters height and / or associated size of the area under the resulting curve are determined at the specific melting point for the aptamers. The further evaluation is again according to Example 1.

Example 17: Evaluation strategies

• i) In erhaltenen Kurven Fluoreszenz/Zykluszahl wird für jedes Aptamer der CT-Wert bzw. der threshold cycle-Wert bestimmt und als Kennwert verwendet.
• ii) Es wird eine Kurve Floreszenz/Temperatur bei einer definierten Zykluszahl gemessen. Diese Kurve wird nach der Temperatur differenziert, wodurch Peaks erhalten werden, wobei die Höhe und/oder die Fläche eines Peaks als Kennwert bestimmt wird. Überlappende Peaks können mit aus beispielsweise der Chromatographie bekannten Methoden auseinandergerechnet werden.
• iii) Es wird eine Kurve Floureszenz/Temperatur bei einer definierten Zykluszahl gemessen. Diese Kurve wird nach der Temperatur differenziert, wodurch Peaks erhalten werden, wobei die Höhe und/oder die Fläche eines Peaks bestimmt wird. Für einen Peak wird dies fürverschiedene Zykluszahlen durchgeführt, wobei eine Kurve Höhe bzw. Fläche/Zykluszahl erhalten wird. Dann wird ein definiertes Zykluszahlintervall ausgewählt und das Integral über dieses Zykluszahlintervall gebildet und als Kennwert verwendet.

Within the scope of the invention, the following different evaluation techniques can be used for the aptamer quantities to be measured.

• i) In the fluorescence / cycle number curves obtained, the CT value or the threshold cycle value is determined for each aptamer and used as the characteristic value.
• ii) Measure a florescence / temperature curve for a defined number of cycles. This curve is differentiated according to the temperature, whereby peaks are obtained, whereby the height and / or the area of a peak is determined as a characteristic value. Overlapping peaks can be calculated by methods known from, for example, chromatography.
• iii) Measure a fluorescence / temperature curve for a defined number of cycles. This curve is differentiated according to the temperature, whereby peaks are obtained, whereby the height and / or area of a peak is determined. For a peak, this is done for different cycle numbers, where a curve height / area / cycle number is obtained. Then, a defined cycle number interval is selected and the integral is formed over this cycle number interval and used as the characteristic value.

In all cases the measurements as well as the standard measurements should be carried out under the same defined conditions.

Claims (6)

Method for the quantitative determination of an unknown amount of a target molecule species with the following method steps: a) a system containing a defined amount of labeled target molecules of the Zielmolekülspezies and an unknown amount of non-labeled target molecules of the same Zielmolekülspezies is contacted with a defined amount of specific for the target molecule specific reporter group-free aptamer molecules, which is by binding the Aptamermoleküle to the target molecules of the Zielmolekülspezies Form aptamer / unlabelled target molecule complexes as well as aptamer / labeled target molecule complexes, and where there is a distribution between the amount of unbound aptamer molecules, aptamer / unlabeled target molecule complexes, and aptamer / labeled target molecule complexes, b) the aptamer / labeled target molecule complexes are separated from unbound aptamer molecules and aptamer / unlabeled target molecule complexes, c1) the amount of unbound aptamer molecules is determined quantitatively by means of a quantitative amplification method, and / or c2) the aptamer / labeled target molecule complexes are dissociated and the amount of released aptamer molecule complexes is quantified by a quantitative amplification method, d) the values obtained in step c1) and / or c2) are entered into a standard curve which can be obtained by carrying out steps a to c1) and / or c2) under the same conditions but using different defined amounts of unlabeled target molecules by entering the values obtained in the standard curve, the unknown amount of unlabeled target molecules is obtained. The method of claim 1, wherein the quantitative amplification method is RT (Real-Time) PCR. The method of claim 1 or 2, wherein aptamer / target molecule complexes, labeled and / or unlabeled, are separated from unbound aptamer molecules by molecular weight selective separation methods. Method according to one of claims 1 to 3, wherein the label of the target molecules is selected from the following group: A) binding to a solid phase, in particular to an immobile solid phase or to mobile particles, B) binding to an anchor group for immobilization to a solid phase, and C) binding to a selection group for a physical and / or physical-chemical separation process. Test system for carrying out a method according to one of claims 1 to 4 containing at least one preparation with a defined amount of reporter group-free aptamer molecules which bind specifically to target molecules of a defined target molecule species, at least one preparation with a defined amount of labeled target molecules of the same target molecule species, Means for separating aptamer molecules from unbound aptamer molecules / labeled target molecule complexes and / or aptamer / unlabeled target molecule complexes, and Means for the quantitative determination of unbound aptamer molecules in a quantitative amplification method. Test system according to claim 5, additionally containing means for dissociating aptamer / target molecule complexes, labeled or non-labeled.