EP1642130A1

EP1642130A1 - Device and method for the direct quantitative in vitro determination of a substance that is contained in a sample

Info

Publication number: EP1642130A1
Application number: EP04740874A
Authority: EP
Inventors: Michael Schirner; Kai Licha
Original assignee: Schering AG
Current assignee: Bayer Pharma AG
Priority date: 2003-07-09
Filing date: 2004-07-09
Publication date: 2006-04-05
Also published as: WO2005005985A1; DE10331093A1; JP2009513940A; US20050037402A1

Abstract

The invention relates to a device and a method for the direct quantitative in vitro determination of a substance that is contained in a sample. The inventive device comprises agents, immobilized on a surface, for identifying the substance, a free substance/emitter conjugate, and agents, immobilized on a surface, for identifying the emitter. The emitter used in the device comprises a portion that reacts to an interaction with the agents for identifying the emitter by changing its emission properties. The inventive device allows the direct quantitative determination in e.g. full blood samples of substances, selected from antigens, such as proteins, peptides, nucleic acids, oligonucleotides, blood components, serum components, lipids, pharmaceutical agents and low molecular compounds or, in a different embodiment, substance-identifying agents, such as antibodies or the fragments thereof.

Description

Device and method for the direct quantitative determination in viϊro of a substance contained in a sample

The present invention relates to a device and a method for the direct quantitative in vitro determination of a substance contained in a sample. The device according to the invention comprises means for substance detection immobilized on a surface, free substance-emitter conjugate, and means for detection of the emitter immobilized on a surface. The emitter of the device used comprises a part which responds to an interaction with the means for detecting the emitter with a change in the emission properties. By means of the device according to the invention, substances selected from antigens such as proteins, peptides, nucleic acids, oligonucleotides, blood components, serum components, lipids, pharmaceuticals and compounds of low molecular weight or antibodies or their fragments can be quantified directly in e.g. Whole blood samples are determined.

Background of the Invention

For the diagnostic detection of substances and their concentration determination in-vitro diagnostic measuring methods are used in many cases today, which are based on biological molecules, such as. B. peptides, proteins, antibodies or oligonucleotides are based, which have a high affinity for a substance to be determined. For this purpose, proteins and peptides are preferably used and particularly preferably antibodies and antibody fragments.

The anti-substance antibodies used serve different purposes. On the one hand they are used for the separation of the substance to be determined from the sample, on the other hand they also perform the task of localizing or positioning different signal transmitters used on the substance to be examined. For the detection of, for example, an antibody in a sample, optical and radioactive measurement methods in particular have become established, but also acoustic [see, for example, Cooper MA, et al. Direct and sensitive detection of a human virus by rupture event scanning. Nat Biotechnol. 2001 Sep; l 9 (9): 833 -7.] And magnetic measuring methods are known. Optical measuring proceedings acquired [Nakamura, RM, Dito, WR, Tucker, ES (Eds.). Immunoassays: Clinical Laboratory Techniques for the 1980s. AR Liss, New York. Edwards, R. (ed.). Immunoassays: Essential Data, 1996, Wiley Europe].

Antibodies and peptides directed against molecules of small molecular weight are already known. This also includes antibodies and peptides against dye molecules [Simeonov A. et al., Science 2000, 290, 307-313; Watt R.M. et al., Immunochemistry 1977, 14, 533-541; Rozinov M.N. et al., Chem. Biol. 1998, 5, 713-728]. Furthermore, antibodies against various dyes are already commercially available, e.g. B. against fluorescein, tetramethylrhodamine, Texas Red, Alexa Fluor 488, BODIPY FL, Lucifer yellow and Cascade Blue, Oregon Green (Molecular Probes, Inc., USA). However, these are polyclonal IgG antibodies for bioanalytical purposes, some of which have uncontrollable cross-reactivities and have not resulted from a strict selection process.

Certain in vitro diagnostic procedures, such as The electrochemiluminescence is based on the combination of different antibodies against the substance to be determined, one antibody being used to separate the substance to be determined from the test sample and the other antibody carrying the diagnostically proven signal molecule. In the case of the diagnostic method of electrochemical luminescence, the labeled antibody is optically detected [Grayeski, M. L., Anal. Chem. 1987, 59, 1243].

In addition to electroluminescence, light-induced phosphorescence and fluorescence can also be used as the optical property of molecules for diagnostic measurement processes. Compared to electroluminescence and phosphorescence, fluorescence in particular as the optical property of molecules offers the advantage of high detection sensitivity and high linearity of the measurement signal over a large dynamic range.

To measure the fluorescence of a fluorophore, various measuring methods have been developed that use different principles within the fluorescence processes. Established measuring methods use e.g. B. the attenuation of polarized light (fluorescence polarization - FP), the measurement of the photon lifetime (fluorescence lifetime measurement - FLM), the bleaching properties (fluorescence photobleaching recovery - FPR) and the energy transfer between different fluorophores (fluorescence resonance energy) Transfer - FRET) [Williams, AT, et ab, Methods Immunol. Anal. 1993, 1, 466; Youn, HJ et al., Anal. Biochem. 1995 Oswald, B. et al., Anal. Biochem. 2000, 280, 272; Szollosi, J. et. al., Cytometry 1998, 34, 159].

Other detection methods are based on a change in the polarization level or the detection of phosphorescence.

In most of the measurement methods already available, the anti-substance antibody is labeled with a fluorophore. This marking takes place through specific and non-specific chemical coupling. The labeled antibody is added in excess to the test sample. This is necessary to bind all substance molecules to be examined. When measuring the fluorescence intensity as the most sensitive measurement parameter of fluorophores, however, it must be taken into account that the unbound, labeled anti-substance antibody also emits a fluorescence signal. For this reason, it is necessary to separate the anti-substance antibody specifically bound to the substance from the unbound portion.

This method is further generally based on the fact that the one anti-substance antibody is used to separate the substance to be examined and the second anti-substance antibody, which recognizes another binding site of the test substance, is labeled with a signaling molecule. In this way, falsification of the measurement result by the unbound, but signaling antibody can be avoided. However, due to the separation step, this procedure is associated with increased methodological and technical effort and higher costs. However, the high technical effort, which prevents this method from being established for rapid diagnostics, proves to be particularly disadvantageous.

Newer fluorescence-based measurement methods, e.g. Fluorescence polarization and fluorescence resonance energy transfer (FRET) are methods which make it possible to determine the content of the specifically bound anti-substance antibodies without separating the proportion of unbound, fluorophore-labeled antibodies.

This enables the content of a substance of unknown concentration to be determined to be measured in one step. However, both methods mentioned here also point have decisive disadvantages that prevent widespread use in in vitro diagnostics. The fluorescence polarization thus has a high detection limit in comparison to the fluorescence intensity measurement and is therefore not able to determine very small amounts of an unknown substance. A further disadvantage is that the measurement method cannot be used in a whole blood sample or serum sample without interference sources, since the polarization light is influenced by a large number of proteins and the blood sample cannot therefore be read without errors.

FRET is also a method that can be used to detect the specifically bound, fluorophore-labeled antibodies without separation steps, although in contrast to fluorescence polarization the fluorescence intensity is recorded as a measurement signal, application to a whole blood sample or serum sample is also not possible. The reason for this lies in the strong absorption and autofluorescence of the measurement sample at the wavelengths which are currently used for the fluorescence resonance energy transfer measurement method (visible spectral range up to approx. 550 nm) [Mathis, G., J. Clin. Ligand Assay, 1997, 20, 141; Mathis, G., Clin. Chem. 1995, 41, 1391; Mathis, G., et. al., Clin. Chem. 1993, 39, 1251 Clarke, EE ₅ et. al., J. Neuroscience Methods 2000, 102, 61; Leblanc, V., et. al., Anal. Biochem. 2002, 308, 247].

It is therefore an object of the present invention to provide a device and an optical measuring method for the detection reagents necessary for this purpose for determining the content of an unknown substance without a separation step, which can also be used for the quantitative determination in a whole blood sample.

This object is achieved according to the invention by the measurement method according to the invention and by the provision of a device for carrying out the measurement method according to the invention according to independent claims 1, 18 and 19. Appropriate configurations are listed in the dependent claims.

According to a first aspect of the present invention, there is thus provided a device (1) for the direct quantitative in vitro determination of a substance (3) contained in a sample, comprising: a) agents for immobilizing substances on a surface (6) Detection (5), b) free substance-emitter conjugate (2), and c) means immobilized on a surface for detection of the emitter (4), the emitter used being a Part comprises, which reacts to an interaction with the means for detecting the emitter (4) with a change in the emission properties. The device according to the invention for the direct quantitative in / tro determination of a substance contained in a sample preferably further comprises d) means for measuring the change in the emission properties of the emitter.

A device according to the invention is preferred, the substance being selected from antigens such as proteins, peptides, nucleic acids, oligonucleotides, blood components, serum components, lipids, pharmaceuticals and compounds of low molecular weight, in particular sugars, dyes or other compounds with a molecular weight of less than 500 Daltons.

A device according to the invention is further preferred, the substance being an antibody or antibody fragment. A device (10) according to the invention is used for the direct quantitative in-v / tro determination of an antibody or antibody fragment (13) contained in a sample, comprising: a) antigen (15) immobilized on a surface (16), b) free antibody (or antibody fragment) emitter conjugate (12), and c) means for immobilizing the emitter (14) immobilized on a surface, the emitter used comprising a part which interacts with the means for recognition of the emitter reacts with a change in the emission properties. Preferably, this device according to the invention for direct quantitative in vitro determination of an antibody or antibody fragment contained in a sample further comprises means for measuring the change in the emission properties of the emitter.

A device according to the invention is further preferred, the change in the emission properties of the part of the emitter being selected from a change in the polarization plane, the fluorescence intensity, the phosphorescence intensity, the fluorescence lifetime and a bathochromic shift in the absorption maximum and / or the fluorescence maximum. However, the invention is not restricted to these special phenomena; the term “change in emission properties” in the context of the present invention is intended to encompass all physical phenomena or effects in which the energy-rich radiation striking the emitter is changed in its properties and this change is thereby changed by the binding / non-binding of the substance-emitter conjugate with its emitter binding partner and the substance is quantitatively dependent The device according to the invention is the substance, for example, a peptide, protein, oligonucleotide and in particular an antibody or an antibody fragment. In the context of the present invention, the antibody fragments are fragments which comprise at least the antigen-binding regions which contain the so-called "complementarity-determining regions"("CDRs"). The antigen-binding regions preferably comprise the complete variable chains VH and VL.

In a particularly preferred aspect of the device according to the present invention, the antibody or the antibody fragment is selected from polyclonal or monoclonal antibodies, humanized antibodies, Fab fragments, in particular monomeric Fab fragments, scFv fragments, synthetic and recombinant antibodies, scTCR- Chains and mixtures thereof.

The anti-substance antibody or the anti-substance antibody fragment of a device of the present invention optimally has a higher antigen binding affinity than the anti-emitter antibody or the anti-emitter antibody fragment to the emitter. According to the invention, this affinity is chosen such that the anti-substance antibody or the anti-substance antibody fragment has an antigen binding affinity which is at least twice as high as that of the anti-emitter antibody or the anti-emitter antibody fragment for the emitter. It is further preferred that the anti-substance antibody or the anti-substance antibody fragment has at least ten times higher antigen binding affinity than the anti-emitter antibody or the anti-emitter antibody fragment for the emitter. A binding affinity of the antibodies of less than 50 nM is preferred and more preferably less than 10 nM. The sensitivity of the test can be optimally selected by choosing the affinities. For this purpose, it is favorable that the optimal setting can be determined directly by means of conventional test series, without the need for expensive removal steps. The same applies to the second embodiment of the test of the invention, in which an adjustment is then made using the amount of components.

Thus, in the device according to the invention, the anti-emitter antibody immobilized on the surface can be present in a molar excess over the anti-substance antibody or over the immobilized antigen, the ratio preferably being from 1: 2 to 1:50. A further aspect of the present invention relates to a device according to the invention, the emitter comprising a dye which has at least one absorption maximum and / or fluorescence maximum within the spectral range from 700 to 1000 μm, preferably at least one absorption maximum and fluorescence maximum within the spectral range from 750 to 900 nm , The bathochromic shift of the dye is chosen such that the shift of the absorption and / or fluorescence maximum to higher wavelengths after interaction with the means for recognizing the emitter is greater than 15 nm, preferably greater than 25 nm, and most preferably approximately 30 nm takes place. A shift that is a property of the dye need not necessarily be considered as such. Usually, the shift would be measured as a change in an emission value adapted to the dye, that is to say at a certain singular wavelength. For this purpose, the device according to the invention is preferably provided, for example, with suitable optical means for measurement which are known to the person skilled in the art. This also applies to the measurement of the change in the polarization plane, the fluorescence intensity, the phosphorescence intensity, the fluorescence lifetime and a bathochromic shift in the absorption maximum and / or the fluorescence maximum.

For the device according to the invention it is preferred that the emitter used comprises a dye which is selected from the group of polymethine dyes, such as dicarbocyanine, tricarbocyanine, indotricarbocyanine, merocyanine, styryl, squarilium and oxonol dyes and Rhodamine dyes, phenoxazine or phenothiazine dyes. In general, the emitter of the substance-emitter conjugate of the device according to the invention can be a cyanine dye of the general formula (I)

in which D represents a radical (II) or (III)

stands, the position marked with the asterisk being the point of attachment to the radical B and for group (IV), (V), (VI), (VII) or (VIII)

(VIII)

may be in which R ¹ and R ² independently represent a Ci-C ₄ sulfoalkyl chain, a saturated or unsaturated, branched or straight-chain d-Cso-alkyl chain, optionally with from 0 to 15 oxygen atoms and / or from 0 to 3 Carbonyl groups is interrupted and / or can be substituted with 0 to 5 hydroxyl groups, means R ³ and R ⁴ independently of one another for the group -COOE ¹ , -CONE ^! E ² ₅ -NHCOE ¹ , -NHCONHE ¹ , - NE ^ ² , -OE ¹ , -OSOsE ¹ , -SO ₃ E ¹ , -SO ₂ NHE ¹ or -E ¹ , where E ¹ and E ^{2 are} independent of each other Hydrogen atom, a CrC-i-sulfoalkyl chain, a saturated or unsaturated, branched or straight-chain Ci-Cso-alkyl chain, which is optionally interrupted by 0 to 15 acidic atoms and / or by 0 to 3 carbonyl groups and / or with 0 to 5 hydroxyl groups is substituted, R ^{5 represents} a hydrogen atom, a methyl, ethyl or propyl group or a fluorine, chlorine, bromine or iodine atom, b represents the number 2 or 3, and X and y independently of one another represent O, S, = C (CH ₃ ) ₂ or - (CH = CH) -, and salts and solvates of these compounds. Surprisingly, it was found that after high-affinity binding of an antibody to a cyanine dye with absorption and fluorescence in the near infrared spectral range (> 750 nm), the absorption maximum and fluorescence maximum were shifted by approximately 30 nm to higher wavelengths (bathochromic shift). Using this principle, it is thus possible, for example, to detect a signal from a whole blood sample directly and spectrally separated over a large concentration range, the signal being linear with the concentration of the substance to be determined.

In the context of the present invention, those of the general formula are used as substance-emitter conjugates

S-E

used, wherein S stands for a substance to be investigated and E for an emitter which comprises a part which reacts to an interaction with the means for detecting the emitter with a change in the emission properties. Suitable structural components of the conjugates according to the invention include Dyes which have at least one absorption maximum and fluorescence maximum within the spectral range from 600 to 1200 nm. Dyes with at least one absorption maximum and fluorescence maximum within the spectral range from 700 to 1000 nm are preferred. Dyes that meet these criteria are, for example, those from the following classes: polymethine dyes, such as dicarbocyanine, tricarbocyanine, merocyanine and oxonol dyes, rhodamine dyes, phenoxazine or phenothiazine dyes, tetrapyrrole dyes, in particular benzoporphyrins, chlorines, bacteriochlorides, pheophores Bacteriopheophorbide, Purpurine and Phthalocyanine.

Preferred dyes are the cyanine dyes with absorption maxima between 750 and 900 nm, with particular advantage indotricarbocyanines. Structural components of the conjugates according to the invention are also the substances whose concentration is to be determined using the method according to the invention.

These are selected, for example, from antigens such as proteins, peptides, nucleic acids, oligonucleotides, blood components, serum components, lipids, pharmaceuticals and compounds fertilize low molecular weight, especially sugars, dyes or other compounds with a molecular weight of less than 500 daltons.

Analogously, within the scope of the present invention, the substance-recognizing agent-emitter conjugates are those of the general formula

SEM-E

used, in which SEM stands for a substance-recognizing agent to be investigated and E for an emitter which comprises a part which reacts to an interaction with the means for recognizing the emitter with a change in the emission properties. Suitable structural components of the conjugates according to the invention include Dyes which have at least one absorption maximum and fluorescence maximum within the spectral range from 600 to 1200 nm. Dyes with at least one absorption maximum and fluorescence maximum within the spectral range from 700 to 1000 nm are preferred. Dyes that meet these criteria are, for example, those from the following classes: polymethine dyes, such as dicarbocyanine, tricarbocyanine, merocyanine and oxonol dyes, rhodamine dyes, phenoxazine or phenothiazine dyes, tetra pyrrole dyes, in particular benzoporphyrins, chlorines, bacteriochlorins, pheophorbide, bacterophorbides, Purpurins and phthalocyanines.

Preferred dyes are the cyanine dyes with absorption maxima between 750 and 900 nm, with particular advantage indotricarbocyanines. The structural components of the conjugates according to the invention are also the substance-recognizing agents, the concentration of which is to be determined by means of the method according to the invention.

These are selected, for example, from peptides, proteins, oligonucleotides and in particular antibodies or antibody fragments.

The dyes contain structural elements via which the covalent coupling to the substance structures or the substance-recognizing structures takes place. These are e.g. B. Linkers with carboxy groups, amino groups, hydroxy groups. In the case of an antigen-antibody binding, the conjugate of the substance to be examined and the emitter has a binding affinity on the one hand to the antibody against the substance to be examined and on the other hand to the antibody against the emitter part (eg fluorophore). After the anti-fluorophore antibody has bound to the fluorophore, the absorption and / or fluorescence maximum is shifted. In this case, the absorption and fluorescence maximum is preferably shifted to higher wavelengths by a value greater than 15 nm. This value is particularly preferably greater than 25 nm.

In a further aspect of the device according to the invention, the agents and / or the antigen (substance) are present directly or indirectly on a surface, randomly or randomly immobilized. "Indirect" immobilization is understood here to mean the coupling of the means via a suitable "linker". Such linkers are e.g. widely used in the art of biological "chips" and are well known to the person skilled in the art. The general function of the linker is the fixation and optional exact positioning of the agents on the surface. The fixation can take place covalently or non-covalently. Usually the linker is on The link between the substance to be detected and the immobilized agent is not involved.Examples of linkers range from, for example, PNA oligomers, silane-containing groups and succinimide groups to groups which form peptide bonds. The "direct" immobilization on the surface takes place without further ado Intermediate groups, for example by an activated terminal group of the agent. Such chemically activated groups are also well known to those skilled in the art. By means of the above-mentioned immobilizing groups, the distribution of the agents on the surface can be statistically random or directed. Directional immobilization allows, for example, the application of various means of detection to a surface and thus e.g. the production of a surface that can be suitable for several tests at the same time.

According to the invention, the device comprises a membrane, ball (pearl or "bead") or solid flat surface, these supports made of nylon, cellulose and their derivatives, resin matrices, silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold, which are conventional materials for such surfaces that are easy to manufacture.

Another aspect of the present invention then relates to a method for the direct quantitative in vt ^{'tro-determination} of a substance contained in a sample (3), comprising the steps of, a) providing a device (1) comprising, i) a surface (6) immobilized agent for substance detection (5), ii) free substance emitter Conjugate (2), and iii) immobilization means for detecting the emitter (4) immobilized on a surface, the emitter used comprising a part which reacts to an interaction with the means for recognizing the emitter with a change in the emission properties, b) in Contacting the device with a sample containing a substance to be quantified and c) measuring the change in the emission properties of the emitter.

A further aspect of the present invention then relates to a method for the direct quantitative determination in v trσ of a substance-recognizing agent present in the sample, comprising the steps of, a) providing a device (10), comprising, i) on a surface (16) immobilized substance (15), ii) free substance-recognizing agent-emitter conjugate (12), and iii) on a surface immobilized agent for recognizing the emitter (14), the emitter used comprising a part which responding to an interaction with the emitter detection means with a change in emission properties, b) contacting the device with a sample containing a substance to be quantified or a substance-detecting agent, and c) measuring the change in emission properties of the emitter. Here too, the device preferably further comprises means for measuring the change in the emission properties of the emitter.

A method according to the invention for quantitative in-vitro determination of a substance contained in a sample is preferred, which further comprises d) quantifying the substance contained in the sample or the substance-recognizing agent by means of the measured change in the emission properties of the emitter.

In a preferred embodiment (see FIG. 1), the measurement method according to the invention is based on the use of a conjugate (2) consisting of the substance to be determined and an emitter (in particular fluorophore) in combination with two antibodies (4, 5), one of which is the antibody substance to be determined binds as antigen (anti-substance antibody, 5) and the other antibody binds the emitter as antigen (anti-emitter antibody, 4). The conjugate (2) according to the invention from the substance to be determined and the emitter competes with the free substance (3) to be determined in the sample for the binding sites of the substance-binding antibody (5). The higher the content of the substance (3) to be determined in the test sample, the more the conjugate (2) is displaced from the antigen binding site of the anti-substance antibody and binds in increasing concentration on the anti-emitter antibody (4) (see FIG. 1). The anti-emitter antibody (5) is characterized in that the spectral properties of the emitter change characteristically due to the binding of the emitter in the antigen binding pocket of the antibody. This makes it possible to determine the proportion of the substance-emitter conjugate (2) which is bound to the anti-emitter antibody (4), using the spectral difference from the proportion which is bound to the anti-substance antibody (5) to determine separately.

The substance-emitter conjugate (2) competes with the substance to be examined (3) for the binding sites on the antibody which is directed against the substance to be examined (5). Increasing concentrations of the substance to be examined (3) in the test sample shift the binding of the conjugate towards the antibody (4), which is directed against the emitter.

In another preferred embodiment (see FIG. 2), the measuring method according to the invention is based on the use of a conjugate (12) consisting of a certain substance-recognizing agent, e.g. an antibody, and an emitter (especially fluorophore) in combination with an antibody and antigen (13, 14), the antigen being the particular substance, e.g. Antibody binds (anti-substance antibody, 13) and the antibody binds the emitter as an antigen (anti-emitter antibody, 14). The conjugate of the substance-recognizing agent and the emitter competes with the free antibody (13) to be determined in the sample for the immobilized substance (15). The higher the content of the substance-recognizing agent to be determined, e.g. Antibody (13) in the test sample, the more the conjugate is displaced from the antigen binding site of the anti-substance antibody (13) and binds to the anti-emitter antibody (14) in increasing concentration (FIG. 2). The anti-emitter antibody (14) is characterized in that the spectral properties of the emitter change characteristically due to the binding of the emitter in the antigen-binding pocket of the antibody. This makes it possible to determine the proportion of the substance-recognizing agent (for example antibody) -emitter conjugate (12) which is bound to the anti-emitter antibody (14), using the spectral difference from the proportion which is in the substance ( 15) is bound to determine separately.

The substance-recognizing-agent-emitter conjugate (12) competes with the substance-recognizing-agent to be examined (13) for the binding sites on the substance (5). Rising concentrations of the substance-recognizing agent to be examined (13) in the test sample shift the binding of the conjugate towards the antibody (14), which is directed against the emitter.

A method according to the invention is preferred, the substance to be determined being selected from antigens, such as proteins, peptides, nucleic acids, oligonucleotides, blood components, serum components, lipids, pharmaceuticals and compounds of low molecular weight, in particular sugars, dyes or other compounds with a molecular weight of below 500 daltons. The substance-recognizing agents are selected, for example, from peptides, proteins, oligonucleotides and in particular antibodies or antibody fragments.

A method according to the invention is further preferred, the change in the emission properties of the part of the emitter being selected from a change in the polarization plane, the fluorescence intensity, the phosphorescence intensity, the fluorescence lifespan and a bathochromic shift in the absorption maximum and / or the fluorescence maximum. However, the invention is not restricted to these special phenomena; the term “change in emission properties” in the context of the present invention is intended to encompass all physical phenomena or effects in which the energy-rich radiation striking the emitter is changed in its properties and this change is thereby changed by the binding / non-binding of the substance-emitter conjugate or substance-recognizing agent-emitter conjugate with its emitter binding partner and the substance is quantitatively dependent In one embodiment of the device according to the invention the substance is for example a peptide, protein, oligonucleotide and in particular an antibody or an antibody fragment.

In the case of an optical measurement, this can be done in different ways and depends mainly on the type of characteristic change in the spectral properties of the emitter (eg fluorophore). It is generally preferred to detect the shift in the absorption wavelength and emission wavelength or to measure the absorption and / or fluorescence intensity at a wavelength which for the most part detects the portion of the emitter bound to the antibody. Depending on the change in the spectral properties of the antibody-bound emitter, other properties, such as. B. the photon lifetime, the polarization and the fading behavior can be used for optical measurement. The particular advantage of fluorophores in the spectral range of near-infrared light lies in the low coverage by components of the blood. This enables deep penetration without the signal to be detected being disproportionately changed.

A method according to the invention is preferred in which peptides, proteins and in particular antibodies or antibody fragments are brought into contact with the sample as the substance-recognizing agent. In a particularly preferred aspect of the method according to the present invention, the antibody or the antibody fragment is selected from polyclonal or monoclonal antibodies, humanized antibodies, Fab fragments, in particular monomeric Fab fragments, scFv fragments, synthetic and recombinant antibodies, scTCR chains and mixtures thereof.

The antibodies used can be bivalent total immunoglobulins, but monomeric Fab fragments are preferably used in the test system. After blocking free binding sites on the solid phase, the system is calibrated with a constant substance fluorophore concentration with a saturating concentration and increasing substance concentration. The sample to be determined is either diluted, but preferably used undiluted for the measurement.

Antibodies which are directed against substances to be examined (anti-substance antibodies) are already known. Anti-substance antibodies with a high affinity for the substance are preferred according to the invention. Simeonov A. et al. describe antibodies against stilbenes ("blue-fluorescent antibodies") in Science 2000, 290, 307-313. The antibodies catalyze specifically photochemical isomerization processes and lead to red-shifted absorption and fluorescence maxima in the UV-VIS spectral range (absorption shift maximum 12 nm, fluorescence shift 22 nm) Simeonov A. et al. Give no indication of a red shift while preserving the fluorescence quantum yield for cyanine dyes in the wavelength range from 600-1200 nm.

Watt RM et al. (Immunochemistry 1977, 14, 533-541) describe the spectral properties of the already known anti-fluorescein antibody construct. After binding of the fluorescein, the antibody causes a shift in the absorption and fluorescence max murns in the visible spectral range, but only around 12 nm or 5 nm. In addition, the fluorescence quantum yield is greatly reduced (by approx. 90%). The red shifts of the antibody-dye constructs used according to the invention are> 15 nm in the NIR spectral range while maintaining the fluorescence quantum yield.

Rozinov M.N. et al. (Chem. Biol 1998, 5, 713-728) recently describe the selection of 12-mer peptides from phage libraries which bind the dyes Texas Red, Rhodamine Red, Oregon Green 514 and fluorescein. A red shift in absorption and fluorescence was observed for Texas Red, but only around 2.8 nm and 1.4 nm, respectively. Rozinov et al. do not, however, suggest that antibodies against cyanine dyes lead to larger shifts in the preservation of the fluorescence quantum yield and are therefore suitable for the process according to the invention.

In addition, the person skilled in the art is also already aware of antibodies against fluorophores which are capable of changing its spectral properties in the UV range after binding the fluorophore. By binding a fluorophore in the antigen binding pocket of an antibody, the fluorescence intensity, the absorption maximum, the emission maximum, and the photon lifetime can be changed [Simeonov A., et al., Science (2000) 307-313]. However, these known antibodies are directed against emitters (fluorophores), which have their absorption and fluorescence emission in the visible and UV range of light.

The anti-substance antibody or the anti-substance antibody fragment in the process of the present invention optimally has a higher antigen binding affinity than the anti-emitter antibody or the anti-emitter antibody fragment to the emitter. According to the invention, this affinity is chosen such that the anti-substance antibody or the anti-substance antibody fragment has an antigen binding affinity which is at least twice as high as that of the anti-emitter antibody or the anti-emitter antibody fragment for the emitter. It is further preferred that the anti-substance antibody or the anti-substance antibody fragment has at least ten times higher antigen binding affinity than the anti-emitter antibody or the anti-emitter antibody fragment to the emitter. A binding affinity of the antibodies of less than 50 nM is preferred and more preferably less than 10 nM. The sensitivity of the test can be optimally selected by choosing the affinities. For this purpose, it is favorable that the optimal setting can be determined directly by means of conventional test series without the need for complex separation reached. The same applies to the second embodiment of the test of the invention, in which an adjustment is then made using the amount of components.

The measurement method according to the invention thus preferably uses anti-substance antibodies which have a higher antigen binding affinity than the anti-fluorophore antibody. Preference is given to anti-substance antibodies with an at least twice higher binding affinity than the anti-fluorophore antibody. Anti-substance antibodies with a binding affinity that is more than ten times higher are particularly preferred. The anti-fluorophore antibodies are preferably directed against fluorophores which absorb and emit in the spectral range of the near-infrared light and whose spectral properties are changed by the binding to the antibody in such a way that the emission signal of the antibody bound is changed Portion of the fluorophore spectrally separated from the free portion of the fluorophore.

Thus, in the process according to the invention, the anti-emitter antibody immobilized on the surface can be in a molar excess compared to the anti-substance antibody or to the immobilized antigen, the ratio preferably being from 1: 2 to 1:50.

Another aspect of the present invention relates to the use of a device according to the invention for in vitro diagnostics. Yet another aspect relates to the use of a substance-emitter conjugate or emitter-recognizing agent, in particular a substance-fluorophore conjugate or anti-fluorophore antibody for in vitro diagnostics.

For this purpose, the device according to the invention can also be present in a diagnostic kit in which the components of the device, optionally together with other auxiliaries, are made available together or in separate containers. A further possibility consists in a first kit that provides the basic elements of the device of the invention (eg suitable surface and antibodies and / or substance coupled to it), which then together with the ingredients of a second kit (containing substance for calibration and / or other antibodies) is "specialized" for the respective application. Such a second kit could, for example, Emitter conjugate included. All of these kits can also contain special instructions and documents (e.g. calibration curves, instructions for quantification, etc.).

The invention will now be described in more detail below by means of examples with reference to the attached figures, without, however, being restricted thereto. It shows:

1 shows a first embodiment of the device of the invention, FIG. 2 shows a second embodiment of the device of the invention, and FIG. 3 shows the absorption spectrum (left) and fluorescence spectrum of the dye without and in the presence of antibody MOR02965 in PBS from Example 2.

Examples:

Example 1: Selection, production and characterization of emitter-binding antibodies: Selection of HuCAL GOLD antibody fragments against the cyanine dye Fuji 6-4 (ZK203468) [trisodium-3,3-dimethyl-2- {4-methyl-7 - [3,3-dimethyl-5-sulfonato-l- (2-sulfonatoethyl) -3H-indohum-2-yl] hepta-2,4,6-trien-l-ylidene} -l- (2-sulfonatoethyl) -2,3-dihydro-lH-indole-5-sulfonate, inner Sak]

HuCAL GOLD Antibody Library:

Antibody library HuCAL GOLD. HuCAL GOLD is a fully synthetic, modular human antibody library in Fab antibody fragment format. HuCAL GOLD is based on the HuCAL consensus antibody genes which have been described for the HuCAL-scFvl library (WO 97/08320; Knappik, (2000), J. Mol. Biol 296, 57-86; Krebs et al. J Immunol Me - thods. 2001 Aug l; 254 (l-2): 67-84). In HuCAL GOLD all six CDR areas are diversified through the use of so-called trinucleotide mutagenesis (Virnekäs et al. (1994) Nucleic Acids Res. 1994 Dec 25; 22 (25): 5600-7) according to the composition of these areas in human antibodies, whereas in earlier HuCAL libraries (HuCAL-scFvl and HuCAL-Fabl only the CDR3 areas in VH and VL had been diversified according to the natural composition (see Knappik et al., 2000). In addition, HuCAL also found GOLD a modified screening method use, the so-called CysDisplay (WO 01/05950). Vλ positions 1 and 2. The original HuCAL master genes were constructed with their authentic N-termini: VLλl: QS (CAGAGC), VLλ2: QS (CAGAGC), and VLλ3: SY (AGCTAT). These sequences can be found in WO 97/08320. When the HuCAL scFvl library was produced, these two amino acid residues were changed to "DI" in order to facilitate cloning (EcoRI site). These residues were retained in the production of HuCAL-Fabl and HuCAL GOLD. Therefore, all HuCAL libraries contain VLλ genes with the EcoR interface GATATC (DI) at the 5 'end. All HuCAL kappa genes (master genes and all genes in the libraries) contain DI at the 5 'end anyway, since these represent the authentic N-termini (WO 97/08320).

VH position 1. The original HuCAL master genes were produced with their authentic N-termini: VH1A, VH1B, VH2, VH4, and VH6 with Q (= CAG) as the first amino acid residue and VH3 and VH5 with E (= GAA). The corresponding sequences can be found in WO 97/08320. When the HuCAL-Fabl and the HuCAL GOLD library were cloned, the amino acid Q (CAG) was inserted in this position 1 in all VH genes.

Phagemid production

Infection of E. coli TOP 10F 'cells from the HuCAL GOLD Antiköφer library or from the maturation libraries using Helfeφhagen produced and enriched large amounts of phagemids. For this purpose, the HuCAL GOLD and the maturation libraries (in the TOP 10F 'cells) in 2xYT medium with 34 μg / ml chloramphenicol / 10 μg / ml tetracycline / 1% glucose at 37 ° C up to an OD ₆ oo cultivated by 0.5. The infection was then carried out with VCSM13 Helfeφhagen at 37 ° C. The infected cells were pelleted and resuspended in 2xYT / 34 μg / ml chloramphenicol / 10 μg / ml tetracycline / 50 μg / ml kanamycin / 0.25 mM IPTG and cultivated at 22 ° C. overnight. The phages were precipitated twice from the supernatant with PEG and harvested by centrifugation (Ausubel (1998) Current protocols in molecular biology. John Wiley Sons, Inc., New York, USA). The phages were resuspended in PBS / 20% glycerin and stored at -80 ° C.

The phagemid amplification between the individual selection rounds was carried out as follows: log phase E. coli TG 1 cells were infected with the selected phages and plated on LB agar plates with 1% glucose / 34 μg / ml chloramphenicol. After overnight incubation, the bacterial colonies were scraped off, re-cultivated and infected with VCSM13 Helfeφhagen. Primary selection of antibodies against the Fuji 6-4 dye (ZK203468)

The purified and concentrated phagemids from the HuCAL GOLD Antiköφer library were used in a standard selection process. ZA203468 or transferrin-coupled ZK203468 were used alternately as antigens. The antigens were taken up in PBS and applied in concentrations of 50 μg / ml to Maxisoφ ™ Mikrotiteφlatten F96 (Nunc). The Maxisoφ plates were incubated at 4 ° C. overnight ("coating"). After blocking the Maxisoφ plates with 5% milk powder in PBS, approx. 2E + 13 HuCAL GOLD phages were added to the antigen-loaded, blocked spots and incubated there overnight or for two hours at room temperature. After several washing steps, which became more stringent as the selection rounds progressed, bound phages were eluted with 20 mM DTT or 100 μM unconjugated ZK203468. A total of three successive selection rounds were carried out, the phage amplification taking place between the selection rounds, as described above.

Sub-cloning of selected Fab fragments for expression

After the three-round antibody selection, the Fab-coding inserts of the isolated HuCAL clones were sub-cloned into the expression vector pMORPHX9_MS in order to facilitate the subsequent expression of the Fab fragments. For this purpose, the purified plasmid DNA of the selected HuCAL Fab clones was digested with the restriction enzymes Xbal and EcoRI. The Fab-coding inserts were purified and ligated into the correspondingly digested vector pMORPHX9_MS. This cloning step leads to the Fab-expressing vector pMORPHX9_Fab_MS. Fab fragments that are expressed by this vector carry two C-terminal tags (Myc-Tag and Strep-Tag II) for purification and detection.

Sereening mmά Characterization of E-20346δ-binding Fab Fragments Several thousand clones were isolated after the selection and sub-cloning and tested by ELISA in 384-well format for specific recognition of the antigens ZK203468-BSA and transferrin used in the panning. Clones identified here were examined in an inhibition ELISA for efficient binding of the unconjugated dye. This resulted in the parenteral Fab fragments MOR02628 (protein sequences SEQ-ID NO: 1 (VH-CH) and SEQ-ID NO: 2 (VL-CL); DNA sequences SEQ-ID NO: 3 (VH-CH) and SEQ-ID NO: 4 (VL-CL)), MOR02965 (protein sequences SEQ-ID NO: 5 (VH-CH) and SEQ-ID NO: 6 (VL- CL); DNA sequences SEQ-ID NO: 7 (VH-CH) and SEQ-ID NO: 8 (VL-CL)) and MOR02977 (protein sequences SEQ-ID NO: 9 (VH-CH) and SEQ-ID NO: 10 ( VL-CL); DNA sequences SEQ-ID NO: 11 (VH-CH) and SEQ-ID NO: 12 (VL-CL)), which efficiently bind the non-conjugated dye ZK203468.

Example 2: Photophysical characterization of the dye-antibody complexes and determination of the spectral shifts / fluorescence quantum yields. Dye-antibody complexes based on antibodies with the indotricarbocyanine dye trisodium-3,3-dimethyl-2- {4-methyl-7- 7- [3,3-dimethyl-5-sulfonato-1 - (2-sulfonatoethyl) -3H-indolium-2-yl] hepta-2,4,6-triene-1-ylidene} - 1 - (2-sulfonatoethyl) - 2,3-dihydro-lH-indole-5-sulfonate, inner salt examined (see Example 1). Solutions with a concentration of 1 μmol / 1 of the above were used. Dye and 2.4 μmol / 1 of each antibody in PBS and incubated for 2 h at room temperature. The absorption maxima were determined with a spectrophotometer (Perkin-Elmer, Lambda2). The fluorescence maxima and fluorescence quantum yields were determined using a SPEX fluorolog (wavelength-dependent sensitivity of lamp and detector calibrated) relative to indocyanine green (Q = 0.13 in DMSO, J Chem Eng Data 1977, 22, 379, Bioconjugate Chem 2001, 12, 44 ). The spectral shifts relative to the maxima of a solution of the above were determined from the absorption and fluorescence maxima. Dye without antibodies in PBS (1 μmol / 1) calculated (Absoφtionsmax. 754 nm, fluorescence max. 783 nm, fluorescence quantum yield 10%).

The results are summarized in the following Table 1: Table 1

Example 3: Synthesis of the conjugate from substance-recognizing agent and emitter: anti-ED-B fibronectin antibody / indotricarbocyanine conjugate

Antibodies to the highly pure recombinant ED-B domain of fetal fibronectin are preferably used as scFv, Fab, (Fab) ₂ or as total IgG. In the present example, a Fab with a C-terminal cysteine tag is used and with a linker-modified derivative of the indotricarbocyanine dye trisodium-3,3-dimethyl-2- {4-methyl-7- [3,3-dimethyl-5-sulfonato - 1 - (2-sulfonatoethyl) -3H-indolium-2-yl] hepta-2,4,6-triene-1-ylidene} - 1 - (2-sulfonatoethyl) -2,3-dihydro-1H-indole- 5-sulfonate, inner salt covalently conjugated. The following work steps are carried out:

Synthesis of trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-l- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (5 - {[2nd - (2,5-dioxo-2,5-dihydro-lH-pyrrole-l-yl) ethyl] carbamoyl} -3-oxapentyl) hepta-2,4,6-trien-l-ylidene} -l- (2-sulfonatoethyl) -2,3-dihydro-lH-indoI-5-sulfonate, inner salt for conjugation to Fab

a) 3-Oxa-6- (4-pyridinyl) hexanoic acid tert-butyl ester A solution of 75 g (0.4 mol) of 3- (4-pyridnyl) -l-propanol in 400 ml of toluene / 50 ml of THF is mixed with 10 g of tetrabutylammonium sulfate and 350 ml of 32 percent. Sodium hydroxide solution added. Then 123 g (0.68 mol) of bromoacetic acid ter / butyl ester is added dropwise and 18 h at room temperature. touched. The organic phase is separated off and the aqueous phase is extracted three times with diethyl ether. The combined organic phases are washed with NaCl solution, dried over sodium sulfate and concentrated. After chromatographic purification (silica gel; eluent hexane: ethyl acetate), 56 g of product (41% of theory) are obtained as a brownish oil. b) 3 - [4-Oxa-5- (tert-Butyloxycarbonyl) pentyl] glutaconaldehyde dianilide hydrobromide

A solution of 5.0 g (20 mmol) of 3-oxa-6- (4-pyridinyl) hexanoic acid tert-butyl ester in 60 ml of diethyl ether is mixed with 3.7 g (40 mmol) of amiine and then at 0 ° C. with a Solution of 2.2 g (20 mmol) cyanogen bromide in 8 ml diethyl ether. After stirring at 0 ° C. for 1 h, 50 ml of diethyl ether are added, and the red solid formed is filtered off, washed with ether and vacuum-dried. Yield: 8.5 g (85% of theory) of a violet solid.

c) Trinattiιm 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-l- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (6-carboxy-4 -oxahexyl) hepta-2,4,6-triene-1-ylidene} - 1 - (2-sulfonatoethyl) -2,3-dihydro-lH-indole-5-sulfonate, inner salt A suspension of 3.0 g ( 6 mmol) of 3- [2- (tert-butyloxycarbonyl) ethyl] glutaconaldehyde dianilide hydrobromide (Example 10b) and 4.2 g (12 mmol) of 1- (2-sulfonatoethyl) -2,3,3-trimethyl-3H -indolenine-5-sulfonic acid (example la) in 50 ml of acetic anhydride and 10 ml of acetic acid is mixed with 2.5 g (30 mmol) of sodium acetate and stirred at 120 ° C. for 50 min. After cooling, diethyl ether is added, the precipitated solid is filtered off, stirred in acetone and dried in. After chromatographic purification (RP-C18 silica gel, mobile phase water / methanol), removal of the methanol in vacuo and freeze drying, the title compound is obtained directly. Yield: 2.3 g (41% of theory) of a blue lyophilizate.

d) Trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-l - (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (5- {[2nd - (2,5-dioxo-2,5-dihydro-lH-pyrrole-1-yl) ethyl] carbamoyl} -3-oxapentyl) hepta-2,4,6-triene-1-ylidene} - 1 - (2-sulfonatoethyl) -2,3-dihydro-l H-indole-5-sulfonate, inner salt

1.0 g (1.1 mmol) of the title compound from Example Xe and 0.1 g (1.1 mmol) of triethylamine are dissolved in 15 ml of dimethylformamide, at 0 ° C. with 0.37 g (1.1 mmol) of TBTU added and stirred for 15 min. A solution of 0.42 g (1.7 mmol) of N- (2-aminoethyl) maleimide trifluoroacetate (Int J Pept Protein Res 1992, 40, 445) and 0.17 mg (1.7 mmol) of triethylamine is then added 1.0 ml of dimethylformamide added and 1 h at room temperature. touched. After adding 30 ml of diethyl ether, the solid is centrifuged off, dried and purified by chromatography (RP C-18 silica gel, gradient methanol / water). Yield: 0.85 g of a blue lyophilizate (73% of theory). Synthesis of an indotricarbocyanine-Fab conjugate

0.3 ml of a solution of Fab Antiköφer in PBS (conc. 0.8 mg / ml) is mixed with 60 μL of a solution of tris (carboxyethyl) phosphine (TCEP) in PBS (2.8 mg / ml) and under nitrogen Incubated for 1 h at 25 ° C. Excess TCEP is separated off by gel filtration on a NAP-5 column (eluent: PBS). The amount of Fab obtained (OD _{280 nm} ⁼ 1.4) obtained by means of photometry is _230-250 μg (volume 0.5-0.6 ml). The solution is mixed with 0.03 μmol trisodium 3,3-dimethyl-2- {7- [3,3-dimethyl-5-sulfonato-l- (2-sulfonatoethyl) -3H-indolium-2-yl] -4- (5 - {[2- (2,5-dioxo-2,5-dihydro-1 H-pyrrole-1-yl) ethyl] carbamoyl} -3 -oxapentyl) hepta-2,4,6-triene- 1 -ylidene} - 1 - (2-sulfonatoethyl) -2,3-dihydro-1 H -indole-5-sulfonate, internal salt added (stock solutions of 0.5 mg / ml in PBS) and 30 min at 25 ° C incubated. The conjugate is purified by gel chromatography on a NAP-5 column (eluent: PBS / 10% glycerin). The immunoreactivity of the conjugate solution is determined by means of affinity chromatography (ED-B fibronectin resin) (JImmunol Meth 1999, 231, 239) and is ~ 75%.

Example 4: In vitro assay for the quantification of embryonic fibronectin (ED-B fibronectin) in human serum

The quantification of circulating fetal fibronectin (ED-B - fibronectin) (Curr Opin Drug Discov Devel. 2002, 5, 204) is based on ELISA techniques. For this purpose, transparent 96-well Immunosorb ELISA plates, but preferably white Immunosorb ELISA plates (Nunk, Denmark) are used for chemiluminescence detection. The following work steps are carried out:

Coating the ELΪSA plates The highly pure recombinant ED-B domain of fetal fibronectin (ED-B-FN) is used as an antigen together with the highly pure anti-emitter antibody (see Example 1), which is preferably called scFv, Fab, (Fab ) or as total IgG, immobilized on the surface of the ELISA plates. PBS, but preferably alkaline coupling buffer, is used as the coupling buffer. This consists of a mixture of 17 ml of a 0.2 M Na ₂ CO ₃ solution and 8 ml of a NaHCO ₃ solution, which is made up to 100 ml with bidistilled water. Add the ready-to-use coupling buffer to water. The concentrations of recombinant antigen and anti-emitter antibodies are in the range of 1-10 μg / ml, the optimal molar ratio of recombinant antigen and anti-emitter antibodies having to be determined empirically. However, ratios in the range from 1: 5 to 1: 100 are preferred selected. The coupling takes place either for 2 hours at 37 ° C or at 4 ° C overnight in a volume of 100 μl per well.

Blocking free binding sites

After coupling, free binding sites on the ELISA plates are blocked with PBS containing 2% (w / v) bovine serum albumin or gelatin, but preferably with blocking buffer with 2% (w / v) bovine albumin or gelatin. For this purpose, the plate is knocked out after the coupling and incubated with 200 μl / well blocking buffer for 2 hours at 37 ° C.

calibration

To calibrate the system, a calibration series of the high-purity ED-B-FN is pipetted into human serum. For this purpose, an ED-B-FN sample with a concentration of 10 μg / ml is serially diluted in 1: 2 steps, each sample having a constant concentration of anti-substance antibody labeled with emitter (anti-ED-B FN Fab-indotricarbocyanine conjugate from Example 3) contains. According to the test system, this can range from 0.1 μg / ml to 10 μg / ml.

Pipetting the quantitative ELISA The blocked ELISA plate is knocked out and 100 μl of the calibration protein series are pipetted into the respective holes of the ELISA plate in triplicates. Likewise, the serum or whole blood samples to be determined are applied to the plate in triplicates and incubated for 30 minutes at 37 ° C.

Measurement of the samples

The samples are measured in a spectral fluorometer with two monochromators (SPEX-Fluorog, Jobin Yvon). The wavelength of the excitation light and the detection wavelength for the emitted fluorescence can be chosen freely. The samples are examined in an ELISA plate module. The present example is chosen as the excitation wavelength 790 nm. The fluorescence is detected in a bandpass of 805 - 860 nm. This causes the increase in the fluorescence signal at the red-shifted wavelength (see illustration) due to the increase in the proportion of anti-substance-emitter conjugate, which is immobilized via the dye to immobilized anti-emitter -Antiköφer binds, recorded. typically, one finds a sigmoid curve, the linear measuring range of the calibration series being used for the quantitative determination of the measuring samples.

Example 5: Antibodies against ED-B fibronectin Antibodies against ED-B fibronectin were generated analogously to the procedure described in Example 1 from the HuCAL GOLD antibody library against ED-B fibronectin as antigen.

LIST OF REFERENCE NUMBERS

(1) Device for measuring a substance in a sample

(2) substance-emitter conjugate

(3) substance

(4) Emitter Detecting Agent

(5) Substance-recognizing agent

(6) surface

(10) Device for measuring an antibody-recognizing agent in a sample

(12) Antibody-emitter conjugate

(13) Antibodies

(14) Emitter Detecting Agent

(15) immobilized antigen

(16) surface

Claims

claims

1. A device for the direct quantitative in / tro determination of a substance contained in a sample, comprising: a) agent for immobilization on a surface for substance recognition, b) free substance-emitter conjugate, and c) agent immobilized on a surface for detecting the emitter, the emitter used comprising a part which reacts to an interaction with the means for detecting the emitter with a change in the emission properties.

2. Device for the direct quantitative in vitro determination of a substance contained in a sample according to claim 1, further comprising d) means for measuring the change in the emission properties of the emitter.

3. Device according to one of claims 1 or 2, wherein the substance is selected from antigens, such as proteins, peptides, nucleic acids, oligonucleotides, blood components, serum components, lipids, pharmaceuticals and compounds of low molecular weight, or antibodies and antibody fragments.

4. Device according to one of claims 1 to 3, wherein the change in the emission properties of the part of the emitter is selected from a change in the polarization plane, the fluorescence intensity, the phosphorescence intensity, the fluorescence lifetime and a bathochromic shift of the absorption maximum and / or the fluorescence maximum.

5. Device according to one of claims 1 to 4, wherein the substance-recognizing agents are peptides, proteins, oligonucleotides and in particular Antiköφer or Antiköφerfrag- ment.

6. Device according to one of claims 3 to 5, wherein the Antiköφer or Antiköφer fragments are selected from polyclonal or monoclonal antibodies, humanized antibodies, Fab fragments, in particular monomeric Fab fragments, scFv- Fragments, synthetic and recombinant antibodies, scTCR chains and mixtures thereof.

7. The device according to claim 5 or 6, wherein the anti-substance antibody or the anti-substance antibody fragment has a higher antigen binding affinity than the anti-emitter antibody or the anti-emitter antibody fragment to the emitter.

8. The device according to claim 7, wherein the anti-substance antibody or the anti-substance antibody fragment has an antigen binding affinity which is at least twice higher than that of the anti-emitter antibody or the anti-emitter antibody fragment to the emitter.

9. The device according to claim 7 or 8, wherein the anti-substance antibody or the anti-substance antibody fragment has at least ten times higher antigen binding affinity than the anti-emitter antibody or the anti-emitter antibody fragment to the emitter.

10. Device according to one of claims 7 to 9, wherein the binding affinity of the anti-substance-Antiköφer or the anti-substance-Antiköφerfragments is less than 50 nM and preferably less than 10 nM.

11. The device according to one of claims 1 to 10, wherein the immobilized on a surface anti-emitter Antiköφer is in molar excess compared to the anti-substance antibody or compared to the antigen, the ratio preferably from 1: 2 to 1:50 is.

12. Device according to one of claims 1 to 11, wherein the emitter comprises a dye which has at least one absorption maximum and / or fluorescence maximum within the spectral range from 700 to 1000 nm, preferably at least one absorption maximum and fluorescence maximum within the spectral range from 750 to 900 nm.

13. Device according to one of claims 1 to 12, wherein the shift of Absoφti- ons and / or fluorescence maximum to higher wavelengths after interaction with the means for recognizing the emitter by a value greater than 15 nm, preferably greater than 25 nm, and most preferably approximately 30 nm.

14. Device according to one of claims 1 to 13, wherein the emitter used comprises a dye which is selected from the group of polymethine dyes such as dicarbocyanine, tricarbocyanine, indotricarbocyanine, merocyanine, styryl, squarilium and oxonol dyes and rhodamine dyes , Phenoxazine or phenothiazine dyes.

15. The device according to one of claims 1 to 14, wherein the emitter of the substance-emitter conjugate is a cyanine dye of the general formula (I)

in which D represents a radical (II) or (III)

(VIII) can be in which R ¹ and R ² independently of one another represent a C ₁ -C ₄ sulfoalkyl chain, a saturated or unsaturated, branched or straight-chain Ci-Cso-alkyl chain, optionally with from 0 to 15 oxygen atoms and / or from 0 to 3 carbonyl groups is interrupted and / or can be substituted with 0 to 5 hydroxyl groups, R ³ and R ⁴ mean independently of one another for the group -COOE ¹ , -CONE ^ ² , -NHCOE ¹ , - NHCONHE ¹ , -NE'E ² , -OE ¹ , -OSO ₃ .E ¹ , -SOsE ¹ , -SOzNHE ¹ or -E ¹ , where E ¹ and E ² independently of one another are a hydrogen atom, a C ₁ -C ₄ sulfoalkyl chain, a saturated or unsaturated, represents branched or straight-chain Ci-Cso-alkyl chain which is optionally interrupted by 0 to 15 oxygen atoms and / or by 0 to 3 carbonyl groups and / or is substituted by 0 to 5 hydroxyl groups, R ⁵ for a hydrogen atom, a methyl, ethyl or propyl radical or a fluorine, chlorine, bromine or iodine atom, b di e is 2 or 3, and X and Y are independently O, S, = C (CH ₃ ) ₂ or - (CH = CH) -, and salts and solvates of these compounds.

16. Device according to one of claims 1 to 15, wherein the substance-recognizing agents and / or the substance are present directly or indirectly immobilized randomly or randomly on a surface.

17. The device according to any one of claims 1 to 16, wherein the surface comprises a membrane, ball (bead) or solid flat surface made of resin matrices, silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver or gold consists.

18. Use of a device according to one of claims 1 to 17 for in vitro diagnostics.

19. A method for the direct quantitative in / tro determination of a substance contained in a sample, comprising the steps of, a) making available a device, comprising i) substance-detection agent immobilized on a surface, ii) free substance-emitter conjugate, and iii) agent-immobilization detection agent immobilized on a surface, wherein the emitter used comprises a part that interacts with the agent for Detection of the emitter reacts with a change in the emission properties, b) bringing the device into contact with a sample which contains a substance to be quantified, and c) measuring the change in the emission properties of the emitter.

20. A method for direct quantitative in vt ^{'tro-determination} of a substance contained in a sample, comprising the steps of, a) providing a device according to one of claims 1 to 17, b) contacting the device with a sample, which contains a substance to be quantified, and c) measuring the change in the emission properties of the emitter.

21. The method for quantitative in-v / tro determination of a substance contained in a sample according to claim 19 or 20, further comprising d) quantifying the substance contained in the sample by means of the measured change in the emission properties of the emitter.

22. The method according to any one of claims 19 to 21, wherein the substance is selected from antigens such as proteins, peptides, nucleic acids, oligonucleotides, blood components, serum components, lipids, pharmaceuticals and compounds of low molecular weight, or antibodies and antibody fragments.

23. The method according to any one of claims 19 to 22, wherein the change in the emission properties of the part of the emitter is selected from a change in the polarization level, the fluorescence intensity, the phosphorescence intensity, the fluorescence lifetime and a bathochromic shift of the absorption maximum and / or the fluorescence maximum.

24. The method according to any one of claims 19 to 23, wherein as the substance-recognizing agent peptides, proteins and in particular Antiköφer or Antiköφerfragmente are brought into contact with the sample.

25. The method according to any one of claims 19 to 24, wherein the Antiköφer or Antiköφerfragmente are selected from polyclonal or monoclonal antibodies, humanized antibodies, Fab fragments, in particular monomeric Fab fragments, scFv fragments, synthetic and recombinant antibodies, scTCR chains and Mixtures of these.

26. The method according to claim 24 or 25, wherein the anti-substance antibody or the anti-substance antibody fragment has a higher antigen binding affinity than the anti-emitter antibody or the anti-emitter antibody fragment to the emitter.

27. The method according to claim 26, wherein the anti-substance-Antiköφer or the antiSubstanz- Antiköφerfragment has at least twice higher and in particular ten times higher antigen binding affinity than the anti-emitter Antiköφer or the anti-emitter Antiköφerfragment to the emitter.

28. The method according to claim 24, wherein the immobilized anti-emitter antibody is brought into contact with the sample in molar excess over the anti-substance antibody or over the antigen, the ratio preferably being 1: Is 2-1: 50.

29. The method according to any one of claims 24 to 28, wherein the emitter comprises a dye which has at least one absorption maximum and / or fluorescence maximum within the spectral range from 700 to 1000 nm, preferably at least one absorption maximum and fluorescence maximum within the spectral range from 750 to 900 nm ,

30. The method according to any one of claims 24 to 29, wherein the shift of the absorption and / or fluorescence maximum to higher wavelengths after interaction with the means for detecting the emitter by a value greater than 15 nm, preferably greater than 25 nm, and most preferably takes place around 30 nm.

31. The method according to any one of claims 24 to 30, wherein the emitter used comprises a dye which is selected from the group of polymethine dyes such as dicarbocyanine, tricarbocyanine, indotricarbocyanine, merocyanine, styryl, squarilium and oxonol dyes and rhodamine dyes , Phenoxazine or phenothiazine dyes.

32. The method according to any one of claims 24 to 30, wherein the emitter of the substance-emitter conjugate is a cyanine dye of the general formula (I)

in which D represents a radical (II) or (III)

(VIII) may be in which R ¹ and R ² independently of one another represent a C ₁ -C sulfoalkyl chain, a saturated or unsaturated, branched or straight-chain CrCso-alkyl chain which may be interrupted with from 0 to 15 oxygen atoms and / or from 0 to 3 carbonyl groups is and / or can be substituted with 0 to 5 hydroxyl groups, R ³ and R ⁴ independently of one another mean for the group -COOE ¹ , -CONE'E ² , -NHCOE ¹ , - NHCONHE ¹ , -NE ^ ² , -OE ¹ , -OSO ₃ E ¹ , -SO ₃ E \ -SO ₂ NHE ¹ or -E ¹ , where E and E independently of one another are a hydrogen atom, a C 1 -C 4 -sulfoalkyl chain, a saturated or unsaturated, branched or straight-chain CrCso- Represents alkyl chain, which is optionally interrupted by 0 to 15 oxygen atoms and / or by 0 to 3 carbonyl groups and / or is substituted by 0 to 5 hydroxyl groups, R ⁵ for a hydrogen atom, a methyl, ethyl or propyl radical or a fluorine - chlorine, bromine or iodine atom, b is the number 2 o which means 3, and X and Y independently of one another are O, S, = C (CH ₃ ) ₂ or - (CH = CH) -, and salts and solvates of these compounds.

33. Use of substance-emitter conjugates in a method for direct quantitative in / tro determination according to one of claims 19 to 32.

34. Diagnostic kit comprising means for carrying out the method according to one of claims 19 to 32, optionally together with other auxiliaries and / or instructions, together or in separate containers.