Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching

Definitions

• 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.
• 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.
• peptides, proteins, antibodies or oligonucleotides are based, which have a high affinity for a substance to be determined.
• 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.
• the labeled antibody is optically detected [Grayeski, M. L., Anal. Chem. 1987, 59, 1243].
• light-induced phosphorescence and fluorescence can also be used as the optical property of molecules for diagnostic measurement processes.
• 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.
• Other detection methods are based on a change in the polarization level or the detection of phosphorescence.
• 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.
• 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.
• this procedure is associated with increased methodological and technical effort and higher costs.
• 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.
• FRET Fluorescence polarization and fluorescence resonance energy transfer
• 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].
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the emitter comprising a dye which has at least one absorption maximum and / or fluorescence maximum within the spectral range from 700 to 1000 ⁇ m, 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.
• 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.
• 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.
• 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.
• the emitter of the substance-emitter conjugate of the device according to the invention can be a cyanine dye of the general formula (I)
• 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 !
• 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.
• 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
• 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.
• 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.
• the substance-recognizing agent-emitter conjugates are those of the general formula
• 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.
• 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, bacterophor
• 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.
• peptides 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.
• B. Linkers with carboxy groups, amino groups, hydroxy groups are e.g. B. Linkers with carboxy groups, amino groups, hydroxy groups.
• 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).
• the absorption and / or fluorescence maximum is shifted.
• 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.
• 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".
• 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.
• linker is on
• the link between the substance to be detected and the immobilized agent is not involved.
• 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.
• 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.
• 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 tr ⁇ 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.
• 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.
• 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 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.
• 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.
• 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 for example antibody
• 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.
• 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.
• 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.
• 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
• the substance is for example a peptide, protein, oligonucleotide and in particular an antibody or an antibody fragment.
• 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.
• 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 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.
• 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.
• 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.
• 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.
• 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.
• 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.).
• FIG. 1 shows a first embodiment of the device of the invention
• FIG. 2 shows a second embodiment of the device of the invention
• 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.
• 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 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 (Virnekals et al. (1994) Nucleic Acids Res.
• HuCAL also found GOLD a modified screening method use, the so-called CysDisplay (WO 01/05950). V ⁇ positions 1 and 2.
• the original HuCAL master genes were constructed with their authentic N-termini: VL ⁇ l: QS (CAGAGC), VL ⁇ 2: QS (CAGAGC), and VL ⁇ 3: SY (AGCTAT). These sequences can be found in WO 97/08320.
• the infected cells were pelleted and resuspended in 2xYT / 34 ⁇ g / ml chloramphenicol / 10 ⁇ g / ml tetracycline / 50 ⁇ g / 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 ⁇ g / ml chloramphenicol. After overnight incubation, the bacterial colonies were scraped off, re-cultivated and infected with VCSM13 Helfe ⁇ hagen. Primary selection of antibodies against the Fuji 6-4 dye (ZK203468)
• the purified and concentrated phagemids from the HuCAL GOLD Antikö ⁇ er 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 ⁇ g / ml to Maxiso ⁇ TM Mikrotite ⁇ latten F96 (Nunc).
• the Maxiso ⁇ plates were incubated at 4 ° C. overnight ("coating"). After blocking the Maxiso ⁇ 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.
• bound phages were eluted with 20 mM DTT or 100 ⁇ M unconjugated ZK203468. A total of three successive selection rounds were carried out, the phage amplification taking place between the selection rounds, as described above.
• 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.
• 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.
• 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).
• 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.
• 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:
• 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:
• 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.
• concentrations of recombinant antigen and anti-emitter antibodies are in the range of 1-10 ⁇ g / 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 ⁇ l per well.
• a calibration series of the high-purity ED-B-FN is pipetted into human serum.
• an ED-B-FN sample with a concentration of 10 ⁇ g / 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 ⁇ g / ml to 10 ⁇ g / ml.
• 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 -Antikö ⁇ er 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.

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