DE3912046A1 - New luminescent dyes - of cyanine, merocyanine or styryl type, contg. sulpho gps., useful for labelling components in aq. media - Google Patents

Abstract

(A) Luminescent dyes of formula (I)-(III) are new; where X and Y = O, S or CMe2; m = 1-4; at least one of R1-R7 contains a NCS, NCO, mono- or dichlorotriazinyl; mono- or dihalopyridyl, mono- or dihalodiazinyl, maleimido, aziridinyl, halosulphonyl, halocarbonyl, succinimidyloxycarbonyl, sulphosuccinimidyloxycarbonyl, imido ester, hydrazino, azidonitrophenyl, N3, 3-(2-pyridyldithio)propionamido, COCHO or CHO gp.; at least one of R8 and R9 contains a sulpho gp., opt. in salt form, bonded to an aromatic ring; apart from the above characteristics, R1-R9 are not specifically defined; the significance of the dotted lines is not defined, but in subsidiary claims the left-hand dotted line represents an o-phenylene or 1, 2-naphthylene gp. in (I)-(III) and the right-hand dotted line represents an o-phenylene or 1, 2-naphthylene gp. in (I) and completes and 2-Z-hexahydro-4, 6-dioxo-5-pyrimidylidene gp. in (II), where Z = O or S. (B) New prods. are prepd. by reacting a component contg. an amine, CHO, SH or OH gp. with a water-soluble sulphoindolenin- or naphthosulphoindolenin-based polymethine dye contg. at least one substit. covalently reactive with the amine, CHO, SH or OH gp. The individual dye mols. on the reaction prod. have a mean molar extinction coefft. of at least 50,000 l/mol-cm and a mean quantum efficiency of at least 2% and have absorption and emission maxima in the 400-850 nm range in aq. media. USE/ADVANTAGE - The process may be used to label proteins, nucleic acids, drugs, toxins, cells, particles, plastics or glass surfaces, polymer membranes, etc., for use in assay or flow cytometry systems. The SO3H gps. increase the water-solubility of the dyes and reduce luminescence quenching due to dye/dye interactions.

Description

Cyanine dyes and related polymethine dyes with light absorbing properties have been used in photographic films. Although such dyes require light-absorbing properties, they do not require luminescence (fluorescence or phosphorescence) properties. Cyanine dyes with luminescence properties have so far only been used to a very limited extent. One such use is the labeling of the sulfhydryl group of proteins. In a report, Salama, G., Waggoner, AS and Abramson J., entitled "Sulfhydryl reagent dyes trigger the rapid release of Ca ²⁺ from sarcoplasmin reticulum vesicles (SR)" from Biophysical Journal, 47, 456a (1985), found that cyanine chromophores with an iodoacetyl group were used to form covalent bonds with sulfhydryl groups on the sarcoplasmin reticulum protein at pH 6.7 to induce Ca ²⁺ release. The report also states that fluorescent dyes have been used to label and isolate these proteins.

In a report by Waggoner, A.S., Jenkins, P.L., Carpenter, J. P. and Gupta, R. entitled "Kinetics of Conformational Changes in a region of the rhodopsin molecule, from the retinylidene binding site removed "in Biophysical Journal, 33, 292a (1981), the authors state that the Sulfhydryl group on the F1 range of cattle rhodopsin with a cyanine dye covalently absorbed at 660 nm has been marked. Also according to this report Cyanine dyes for specific labeling of the sulfhydryl group of a protein used. The use of fluorescent  However, dyes are not described in this report.

An article entitled "International Workshop on the application of fluorescence photobleaching techniques Problems of Cell Biology "by Jacobson K., Elson E., Koppel D., Webb W. in Fed. Proc. 42: 72-79 (1983) an article submitted by A. Waggoner. This refers to fluorescent probes of the cyanine type, that can be conjugated to proteins and those in the deep one Red range of the spectrum can be excited.

In the above three reports, the only cyanine Probes mentioned only those that are covalently specific attach the sulfhydryl group of a protein. The only specifically named cyanine compound is one that has a Has iodoacetyl group, which group causes the Cyanine dye covalently reactive towards the sulfhydryl group becomes. In none of the reports above is the covalent reaction of a cyanine dye with any material other than a protein or with any other Group described on a protein as a sulfhydryl group.

However, many non-protein materials do not have sulfhydryl groups, and many proteins do not have a sufficient number from sulfhydryl groups to these groups for the purpose suitable for fluorescence probing. Additional to that, that sulfhydryl groups (-SHSH-) easily to disulfides (-S-S-) be oxidized in the presence of air and therefore for the covalent Attachment to a fluorescent probe is no longer available will.

According to the invention, cyanine and related polymethine Developed dyes that have substituent groups, not only with the appropriate reaction conditions Sulfhydryl groups, but also with amine - (- NH₂ -) - and hydroxy (-OH-) groups or other groups, such as aldehyde - (- CHO-) groups, covalently reactive on proteins and other materials are for fluorescence and phosphorescence detection to enable these materials. Through the invention significant advantages over the use of iodoacetyl Cyanine dyes according to the prior art and their specific Realized reactivity with sulfhydryl groups. Amine and hydroxyl groups are found in proteins and other materials stronger than sulfhydryl groups and they are more stable. Therefore, if fluorescent cyanine dyes are used to detect the Presence of certain proteins are used becomes a stronger fluorescent or phosphorescent light intensity signal given because a larger number of Dye molecules attached to the protein to be probed can be. Furthermore, amine and hydroxy groups become lighter on components that are to be labeled, such as polymer particles, which by nature are neither sulfhydryl, amine or Contain hydroxyl groups attached.

The invention also relates to a method in which luminescence Cyanine dyes that contain a group associated with Amine groups or hydroxyl groups or other reactive Groups are covalent, used to Proteins or other materials with an amine or hydroxy group or another group that is able to reacting with the dye in a mixture used so that the presence and extent of the marked Protein or other material can be detected after the marked components by chromatographic Methods have been separated. According to the above  The sulfhydryl group was apparently special chosen for the covalent reaction because there are so few of these groups on a protein molecule and because in some cases the sulfhydryl group is functional of the protein plays a significant role. the authors could therefore assume that the specific location of a sulfhydryl group be found on a protein structure can. According to these documents, that for the sulfhydryl group specific dye also used as a probe to detect structural changes in a special protein or generate. To change the light absorption by the dye or that released by the dye bond To interpret calcium ion, it was necessary knowing where the probe is bound.

Because there are so few sulfhydryl groups on most protein molecules these groups cannot be numerous enough adequate total luminescence for acquisition exams to surrender. In contrast, amine and hydroxy groups significantly more numerous, and they are on one Protein molecule widely dispersed, which enables that a fluorescence probe in multiple places on the Molecule is added, thereby interpreting the light absorption and fluorescence changes through relief the detection of the protein is excluded.

The present invention relates to labeling with luminescent Polymethine cyanine and related polymethine dyes, such as merocyanine and styryl dyes, proteins and other materials including nucleic acids, DNA, Drugs, toxins, blood cells, microbial materials, Particles, plastic or glass surfaces, polymer membranes etc. at an amine or hydroxy site on the materials mentioned. The dyes are advantageously all in one  aqueous or another medium in which the labeled Material included is soluble. The present invention relates to a two-step marking process in addition to a one-step marking process. With the two-stage Marking process can be a primary component, such as a Antibodies, including amine, Hydroxy, aldehyde or sulfhydryl sites are marked, and the labeled component is used as a probe for the secondary Component, such as an antigen, for which the antibody is specific is used.

According to the prior art discussed above, the specificity the site of attachment by a cyanine probe obtained by using a probe opposite a sulfhydryl group is covalently reactive. According to the Two-stage processes according to the invention can be cyanine and related probes in a first stage with amine, Aldehyde, sulfhydryl, hydroxy or other groups a first component, such as an antibody, are implemented, whereupon the antibody has the desired specificity a second component, such as an antigen, in a second or staining level, the specificity determined by the antigen site of attachment to the antibody becomes.

The present invention also relates to luminescence polymethine Cyanine compounds and related compounds that Contain groups that enable them to be covalent on amine, hydroxyl, aldehyde or sulfhydryl groups to be attached to a target molecule. The invention aims continue to focus on monoclonal antibodies and others Components with these luminescent cyanine compounds are labeled and capable of probes for antigens  to be. If the target is a cell type, then the present invention can be used to control the amount of to measure labeled antibodies to the named cell type is pinned. The measurement can be done in the way that one has the relative brightness or attenuation of the luminescence of the cells.

The present invention can be used to control the concentration of a particular protein or other component to determine in a system. If the number of reactive groups on a protein using a probe can be implemented, is known, then the fluorescence known per molecule, and the concentration of these molecules in the system can be based on the total luminescence intensity of the system can be determined.

The method can be applied to a variety of proteins or other materials in a system quantitative to determine by all of a mixture of Proteins labeled in the system and then the labeled ones Proteins by any means, such as chromatographic Measures, disconnects. The amount of proteins separated that luminescence can be determined. In chromatographic Detection systems can identify the location of the dye on the marked Material can be determined.

The invention can also be used to increase the number of different cells labeled with an antibody are to be determined. This provision can be done in the way done that you have a variety of types of cells in labeled in a system and then the labeled cells outside the system. You can also use the labeled Cells from unlabeled cells outside the  Systems are separated.

Another embodiment of the present invention comprises a multi-parameter process in which a variety of Luminescent cyanine or related dyes are used is, each of a variety of different primary Components, such as antibodies, are attached, each Dye for a different secondary component, such as an antigen that is specific to any of a variety of the above Identify antigens in a mixture of antigens. According to this embodiment, each of the above Antibodies separated with a dye with different Light absorption and various luminescence wavelengths Properties than the dye used to mark the others Probes used are marked. Then everyone will labeled antibodies to a biological to be analyzed Given preparation that contains secondary components such as antigens contains, each by certain labeled antibodies can be stained. Any unreacted dye materials can, for example, from the preparation Washouts are removed if they interfere with the analysis. The biological preparation is then a variety of excitation wavelengths exposed, each excitation wavelength used the excitation wavelength of a particular conjugate Dye. A luminescence microscope or another Luminescence detection system, such as a flow cytometer or a fluorescence spectrophotometer, the filter or monochrometer to select the rays of the excitation wavelength and for the selection of the wavelengths of luminescence, is used to measure the intensity of the rays of the emission wavelength, which corresponds to the excitation wavelength, to determine. The intensity of the luminescence at wavelengths, that of the emission wavelength of a particular conjugate Dye, indicates the amount of antigen  which is bound to the antibody to which the dye is attached is. In certain cases, a single wavelength The suggestion used to make luminescence from two or to excite several materials in a mixture, wherein any fluorescence at a different wavelength and the amount of each labeled species can be measured that their individual fluorescence intensity at the respective Fluorescence wavelength detected. If desired, can a light absorption detection method can be used. The two-stage process according to the invention can be based on any one System to be applied in which one with a dye conjugated primary material and the dye or light absorption detection system is used to control the To detect the presence of another material on which the conjugate of the primary material is directed into the dye is. For example, the dye on Fragment of DNA or RNA conjugated to a dye to form a conjugated DNA or RNA fragment, which then is aimed at a main strand of DNA or RNA, to which the piece is complementary. The same test procedure can be used to check for the presence of any to capture complementary main strand of DNA.

The cyanine and related dyes according to the invention are particularly good for analyzing a mixture of components suitable for dyes with a variety of excitation and emission wavelengths are required because special cyanine and related dyes synthesized can be a wide range of excitation and Have emission wavelengths. Special cyanine and related Dyes with specific excitation and emission wavelengths can be synthesized by the number of the methine groups is varied or by the cyanine ring structures be modified. In this way it is possible  Synthesize dyes with special excitation wavelengths, a special excitation light source, like one Lasers, for example a HeNe laser or a diode laser, correspond to.

The invention relates to the covalent reaction of highly luminescent and highly light-absorbing cyanine and related dye molecules under reaction conditions with amine, hydroxyl, aldehyde, sulfhydryl or other groups on proteins, peptides, carbohydrates, nucleic acids, derivatized nucleic acids, lipids, certain other biological Molecules, biological cells and non-biological materials, for example soluble polymers, polymer particles, polymer surfaces, polymer membranes, glass surfaces and other particles and surfaces. Because luminescence involves highly sensitive optical techniques, the presence of these dye "labels" can be detected and quantified even if the "label" is only present in very small amounts. Thus, the dye-labeling reagents can be used to measure the amount of a material that has been labeled. The most suitable dyes are highly light-absorbing ( ε = 70,000 to 250,000 l / mol-cm or higher) and very luminescent, and they have quantum yields of at least 5% to 80% or more. The qualitative properties concern the dyes themselves and dyes that are conjugated to a marked material.

An important application for these color marking reagents is the production of luminescent monoclonal antibodies. Monoclonal antibodies are protein molecules that very closely and very specifically to certain chemical sites or "markers" on cell surfaces or within Bind cells. These antibodies therefore have an enormous  Research suitability and clinical suitability for identification of certain cell types (for example HLA classification, T cell subsets, bacterial and virus classification etc.) and diseased cells. In the past, the amount of antibody bound to a cell thereby determined quantitatively that you can get the antibody in different ways has marked. The marking is with a radioactive marking (Radioimmunoassay), an enzyme (ELISA techniques) or a fluorescent dye (usually fluorescein, Rhodamine, Texasrot® or Phycoerythrin) have been accomplished. Most manufacturers and users of clinical antibody Reagents like those with the use of radioactive Tracers inherent problems get away, so the Luminescence as one of the most promising alternatives is seen. In fact, many companies are now delivering Fluorescein, Texasrot®, Rhodamine and Phycoerythrin labeled monoclonal antibodies.

In recent years, optical / electronic instrumentation for the detection of fluorescent antibodies on cells got more complicated. For example Flow cytometry can be used to measure the amount of fluorescent antibody on individual cells with a Determine rate of up to 5000 cells per second. Also have microscopic and solution fluorescence techniques Progress made. These instruments can fluoresce at many wavelengths of UV, visible and near Excite the IR range of the spectrum. But most can the currently available, usable fluorescent labeling Reagents only excited in the 400 to 580 nm range of the spectrum will. The exceptions are some of the pigments from Phycobiliprotein-type that has been isolated from marine organisms are and which can be covalently attached to proteins. These can be excited at slightly lower wavelengths  will. There is therefore a large spectral window in the range from 580 to about 900 nm where it is necessary that new ones Labeling reagents for labeling biological and non-biological materials become available, the Analysis carried out with the currently available instrumentation shall be. New reagents in this spectral range are stimulable, would enable multi-color To perform luminescence analyzes of markers on cells because Antibodies with different specificities marked with a different colored fluorescent dye could become. Thus, the presence of several Markers can be determined simultaneously for each analyzed cell.

The invention also relates to luminescent (fluorescent or phosphorescent) cyanine, merocyanine and styryl Dyes themselves, which are biological and non-biological Materials can be covalently attached. Merocyanine and styryl dyes are used for the purposes of this invention considered related to cyanine dyes. The new Labeling reagents themselves, but especially if they are conjugated to a marked component by light of the first defined wavelengths, for example by light in wavelength ranges of the spectrum of 450 up to 900 nm. The background fluorescence from Cells are generally made at a low wavelength. Therefore, the labeling reagents will face each other distinguish the background fluorescence. Of special interest are the derivatives that absorb light at 633 nm, because they are cheap, intense, stable and durable HeNe- Laser sources can be excited. Light defined by second Wavelengths by the highlighted component fluorescent or phosphorescent, can then be detected will. That has fluorescent or phosphorescent light  generally a longer wavelength than the excitation light. In the detection stage, a luminescence microscope can be used used a filter to absorb stray light the excitation wavelength and the passage of the wavelength that corresponds to the luminescence, that of the respective The dye label corresponds to that used with the sample becomes. Such an optical microscope is for example described in US Pat. No. 4,621,911.

Not all cyanine and related dyes are luminescent. However, the dyes of the invention include those cyanine and related dyes that are luminescent. They are relatively photostable and many are soluble in the reaction solution, preferably an aqueous solution. The conjugated dyes themselves, but especially if they are conjugated with a labeled component, have molar extinction coefficients ( ε ) of at least 50,000 and preferably at least 100,000 liters per mol-cm. The extinction coefficient is a measure of the ability of the molecules to absorb light. The conjugated dyes according to the invention have quantum yields of at least 2% and preferably at least 10%. For this purpose, the conjugated dyes according to the invention absorb and emit light in the spectral range from 400 to 900 nm and preferably in the spectral range from 600 to 900 nm.

Arylsulfonated dyes

It has now been found that the aryl sulfonate described herein or arylsulfonic acid dyes by their nature are more fluorescent and improved photostability and have water solubility properties than similar dyes without aryl sulfonate or aryl sulfonic acid groups. The Terms used herein include aryl sulfonate or aryl sulfonic acid  are said to be arylsulfonic acid groups or arylsulfonate groups denote, where the groups mentioned to aromatic Ring structures are attached, including a single one aromatic ring structure or a condensed ring structure, for example a naphthalene structure. The only aromatic ring structure or the condensed aromatic Ring structure can be in polymethine, cyanine, or merocyanine Styryl dyes may be present.

Many dyes with planar molecular structures with inclusion of common cyanine dyes tend to be aqueous Solution to form dimers and higher order aggregates and especially when also inorganic Salts are present, such as in buffered Solutions and physiological saline is the case. These Aggregates usually have absorption bands that go to that short wavelength side of the monomer absorption shifted are, and they are generally very weakly fluorescent Species. The tendency of cyanine dyes, in aqueous Solution to form aggregates is particularly easy in the Photography well known (West, W., and Pierce S., J. Phys. Chem., 69: 2894 (1965); Sturmer, D.M., Spec. Top in heterocyclic Chemistry, 30 (1974)).

Many dye molecules, and especially cyanine dye molecules, tend to form aggregates in aqueous solution. The aryl sulfonate dyes have been found to have a minimal tendency to form these aggregates. When used to form fluorescent labeling reagents, the aryl sulfonate dyes have a reduced tendency to form aggregates when bound to proteins or other molecules such as antibodies with high surface densities. The tendency of a particular dye molecule to form aggregates in a saline solution (e.g. 150 mM sodium chloride solution) can be taken as a measure of the tendency of the same dye molecule to form aggregates on the surface of proteins. It is therefore desirable for dye molecules to have a minimal tendency to form aggregates in aqueous salt solutions. The values of Figure 2 show that a particular arylsulfonated dye has only a low tendency to form aggregates even at high concentrations in aqueous saline.

Fig. 1 shows the monomer absorption spectrum and the dimer absorption spectrum of a typical cyanine dye, dissolved in an aqueous buffer. The dye used to generate these spectra, N, N'-disulfobutylindodicarbocyanine, has no arylsulfonate groups and easily forms dimers even at concentrations in the submillimolar range. The dimer spectrum was calculated from the spectra of the dye at various concentrations (cf. West and Pearce's method, 1965). At a 3 mM concentration in a phosphate buffered saline, the absorptions of the monomer and dimer bands were approximately the same.

The spectrum of the improved sulfoindodicarbocyanine, N, N'-diethyl-indodicarbocyanine-5,5'-disulfonic acid, is shown in FIG. 2. This dye showed no evidence of aggregation in the saline at concentrations up to 10 mM.

The effectiveness with which a certain reactive dye couples to a protein, for example an antibody, is usually determined under defined reaction conditions. The dye tested was the bis-N-hydroxysuccinimide ester of N, N'-di-carboxypentyl-indodicarbocyanine-5,5'-disulfonic acid. Fig. 3 shows that this active sulfocyanine dye ester reacts effectively with sheep immunoglobulin in a carbonate buffer at pH 9.2, thereby forming covalently labeled antibody molecules that have a dye to antibody molar ratio in the range of less than 1 to more than 20, depending on the relative dye and antibody concentrations in the reaction solution. The slope of the linear least squares of the values shows that under these conditions the labeling efficiency of this dye is about 80%. In similar studies, fluorescein isothiocyanate (FITC) reacted with an effectiveness of about 20%.

The reactivity of the active ester of the new sulfoindodicarbocyanine dye was examined by labeling sheep immunoglobulin (IgG). The protein (4 mg / ml) was dissolved in 0.1 M carbonate buffer (pH = 9.2). Aliquots of the reactive dye, dissolved in anhydrous dimethylformamide, were added to the protein samples to give the original dye: protein molar ratios. After 30 minutes, the protein was separated from the unconjugated dye by gel permeation chromatography (Sephadex G-50). The resulting dye: protein molar ratios were determined spectrophotometrically and are shown in FIG. 3.

At low dye to protein ratios, the absorption spectra of the labeled proteins show bands that closely correspond to the spectra of the free monomeric dye. Antibody molecules that have been strongly labeled (high dye: protein ratios) or that have been labeled with dyes with a high tendency to aggregate in aqueous solutions are often found to have new absorption peaks that appear at shorter wavelengths than the absorption bands of the monomeric dye in aqueous solution. The wavelength of the new absorption peak often falls in a range which is characteristic of the dimer absorption spectrum of the dye (cf. FIG. 1).

More strongly labeled antibodies have higher ratios of short to long wavelength absorption peaks. This shorter wavelength absorption peak can be seen in Fig. 4 at approximately 590 nm. The longer wavelength peak (at 645 nm) in Fig. 4 is due to monomeric dye molecules bound to the antibody. The labeling reagent used to prepare the antibody absorption spectrum in Figure 4 (the bis-N-hydroxysuccinimide ester of N, N'-disulfobutyl-indodicarbocyanine-5,5'-acetic acid) has no aryl sulfonate groups and forms easily in aqueous salt solutions and on antibodies with which it has been reacted, dimers. It is of crucial importance that the fluorescence excitation spectra of these antibodies show that the excitation of the labeled antibodies at the short wavelength peak does not generate as much fluorescence as when the excitation at the longer wavelength peak. This observation is consistent with the idea that the shorter wavelength absorption peak is due to the formation of non-fluorescent dimers and aggregates on the antibody molecules.

It has been found that the labeling reagent used to obtain the values of FIG. 3, consisting of an aryl sulfonate cyanine dye, does not readily aggregate on the antibody molecules. This results from the considerably smaller absorption peak at wavelengths at which dimers characteristically absorb (cf. FIG. 5). This is important because antibodies and other proteins labeled with these "non-aggregating" labeling reagents should result in more fluorescent labeled proteins. In fact, the arylsulfocyanines produce glossy fluorescent antibodies even when the average dye / antibody ratio is relatively high (see FIG. 6).

Sheep immunoglobulin (IgG) labeled with a carboxyindodicarbocyanine dye is shown in FIG. 4. Figure 5 shows the protein that has been conjugated to the new sulfiindodicarbocyanine dye. The presence of increased dimers (see Fig. 1) in the former sample is evident. Although the molar ratio of dye: protein was approximately the same in both preparations, the protein shown in FIG. 5 was considerably more fluorescent than the sample shown in FIG. 4.

In order to obtain shiny or brightly fluorescent antibodies or other proteins which have been labeled with fluorescent dyes, it is important that the mean quantum yield per dye molecule on the protein is as high as possible. It has generally been found that as the surface density of the dye molecules on the protein increases (ie, when the ratio of dye to protein increases), then the mean quantum yield of the dyes is reduced. This effect has sometimes been attributed to the extinction that occurs as a result of a dye-dye interaction on the surface of more labeled proteins. The formation of non-fluorescent dimers on protein surfaces can certainly contribute to this quenching. Figure 6 shows that the mean quantum yield of an arylsulfocyanine dye slowly decreases as the dye / protein ratio increases (curve with diamond symbols). In contrast, the short one with the round symbols shows that a very rapid decrease in the mean quantum yield for the conjugate of a non-arylsulfocyanine dye (N, N′-disulfobutylindodicarbocyanine 5-isothiocyanate) takes place when the dye / protein Ratio increases. Therefore, the sulfocyanine dye shown in Fig. 6 gives a more glossy fluorescent antibody than the other dye, particularly in the range of 1 to 10 dye molecules per antibody molecule.

The mean fluorescence quantum yield of the individual dye molecules on the labeled proteins is a measure of the fluorescence signal available from these biomolecules. Values of sheep immunoglobulin (IgG) labeled with the new sulfoindodicarbocyanine dye in phosphate buffered saline are shown in the curve with diamond symbols of FIG. 6. The curve with round symbols in FIG. 6 shows proteins which have been labeled with a reactive indodicarbocyanine isothiocyanate dye for comparison. In Fig. 6, the quantum yields with a dye / protein ratio of zero represent the values for the methylamine adducts of the reactive dyes (free dye) in the buffer.

Background procedure

Luminescence probes are valuable reagents for analysis and separation of molecules and cells and for detection and quantitative determination of other materials. A very small number of luminescent molecules can under optimal circumstances. Barak and Webb see less than 50 fluorescence lipid analogs related to the LDL reception of cells using a SIT camera, J. Cell. Biol. 90: 595-605 (1981). The flow cytomerism can used to make less than 10,000 fluorescein molecules to grasp that with particles or certain cells are associated (Muirhead, Horan and Poste, Bio / Technology  3: 337-356 (1985)). Some specific examples of the application of fluorescence probes are (1) the identification and Separation of subpopulations of cells in a mixture of Cells by the techniques of fluorescence flow cytometry, fluorescence-activated cell sorting and fluorescence Microscopy; (2) determining the concentration a substance that binds to a second species (for example Antigen-antibody reactions), in the technique of Fluorescence immunoassays; (3) the localization of substances in gels and other insoluble carriers by the techniques fluorescence staining. These techniques are used by Herzenberg et al., "Cellular Immunology", 3rd edition, chapter 22; Blackwell Scientific Publications, 1978 (fluorescent activated cell sorting); and by Goldman, "Fluorescence Antibody Methods ", Academic Press, New York, 1968 (fluorescent Microscopy and fluorescence staining); and in "Applications of Fluorescence in the Biomedical Sciences " by Taylor et al., Alan Liss Inc., 1986.

When using fluorescent agents for the above Purposes exist with regard to the choice of the fluorescent agent many restrictions. One limitation is the absorption and emission properties of the fluorescent agent, because there are many ligands, receptors and materials in the test sample, for example blood, urine, cerebrospinal fluid, fluoresce and the exact determination of the fluorescence of the disturb fluorescent sample. This phenomenon is called Auto fluorescence or background fluorescence. A Another aspect is the ability of the fluorescent agent with ligands and receptors and other biological and conjugate nonbiological materials, and the Effect of such conjugation on the fluorescent agent. In many situations, the conjugation to another Molecule to a significant change in fluorescence  Properties of the fluorescent agent lead and in some Cases even the quantum performance of the fluorescent agent is essential destroy or diminish. It is also possible that the conjugation with the fluorescent agent the function of inactivated molecule to be labeled. A third consideration lies in the quantum power of the fluorescent agent, which for sensitive detection should be high. A fourth Consideration is the light absorption capacity or the extinction coefficient of the fluorescent agent that is as large as should be possible. It is also important whether the React fluorescent molecules with each other when they are in close proximity, which can lead to self-extinguishing would. Another fear is whether a non-specific one Binding of the fluorescent agent to other compounds or container walls either yourself or in conjunction with the compound to which the fluorescent agent conjugates is done.

The applicability and value of the methods described above are closely related to the availability of suitable fluorescent Connected paired. In particular, there is a need for fluorescent substances in the longer wavelength range of the visible range (yellow to near infrared) emit as the excitation of these chromophores less autofluorescence and also results in multiple chromophores, which at different wavelengths that fluoresce simultaneously can be analyzed if the total visible and the near infrared regions of the spectrum can be used. Fluorescein, a widely used fluorescent compound, is a suitable emission link in the green Range, although with certain immunoassays and cell analysis systems the background autofluorescence, which by excitation is generated at fluorescein absorption wavelengths limits the sensitivity of detection. However  the conventional red fluorescent marker Rhodamine has been shown to be less effective than fluorescein. Texasrot® is a suitable marking agent, which at 578 nm can be excited and fluoresces at a maximum of 610 nm.

Phycobiliproteins make an important contribution because of their high extinction coefficients and the high quantum yield made. These chromophore-containing proteins can be covalent are bound to many proteins and they are in Fluorescence antibody assays in microscopy and the Flow cytometry is used. However, the phycobiliproteins have following disadvantages: (1) the protein labeling procedure is relatively complex; (2) protein labeling performance is usually not high (typically a medium of 0.5 phycobiliprotein molecules per protein); (3) that Phycobiliprotein is a natural product, and its manufacture and cleaning is complex; (4) which are phycobiliproteins expensive; (5) there are no phycobiliproteins as labeling Reagents available that continue to the red area of the spectrum fluoresce as allophycocyanin, which maximum at 680 nm fluorescent; (6) the phycobiliproteins are chemical relatively unstable; (7) They are easily photo bleached subjected; (8) the phycobiliproteins are large proteins with molecular weights in the range of 33,000 to 240,000, and they are larger than many materials that are marked are said to be derivatized, such as metabolites, drugs, hormones Nucleotides and many proteins including antibodies. The latter disadvantage is of particular importance because antibodies, avidin, DNA hybridization probes, hormones and small molecules with the big phycobiliproteins are marked, may not be able to bind to their targets because of steric constraints, imposed by the size of the conjugated complex will. The rate of binding of conjugates to targets is opposite  low molecular weight conjugates slowly.

By other techniques such as histology, cytology, immunoassays, would also have significant benefits from using one Fluorescent agent with high quantum efficiency, absorption and emission properties at longer wavelengths and with simple execution of the conjugation by a non-specific interference are essentially free.

According to the invention, reactive fluorescent arylsulfonated Cyanine and related dyes with relative large extinction coefficients and high quantum yields for the purpose of recording and quantifying marked components used.

Fluorescent cyanine and related dyes can for marking biological materials such as antibodies, Antigens, avidin, streptavidin, proteins, peptides, derivatized nucleotides, carbohydrates, lipids, biological Cells, bacteria, viruses, blood cells, tissue cells, Hormones, lymphokines, biological trace molecules, toxins and drugs. Fluorescent dyes can also be used to mark non-biological materials, such as soluble polymers and polymers and glass particles, drugs, Conductors, semiconductors, glass and polymer surfaces, Polymer membranes and other solid particles can be used. The component to be marked can also be in a mixture with other materials. The mixture in which the Labeling reaction takes place, a liquid mixture, in particular an aqueous mixture. The capture level can with the mixture in liquid or dry state, for example, a slide.  

According to the invention it is necessary that cyanine dyes by incorporating a reactive group into the cyanine molecule be modified. This reactive group staples covalently to a target or target molecule, preferably to an amine or hydroxy site, and in some cases a sulfhydryl or aldehyde site. The invention uses also the modification or use of cyanine and thus related dye structures to their solubility in the Increase test fluid to (1) handle it To make labeling reactions easier, (2) the aggregation of the dye on the surface of the proteins to be labeled help prevent and (3) non-specific binding from labeled materials to biological materials and to prevent surfaces and the assay device help.

The cyanine and related dyes offer one important advantage over currently available fluorescent Labeling reagents. The first is cyanine and therefore related dyes have been synthesized in one Absorb range of the spectrum from 400 to almost 1100 nm and emit. Thus, reactive derivatives of these dyes be prepared for assays that require simultaneous Require measurement of a number of marked materials. Multi-color (or multi-parameter) analysis of this type can in the interest of simplicity, cost effectiveness or to determine ratios of different marked Species on each particle in a complex mixture of Particles may be appropriate (for example the ratios of Antigen markers on individual blood cells in a complex Mixture by multi-parameter flow cytometry or Fluorescence microscopy). Second, absorb and fluoresce many cyanine and related dyes strongly. Third, there are many cyanine and related dyes  relatively photostable, and they do not bleach easily in the fluorescence microscope. Fourth, cyanine and related derivatives are made, the simple and are effective coupling reagents. Fifth, there are many Structures and synthetic procedures available, and the class of dyes is diverse. It can therefore many structural modifications are made to to make the reagents more or less water soluble. Their charge can be changed so that they are the molecule do not disturb to which they are attached, making the non-specific Bond can be reduced. For the sixth in contrast to the phycobiliproteins are the cyanine dyes relatively small (molecular weight = 1000) so that it not significantly with the ability of the marked Interfering molecule sterically, their binding site quickly to achieve or perform their function.

Thus, cyanine dye markers bring many potential Advantages with itself. These dyes can do this used to make one or more components in one Liquid, especially an aqueous liquid, selective to mark. The marked components can then can be detected by optical or luminescence methods. Alternatively the marked component can be used to to stain a second component for which they are strong Has affinity. The presence of the second component is then determined by optical or luminescence methods. In this case, the dye is treated with an amine, Hydroxy, aldehyde or sulfhydryl group on the marked Component implemented. For example, the highlighted Component be an antibody, and the stained component, for which she has a strong affinity can be a biological one Cell, an antigen or a hapten or a biological Cell or particle called antigen or  Contains hapten. According to another example, the labeled component avidin, and the stained component can be a biotinylated material. Lectins, conjugated to polymethine-cyanine dyes used to capture specific carbohydrate groups and determine quantitatively. Furthermore, luminescence Cyanine and related dyes on fragments be pinned by DNA or RNA. The marked fragments of DNA or RNA can be used as fluorescence hybridization probes used to the presence and the amount of specific complementary nucleotide sequences in Identify samples that contain DNA or RNA. Also the dye can be attached to a hormone or to a ligand (for Example a hormone, a protein, a peptide, a lymphokine, a metabolite), which is added afterwards a receptor is added.

Reactive described in patent specifications for other purposes Cyanine dyes

According to US Pat. No. 4,337,063 (Miraha et al.), 44 04 289, 44 05 711 and 44 14 325 (Masuda et al.) Is a variety of Cyanine dyes have been synthesized, the active N-hydroxysuccinimide ester Groups included. These patents show that these reagents are used as photographic sensitizers can be. The possible fluorescence properties these reagents are not mentioned in the patents. Indeed are not fluorescent properties for these processes either required. Most of those in these patents mentioned dyes are only weakly fluorescent, and they are not particularly photostable. Furthermore are their solubility properties for many applications not optimal, which marked a fluorescence detection of Materials would include.  

In GB-PS 15 29 202 (Exekiel et al.) Are numerous Cyanine dye derivatives described as covalently reacting Molecules are used. The ones in these reagents reactive group used is an azine group to which mono- and belong to dichlorotriazine groups. This publication relates focus on the development and use of this Reagents as sensitizers for photographic films. No fluorescence is required for this process, and most of the reagents described therein are not fluorescent. Finally, this publication refers not on the development and use of reactive Cyanine dyes for the purpose of detection and quantitative Determination of marked materials.

The present invention relates to covalent methods Attaching luminescent cyanine and cyanine-like dyes of biological materials, non-biological materials and macromolecules and particles so these materials be marked luminescent. That way it can marked material by luminescence detection methods recorded and / or determined quantitatively.

The invention relates to a method for detecting a component in a liquid that is characterized is that you add a dye to the liquid mentioned adds that from the group consisting of cyanine, merocyanine and styryl dyes is selected and that in the liquid is soluble and contains a substituent so that it with amine and hydroxy groups and possibly with aldehyde and sulfhydryl groups on said component covalently becomes reactive so that the named component is marked becomes. The marked component is then luminescent or light absorption methods detected and / or quantitative certainly. If the labeled component is an antibody, a  DNA fragment, a hormone, a lymphokine or a medicine then the marked component can be used to to the presence of a second component to which it is bound to identify what the second component is can be recorded and / or determined quantitatively.

Any level of luminescence or light absorption detection can be applied. For example, the Detection level can be an optical detection level at which the liquid with light of the first defined wavelengths is irradiated. Light with second defined wavelengths, that fluoresces from the labeled component or is phosphoresced, is then detected. The capture can also be done by optical light absorption. So For example, the detection level can be carried out in this way be that light with first defined wavelengths through the liquid and then the wavelength of the light that has passed through the liquid is determined.

If desired, the detection level can also be a chemical one Analysis involves chemically attaching the cyanine or a related chromophore to detect the component.

The basic structures of the cyanine, merocyanine and styryl dyes, that can be modified to provide covalent labeling Generating reagents are shown below:  

More specific examples of polymethine cyanine dyes are as follows:

Are in these structures
X and Y from the group

selected;
Z is selected from the groups O and S and
m is an integer selected from the group consisting of 1, 2, 3 and 4.

In the above formulas, the number of methine groups partly determines the excitation color. The cyclic azine structures can also determine the excitation color in part. Higher values of m often contribute to increased luminescence and absorption. At values of m above 4, the connection becomes unstable. A further luminescence can be given by modifications of the ring structures. If m = 2, the excitation wavelength is about 650 nm and the compound is very fluorescent. Maximum emission wavelengths are generally 15 to 100 nm higher than the maximum excitation wavelengths.

At least one, preferably only one, and possibly two or more of the groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇ in each molecule is a reactive group for attaching the dye to the marked component. For certain reagents, at least one of the groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇ also have a group on each molecule which increases the solubility of the chromophore or which Selectivity of the labeling of the labeled component is impaired or the position of the marking of the marked Component affected by the dye.

In the formulas mentioned, at least one of the above includes Groups R₈, R₉ (if available) and R₁₀ (if available) at least one sulfonate group. The name sulfonate is said to  Include sulfonic acid because the sulfonate group is only one represents ionized sulfonic acid.

Reactive groups attached directly or indirectly to the chromophore can be added to the groups R₁, R₂, R₃, R₄, R₅, Forming R₆ and R₇ can include reactive groups, for example groups containing isothiocyanate, isocyanate, Monochlorotriazine, dichlorotriazine, mono- or dihalo- substituted pyridine, mono- or dihalo-substituted Diazine, maleimide, aziridine, sulfonyl halide, acid halide, Hydroxysuccinimide ester, hydroxysulfosuccinimide ester, Imidoester, hydrazine, azidonitrophenyl, azide, 3- (2- Pyridyldithio) propionamide, glyoxal and aldehyde included.

Specific examples of the groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇, which are particularly good for marking components with available Amino, hydroxy and sulfhydryl groups are suitable are the following groups:

where at least one of Q or W is a leaving group, such as I, Br, Cl, is:

Specific examples of groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇, which is particularly good for marking components with available ones Sulfhydryl groups are suitable and which in one Two-step procedure used for labeling antibodies are the following groups:

where Q is a leaving group such as I or Br:

where n is 0 or an integer.

Specific examples of the groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇, which are particularly good for marking components through light-activated networking are suitable the following groups:

For the purpose of increasing the water solubility or reducing an undesirable non-specific binding of the labeled component to unsuitable components in the sample or to reduce reactions between two or more reactive chromophores on the labeled component, which could lead to the extinction of the fluorescence, the groups R 1 , R₂, R₃, R₄, R₅, R₆ and R₇ can be selected from the well-known polar and electrically charged chemical groups. Examples include -EF, where F is hydroxy, sulfonate, sulfate, carboxylate, substituted amino or quaternary amino and where E is a spacer group, such as - (CH₂) _n -, where n is 0, 1, 2, 3 or 4. Suitable examples include alkyl sulfonate, - (CH₂) ₃SO³⊖ and (CH₂) ₄-SO³⊖.

The polymethine chain of the luminescent dyes according to the invention can also have one or more cyclic chemical groups contain bridges between two or more of the Form carbon atoms of the polymethine chain. These bridges could serve chemical or photostability of the dye and they could be used the absorption and emission wavelengths of the dye to change or its extinction coefficient or to change its quantum yield. Improved solubility properties could get through this modification will.

According to the invention, the marked component can be one of the following Substances are selected: antibodies, proteins, peptides, Enzyme substrates, hormones, lymphokines, metabolites, receptors, Antigens, haptens, lectins, avidin, streptavidin, Toxins, carbohydrates, oligosaccharides, polysaccharides, Nucleic acids, deoxynucleic acids, derivatized nucleic acids, derivatized deoxynucleic acids, DNA fragments, RNA fragments, derivatized DNA fragments, derivatized RNA fragments, natural drugs, virus particles,  Bacterial particles, virus components, yeast components, blood cells, Blood cell components, biological cells, non-cellular Blood components, bacteria, bacterial components, natural and synthetic lipid vesicles, synthetic Drugs, poisons, polluting substances, Polymers, polymer particles, glass particles, glass surfaces, Plastic particles, plastic surfaces, polymer membranes, Conductors and semiconductors.

A cyanine or a chromophore related to it can also be made when implemented with a component will absorb light at 633 nm. At the registration level a helium-neon laser can be used that Light is emitted at this wavelength of the spectrum. Also can produce a cyanine or related dye which, when implemented with a component, Can absorb maximum light between 700 nm and 900 nm. In this case, a laser diode can be used in the detection stage used the light in this area of the spectrum emitted.

selectivity

The reactive groups given above are relatively specific for marking certain functional groups Proteins and other biological or non-biological Molecules, macromolecules, surfaces or particles, provided that suitable reaction conditions including suitable pH conditions.

Properties of the reactive cyanine, merocyanine and styryl Dyes and their products

The spectral properties of the dyes according to the invention are not significantly changed by the functionalization described here. The spectral properties of the labeled proteins and other compounds are also not very different from those of the base dye molecule that has not been conjugated to a protein or other material. The dyes described herein, alone or conjugated to a labeled material, generally have large extinction coefficients ( ε = 100,000 to 250,000), high quantum yields as high as 0.4 in certain cases, and they absorb and emit light in the 400 spectral range up to 900 nm. They are therefore of particular value as labeling reagents for luminescence detection.

Optical detection methods

For the detection or determination of a marked or colored Component can use any method. The detection method can use a light source that the mixture containing the marked material with Illuminated light with the first defined wavelengths. Known Devices are used that emit light with second wavelengths detect what light is let through the mixture or that is fluorescent from the mixture or is luminescent. Such detection devices are for example fluorescence spectrometer, absorption spectrophotometer, Fluorescence microscope, translucent Microscopes and flow cytometers, optical fiber sensors and Immunoassay instruments.

Chemical analysis methods can also be used in the method according to the invention used to attach the dye to the selected component or components capture. Examples of chemical analysis methods  are infrared spectrometry, NMR spectrometry, absorption spectrometry, fluorescence spectrometry, mass spectrometry and chromatography.

The invention is illustrated in the examples.

example 1 Effect of pH on the conjugation of Sulfoindodicarbocyanine with protein

Samples of sheep γ- globulin (4 mg / ml) in 0.1 M carbonate buffers (pH = 8.5, 8.9 and 9.4) were at room temperature with a 10-fold molar excess of the following active sulfoindodicarbocyanine ester ( m = 2) mixed.

At appropriate times, ranging from 5 seconds to 30 minutes protein samples were noncovalent attached dye by gel permeation chromatography separated on Sephadex® G-50. The maximum mark of the Proteins took place after 10 minutes and gave final molar ratios  of dye / protein of 5.8, 6.4 and 8.2 for the samples, which were incubated at pH values of 8.5, 8.9 and 9.4 were. The times it took to dye / Protein ratio of 5 and quantum yields of Products are to be obtained at the different pH values compiled in the following table:

These values show that the protein labeling with this Dye is better at pH 9.4 than at one pH below 9. In the buffer with higher pH the conjugation reaction was very fast, but the labeling effectiveness was excellent, and the product was more fluorescent. The value of the quantum yield gives the average quantum yield per dye molecule on the labeled Protein again.

Example 2 Conjugation of the active sulfoindocarbocyanine ester with protein

Sheep γ- globulin (1 mg / ml), dissolved in phosphate-buffered saline (PBS) with a pH of 7.4, was adjusted to a pH of 9.4 with 0.1 M sodium carbonate. A cyanine dye marker (structure in Example 1, m = 1) was added to aliquots of this protein solution to give different dye / protein molar ratios. After 30 minutes of incubation at room temperature, the mixtures were separated by gel permeation chromatography on Sephadex® G-50, eluting with PBS. The molar ratio of the dyes covalently attached to the proteins in the products was 1.2, 3.5, 5.4, 6.7 and 11.2 for initial dye / protein ratios of 3, 6, 12, 24 or 30.

Example 3 Labeling of AECM dextran with a sulfoindodicarbocyanine

N-Aminoethyl-carboxamidomethyl (AECM) dextran with an average of 16 amino groups per dextran molecule was synthesized from dextran with an average MW of 70,000 (Inman, JK, J. Immunol. 114: 704-709 (1975)). Part of the AECM-Dextran (1 mg / 250 µl), dissolved in 0.1 m carbonate buffer, with a pH of 9.4 became 0.2 mg of the active sulfoindodicarbocyanine ester (structure in Example 1, m = 2), whereby a dye / protein molar ratio of 10 was obtained. The mixture was stirred at room temperature for 30 minutes. The dextran was then separated from non-conjugated dye by Sephadex® G-50 gel permeation chromatography using ammonium acetate (50 mM) as the elution buffer. An average of 2.2 dye molecules were covalently attached to each dextran molecule.

Example 4 Labeling of a specific antibody with active Sulfoindodicarbocyanine esters

Sheep γ- globulin (1 mg / ml) specific for murine IgG in 0.1 m carbonate buffer (pH = 9.4) was mixed with the active sulfoindodicarbocyanine ester (structure in Example 1, m = 2) at a ratio of 8 Dye molecules mixed per protein molecule. After incubating at room temperature for 30 minutes, the labeling mixture was separated by gel filtration on Sephadex® G-50, which had been equilibrated with phosphate-buffered saline (pH = 7.4). The protein obtained contained an average of 4.5 dye molecules covalently attached to each protein molecule.

Example 5 Staining and microscopic visualization of human Lymphocytes with sulfoindodicarbocyanine dye, the was conjugated to sheep anti-mouse IgG antibody

Freshly isolated peripheral blood lymphocytes were treated with mouse anti- β 2-microglobulin (0.25 µg / 10⁶ cells) at 0 ° C for 30 minutes. The cells were washed twice with DMEM buffer and then treated with the sheep anti-mouse IgG antibody (1 ug / 10⁶ cells) labeled with sulfoindodicarbocyanine. After incubation at 0 ° C for 30 minutes, excess antibody was removed by centrifuging the cells and the cells were washed again twice with DMEM buffer. Aliquots of the cells were attached to slides for analysis by fluorescence microscopy. Under the microscope, the lymphocytes on the slide were irradiated with light at 610 to 630 nm, and the fluorescence at 650 to 700 nm was recorded with a red-sensitive amplified COHU television camera, which was connected to an image digitizer and a television monitor. The cells stained by this method showed fluorescence under the microscope. In a control experiment, the use of the mouse primary anti- β 2 microglobulin antibody was omitted, but the staining and analysis were otherwise performed as described above. The control sample showed no fluorescence under the microscope, indicating that the sheep anti-mouse antibody labeled with sulfoindodicarbocyanine does not result in significant non-specific binding to lymphocytes.

Claims (51)

1. A method for marking a component of an aqueous liquid, characterized in that

• (a) a luminescent dye from the group consisting of cyanine, merocyanine and styryl dyes, which contains at least one sulfonic acid or sulfonate group attached to an aromatic nucleus, is added to the liquid containing said component, said dye being reactive with said component is and
• (b) reacting the dye with said component so that the dye marks said component.

2. The method according to claim 1, characterized in that that the dye is more than one sulfonic acid or contains sulfonate group. 3. The method according to claim 1, characterized in that said dye has a substituent contains to him with an amine, aldehyde, sulfhydryl  or hydroxy group on the component mentioned covalently reactive to make, and that the dye mentioned with a or several of the groups mentioned. 4. The method according to claim 1, characterized in that the sulfonic acid or sulfonate group the water solubility of the dye increases. 5. The method according to claim 1, characterized in that the sulfonic acid or sulfonate group the dye / dye interactions decreased that a Induce quenching of fluorescence. 6. The method according to claim 1, characterized in that the dye is a cyanine dye is. 7. The method according to claim 1, characterized in that the dye is a merocyanine dye is. 8. The method according to claim 1, characterized in that the component from the group antibodies, Proteins, peptides, enzyme substrates, hormones, lympokines, Metabolites, receptors, antigens, haptens, lectins, Toxins, carbohydrates, oligosaccharides, polysaccharides, Nucleic acids, deoxynucleic acids, derivatized nucleic acids, derivatized deoxynucleic acids, DNA fragments, RNA fragments, derivatized DNA fragments, derivatized RNA fragments, natural drugs, virus particles, virus components, Yeast components, blood cells, blood cell components, biological cells, non-cellular blood components, Bacteria, bacterial components, natural and synthetic Lipid vesicles, synthetic drugs and natural drugs,  Poisons, polluting substances, polymers, Polymer particles, glass particles, glass surfaces, plastic particles, Plastic surfaces, polymer membranes, conductors and semiconductor is selected. 9. The method according to claim 1, characterized in that it is the stage of optical detection of the marked component mentioned. 10. The method according to claim 9, characterized in that said detection level is radiation the marked component with light and the subsequent one Detection of light by the said labeled component is fluoresced. 11. The method according to claim 9, characterized in that the detection level mentioned excites the light with a laser. 12. The method according to claim 9, characterized in that in the mentioned registration level Fluorescence microscopy is used. 13. The method according to claim 9, characterized in that that in the mentioned registration level Flow cytometry is used. 14. The method according to claim 9, characterized in that the mentioned detected component is quantitative is determined. 15. The method according to claim 1, characterized in that the dye mentioned is a styryl Dye is.   16. A method for marking a component in an aqueous liquid, characterized in that

• (a) to the liquid containing said component, a luminescent dye from the group of polymethine dyes based on sulfoindolenine and naphthosulfoindolenine, which contains at least one sulfonic acid or sulfonate group attached to one or more rings of a heterocyclic nucleus, wherein said dye is reactive with said component;
• (b) reacting said dye with said component so that said dye marks said component; and that one
• (c) said labeled component is detected by optical methods.

17. The method according to claim 16, characterized in that that the dye mentioned more than contains a sulfonic acid or sulfonate group. 18. A method for marking a first component and using the marked first component for binding with a second component to form a marked second component, characterized in that

• (a) a luminescent dye from the group consisting of cyanine, merocyanine and styryl dyes, which contains at least one sulfonic acid or sulfonate group attached to an aromatic nucleus, is added to an aqueous liquid which contains said first component, the dye being opposite the first component is reactive;
• (b) reacting said dye with said first component so that said dye labels said first component by luminescence, thereby obtaining a labeled first component;
• (c) attaching said labeled first component to said second component to form a labeled second component; and that one
• (d) said labeled second component is detected by an optical method.

19. The method according to claim 18, characterized in that the dye mentioned more than contains a sulfonic acid or sulfonate group. 20. The method according to claim 18, characterized in that the first component is an antibody and that the second component is a substance from the Group of antigens, haptens, biological cells, viruses and Is particles that carry an antigen and a hapten. 21. The method according to claim 18, characterized in that said first component Lectin is and that said second component is a Substance from the group carbohydrates, macromolecular substances, that carry a carbohydrate, and cells that carry a carbohydrate wear is. 22. The method according to claim 18, characterized in that that said first component avidin or streptavidin and that said second component is a biotinylated material.   23. The method according to claim 18, characterized in that that said first component DNA fragment and that said second component is a is a complementary strand of DNA or RNA. 24. The method according to claim 18, characterized in that said first component Is RNA fragment and that said second component is a complementary strand of DNA or RNA. 25. The method according to claim 18, characterized in that said first component Ligand from the group hormones, proteins, peptides, lymphokines and is metabolite and that said second component is a receptor. 26. The method according to claim 18, characterized in that said first component Polymer particles, which is a substance from the group of antibodies, Nucleic acid fragments, drugs, toxins, hormones, Metabolites, biological cells, bacteria, virus particles, Contains antigens and haptens and that said second component a corresponding antigen, a complementary one Nucleic acid sequence, an antibody or a receptor is. 27. The method according to claim 18, characterized in that said second component Is antibody. 28. The method according to claim 18, characterized in that that said first component is a Substance from the group of antigens, haptens or cells that contain an antigen or hapten, and particles containing a  Containing antigen or hapten is and that the said second component is an antibody. 29. The method according to claim 18, characterized in that that the dye mentioned with an amine, hydroxy or sulfhydryl group on the first Component. 30. The method according to claim 18, characterized in that the dye mentioned with an aldehyde group on said first component. 31. The method according to claim 18, characterized in that the dye mentioned is a cyanine Is dye and deals with an amine or hydroxy group implemented on the first component mentioned. 32. The method according to claim 18, characterized in that the dye mentioned is a cyanine Is dye and deals with an aldehyde or sulfhydryl group implemented on the first component mentioned. 33. The method according to claim 18, characterized in that that said second component the group blood cells, tissue cells, bacteria, viruses, Chromosomes, DNA, RNA, toxins, drugs, hormones and Polymer particles is selected. 34. Luminescence photostable reaction product from a Component which is an amine, aldehyde, sulfhydryl or Contains hydroxyl group and a water-soluble dye from the group polymethine dyes on sulfoindolenine Foundation and naphthosulfoindolenine foundation, the  contains at least one substituent to make it opposite the amine, aldehyde, sulfhydryl or hydroxyl group mentioned on the component mentioned covalently reactive make, with the individual dye molecules on the mentioned reaction product an average molar extinction coefficient of at least 50,000 l per mol-cm, have an average quantum yield of at least 2% and Absorb light in a maximum of 400 to 850 nm range and emit when the marked component is in a aqueous environment. 35. Reaction product according to claim 34, characterized in that the component mentioned Is antibody. 36. Reaction product according to claim 34, characterized in that that the component mentioned the group proteins, peptides, drugs, toxins, metabolites, Lymphokines, carbohydrates, lipids, blood cells, bacterial particles, Virus particles, antigens, haptens, polymer particles, Glass surfaces and polymer surfaces selected is. 37. Reaction product according to claim 22, characterized in that said second component is a DNA or RNA fragment. 38. The method according to claim 1, characterized in that said dye from the group: is selected, where
X and Y from the group are selected;
m is an integer from group 1, 2, 3 and 4;
at least one of said groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇ at least one group from the group of isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or dihalogen-substituted pyridine, mono- or dihalogen-substituted diacine, maleimide, Aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3- (2-pyridyl-dithio) propionamide, glyoxal and aldehyde; and where
at least one of the groups R₈ and R₉ contains at least one arylsulfonyl or arylsulfonate grouping. 39. The method according to claim 38, characterized in that at least one of the groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇ at least one group from the group: contains, wherein at least one of Q or W is selected from the group Cl, Br and I and where n is zero or any integer. 40. The method according to claim 38, characterized in that at least one of said groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇ at least one group from the group: contains. 41. The method according to claim 38, characterized in that at least one of the groups mentioned R₁, R₂, R₃, R₄, R₅, R₆ and R₇ also have at least one grouping from the group hydroxy, sulfonate, sulfate, carboxylate and contains substituted amine or quaternary amine groups. 42. luminescent dye from the group: in which
X and Y from the group are selected;
m is an integer from group 1, 2, 3 and 4;
at least one of said groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇ at least one group from the group of isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or dihalogen-substituted pyridine, mono- or dihalogen-substituted diacine, maleimide, Aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3- (2-pyridyl-dithio) propionamide, glyoxal and aldehyde; and where
at least one of the groups R₈ and R₉ contains at least one arylsulfonyl or arylsulfonate grouping. 43. Luminescent dye according to claim 42, characterized characterized that it is water soluble. 44. Luminescent dye according to claim 42, characterized characterized that he has a molar in water Extinction coefficient of at least 50 000 l per mol-cm, has a quantum yield of at least 2% and that it absorbs light in the spectral range from 400 to 900 nm and emitted. 45. Luminescent dye according to claim 42, characterized characterized that he has a molar in water Extinction coefficient of at least 100 000 l per mol-cm, has a quantum yield of at least 10% and that it absorbs light in the spectral range from 600 to 900 nm or emitted.   46. luminescent dye from the group: wherein
X and Y from the group are selected;
Z is selected from the group O and S;
m is an integer from the group 1, 2, 3 and 4 and at least one of the groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇ is at least one group from the group: contains, where
at least one of Q or W is selected from the group I, Br and Cl and n is 0 or an integer; and where
at least one of the groups R₈ and R₉ (if present) contains at least one sulfonate grouping. 47. luminescent dye from the group: in which
X and Y from the group are selected;
Z is selected from the group O and S;
m is an integer from group 1, 2, 3 and 4;
at least one of the groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇ at least one group from the group: contains, where
n is 0 or an integer; and where
at least one of the groups R₈, R₉, R₁₀ (if present) and R₁₁ (if present) contains at least one sulfonic acid or sulfonate group. 48. luminescent dye from the group: in which
X and Y from the group are selected;
m is an integer from group 1, 2, 3 and 4;
at least one of the groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇ at least one group from the group: contains; and where
at least one of the groups R₈ and R₉ contains at least one arylsulfonic acid or arylsulfonate grouping. 49. Luminescent dye according to claim 42, characterized characterized in that at least one of the Groups R₁, R₂, R₃, R₄, R₅, R₆ and R₇ also groupings to increase water solubility and reduce the contains non-specific binding, the groupings  from hydroxy, sulfonate, sulfate, carboxylate and substituted Amine or quaternary amine groups selected are. 50. A method, characterized in that a material labeled with a sulfocyanine dye with one or more other fluorescent materials, which all light significantly at a single excitation wavelength absorb, but at different wavelengths fluoresce, mixed and then all of the above fluorescent materials with a single Excites wavelength of light and then the amount of each of the fluorescent materials mentioned quantified, by adding their individual fluorescence signals their different fluorescence wavelengths detected. 51. The method according to claim 50, characterized in that with the sulfocyanine dye labeled material an antibody or a DNA hybridization probe is.