DE3943870B4 - Luminescently labeled component of an aqueous liquid - Google Patents

Abstract

luminescent labeled component of an aqueous Liquid, comprising a luminescent dye selected from the group consisting of Cyanine, merocyanine, and styryl dyes, said Dye covalently reactive with the said component and to this is bound, wherein the component is a molecule selected from the group consisting of biological cells, antibodies, proteins, Peptides, enzyme substrates, hormones, lymphokines, metabolites, receptors, Antigens, haptens, lectins, toxins, carbohydrates, sugars, Oligosaccharides, polysaccharides, nucleotides, derivatized Nucleotides, nucleic acids, Deoxynukleinsäuren, derivatized nucleic acids, derivatized deoxynucleic acids, DNA fragments, RNA fragments, derivatized DNA fragments, derivatized RNA fragments and drugs, or wherein the component is selected from the group consisting of soluble Polymers, plastic particles, plastic surfaces, glass particles and glass surfaces.

Description

Cyanine dyes and related polymethine dyes having 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 luminescent properties have so far experienced only a very limited recovery. One such use is, for example, the labeling of the sulfhydryl group of proteins. In a report, Salama, G., Wagoner, AS and Abramson J., entitled "Sulfhydryl Reagent Dyes Solve Rapid Release of Ca ²⁺ from Sarcoplasmin Reticulum Vesicles (SR)" from Biophysical Journal, 47, 456a (1985), found that cyanine chromophores having 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 were used to label and isolate these proteins.

In a report by Wagoner, A.S., Jenkins, P.L., Carpenter, J.P. and Gupta, R., entitled "Kinetics of conformational 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 area of cattle rhodopsin with a cyanine dye has been covalently labeled with an absorbance at 660 nm. Also according to this Report cyanine dyes for specific labeling of the sulfhydryl group a protein used. The use of fluorescence dyes however, is not described in this report.

One Article titled "International Workshop on the Application of fluorescence photobleaching techniques to problems Cell Biology "by Jacobson K., Elson E., Koppel D., Webb W. in Fed. Proc. 42: 72-79 (1983), reports about an article submitted by A. Wagoner.

This refers to cyanine-type fluorescence probes attached to proteins can be conjugated and which can be excited in the deeper red region of the spectrum.

In the three reports mentioned above are the only cyanine probes mentioned, covalently specific to the sulfhydryl group of a protein to pin. The only specifically mentioned cyanine compound is one those having an iodoacetyl group which causes group that the Cyanine dye over the sulfhydryl group becomes covalently reactive. In none of the above Reports the covalent reaction of a cyanine dye with any material other than a protein or any one another group described on a protein as a sulfhydryl group.

Lots However, non-proteinaceous materials have no sulfhydryl groups, and many proteins do not have enough Number of sulfhydryl groups on these groups for the purpose to make the fluorescent probing suitable. Additional to that, that sulfhydryl groups (-SHSH-) readily oxidized to disulfides (-S-S-) in the presence of air and therefore for covalent attachment to a fluorescent probe becomes unavailable.

According to the invention, cyanine and related polymethine dyes have now been developed which have substituent groups which, under suitable reaction conditions, have not only sulfhydryl groups but also amine (-NH ₂ ) and hydroxy (-OH) groups or other groups. like covalently reactive aldehyde (- CHO) groups on proteins and other materials to allow fluorescence and phosphorescence detection of these materials. The invention provides significant advantages over the use of the prior art iodoacetyl-cyanine dyes and their specific reactivity with sulfhydryl groups. Amines and hydroxy groups are more prevalent in proteins and other materials than sulfhydryl groups, and they are more stable. Therefore, when fluorescence cyanine dyes are used to detect the presence of certain proteins, a stronger fluorescence or phosphorescence light intensity signal is emitted because a larger number of dye molecules can be attached to the protein to be probed. Furthermore, amine and hydroxy groups are more likely to be attached to components that are to be labeled, such as polymer particles that inherently contain neither sulfhydryl, amine or hydroxy groups.

The invention also relates to a process in which luminescent cyanine dyes containing a group covalently reactive with amine groups or hydroxy or other reactive groups are used to utilize proteins or other materials having an amine or hydroxy group or other group capable of reacting with the dye in a mixture such that the presence and extent of the labeled protein or protein other material can be detected after the labeled components have been separated by chromatographic methods. Apparently, according to the above references, the sulfhydryl group was 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 plays a significant role in the function of the protein. The authors were therefore able to conclude that the specific location of a sulfhydryl group on a protein structure can be determined. According to these references, the dye specific for the sulfhydryl group has also been used as a probe to detect or produce structural changes in a particular protein. In order to interpret a change in light absorption by the dye or the calcium ion released by dye binding, it was necessary to know where the probe was bound.

There on most protein molecules as few sulfhydryl groups are, these groups can not be sufficiently numerous to provide adequate overall luminescence for detection studies to surrender. In contrast, For example, amine and hydroxy groups are significantly more numerous and they are on a protein molecule widely dispersed, which makes it possible will that one Fluorescence probe is attached to multiple sites on the molecule, whereby an interpretation of the light absorption and fluorescence changes by Facilitation of the capture of the protein is excluded.

The The present invention relates to luminescent polymethine cyanine labeling. and related polymethine dyes, such as merocyanine and styryl dyes, of proteins and other materials including nucleic acids, DNA, Medicines, toxins, blood cells, microbial materials, particles, plastic or glass surfaces, Polymer membranes etc. at an amine or hydroxystelle on the mentioned materials. The dyes are advantageously in an aqueous or another medium containing the labeled material, soluble. The present invention relates to a two-stage labeling method additionally to a one-step marking procedure. In the two-stage Labeling method may involve a primary component, such as an antibody Include on it including Amine, hydroxy, aldehyde or sulfhydryl sites, and the labeled component is used as a probe for the secondary component, like an antigen, for the antibody is specific.

According to the above The prior art discussed was the specificity of the site the attachment by a cyanine probe obtained by a probe was used opposite covalently reactive with a sulfhydryl group. According to the two-stage according to the invention Procedures can Cyanine and related probes in a first stage with amine, Aldehyde, sulfhydryl, hydroxy or other groups on a first Component, such as an antibody, whereupon the antibody has the desired specificity in one second component, such as an antigen, in a second or staining step the specificity by the antigenic site of the Attach to the antibody is determined.

The The present invention also relates to luminescent polymethine cyanine compounds and related compounds that contain groups that they covalently attach to amine, hydroxy, aldehyde or Sulfhydryl groups to be attached to a target molecule. The Invention is further directed to monoclonal antibodies and other components containing these luminescent cyanine compounds are labeled and capable of detecting probes for antigens be. If the target is a cell type, then the present one Be used to increase the amount of labeled antibody measure that is attached to said cell type. The measurement can be done in such a way that one the relative brightness or attenuation of the luminescence of the cells detected.

The The present invention can be used to determine the concentration a particular protein or component in one System to determine. If the number of reactive groups on one Protein that can be reacted with a probe is known then the fluorescence per molecule is known, and the concentration of these molecules in the system can be determined by the total luminescence intensity of the system be determined.

The Procedure can be applied to a variety of proteins or to quantify other materials in a system, by marking all of a mixture of proteins in the system and then the labeled proteins by any means, like chromatographic measures, separates. The amount of separated proteins that luminesce, can be determined. In chromatographic detection systems the location of the dye on the labeled material can be determined.

The Invention may also be used to determine the number of different ones Cells with an antibody are labeled to determine. This provision may be in the way done that one a variety of types of cells labeled in a system and then the labeled cells are separated outside the system. Also can the labeled cells of unlabelled cells outside be separated from the system.

A another embodiment of the present invention a multiparameter method in which a plurality of luminescence cyanine or related dyes used, each to a variety of different primary Components, such as antibodies, attached, each dye being for a different secondary component, as an antigen, is specific to each of a variety of Identify antigens in a mixture of antigens. According to this embodiment is any of the mentioned antibodies Separately with a dye with different light absorption and various luminescence wavelength characteristics as the dye, the used to mark the other probes marked. thereupon become all labeled antibodies given to a biological preparation to be analyzed, the secondary components, like antigens, contains, which can each be stained by specific labeled antibodies. any unreacted dye materials can be obtained from the preparation, for example be removed by washing out if they interfere with the analysis. The biological preparation is then exposed to a variety of excitation wavelengths, with each used excitation wavelength the excitation wavelength a particular conjugated dye. A luminescence microscope or another luminescence detection system, such as a flow cytometer or a fluorescence spectrophotometer, the filter or monochrometer for selecting the excitation wavelength rays and for selecting the wavelengths of the Luminescence is used to determine the intensity of the rays the emission wavelength, that of the excitation wavelength corresponds to determine. The intensity of luminescence at wavelengths that the emission wavelength corresponds to a particular conjugated dye, the amount shows of the antigen bound to the antibody to which the Dye is added. In certain cases can be a single wavelength the excitation can be used to detect luminescence from two or more excite multiple materials in a mixture, each fluorescence at a different wavelength and the amount of each labeled species can be measured thereby that he their individual fluorescence intensity detected at the respective fluorescence wavelength. If desired, For example, a light absorption detection method can be used. The two-stage process according to the invention can be applied to any system in which one with a dye conjugated primary material and the dye ge or light absorption detection system used to indicate the presence of another material too detect the conjugate of the primary material in the dye is directed. For example, the dye may be attached to a fragment of DNA or RNA conjugated to a dye conjugate DNA or RNA fragment, which then on a main strand of DNA or RNA to which the piece is complementary. The same test method can be used to detect the presence from any complementary Main strand of DNA to capture.

The cyanine according to the invention and related dyes are particularly good for the analysis of a Mixture of components suitable in which dyes of a variety of excitation and emission wavelengths are required because special cyanine and related dyes can be synthesized which have a wide range of excitation and emission wavelengths. Special cyanine and related dyes with specific Excitation and emission wavelengths can be synthesized by varying the number of methine groups or by modifying the cyanine ring structures. On This way it is possible dyes with special excitation wavelengths to synthesize to a special excitation light source, such as a laser, for example a HeNe laser or a diode laser, correspond to.

The invention relates to the covalent reaction of highly luminescent and high-light absorbing cyanine and related dye molecules under reaction conditions with amine, hydroxy, aldehyde, sulfhydryl or other groups on proteins, peptides, carbohydrates, nucleic acids, derivatized nucleic acids, lipids, certain other biologicals Molecules, biological cells and non-biological materials, such as soluble polymers, polymer particles, polymer surfaces, polymer membranes, glass surfaces and other particles and surfaces. Since luminescence involves highly sensitive optical techniques, the presence of these dye "labels" can be detected and quantified even if the "label" is present only in very small amounts. Thus, the dye-labeling reagents can be used to measure the amount of 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 have quantum yields of at least 5% to 80% or more. Qualitative properties concern the dyes themselves and dyes that correspond to a labeled material are greedy.

An important application for these color-labeling reagents is the production of luminescent monoclonal antibodies. Monoclonal antibodies are protein molecules that bind very tightly and very specifically to particular chemical sites or "markers" on cell surfaces or within cells. These antibodies therefore have tremendous research capability and clinical utility to identify particular cell types (for example, HLA classification, T-cell subsets, bacterial and viral classification, etc.) and diseased cells. In the past, the amount of antibody bound to a cell has been quantified by labeling the antibody in various ways. The marking has been accomplished with a radioactive label (radioimmunoassay), an enzyme (ELISA techniques) or a fluorescent dye (usually fluorescein, rhodamine, Texas Red ^®, or phycoerythrin). Most manufacturers and users of clinical antibody reagents wish to avoid the inherent problems with the use of radioactive tracers, so that luminescence is considered to be one of the most promising alternatives. In fact, now provide many companies with fluorescein, Texas Red ^®, rhodamine and phycoerythrin labeled monoclonal antibodies.

In In recent years, the optical / electronic instrumentation for the Capture of fluorescent antibodies to cells more complicated become. For example, flow cytometry can be used be to the amount of fluorescent antibody on individual cells at a rate of up to 5,000 cells per second. Also microscopic and solution fluorescence techniques have made progress. These instruments can fluoresce in many wavelength of the UV, visible and near IR region of the spectrum. But you can most of the currently available, usable fluorescence labeling reagents only in the 400 to 580 nm range of the spectrum are excited. The exceptions are some of the pigments of the phycobiliprotein type isolated from marine organisms and which can be covalently attached to proteins. These can at slightly lower wavelengths be stimulated. There is therefore a large spectral window in the area from 580 to about 900 nm, where it is necessary that new Labeling reagents for labeling of biological and non-biological Materials available using the analysis currently available should. New reagents that are excitable in this spectral region, would make it possible Multi-Color Lumineszenzanalysen from markers to cells, there antibodies with different specificities each with a different colored fluorescent dye could be marked. Thus, that could Presence of multiple markers simultaneously for each analyzed Cell to be determined.

The Invention also relates to luminescent (fluorescent or phosphorescent) Cyanine, merocyanine and styryl dyes themselves covalently attached to biological and nonbiological materials be linked can. Merocyanine and Styryl dyes are used for the purposes of this invention as related to cyanine dyes considered. The new labeling reagents themselves, but especially when conjugated to a labeled component, can by light of first defined wavelengths, for example by light in wavelength ranges of the spectrum from 450 to 900 nm. The background fluorescence Cells are generally at a lower wavelength. Therefore, be the labeling reagents are opposite to the background fluorescence differ. Of particular interest are the derivatives, the light Absorb at 633 nm, as they pass through cheap, intense, stable and durable HeNe laser sources can be stimulated. Light of second defined wavelengths, which is marked by the Component is fluorescent or phosphoresced, can then be detected. The fluorescent or phosphorescent light generally has a larger wavelength than the excitation light. In the detection stage, a luminescence microscope be used, which has a filter for absorbing stray light the excitation wavelength and to the passage of the wavelength has, corresponding to the luminescence, that of the respective dye marking corresponds to that used with the sample. Such an optical Microscope is described, for example, in US Pat. No. 4,621,911.

Not all cyanine and related dyes are luminescent. However close the dyes of the invention such cyanine and related dyes which are luminescent are. They are relatively photostable, and many are in the reaction solution, preferably an aqueous solution, soluble. The conjugated dyes themselves, but especially when combined with conjugated to 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 molecules' ability to light to absorb. The conjugated dyes of the invention have quantum yields of at least 2% and preferably at least 10%. For this purpose, the conjugated compounds according to the invention absorb and emit Dyes light in the spectral range from 400 to 900 nm and preferably in the spectral range from 600 to 900 nm.

Arylsulfonierte dyes

It has now been found that the arylsulfonate or arylsulfonic acid dyes of theirs described herein Nature after stronger are fluorescent and have improved photostability and water solubility properties have as similar Dyes without arylsulfonate or arylsulfonic acid groups. The ones used herein Designations of arylsulfonate or arylsulfonic acid are arylsulfonic acid groups or aryl sulfonate groups, said groups are added to aromatic ring structures, with inclusion of a single aromatic ring structure or a fused ring structure, for example, a naphthalene structure. The only aromatic Ring structure or the fused aromatic ring structure can present in polymethine, cyanine, merocyanine or styryl dyes be.

Lots Dyes with planar molecular structures including customary Cyanine dyes tend to form dimers and higher order aggregates in aqueous solution to form, especially if also inorganic Salts are present, as for example in buffered solutions and physiological Saline the case is. These aggregates usually have absorption bands, the to the short wavelength side of monomer absorption, and they are in general very weakly fluorescent species. The tendency of cyanine dyes, in aqueous solution easily Forming aggregates is well known, especially in photography (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 in particular cyanine dye molecules, tend to form aggregates in aqueous solution. It has been found that the aryl sulfonate dyes have a minimal tendency to form these aggregates. When used to form fluorescent label reagents, the aryl sulfonate dyes have a reduced tendency to form aggregates when bound with high surface densities to proteins or other molecules such as antibodies. The tendency of a particular dye molecule to form aggregates in a saline solution (for example, 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. For dye molecules, it is therefore desirable that they have a minimal tendency to form aggregates in aqueous salt solutions. The values of 2 show that a certain arylsulfonated dye has only a low tendency to form aggregates, even at high concentrations in aqueous saline.

The 1 Figure 4 shows the monomer absorption spectrum and the dimer absorption spectrum of a typical cyanine dye dissolved in an aqueous buffer. The dye used to make these spectra, N, N'-di-sulfobutyl-indodicarbocyanine, has no aryl sulfonate groups and readily forms dimers even at submillimolar concentrations. The dimer spectrum was calculated from the spectra of the dye at various concentrations (see the method of West and Pearce, 1965). At a 3 mM concentration in a phosphate buffered saline solution, the absorptions of the monomer and dimer bands were about the same.

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

Usually, the effectiveness with which a particular reactive dye couples to a protein, such as an antibody, is determined under defined reaction conditions. The dye tested was the bis-N-hydroxysuccinimide ester of N, N'-di-carboxypentyl-indodicarbocyanine-5,5'-disulfonic acid. The 3 shows that this sulfo cyanine active dye ester reacts effectively with sheep immunoglobulin in a carbonate buffer at pH 9.2 to form covalently labeled antibody molecules having 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 indicate that under these conditions the labeling efficiency of this dye is about 80%. In similar studies, fluorescein isothiocyanate (FITC) reacted to an efficacy 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 added to the protein samples to give the original molar ratios of dye: protein. After 30 minutes, the protein was separated from the unconjugated dye by gel permeation chromatography (Sephadex G-50). The resulting molar ratios of dye: protein were determined spectrophotometrically and they are in 3 shown.

At low dye to protein ratios, the absorption spectra of the labeled proteins show bands that closely match the spectra of the free monomeric dye. Antibody molecules that have been highly labeled (high dye: protein ratios) or that have been labeled with dyes that have 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 within a range that is characteristic of the dimer absorption spectrum of the dye (cf. 1 ).

More strongly labeled antibodies have higher ratios of short to long wavelength absorption peaks. This shorter wavelength absorption peak can be found in 4 be seen at about 590 nm. The longer wavelength peak (at 645 nm) in 4 is due to monomeric dye molecules bound to the antibody. The labeling reagent used to measure the antibody absorption spectrum in 4 (the bis-N-hydroxysuccinimide ester of N, N'-di-sulfobutyl-indodicarbocyanine-5,5'-acetic acid) has no arylsulfonate groups and forms dimers readily in aqueous salt solutions and on antibodies with which it has been reacted. Of crucial importance is that the fluorescence excitation spectra of these antibodies show that the excitation of the labeled antibodies does not produce as much fluorescence at the short wavelength peak as it does with longer wavelength peak excitation. This observation is consistent with the idea that the shorter wavelength absorption peak is due to the formation of nonfluorescent dimers and aggregates on the antibody molecules.

It has been found that this is necessary to obtain the values of 3 used labeling reagent consisting of an arylsulfonate cyanine dye to which antibody molecules are not readily aggregated. This results from the significantly smaller absorption peak at wavelengths characteristically absorbing dimers (cf. 5 ). This is of importance because antibodies and other proteins labeled with these "non-aggregating" labeling reagents should yield more fluorescent labeled proteins. In fact, the arylsulfocyanines give glossy fluorescent antibodies even when the average dye to antibody ratio is relatively high (cf. 6 ).

Sheep immunoglobulin (IgG) which has been labeled with a carboxyindodicarbocyanine dye is described in U.S. Pat 4 shown. The 5 shows the protein that has been conjugated with the new sulfindindicarbocyanine dye. The presence of increased dimer (cf. 1 ) in the former sample is obvious. Although the dye: protein molar ratio was approximately equal in both preparations, this was in 5 represented protein significantly more fluorescent than those in 4 shown sample.

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

The average fluorescence quantum yield of the individual dye molecules on the labeled proteins is a measure of the fluorescence signal obtainable from these biomolecules. Levels of sheep immunoglobulin (IgG) labeled with the new sulfoindodicarbocyanine dye in phosphate buffered saline are shown in the curve with diamond symbols 6 shown. The curve with round sym bolen the 6 Figure 4 shows proteins labeled with a reactive indodicarbocyanine isothiocyanate dye for comparison. In 6 For example, at a dye / protein ratio of zero, the quantum yields represent the values for the methylamine adducts of the reactive dyes (free dye) in the buffer.

Background method

Luminescence probes are valuable reagents for the analysis and separation of molecules and cells and for detection and quantitative determination of other materials. A very small one Number of luminescent molecules can be detected under optimal circumstances. Barak and Webb see less than 50 fluorescence lipid analogues in the Related to the LDL reception of cells using a SIT camera, J. Cell. Biol. 90: 595-605 (1981). Flow cytometry can be used to detect less than 10,000 fluorescein molecules, associated with particles or certain cells (Muirhead, Horan and Poste, Bio / Technology 3: 337-356 (1985)). Some special examples for the application of fluorescence probes are (1) the identification and separating subpopulations of cells in a mixture of Cells through the techniques of fluorescence flow cytometry, the fluorescence-activated Cell sorting and fluorescence microscopy; (2) the provision the concentration of a substance that binds to a second species (for example, antigen-antibody reactions), in the technique of fluorescence immunoassay; (3) the localization of substances in gels and other insoluble carriers by the techniques of Fluorescence staining. These techniques are described by Herzenberg et al., Cellular Immunology, 3rd Edition, Chapter 22; Blackwell Scientific Publications, 1978 (Fluorescence activated cell sorting); and Goldman, "Fluorescence Antibody Methods ", Academic Press, New York, 1968 (fluorescence microscopy and fluorescence staining); and in "Applications of Fluorescence in the Biomedical Sciences ", edited by Taylor et al., Alan Liss Inc., 1986.

at the use of fluorescers for the above purposes many restrictions on the choice of fluorescer. A limitation consists in the absorption and emission properties of the fluorescer, since many ligands, receptors and materials in the test sample, For example, blood, urine, cerebrospinal fluid, fluoresce and the accurate determination of the fluorescence of the fluorescent sample to disturb. This phenomenon is called autofluorescence or background fluorescence designated. Another aspect is the ability of the fluorescer to with ligands and receptors and other biological and non-biological Conjugate materials, and the effect of such conjugation on the fluorescent agent. In many situations, the conjugation to another molecule to a significant change the fluorescence properties lead the fluorescent agent and in some cases even significantly destroy the quantum efficiency of the fluorescer or Reduce. It is also possible, that the Conjugation with the fluorescent agent the function of the to be labeled molecule inactivated. A third consideration lies in the quantum efficiency of the fluorescer, which is responsible for a sensitive Capture should be high. A fourth consideration is light absorption capability or the extinction coefficient of the fluorescent agent to be as large as possible should. Of importance is also whether the fluorescence molecules are related when they are in close proximity, which would lead to self-extinction. A further fear goes there, whether a non-specific binding of the fluorescer to other connections or container walls either itself or in conjunction with the compound to which the fluorescer is conjugated takes place.

The utility and value of the methods described above are closely linked to the availability of suitable fluorescent compounds. In particular, there is a need for fluorescent substances that emit in the longer wavelength range of the visible region (yellow to near infrared) since the excitation of these chromophores results in less autofluorescence and also multiple chromophores that fluoresce at different wavelengths that can be analyzed simultaneously when the entire visible and near infrared regions of the spectrum can be used. Fluorescein, a widely used fluorescent compound, is a suitable green emitting compound, although in certain immunoassays and cell analysis systems, the background autofluorescence produced by excitation at fluorescein absorption wavelengths limits the sensitivity of detection. However, the conventional red fluorescent marker rhodamine has been found to be less effective than fluorescein. Texas Red ^® is a suitable labeling agent that can be excited at 578 nm and exhibits maximum fluorescence nm at 610th

Phycobiliproteins have made an important contribution because of their high extinction coefficient and high quantum efficiency. These chromophore-containing proteins can be covalently linked to many proteins and used in fluorescence antibody assays in microscopy and flow cytometry. However, the phycobiliproteins have the following disadvantages: (1) the protein labeling ver driving is relatively complex; (2) protein labeling performance is usually not high (typically an average of 0.5 phycobiliprotein molecules per protein); (3) the phycobiliprotein is a natural product and its preparation and purification is complex; (4) the phycobiliproteins are expensive; (5) no phycobiliproteins are available as labeling reagents which fluoresce farther to the red region of the spectrum than allophycocyanin, which fluoresces at 680 nm at most; (6) the phycobiliproteins are chemically relatively unstable; (7) they are easily subject to photobleaching; (8) The phycobiliproteins are large proteins with molecular weights in the range of 33,000 to 240,000 and are larger than many materials to be labeled, such as metabolites, drugs, hormones, derivatized nucleotides and many proteins including antibodies. The latter disadvantage is of particular importance, since antibodies, avidin, DNA hybridization probes, hormones, and small molecules labeled with the large phycobiliproteins may not be able to bind to their targets because of steric constraints imposed by them the size of the conjugated complex can be imposed. The binding rate of conjugates to targets is slow compared to low molecular weight conjugates.

By Other techniques, such as histology, cytology, immunoassays, would also significant advantages through the use of a fluorescer with high quantum efficiency, absorption and emission properties at longer wavelengths and with simple implementation the conjugation, which is characterized by a nonspecific interference in the are essentially free, experienced.

According to the invention be reactive fluorescent arylsulfonated cyanine and thus related dyes with relatively high extinction coefficients and high quantum yields for the purpose of detection and quantitative Determination of labeled components used.

fluorescent Cyanine and related dyes can be used to label biological materials, like 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 for marking non-biological materials, such as soluble Polymers and polymeric and glass particles, drugs, ladders, Semiconductors, glass and polymer surfaces, polymer membranes and others solid particles are used. The component to be marked may 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, be. The detection step can be with the mixture in liquid or dry state, such as a slide, performed.

It is according to the invention required that cyanine dyes modified by the incorporation of a reactive group in the cyanine molecule become. This reactive group attaches itself covalently to a target or target molecule, preferably to an amine or hydroxystelle, and in some cases a sulfhydryl or aldehyde site. The invention also uses the modification or use of cyanine and related Dye structures to their solubility in the test fluid to increase, (1) to make it easier to handle in labeling reactions, (2) the aggregation of the dye on the surface of the help to prevent proteins to be labeled and (3) the non-specific Binding of labeled materials to biological materials and on surfaces and help prevent the assay device.

The Cyanine and its related dyes provide an important Advantage over currently available fluorescent labeling reagents. The first is cyanine and related dyes have been synthesized in one Absorb range of the spectrum from 400 to nearly 1100 nm and emit. Thus, you can reactive derivatives of these dyes are prepared for assays, a simultaneous measurement of a number of marked materials require. The multicolor (or multiparameter) analysis of this kind may be in the interest of simplicity, cost effectiveness or Determination of ratios of different marked species on each particle in a complex Mixture of particles be appropriate (for Example of the conditions of antigen markers on individual blood cells in a complex Mixture by multiparameter flow cytometry or fluorescence microscopy). Secondly, many cyanine absorbs and fluoresces related dyes strong.

Third, many cyanine and related dyes are relatively photostable, and they do not readily bleach in a fluorescence microscope. Fourth, cyanine and related derivatives can be prepared which are simple and effective coupling reagents. Fifth, many structures and synthetic procedures are available, and the class of dyes is diverse. Thus, many structural modifications can be made to render the reagents more or less water-soluble. Their charge can be altered so that they do not disturb the molecule to which they are attached, where can be reduced by the non-specific binding. Sixth, unlike the phycobiliproteins, the cyanine dyes are relatively small (molecular weight = 1000), so that they do not significantly interfere sterically with the ability of the labeled molecule to rapidly reach or perform its binding site.

Consequently Cyanine dye markers bring many potential advantages with himself. These dyes can be used to create one or more components in one Liquid, in particular an aqueous liquid, to mark selectively. The labeled components can then be detected by optical or Lumineszenzmethoden. Alternatively, you can the marked component can be used to create a second component to color, for the she has a strong affinity has. The presence of the second component is then through optical or luminescence detected. In this case will the dye having an amine, hydroxy, aldehyde or sulfhydryl group implemented on the marked component. So, for example the labeled component is an antibody, and the stained component, for you a strong affinity has, can be a biological cell, an antigen or a hapten or a biological cell or a particle containing said antigen or contains hapten, be. According to one another example is the labeled component avidin, and the stained component may be a biotinylated material. Also, lectins containing polymethine cyanine dyes are conjugated to special carbohydrate groups to capture and quantify. Furthermore, luminescence cyanine and attaching related dyes to fragments of DNA or RNA become. The labeled fragments of DNA or RNA can be used as Fluorescence hybridization probes are used to detect the presence and the amount of specific complementary nucleotide sequences in To identify samples containing DNA or RNA. Also can the dye to a hormone or to a ligand (for example, a Hormone, a protein, a peptide, a lymphokine, a metabolite) added which is then added to a receptor.

For others Purpose described in patents reactive cyanine dyes

According to the US-PSen 4,337,063 (Miraha et al.), 4,404,289, 4,405,711 and 4,414,325 (Masuda et al.) a variety of cyanine dyes have been synthesized, containing the active N-hydroxysuccinimide ester groups. These patents show that this Reagents can be used as photographic sensitizers can. The possible Fluorescence properties of these reagents are in the patents not known. Indeed are also for these processes do not require fluorescence properties. The Most of the dyes mentioned in these patents are only weakly fluorescent, and they are not particularly photostable. Furthermore, their solubility properties for many Applications not optimal, the fluorescence detection of would contain marked materials.

In GB Patent 1,529,202 (Exekiel et al.) discloses numerous cyanine dye derivatives described which are used as covalently reacting molecules. In the Reactive group used in these reagents is an azine group, to the mono and Dichlortriazineruppen belong. This document refers to the development and use these reagents as sensitizers for photographic films. For this Procedure does not require fluorescence, and most of the Reagents described therein are not fluorescent. Finally, refers This document does not refer to the development and use of reactive cyanine dyes for the purpose of detection and quantitative Determination of marked materials.

The The present invention relates to methods for covalent attachment of luminescent cyanine and cyanine dyes to biologicals Materials, non-biological materials and macromolecules and particles, so that these materials are labeled luminescent. To this The labeled material can be detected by luminescence detection methods detected and / or quantified.

The The invention relates to a method for detecting a component in a liquid, which is characterized in that one to the said liquid Add a dye selected from the group consisting of cyanine, Merocyanine and Styryl dyes selected is and in the liquid soluble is and contains a substituent, in order to work with amine and hydroxy groups and possibly aldehyde and sulfhydryl groups becomes covalently reactive on said component, so that said Component is highlighted. The marked component is then through Luminescent or Light absorption methods detected and / or quantified. If the marked component is on Antibody, a DNA fragment, a hormone, a lymphokine or a drug is, then the marked component can be used to the presence of a second component to which it is bound to identify what the second component detects and / or can be determined quantitatively.

each Any luminescence or light absorption detection stage can be applied. For example, the detection level can be a be optical detection stage, in which the liquid with light from first defined wavelengths is irradiated. Light with second defined wavelengths, the fluoresced or phosphoresced through the labeled component will be detected. The detection can also be done by optical light absorption. Thus, for example, the detection stage can be carried out in the manner that he Light with first defined wavelengths through the liquid conducts and then the wavelength the light passing through the liquid is passed, determined.

If desired, the detection stage may also include a chemical analysis to chemically attaching the cyanine or a related chromophore to the component.

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

More specific examples of polymethine cyanine dyes are as follows:

ausgewählt;
Z ist aus der Gruppe O und S ausgewählt und
m ist eine ganze Zahl, die aus der Gruppe bestehend aus 1, 2, 3 und 4 ausgewählt ist.In these structures are
X and Y from the group, O, S and

selected;
Z is selected from the group 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 partially determine the excitation color. Often, higher values of m contribute to increased luminescence and absorption. At values of m above 4, the connection becomes unstable. Further luminescence can be imparted by modifications of the ring structures. If m = 2, then the excitation wavelength is about 650 nm, and the compound is very strongly fluorescent rend. 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 said R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R _{7 groups} in each molecule is or are a reactive group for attachment of the dye the marked component. For certain reagents, at least one of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ on each molecule may also be a group which increases the solubility of the chromophore or the selectivity of the label of the labeled component impaired or impaired the position of the label of the labeled component by the dye.

In said formulas, at least one of said R ₈ , R ₉ (if present) and R ₁₀ (if present) comprises at least one sulfonate group. The term sulfonate is intended to include sulfonic acid because the sulfonate group is only an ionized sulfonic acid.

Reactive groups which may be added directly or indirectly to the chromophore to form the groups R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ may include reactive moieties, for example groups which include isothiocyanate , Isocyanate, monochlorotriazine, dichlorotriazine, mono- or dihalogen-substituted pyridine, mono- or dihalogen-substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imidoester, hydrazine, azidonitrophenyl, azide, 3- (2-pyridyldithio) -proprionamide, glyoxal and aldehyde.

wobei mindestens eines von Q oder W eine Austrittsgruppe, wie I, Br, Cl, ist:

Specific examples of the groups R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ , which are particularly suitable for labeling components with available amino, hydroxy and sulfhydryl groups, are the following groups:

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

worin Q eine Austrittsgruppe, wie I oder Br, ist:

worin n 0 oder eine ganze Zahl ist.Specific examples of groups R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ which are particularly well suited for labeling components with available sulfhydryl groups and which are used in a two-step method for labeling antibodies can, are the following groups:

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

where n is 0 or an integer.

Specific examples of the groups R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ , which are particularly well suited for labeling components by photoactivated crosslinking, are the following groups:

For the purpose of increasing the water solubility or reducing undesired nonspecific binding of the labeled moiety to unsuitable components in the sample or to reduce reactions between two or more reactive chromophores on the labeled moiety, which could lead to quenching of the fluorescence, the groups R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R _{7 are selected} from the well known polar and electrically charged chemical groups. Examples are -EF, wherein F is hydroxy, sulfonate, sulfate, carboxylate, substituted amino or quaternary amino and wherein E is a spacer group such as - (CH ₂ ) _n -, where n is 0, 1, 2, 3 or 4 , Suitable examples are, for example, alkylsulfonate, - (CH ₂ ) ₃ SO ^3⊝ and (CH ₂ ) ₄ -SO ^3⊝ .

The Polymethine chain of the luminescent dyes according to the invention may also be a or more cyclic chemical groups containing the bridges between form two or more of the carbon atoms of the polymethine chain. These bridges could serve the chemical or photostability of the dye to increase, and they could to be used, the absorption and emission wavelengths of the dye to change or its extinction coefficient or its quantum yield to change. improved solubility could obtained by this modification.

According to the invention, the labeled component can be selected from the following substances: 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 Medicines, virus particles, bacterial particles, viral components, Yeast components, blood cells, blood cell components, biological cells, non-cellular Blood components, bacteria, bacterial components, natural and synthetic lipid vesicles, synthetic medicines, poisons, pollutants, Polymers, polymer particles, glass particles, glass surfaces, plastic particles, Plastic surfaces, Polymer membranes, conductors and semiconductors.

One Cyanine or a related chromophore can also be made which, when implemented with a component, will light at 633 nm can absorb. In the detection stage can be a helium-neon laser be used, which emits light at this wavelength of the spectrum. Also, a cyanine or related dye can be made which, when implemented with a component, will light maximum between 700 nm and 900 nm can absorb. In this case can be used in the detection stage, a laser diode, the Light emitted in this region of the spectrum.

selectivity

The The above reactive groups are relatively specific for the label certain functional groups on proteins and other biologicals or nonbiological molecules, Macromolecules, surfaces or Particles, provided that suitable Reaction conditions including appropriate pH conditions be applied.

Properties of the reactive Cyanine, merocyanine and styryl dyes and their products

The Spectral properties of the dyes of the invention are characterized by does not appreciably alter the functionalization described herein. The Spectral properties of the labeled proteins and the other compounds are also not very different from those of the basic dye molecule, the not conjugated to a protein or other material is. The dyes described herein have, alone or on a labeled material conjugated, generally 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 spectral range of 400 to 900 nm. Thus, they are of particular value as labeling reagents for the Luminescence detection.

Optical detection methods

to Detection or determination of a marked or colored component Any method can be used. The collection method can use a light source that has the mixture that marked it Contains material, irradiated with light at first defined wavelengths. Known devices are used, which capture light with second wavelengths, which light has passed through the mixture or through the Mixture fluoresces or luminesces. Such detection devices Examples are fluorescence spectrometers, absorption spectrophotometers, Fluorescence microscopes, translucent microscopes and flow cytometers, fiber optic sensors and immunoassay instruments.

at the method according to the invention can Also, chemical analysis methods are used to attach of the dye to the labeled component or components capture. examples for Chemical analysis methods are infrared spectrometry NMR spectrometry, absorption spectrometry, fluorescence spectrometry, the mass spectrometry and the chromatography.

The Invention is illustrated in the examples.

example 1

Effect of pH on the conjugation of sulfoindodicarbocyanine with protein

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

At appropriate times, which ranged from 5 seconds to 30 minutes, protein samples were separated from non-covalently attached thereto a dye by gel permeation chromatography on Sephadex ^® G-fiftieth The maximum labeling of the protein occurred after 10 minutes and gave final dye ratios of dye / protein of 5.8, 6.4, and 8.2 for the samples, which were stable at pH 8.5, 8.9, and 8.2, respectively. 9.4 were incubated. The times required to give a dye / protein ratio of 5 and quantum yields of the products at the various pH values are summarized in the following table:

These data indicate that the protein labeling with this dye is better at pH 9.4 than at pH below 9. In the higher pH buffer, the conjugation was greater action very quickly, but the labeling efficiency was excellent and the product was more fluorescent. The value of the quantum yield represents the average quantum yield per dye molecule on the labeled protein.

Example 2

Conjugation of the active Sulfoindocarbocyanine ester with protein

Sheep γ-globulin (1 mg / ml) dissolved in pH 7.4 phosphate buffered saline (PBS) was adjusted to pH 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 incubation at room temperature, the mixtures by gel permeation chromatography on Sephadex ^® G-50 were separated by 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 ratios of dye / protein of 3, 6, 12, 24 or 30.

Example 3

Labeling of AECM-Dextran with a sulfoindodicarbocyanine

N-aminoethyl carboxamidomethyl (AECM) dextran having 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)). A portion of the AECM-dextran (1 mg / 250 μl) dissolved in 0.1 M carbonate buffer pH 9.4 was added to 0.2 mg of the active sulfoindodicarbocyanine ester (structure in Example 1, m = 2) to give a dye / protein molar ratio of 10. The mixture was stirred at room temperature for 30 minutes. The dextran was then separated from unconjugated dye by Sephadex ^® G-50 gel permeation chromatography using ammonium acetate (50 mM) as an elution buffer. An average of 2.2 dye molecules was covalently bound to each dextran molecule.

Example 4

Marking a specific antibody with active sulfoindodicarbocyanine ester

Murine IgG-specific sheep γ-globulin (1 mg / ml) in 0.1M carbonate buffer (pH = 9.4) was treated with the active sulfoindodicarbocyanine ester (structure in Example 1, m = 2) at a ratio of 8 Dye molecules per protein molecule mixed. After 30 minutes of incubation at room temperature, the labeling mixture by gel filtration on Sephadex ^® G-50 which had been equilibrated with phosphate buffered saline (pH = 7.4) was separated. The recovered protein 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, which conjugates to sheep anti-mouse IgG antibody was

Freshly isolated peripheral blood lymphocytes were treated with mouse anti-β2 microglobulin (0.25 μg / 10 ⁶ cells) at 0 ° C for 30 minutes. The cells were washed twice with DMEM buffer and then treated with the sulfoindodicarbocyanine-labeled sheep anti-mouse IgG antibody (1 μg / 10 ⁶ cells). After incubation at 0 ° C for 30 minutes, excess antibody was removed by centrifugation of the cells, and the cells were again washed twice with DMEM buffer. Aliquots of the cells were mounted on microscope slides for analysis by fluorescence microscopy. Under the microscope, the lymphocytes on the slide were irradiated with light of 610 to 630 nm, and the fluorescence of 650 to 700 nm was detected with a red-sensitive amplified COHU television camera 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 primary mouse anti-β2-microglobulin antibody was omitted, but staining and analysis were otherwise performed as described above. The control sample showed no fluorescence under the microscope, indicating that the sulfoindocyanine-labeled sheep anti-mouse antibody does not give significant non-specific binding to lymphocytes.

Claims (5)

Luminescently labeled component of an aqueous Liquid, comprising a luminescent dye selected from the group consisting from cyanine, merocyanine, and styryl dyes, the said Dye covalently reactive with the said component and to this is bound, wherein the component is a molecule selected from the group consisting of biological cells, antibodies, proteins, Peptides, enzyme substrates, hormones, lymphokines, metabolites, receptors, Antigens, haptens, lectins, toxins, carbohydrates, sugars, Oligosaccharides, polysaccharides, nucleotides, derivatized Nucleotides, nucleic acids, Deoxynukleinsäuren, derivatized nucleic acids, derivatized deoxynucleic acids, DNA fragments, RNA fragments, derivatized DNA fragments, derivatized RNA fragments and drugs, or wherein the Component selected is from the group consisting of soluble polymers, plastic particles, Plastic surfaces, Glass particles and glass surfaces. Luminescent labeled component according to claim 1, wherein said dye is attached to an aromatic nucleus at least one sulfonic acid or sulfonate group. Luminescently labeled component according to claim 1 or 2, labeled with a luminescent dye from the group:

wherein the dotted lines each indicate the carbon atoms necessary to form the dye, X and Y are independently selected from the group consisting of O, S and CH ₃ -C-CH ₃ ; m is an integer from the group 1, 2, 3 and 4; at least one of said R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R _{7 groups is} a reactive group reactive with amino, sulfhydryl, aldehyde or hydroxyl groups; and in the event that one of the groups R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R _{7 is} not a reactive group, these other groups R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R _{7 are} selected from the group consisting of hydrogen and a polar group EF, wherein E is a spacer group of the formula - (CH ₂ ) _n -, where n is 0, 1, 2, 3 or 4 and F is hydroxy, sulfonate, sulfate, carboxylate, substituted amino or quaternary amino, or from the group:

wherein the dotted lines each indicate the carbon atoms necessary to form the dye, X and Y are independently selected from the group consisting of O, S and CH ₃ -C-CH ₃ ; m is an integer from the group 1, 2, 3 and 4; at least one of said R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R _{7 groups is} a reactive group reactive with amino, sulfhydryl, aldehyde or hydroxyl groups; and in the event that one of the groups R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R _{7 is} not a reactive group, these other groups R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R _{7 are} selected from the group consisting of hydrogen and a polar group EF, wherein E is a spacer group of the formula - (CH ₂ ) _n -, where n is 0, 1, 2, 3 or 4 and F is hydroxy, sulfonate, sulfate, carboxylate, substituted amino or quaternary amino; and at least one of R ₈ and R _{9 is} a sulfonate or sulfonic acid group. Luminescent labeled component according to claim 3, wherein the reactive group is selected from the group consisting from isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, Mono or dihalo-substituted pyridine, mono- or dihalo-substituted Diazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, Hydroxysulfosuccinimide ester, imidoester, glyoxal and aldehyde. Protein, nucleic acid, Cell, sugar or carbohydrate with at least one amino or Hydroxy group labeled with a luminescent dye as in claim 1 or 2 defined.