JP2003232735A - Method of labeling or detecting substance using luminescent aryl sulfonate cyanine dye - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substance contained in an aqueous liquid labeled with a dye useful for detection. <P>SOLUTION: A method of labeling a component contained in an aqueous liquid includes a step (a) of adding a luminescent dye selected from among a group composed of cyanine, merocycanine, and styryl dues having at least one sulfonic group or sulfonate group attached to an aromatic nucleus and having reactivity to the component to the aqueous liquid containing the component and a step (b) of causing the dye and component to act upon another so that the dye may label the component. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to the labeling and detection of substances with luminescent aryl sulphonate cyanine dyes.

[0002]

BACKGROUND OF THE INVENTION Cyanine and related dyes having light absorbing properties have been used in photographic films. Such dyes require light-absorption properties, but not luminescence (fluorescence or phosphorescence) properties. So far, cyanine dyes with luminescent properties have had very limited applications. Such uses include labeling the sulfhydryl groups of the protein in particular. “Sulfhydryl reagent dye initiates rapid Ca ²⁺ release from myoplasmic reticulum (SR) (Sulfhydryl Reagent DyesTr
Igger the Rapid Release of
Ca ² from Sacoplastic Reticu
lum Vesicles (SR) ”in Salama G. et al. Waggoner A. S. Ab
ramson J. Reported that a cyanine chromophore bearing an iodoacetyl group was used to form a covalent bond with a sulfhydryl group on the myoplasmic reticulum protein at pH 6.7 to initiate Ca ²⁺ release ( Biophys
ical Journal, 47, 456a (198)
5)). This article also states that fluorescent dyes were used to label or isolate proteins.

"Mechanics of conformational changes in the rhodopsin molecular region away from the retinylidene binding site (Kinetics)
of Conformal Changes
ina Region of the Rhodopsin
Molecular Away From the Reti
Nylidene Binding Site) ", Waggoner A. et al. S. , Jenki
ns P. L. Carpenter J .; P. And Gupta R. et al. Reported that the sulfhydryl group on the F1 region of bovine rhodopsin was covalently labeled with a cyanine dye that has an absorbance at 660 nm [Biop.
physical Journal, 33, 292a (1
985)]. Again, it was reported that cyanine dyes were used to label the sulfhydryl groups of proteins, but the use of fluorescent dyes was not disclosed.

"International seminar on the application of fluorescent photobleaching techniques to problems in cell biology.
Tialal Workshop on the Appl
ication of Fluorescence ph
otobleaching Technologies to
Problems in Cell Biology) "
The article entitled, Jacobson K. et al., Describes a cyanine-type fluorescent probe that can be complexed to proteins and excited in the deep red region of the spectrum. , Elson E. , K
opel D.D. And Webb W.W. Have been reported (Fed. Proc. 42: 72-79 (198).
3)).

The only cyanine probe described in any of the above three papers is especially one that covalently binds to the sulfhydryl groups of proteins. The only specific cyanine compound described is one that has an iodoacetyl group, which covalently reacts a cyanine dye with a sulfhydryl group. None of the above articles disclose a covalent reaction between a cyanine dye and a substance other than a protein or an arbitrary group on a protein other than a sulfhydryl group.

[0006]

However, many non-protein substances do not have sulfhydryl groups, and many proteins do not have such groups sufficiently to make them useful for fluorescence exploration. Moreover, the sulfhydryl group (-SHSH-) readily oxidizes to disulfide (-SS-) in the presence of air, which renders it unacceptable for covalent attachment to fluorescent probes.

[0007]

The present invention provides a method of labeling a component in an aqueous liquid. The method comprises (a) adding a luminescent dye selected from the group consisting of cyanine, merocyanine and styryl dyes having at least one sulfonic acid group or sulfonate group bonded to an aromatic nucleus to the liquid containing the component, The dye is reactive with the component, and (b) comprising reacting the dye with the component such that the dye labels the component.

In another aspect of the invention, a method of labeling a component in an aqueous liquid is provided. The method is (a)
A sulfoindolenin base and a naphthosulfoindolenin base polymethine containing at least one sulfonic acid group or sulfonate group bonded to one or more rings of a heterocyclic nucleus in the liquid containing the above component. Adding a luminescent dye selected from the group consisting of dyes, said dye being reactive with said component, (b) reacting said dye with said component such that said dye labels said component, and (c) Detecting the labeled component by an optical method.

In another aspect of the invention, there is provided a method of labeling a first component and using the labeled first component to bind a second component to form a labeled second component. The method comprises (a) a luminescent dye selected from the group consisting of cyanine, merocyanine and styryl dyes containing at least one sulfonic acid group or sulfonate group bonded to an aromatic nucleus in the aqueous liquid containing the first component. The dye is reactive with the first component, and (b) reacting the dye with the first component such that the dye luminescently labels the first component to provide the labeled first component. ,
(C) binding the labeled first component to the second component to form a labeled second component, and (d) detecting the labeled second component by an optical method.

In yet another embodiment of the present invention, a sulfocyanine dye-labeled substance and one or two different fluorescent substances that all significantly absorb light at a single excitation wavelength but emit fluorescence at different wavelengths. Mixing the above with each other, then exciting all of the fluorophores with light of a single wavelength, and weighing each of the fluorophores by detecting their individual fluorescence signal at their different fluorescence wavelengths. A method including is provided.

[0011]

According to the present invention, not only sulfhydryl groups on proteins and other substances but also amines (--NH 2) can be used under reaction conditions suitable for fluorescence or phosphorescence detection of substances.
₂ ) and hydroxy (OH) group and other aldehydes (-
Cyanine and related polymethine dyes having substituents that covalently react with groups such as CHO) groups have been developed. The present invention provides significant benefits over the use of conventional iodoacetyl cyanine dyes and their specific reactivity with sulfhydryl groups. Amine and hydroxy groups are more predominant and stable in proteins and other substances than sulfhydryl groups. Therefore, when a fluorescent cyanine dye is used for probing the presence or absence of a specific protein, many dye molecules can be bound to the protein to be probed, so that a stronger fluorescence or phosphorescence intensity signal is generated. Moreover, amine or hydroxy groups are, of course, more easily added to the desired component of the label, such as sulfhydryl, polymer particles free of amine or hydroxy groups.

The present invention also provides a group that covalently reacts with an amine or a hydroxy group or another reactive group so that the presence or absence or amount of the labeled protein or other substance can be probed after separating the labeled component by a chromatographic method. A method for labeling proteins or other sources having amine or hydroxy groups or other groups capable of reacting with dyes in a mixture using a luminescent cyanine dye containing. According to the literature cited above, it is clear that sulfhydryl groups were chosen for covalent reactions, especially because there are very few sulfhydryl groups on the protein molecule and in some cases they play a significant role in the function of the protein. It was Therefore, it was possible for the author to try to identify the specific position of the sulfhydryl group on the protein structure. Further, in the above-mentioned literature, the sulfhydryl group was used as a probe for investigating or causing a structural change of a specific protein. Thus, it was necessary to know the binding site of the probe in order to determine the change in absorption by the dye or the change in released calcium ions due to dye binding.

The number of sulfhydryl groups on most proteins is so low that the number of sulfhydryl groups may not be sufficient to provide sufficient total luminescence for probing.
In contrast, the number of amine or hydroxy groups is significantly higher in number and widely dispersed on the protein molecule, allowing fluorescent probes to be attached to multiple sites on the molecule, thereby facilitating protein detection. Therefore, the determination of absorption or fluorescence change is eliminated.

The present invention is directed to proteins containing luminescent polymethine cyanine or related polymethine dyes, and other substances, including nucleic acids, DNAs, drugs, toxins, blood cells, microbial substances, particles, plastic or glass surface materials, polymer films, etc.
It relates to labeling at amine or hydroxy sites on the substance. Advantageously, the dye is soluble in aqueous or other media containing the labeling substance. The present invention relates to a one-step labeling method as well as a two-step labeling method. In the two-label method, a first component, such as an antibody, can be labeled at sites on the component, including amine, hydroxy, aldehyde or sulfhydryl sites. The labeled component is used as a probe for a second component, such as an antibody-specific antigen.

In the above-mentioned prior art, the specificity of the binding site by the cyanine probe was achieved by using a probe that covalently reacts with a sulfhydryl group. According to the two-step method of the present invention, in the first step, cyanine or a related probe is treated with an amine, an aldehyde, or the like on the first component such as an antibody.
It can be reacted with sulfhydryl, hydroxy or other groups, so that in the second or staining step the antibody can achieve the desired specificity with the second component, such as the antigen. This specificity is examined by the site of antigen binding to the antibody.

The present invention also relates to luminescent polymethinecyanines and related compounds containing groups that allow covalent attachment to amine, hydroxy, aldehyde or sulfhydryl groups on the target molecule. The present invention relates to monoclonal antibodies and other components labeled with these luminescent cyanine compounds that can serve as probes for antigens. When the target is a single type of cell, the present invention can be used to measure the amount of labeled antibody bound to the cell. This measurement can be performed by examining the relative light and dark of the luminescence of the cells.

The present invention can be used to determine the concentration of specific proteins or other components of the system. If the number of reactive groups on the protein that can react with the probe is known, the fluorescence per molecule can be known, and the molecular concentration of the system can be determined by the total luminescence intensity of the system.

The present method can be used to quantify various proteins or other substances in the system by labeling all mixtures of proteins in the system and then separating the labeled proteins by any means such as chromatography. . Then, the amount of luminescent protein to be separated can be examined, and the position of the dye on the labeling substance can be confirmed by a chromatographic detection system.

The present invention can also be used to determine the number of different cells labeled by an antibody. This number study can be performed by labeling multiple cells of the system and then separating the labeled cells out of the system. Labeled cells can also be separated from non-labeled cells outside the system.

Another embodiment of the invention is a plurality of luminescent cyanines each of which is a different first component such as an antibody, each bound to a first component specific for a different second component such as an antigen. Alternatively, the relevant dye comprises a multi-parameter method used to identify each of the multiple antigens in the antigen mixture. According to this embodiment, each antibody is separately labeled with a dye that has different absorption and luminescence wavelength properties than the dyes used to label the other probes. Then
All labeled antibodies are added to a biological preparation that is analyzed to contain a second component, such as an antigen, each of which can be stained by the particular labeled antibody. Any unreacted dye material can be removed from the preparation by washing if it interferes with the analysis. The biological preparation is then subjected to various excitation wavelengths. Each excitation wavelength used is the excitation wavelength of the particular conjugated dye. A luminescence microscope or other luminescence such as a flow cytometer or a fluorescence spectrophotometer with a filter or monochromator to select the light of the excitation wavelength and to select the wavelength of the luminescence to examine the luminous intensity of the emission wavelength corresponding to the excitation wavelength. A detection system is used. The luminescence intensity at a wavelength corresponding to the emission wavelength of a particular conjugated dye indicates the amount of antigen bound to the antibody bound by the dye. In some cases, a single excitation wavelength can be used to excite luminescence from two or more substances in the mixture, each emitting fluorescence at a different wavelength, and detecting individual fluorescence intensities at each fluorescence wavelength. By doing so, the amount of each labeled species can be measured. Absorption detection methods can be used if desired. The two-step method of the present invention can be applied to any system that uses a first substance combined with a dye to probe the presence or absence of another substance to which the first substance-dye complex is directed in a luminescence or absorption detection system. For example,
Dye binds to DNA or RNA fragment to bind dye D
An NA or RNA fragment can be formed which is then directed to the DNA or RNA backbone to which the fragment is complementary. The same test method can be used to probe the presence or absence of a complementary backbone of DNA.

The cyanine and related dyes of the present invention are especially:
Since specific cyanines and related dyes with a wide range of excitation and emission wavelengths can be synthesized, they are well suited for component mixture analysis requiring dyes of various excitation and emission wavelengths. Specific cyanines and related dyes with specific excitation and emission wavelengths can be synthesized by changing the number of methine groups or the cyanine ring structure. In this way it is possible to synthesize dyes having a specific excitation wavelength corresponding to a specific excitation light source such as a laser eg HeNe or a diode laser.

The present invention provides cyanine and related dye molecules that are highly luminescent and highly absorbable under reaction conditions to proteins, peptides, carbohydrates, nucleic acids, derived nucleic acids, lipids, other specific biological molecules, and biological cells. Shared with the above amines, hydroxy, aldehydes, sulfhydryls or other groups and non-biological substances such as soluble polymers, polymer particles, polymer surface materials, polymer membranes, glass surface materials and other particles or surface materials Regarding reacting. Luminescence includes a sensitive optical technique that allows probing and quantifying the presence or absence of a dye "label" even when the label is present in very low amounts, thus labeling with dye labeling reagents. It is possible to measure the amount of substances. Most useful dyes have high absorbance (ε = 70,000 to 250,000 liters)
b / mol cm or more), is highly emissive and has a quantum yield of at least 5-80% or more. The amount applies to the dye itself and also to the dye bound to the labeling substance.

An important application of such color labeling reagents is the production of luminescent monoclonal antibodies. Monoclonal antibodies are protein molecules that bind very tightly and specifically to specific chemical sites or cell surface or intracellular "markers". Therefore, these antibodies have enormous research and clinical application in identifying specific cell types (eg HLA, T cells, bacteria and viruses, etc.) and abnormal cells. Conventionally, antibody bound to cells is quantified by labeling the antibody by various methods, and the label is a radioactive label (radioimmunoassay), enzyme (ELISA technique).
Or fluorescent dyes (usually fluorescein, rhodamine, Tex
as Red® or red algae). Most manufacturers or users of clinical antibody reagents desire to eliminate the problems associated with the use of radiotracers, and thus luminescence is considered one of the most promising alternatives. In fact, many vendors nowadays have used clonal antibody-labeled fluorescein, T
exas Red®, Lodamin and red algae are marketed.

In recent years, optical / electronic devices for detecting fluorescent antibodies on cells have become increasingly complex. For example, the amount of fluorescent antibody on an individual cell can be measured at a rate of up to 5,000 cells / second using a flow cytometry method,
There are also advances in microscopy and solution fluorescence techniques. These devices are capable of exciting fluorescence at many wavelengths in the UV, visible and near IR regions of the spectrum. Most of the useful fluorescent labeling reagents accepted today are 400-580 nm
It can be excited in the spectral region. The exception is that it may be covalently bound to the protein and excited at a slightly longer wavelength. Some phycobiliprotein type pigments isolated from marine organisms. Therefore, there is a large spectral window in the 580 to about 900 nm range that necessitates new labeling reagents to be effective in labeling biological or non-biological substances that are analyzed in currently marketed instruments. The new reagents, which can be excited in this spectral region, make it possible to carry out multicolor luminescence analysis of markers on cells, since antibodies with different specificities can each be labeled with different colored fluorescent dyes. In this way it is possible to check simultaneously for the presence of several markers for each cell analyzed.

The invention also relates to the luminescent (fluorescent or phosphorescent) cyanine, merocyanine and styryl dyes themselves, which can be covalently bound to biological or non-biological substances. Merocyanine and styryl dyes are recognized as related to cyanine dyes for this invention. The new labeling reagent itself can be excited with light of a wavelength initially defined, especially when it is bound to a labeling component, for example light of a wavelength in the 450-900 nm spectral range. Background fluorescence of cells generally occurs at lower wavelengths. Therefore, labeling reagents are distinguished from background fluorescence. Particularly advantageous are derivatives that absorb light at 633 nm. Because the derivative is H, which is cheap, strong, stable and has a long life.
It can be excited by the eNe laser source. The light of the second defined wavelength, which is fluorescently or phosphorescently emitted by the labeling component, can then be detected. Fluorescence or phosphorescence generally has a longer wavelength than excitation light. The detection stage is
A luminescence microscope can be used which has a filter for absorbing scattered light of the excitation wavelength and for passing the corresponding wavelength of luminescence corresponding to the particular dye label used with the analyte. Such an optical microscope is described in US Pat. No. 4,621,911.

Not all cyanines and related dyes are luminescent. However, in the dye of the present invention,
Included are cyanine and related dyes that are luminescent. They are relatively stable and often soluble in reaction solutions, preferably aqueous solutions. The bound dye itself has a molecular extinction coefficient (ε) of at least 50,000, preferably 100,000 l / mol cm, especially when it is attached to the labeling component. Extinction coefficient is a measure of a molecule's ability to absorb light. The binding dyes of the invention have a quantum yield of at least 2%, preferably at least 10%. In addition, the binding dyes according to the invention are preferably 400-900 nm, preferably 600
It absorbs and emits light in the ~ 900 nm spectral range.

Aryl Sulfonated Dyes Aryl sulfonate or aryl sulfonic acid substituted dyes such as those described herein are intrinsically more fluorescent and have improved photostability as compared to similar dyes without aryl sulfonate or aryl sulfonate groups. And was found to be water soluble. As used herein, the term aryl sulphonate or aryl sulphonic acid group refers to a compatible aryl sulphonic acid group or aryl sulphonate attached to an aromatic ring structure containing a single ring aromatic structure or a fused ring structure such as a naphthalene structure. Means a group. Single ring aromatic structures or fused ring aromatic structures can be present in polymethine, cyanine, merocyanine or styryl type dyes.

Many dyes having the same planar molecular structure, including usually cyanine dyes, form dimers or more conjugates in aqueous solution, especially when inorganic salts are also present, such as in buffers or saline. Easy to form. These conjugates generally have absorption bands shifted to the shorter wavelength side of monomer absorption, and are generally very weak fluorescent species. The tendency of cyanine dyes to easily form a conjugate in an aqueous solution is well known in the photographic industry [West W. & Pierce S. , J. Ph
ys. Chem . , 69; 1894 (1965); St
urmer D. M. , Spec. Top in He
Terocyclic Chemistry , 30 (1
974)].

Many dye molecules, especially cyanine dye molecules, tend to form conjugates in aqueous solution. Therefore, aryl sulfonate dyes have been found to have minimal tendency to form these conjugates. When an aryl sulfonate dye is used to form a fluorescent labeling reagent, it has a reduced tendency to conjugate when bound to other molecules such as proteins or antibodies at high surface densities. The tendency of a particular dye molecule to form a conjugate in a salt solution (eg 150 mM sodium chloride) can be taken as a measure of the tendency of the same dye molecule to form a conjugate on the surface of a protein. Therefore, it is desirable that the dye molecules have a minimal tendency to form conjugates in aqueous salt solution. The data shown in Figure 2 can be used to illustrate that certain aryl sulfonated dyes have a low tendency to form conjugates even at high concentrations in aqueous salt solutions.

FIG. 1 shows the monomer and dimer absorption spectra of typical cyanine dyes dissolved in aqueous buffer. The dye N, N'-di-sulfobutylindodicarbocyanine used to generate these spectra has no aryl sulfonate groups and readily forms dimers even at concentrations below the millimolar range. Dimer spectra were calculated from dye spectra of different concentrations (see method of West & Pearce, 1965). At a concentration of 3 mM in phosphate buffered saline solution, the absorption of the monomer and dimer bands was approximately equal.

Improved sulfoindodicarbocyanine N, N'-diethylindodicarbocyanine-5,5 '
-The spectrum of disulfonic acid is shown in FIG. This dye showed no sign of conjugate in saline solution up to a concentration of 10 mmol.

It is common practice to measure the efficiency with which a particular reactive dye binds to a protein such as an antibody under defined reaction conditions. The test dye is bis-N of N, N'-dicarboxypentyl indodicarbocyanine-5,5'-disulfonic acid.
-Hydroxysuccinimide. FIG. 3 shows that the sulfocyanine dye active ester efficiently reacts with sheep immunoglobulin in a carbonate buffer of pH 9.2, and ranges from less than 1 to 20 or more depending on the relative dye or antibody concentration in the reaction solution. 3 illustrates forming covalently labeled antibody molecules having a dye: antibody molar ratio of over. The slope obtained by applying the least squares method to the data shows that the labeling efficiency of this dye under these conditions is about 80%. In a similar study, fluorescein isothiocyanate (FITC) reacted with an efficiency of about 20%.

The reactivity of the active ester of the novel sulfoindodicarbocyanine dye is sheep immunoglobulin (IgG).
Was investigated by labeling. Protein (4mg / m
1) was dissolved in 0.1 molar carbonate buffer (pH 9.2). An aliquot of the reactive dye dissolved in anhydrous dimethylformamide was added to the protein sample to obtain the initial dye: protein molar ratio. After 30 minutes, gel permeation chromatography (Se
The protein was separated from the sheep-bound dye by phadex G-50). The resulting dye: protein molar ratio was examined by a spectrophotometer and is shown in FIG.

At low dye: protein ratios, the absorption spectrum of the labeled protein shows a band closely corresponding to that of the free monomer dye. Antibody molecules that are highly labeled (high dye: protein ratio) or labeled with dyes that have a high tendency to aggregate in aqueous solution often show a new absorption peak that appears at a shorter wavelength than the absorption band of the monomer dye in aqueous solution. It has been found to have. The wavelength of the new absorption peak is often
It enters the region showing the dimer absorption spectrum characteristic of the dye (see FIG. 1).

The higher the degree of labeling of the antibody, the higher the short wavelength: long wavelength absorption peak ratio. This shorter wavelength absorption peak can be seen at about 590 nm in FIG. In FIG. 4, the longer wavelength (645 nm) peak is due to the monomer dye molecule bound to the antibody. The bis-N-hydroxysuccinimide ester of the labeling reagent N, N'-dicarbobutylindodicarbocyanine-5,5'-acetic acid used to provide the antibody absorption spectrum of Figure 4 has no aryl sulfonate group. Readily forms a dimer in the aqueous salt solution and also on the antibody with which it has reacted. Importantly, the fluorescence excitation spectra of these antibodies show that the excitation of labeled antibody at the short wavelength peak does not proportionally generate fluorescence as much as the excitation at the long wavelength peak. This discussion is consistent with the notion that the short wavelength absorption peak is due to the formation of non-fluorescent dimers or conjugates on the antibody molecule.

We have determined that the aryl sulphonate cyanine dye used to obtain the data in FIG. 3 is by a much smaller absorption peak at the characteristic absorption wavelength of the dimer (see FIG. 5). As described above, it was found that they do not easily bind on the antibody molecule. This is important because antibodies and other "non-binding" labeling reagents should produce more fluorescently labeled proteins. In fact, arylsulfocyanines produce bright fluorescent antibodies even when the average dye / antibody ratio is relatively high (see Figure 6).

FIG. 4 shows sheep immunoglobulin labeled with a carboxyindodicarbocyanine dye. FIG. 5 shows the protein bound to the novel sulfoindodicarbocyanine dye. The presence of increased dimers in the former sample (see Figure 1) is apparent. Although the dye: protein molar ratios were about the same for both preparations, the protein shown in FIG. 5 was more fluorescent than the sample shown in FIG.

In order to have a bright fluorescent antibody or other protein labeled with a fluorescent dye, it is important that the average quantum yield per dye molecule on the protein is as high as possible. In general, as the surface density of dye molecules on protein increases (ie, the dye / protein ratio increases), the average quantum yield of dye decreases. This effect has sometimes been attributed to quenching resulting from dye interactions on the surface of highly labeled proteins.
Indeed, the formation of non-fluorescent dimers on the protein surface may contribute to this quenching. FIG. 6 shows that the average quantum yield of the arylsulfocyanine dye decreases slowly with increasing dye / protein ratio (curve connecting diamonds). In contrast, the curve connecting the circles shows that as the dye / protein ratio increases, the non-arylsulfocyanine dye (N, N '
-Di-sulfobutyl indodicarbocyanine-5-isothiocyanate), indicating that there is a very rapid reduction in the average quantum yield. Therefore, the sulfocyanine dye illustrated in FIG. 6 produces sharper fluorescent antibodies compared to other dyes, especially in the labeling range of 1 to 10 dye molecules per antibody molecule.

The average fluorescence quantum yield of individual dye molecules on the labeled protein is a measure of the fluorescence signal obtained from these biomolecules. The data from the sheep immunoglobulin (IgG) labeled with the novel sulfoindodicarbocyanine dye in saline solution with phosphate buffer is shown by the curve connecting diamonds in FIG. The curve connecting the circles in FIG. 6 shows the protein labeled with the indodicarbocyanine-isothiocyanate reactive dye for comparison. In Figure 6, the dye / protein ratio is 0.
The quantum yields at are the values relating to the methylamine adduct of the reactive dye (free dye) in the buffer.

Background Method Luminescent probes are useful reagents for the analytical separation of molecules and cells and for the detection and quantification of other substances. A very small number of luminescent molecules can be detected under optimal conditions. B
arak and Webb use the SIT camera to
We visualized less than 50 fluorescent lipid analogs related to DL acceptance (J. Cell Biol. 90: 595-60.
4 (1981)]. 1 related to particles or specific cells
Flow cytometry can be used to detect less than 10,000 molecules of fluorescein [Mirhead,
Horan and Poste, Bio / Technol
3: 337-356 (1985)]. Some specific examples of fluorescent probe applications are (1) identification and separation of subpopulations of cells in a cell mixture by the techniques of fluorescence flow cytometry, fluorescence activated cell sorting and fluorescence microscopy, (2) fluorescence. Localization of substances in gels and other insoluble carriers by techniques of immunoassay techniques, such as densitometry of substances that bind to another species (eg, antigen-antibody reactants) and (3) fluorescent staining techniques. These techniques are described in Herzenberg et al., “Cell Immunology (C
ellular Immunology) ", 3rd edition,
Chapter 22; Blackwell Scientific
Publications, 1978 (fluorescence activated cell classification) and Goldman, “Fluorescent antibody method (Fluor).
escape Antibody Method
s) ”, Academic Pres, New York
s, 1968 (fluorescence microscopy and fluorescence staining (fluor
esence microscopy and flu
orness staining)) and A
applications of Fluorescence
e in the Biomedical Science
ed., Tailor et al., Alan Liss Inc. ,
1986.

When fluorescence is used for the above purpose, there are many restrictions on the selection of the fluorescent substance. One constraint is the absorption and emission properties of fluorescent materials. This is because many ligands, receptors and substances in analytes such as blood, urea, cerebrospinal fluid fluoresce and interfere with the accurate measurement of fluorescence of fluorescent labels. This phenomenon is called autofluorescence or background fluorescence. Another consideration is
The ability of a fluorescent substance to bind to ligands and receptors and other biological or non-biological substances and the effect of binding to such fluorescent substance. In many situations, binding to another molecule can result in a substantial change in the fluorescent properties of the fluorophore, which in some cases substantially impairs or reduces the quantum efficiency of the fluorophore. It is also possible that the binding with a fluorescent substance inactivates the function of the labeled molecule. A third consideration is the quantum efficiency of the fluorescent substance, which should be high for sensitive detection. The fourth consideration is the absorptivity or absorptivity of the fluorescent substance, which should be as large as possible. In addition, whether or not the fluorescent molecules interfere with each other to cause self-quenching when they are in the proximity state is related. It also concerns whether the fluorescent substance non-specifically binds to the container wall in the context of another compound or by the fluorescent substance itself or of the compound to which it is bound.

The applicability and value of the above method is closely tied to the availability of suitable fluorescent compounds. In particular, a fluorescent substance that emits light in the long-wavelength visible region (yellow to near infrared) is necessary.
Because the excitation of these chromophores does not cause much autofluorescence, and a large number of chromophores emitting at different wavelengths can be analyzed simultaneously when the full visible and near infrared regions of the spectrum can be used. Although the widely used fluorescent compound fluorescein is a useful emitter in the green region, background autofluorescence resulting from excitation at the fluorescein absorption wavelength limits detection sensitivity in certain immunoassays and cellular analysis systems. However, the conventional red fluorescently labeled Lodamine was found to be less effective than fluorescein. Texas Re
d® is a useful labeling reagent that can be excited at 578 nm and fluoresces maximally at 610 nm.

Phycobiliprotein has made important contributions due to its high absorptivity and high quantum yield. Such chromophore-containing proteins can be covalently linked to many proteins and are thus used in fluorescent antibody assays in microscopy and flow cytometry. Phycobiliprotein has (1) a relatively complicated protein labeling method, and (2) a protein labeling efficiency is not usually high (typically 0.5 phycobiliprotein molecules / average).
Protein), (3) phycobiliprotein is a natural product, its preparation and purification are complicated, (4) phycobiliprotein is expensive, and (5) red region more than allophycocyanin which emits maximum fluorescence at 680 nm. Phycobiliprotein, which is effective as a labeling reagent that fluoresces in the spectrum, does not exist, (6) phycobiliprotein is relatively chemically unstable, (7) it easily photobleachs, (8) ) Phycobiliprotein is 33,000-24
It is a large protein with a molecular weight in the range of 10,000 and has the disadvantage that labels such as metabolites, drugs, hormones, derivatized nucleotides and many proteins including antibodies are larger than many substances that are desirable. The latter drawback is particularly serious. Because large phycobiliprotein-labeled antibodies, avidin, DNA hybridization probes, hormones and small molecules cannot bind to their target due to the steric restrictions imposed by the size of the complex complex, and bind to the target. This is because the binding rate of is slow with respect to the low molecular weight conjugate.

Other techniques, including histology, cytology, immunoassays, also exhibit high quantum efficiency at long wavelengths, absorption and emission properties, have simple coupling means and are virtually free of non-specific interference. Substantially benefit from the use of fluorescent materials.

[0045] Outline of the Invention The present invention uses fluorescence arylsulfonates cyanine and related dyes have a relatively large extinction ratio and high quantum yield in the assay and quantification purposes of labeling component. Fluorescent cyanine and related dyes include antibodies, antigens, avidin, streptavidin, proteins, peptides, derivatized nucleotides,
Carbohydrates, lipids, biological cells, bacteria, viruses, blood cells, tissue cells, hormones, lymphokines,
It can be used to label biological substances such as trace biomolecules, toxins and drugs. Fluorescent dyes include soluble polymers, polymers or glass particles, drugs,
It can also be used to label non-biological substances such as conductors, semiconductors, glass or polymer facings, polymer membranes and other solid particles. The components to be labeled can be present in mixed form with other substances. The mixture in which the labeling reaction takes place can be a liquid mixture, especially a water mixture. The detection step can occur with a liquid such as a microscope slide or a dry mixture.

The present invention requires a cyanine dye to be modified by incorporation into the cyanine molecule of a reactive group that is covalently attached to the target molecule, preferably at the amine or hydroxy site, and in some cases at the sulfhydryl or aldehyde site. The present invention also enhances the solubility of cyanine and related dye structures in the test liquid to (1) facilitate handling in the labeling reaction and (2) help prevent dye binding on the surface of the labeled protein. And (3) use or modify cyanine and related dye structures to help prevent non-specific binding of labeled substances to biological substances and surface materials or assay devices.

Cyanine and related dyes offer important advantages over existing fluorescent labeling reagents. First of all, 400
Cyanine and related dyes are synthesized which absorb or emit in the spectral region of about 1100 nm. Thus, reactive derivatives of these dyes can be prepared for assays that require simultaneous measurement of multiple labeling substances. This type of multi-color (or multi-parameter) analysis is simple, cost effective, or the ratio of different labeled species on each particle in a complex mixture of particles (eg complex parameters by multi-parameter flow cytometry or fluorescence microscopy). Ratio of antigen markers on individual blood cells in the mixture). Second, many cyanines and related dyes absorb light strongly and fluoresce. Third, many cyanines and related dyes are relatively photostable and do not bleach rapidly under fluorescent microscopy. Fourth, cyanine and related dye derivatives that are simple and effective coupling reagents can be prepared. Fifth, many structures and synthetic methods are effective, and dyes of this kind are versatile. Therefore, many structural modifications can be made to make the reagents somewhat water soluble. The charge may change so as not to disturb the molecule to which it binds and may reduce non-specific binding. Sixth, unlike phycobiliprotein,
Cyanine dye is relatively small (molecular weight = 1,000),
Therefore, it does not noticeably sterically hinder the ability of the labeled molecule to quickly reach its binding region or perform its function.

Thus, cyanine-type dye labeling agents offer many potential advantages. Such dyes can be used for selective labeling of one or more components in liquids, especially aqueous liquids. The labeled component can then be detected by optical or luminescent methods. Alternatively, the labeled component is used to stain another component for which it has a strong affinity, so that the latter component is detected by optical or luminescent methods. In this case the dye reacts with the amine, hydroxy, aldehyde or sulfhydryl groups of the labeling component. For example, the labeling component can be an antibody and the stained component to which it has a strong affinity can be a biological cell, an antigen or hapten, or a biological cell or particle containing an antigen or hapten. In another example, the labeling component can be avidin and the dyed component can be a biotinylated material. Moreover, a specific carbohydrate group can be detected and quantified using a lectin bound to a polymethinecyanine type dye. In addition, the luminescent cyanine and related dyes can bind to DNA or RNA fragments. The DNA or RNA labeled fragment can be used as a fluorescent hybridization probe for examining the presence and amount of a specific complementary nucleotide sequence in a sample containing DNA or RNA. In addition, the dye is a hormone or a ligand (eg, hormone,
Proteins, peptides, lymphokines, metabolites) and then the hormone or ligand may bind to the receptor.

Patents relating to other uses of reactive cyanines
Description Miraha et al. (U.S. Pat. No. 4,337,063) and Masuda et al. (U.S. Pat. No. 4,404,289).
No. 4,405,711 and No. 4,414,
No. 325) synthesizes various cyanine dyes having an N-hydroxysuccinimide active ester group. These patents show that the above reagents can be used as photographic sensitizers. The potential fluorescent nature of the reagent is not described in the above patent, and in fact no fluorescence is required for the process. Most of the dyes listed in the above patents emit only weak fluorescence, are not particularly photostable, and their solubility properties are also not optimal for many applications involving fluorescence detection of labeling substances.

Exekiel et al. (UK Patent No. 1,52
9,202) presents many cyanine dye derivatives that can be used as covalently reactive molecules. The reactive group used in this reagent is an azine group to which the mono- or dichlorotriazine group belongs. The British patent relates to the development and use of the above reagents as photographic film sensitizers. Fluorescence is not required for the process and most of the reagents described do not fluoresce. This British patent does not relate to the development and use of reactive cyanine dyes for the purpose of detecting or quantifying labeled substances.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides luminescent cyanine and cyanine-type dyes as biological agents for rendering a labeled substance luminescent so that it can be detected and / or quantified by a luminescence detection method. , Non-biological and macromolecules and methods for covalent attachment to particles.

The present invention, when detecting a component in a liquid, is a dye soluble in the liquid and contains a substituent group that covalently binds the dye to the amine or hydroxy group on the component, and optionally to an aldehyde or sulfhydryl group. It relates to a method of adding a dye selected from the group consisting of cyanine, merocyanine and styryl dyes so that it labels the above components. The labeled component is detected and / or quantified by luminescence or light absorption methods. When the labeled component is an antibody, DNA fragment, hormone, lymphokine or drug, the labeled component can be used to determine the presence or absence of another component to which it binds, thus detecting the other component. And / or can be quantified.

Any valid luminescence or absorption detection step can be used. For example, the detection stage can be an optical detection stage in which the liquid is illuminated with light of a wavelength originally defined. Then, light at another defined wavelength, which is fluorescent or phosphorescent by the labeling component, is detected. Detection can also be by optical absorption. For example, the detecting step includes passing light of an initially defined wavelength through the liquid and then identifying the light wavelengths conducted by the liquid.

If desired, the detection step can include a chemical assay that chemically detects the binding of cyanine or a related chromophore to the component. The basic structures of cyanine, merocyanine and styryl dyes that can be modified to create covalent labeling reagents are shown below: Cyanine

[0055]

[Chemical 1] Merocyanine

[0056]

[Chemical 2] Styryl

[0057]

[Chemical 3] The following are more specific examples of polymethine type dyes: Cyanine

[0058]

[Chemical 4] Merocyanine

[0059]

[Chemical 5] Styryl

[0060]

[Chemical 6] Cyanine

[0061]

[Chemical 7] Merocyanine

[0062]

[Chemical 8] Styryl

[0063]

[Chemical 9] In the above structure, X and Y are O, S and

[0064]

[Chemical 10] Z is selected from the group consisting of O and S, and m is an integer selected from the group consisting of 1, 2, 3 and 4.

In the above equation, the number of methine groups partly determines the excitation color. The cyclic azine structure can also partially determine the excited structure. Often, higher values of m contribute to increased luminescence or absorbance. At values of m higher than 4, the compound becomes unstable. There, further luminescence may be imparted by modification with the ring structure. m
= 2, the excitation wavelength is about 650 nm and the compound fluoresces correctly. The maximum emission wavelength is approximately 15 to 100 nm larger than the maximum excitation wavelength.

In each molecule R ₁ , R ₂ , R ₃ , R ₄ , R ₅ ,
At least one of the R ₆ and R ₇ groups (preferably only one, optionally two to three or more) is a reactive group for attaching the dye to the labeling component. For some reagents, at least one of the R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ groups on each molecule enhances the solubility of the chromophore or the label of the labeling component. It may be a group which affects the selectivity or the labeling position of the labeling component by the dye.

In the above formula, at least one of R ₈ , R ₉ (when present) and R ₁₀ (when present) contains at least one sulfonate group. The term sulfonate group shall include sulfonic acid. This is because the sulfonate group is only an ionized sulfonic acid.

Directly or indirectly bound to a chromophore
R₁, R₂, R₃, R_Four, R_Five, R₆And R ₇Form a group
As a reactive group, isothiocyanate, isocyanate
G, monochlorotriazine, dichlorotriazine, mono
-Or di-halogen-substituted pyridine, mono- or di-
Halogen-substituted diazine, maleimide, aziridine, sulfone
Honyl halide, acid halide, hydroxysuccinimide
Stell, hydroxysulfosuccinimide ester, a
Midester, hydrazine, azidonitrophenyl, a
Zido, 3- (2-pyridyldithio) propionamide,
Reactions such as groups containing glyoxal and aldehydes
Parts may be included.

R ₁ , R ₂ , which is particularly useful for labeling components having effective amino, hydroxy and sulfhydryl groups,
Specific examples of R ₃ , R ₄ , R ₅ , R ₆ and R ₇ groups include:

[0070]

[Chemical 11] (Here, at least one of Q and W is I, Br or C.
a residue such as l),

[0071]

[Chemical 12] R ₁ , which is particularly useful for labeling components with effective sulfhydryls that can be used to label antibodies in a two-step method,
Specific examples of R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ groups are:

[0072]

[Chemical 13] (Where Q is a residue such as I or Br),

[0073]

[Chemical 14] and

[0074]

[Chemical 15] (Where n is 0 or an integer).

As specific examples of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ groups which are particularly useful for labeling components by light activated crosslinking.

[0076]

[Chemical 16] Is included.

Two or more reactive chromophores on the labeled component that may enhance water solubility or reduce undesired non-specific binding of the labeled component to unsuitable components in the sample, or may also result in fluorescence quenching. In order to reduce the interaction,
The R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ groups can be selected from the known polar or charged chemical groups. Example is −
EF, where F is hydroxy, sulfonate,
Represents a sulfate, a carboxylate, a substituted amino or a quaternary amino, and E is-(CH ₂ ) _n (n = 0, 1, 2, 3
Or a spacer group such as 4). Useful examples of the alkyl sulfonate - (CH _{2) 3} SO ^3- and - (C
H ₂ ) ₄ —SO ^{3 −} is included.

The polymethine chains of the luminescent dyes according to the invention are also
It may contain one or more cyclic chemical groups which form a bridge between two or more carbon atoms of the polymethine chain. These bridges may serve to enhance the chemical or photostability of the dye and may be used to change the absorption or emission wavelength of the dye or change the extinction coefficient of the quantum yield. Improved dissolution properties can be achieved by this modification.

According to the invention, the labeling component is an antibody, a protein,
Peptides, enzyme substrates, hormones, lymphokines, metabolites, receptors, antigens, haptens, lectins, avidin, streptavidin, toxins, carbohydrates, oligosaccharides,
Polysaccharide, nucleic acid, deoxynucleic acid, derivative nucleic acid, derivative deoxynucleic acid, DNA fragment, RNA fragment, derivative DNA fragment, derivative RNA fragment, natural drug, virus particle, bacterial particle, virus component, yeast component, blood cell, blood cell component , Biological cells, non-cellular blood components, bacteria, bacterial components, natural or synthetic lipid sac, synthetic drugs, poisons, environmental pollutants, polymers,
It can be polymer particles, glass particles, glass surfacing, plastic particles, plastic surfacing, polymer membranes, conductors and semiconductors.

A cyanine or related chromophore capable of absorbing light at 633 nm upon reaction with certain components can be prepared,
Thus, in the detection step, a helium neon laser emitting at this spectral wavelength can be used. It is also possible to prepare cyanines or related dyes that are capable of maximally absorbing light between 700 nm and 900 nm upon reaction with certain components, so that the detection process emits light in this region of the spectrum. Laser diode can be used.

Selectivity The reactive groups listed above are specific to proteins or other biological or non-biological molecules, macromolecules, surface materials or particles so long as appropriate reaction conditions including suitable pH conditions are used. It is relatively specific for labeling functional groups.

Reactive Cyanine, Merocyanine and Ste
Properties of Ril Dyes and Their Products The spectral properties of the dyes of this invention are not appreciably changed by the functionalization described herein. The spectral characteristics of labeled proteins and other compounds are also not so different from those of base dye molecules that are not bound to proteins or other substances. The dyes described in the present invention alone or bound to a labeling substance generally have a large extinction coefficient (ε = 100,000 to 250,000).
00) and, in some cases, high quantum yields, on the order of 0.4, and emit light in the spectral range of 400-900 nm. Thus, they are of particular value as labeling reagents for luminescence detection.

Optical Detection Method Any method can be used to detect the labeling or staining component. The detection method can use a light source that illuminates the mixture containing the labeling substance with light having a wavelength that is initially defined. Known devices are used which detect light at a second wavelength which is either transmitted by the mixture or fluoresced or emitted by the mixture. Such detectors include fluorescence spectrometers, absorption spectrophotometers, fluorescence microscopes, transmitted light microscopes or flow cytometers, fiber optic sensors and immunoassay devices.

The method of the present invention may also employ chemical analytical methods to detect dye binding to the label component (s). Chemical analysis methods can include infrared spectrometry, NMR spectrometry, absorption spectrometry, fluorescence spectrometry, mass spectrometry and chromatographic methods.

[0085]

EXAMPLES Example 1 For the binding of sulfoindodicarbocyanine to proteins
Effect of pH Sheep gamma-globulin (4 mg / mg in 0.1 M carbonate buffer)
ml) The sample was mixed at room temperature with a 10-fold molar excess of the following sulfoindodicarbocyanine active ester (m = 2) at room temperature:

[0086]

[Chemical 17] Sephadex G-5, suitable time for 5 seconds to 30 minutes
The protein sample was separated from the non-covalently bound dye by gel permeation chromatography on zero. Maximum protein labeling occurred after 10 minutes and was 5.8, 6.4 and 8. respectively for samples incubated at pH 8.5, 8.9 and 9.4.
A final dye / protein molar ratio of 2 was produced. The time required to bring the dye / protein ratio to 5 and the product quantum yields at different pHs are shown in the following table: pH time (sec) quantum yields 8.5 115 0.09 8.9 53 0.09 9.4 6.5 0.17 The above data shows that the protein labeled with this dye is better at pH 9.4 than below pH 9. The higher the pH of the buffer, the more rapid the binding reaction, but the better the labeling efficiency and the higher the fluorescence of the product. Quantum yield represents the average quantum yield per dye molecule on the labeled protein.

Example 2 Coupling of sulfoindocarbocyanine active ester and protein
Sheep γ- globulin causes dissolved in phosphate buffer containing saline merging pH7.4 (PBS) (1mg / ml ), adjusted to pH9.4 with 0.1M sodium carbonate. Cyanine dye labeling agent (structure of Example 1, m = 1) was added to aliquots of this protein solution in order to obtain different dye / protein molar ratios.
After incubation for 30 minutes at room temperature, separation was performed by Sephadex G-50 gel permeation chromatography using PBS as an eluent. The molar ratios of dyes covalently bound to proteins in the product were 1.2, 3.5, 5.4, 6.7 and 11.2 for the initial dye / protein ratios 3, 6, 12, 24 and 30, respectively. there were.

Example 3 AECM Dext with Sulfoindodicarbocyanine
Labeling orchids N-aminoethyl-carboxamidomethyl (AEC containing an average of 16 amino groups per dextran molecule)
M) Dextran was synthesized from dextran with an average molecular weight of 70,000 [Inman, J. et al. K. J. Immu
nol. 114 : 704-709 (1975)]. pH
9.4, AECM-dissolved in 0.1M carbonate buffer
A portion of dextran (1 mg / 250 μl) was added to a sulfoindodicarbocyanine active ester (structure of Example 1, m
= 2) 0.2 mg in addition to give a dye / protein molar ratio of 10. The mixture was stirred at room temperature for 30 minutes. Dextran was then separated from unbound 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 Sulfoindodicarbocyanine active ester of specific antibody
Labeled murine IgG in 1.1M carbonate buffer (pH 9.4)
(1 mg / ml) specific sheep γ-globulin and sulfoindodicarbocyanine active ester (structure of Example 1,
m = 2) with a dye molecule / protein molecule ratio of 8. After incubation at room temperature for 3 minutes, the labeling mixture was separated by gel filtration on Sephadex G-50 equilibrated with phosphate buffered saline (pH 7.4). The recovered proteins contained an average of 4.4 dye molecules covalently bound to each protein.

Example 5 Sulfoindodicarb conjugated to sheep anti-mouse IgG antibody
Staining of human lymphocytes with cyanine dye or microscopy
Visualization Freshly isolated peripheral blood lymphocytes were treated with mouse anti-β2-microglobulin (0.25 μg10 ⁶ cells) for 30 minutes at 0 ° C. Cells were washed twice with DMEM buffer and then treated with sulfoindodicarbocyanine labeled sheep anti-mouse IgG antibody (1 μg / 10 ⁶ cells). 0
After 30 minutes of incubation at 0 ° C., cells were centrifuged to remove excess antibody and cells were washed again with DMEM buffer. Aliquots of cells were mounted on slides for analysis by fluorescence microscopy. Illuminate the lymphocytes on the slide with light of 610-630 nm under a microscope and use a COTU red-sensitivity enhanced television camera mounted on an image digitizer and television monitor to 650-700 nm.
Fluorescence was detected. Cells stained by this method showed fluorescence under the microscope. In a control experiment, staining and analysis was performed as described above except that the use of primary mouse anti-β2-microglobulin antibody was omitted. The control sample showed no fluorescence under the microscope, thus indicating that the sulfoindocyanine labeled sheep anti-mouse antibody did not result in significant non-specific binding to lymphocytes.

[Brief description of drawings]

FIG. 1 shows the monomer absorption spectrum and dimer spectrum of N, N′-disulfobutylindodicarbocyanine dye.

FIG. 2 shows the spectrum of N, N′-diethylindodicarbocyanine-5,5′-disulfonic acid dye.

FIG. 3 is a dye / protein molar ratio in a labeling reaction of sheep immunoglobulin with a bis-N-hydroxysuccinimide dye of N, N′-dicarboxypentyl indodicarbocyanine-5,5′-disulfonic acid. Indicates.

FIG. 4 shows the absorption spectrum of an antibody labeled with bis-N-hydroxysuccinimide ester of N, N′-dicarbobutylindodicarbocyanine-5,5′-acetic acid.

FIG. 5 shows an absorption spectrum of a protein bound with a novel sulfoindodicarbocyanine dye.

FIG. 6 shows a comparison of changes in the quantum yield of arylsulfocyanine dyes (diamonds) with increasing dye / protein ratio and that of non-arylsulfocyanine dyes (circles).

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 2G043 AA01 BA16 CA04 DA02 EA01                       GA07 GB21 JA01 LA01 MA01                 2G045 DA13 DA36 DA80 FA29 FB12                       GC15                 2G054 AA10 CA22 CA23 CA30 CE02                       EA03 EB01 GA04

Claims (4)

[Claims] 1. A method of quantifying one or more fluorophores, which comprises a luminescent dye-labeled substance and all of which significantly absorb light at a single excitation wavelength but fluoresce at different wavelengths. Mixing one or more phosphors, and all the phosphors,
Excitation with a single wavelength of light and quantification of each of the fluorophores by detecting their individual fluorescence signals at their different fluorescence wavelengths, wherein the luminescent dye is bound to an aromatic nucleus A method as described above, comprising selected from the group consisting of cyanine, merocyanine and styryl dyes having at least one sulfonic acid group or sulfonate group. 2. A method for labeling a component in an aqueous liquid, comprising: (a) cyanine or merocyanine having at least one sulfonic acid group or sulfonate group bonded to an aromatic nucleus in the liquid containing the component. A luminescent dye selected from the group consisting of and a styryl dye, the dye being reactive with the component, and (b) reacting the dye with the component such that the dye labels the component. How to include. 3. The dye comprises a substituent that causes the dye to covalently react with an amine, aldehyde, sulfhydryl or hydroxy group on the component, the dye reacting with a group on the component. Method 2. 4. The component is an antibody, protein, peptide, enzyme substrate, hormone, lymphokine, metabolite, receptor, antigen, hapten, lectin, toxin, carbohydrate,
Oligosaccharide, polysaccharide, nucleic acid, nucleotide, derivatized nucleotide, deoxynucleic acid, derivatized nucleic acid, derivatized deoxynucleic acid, DN
A fragment, RNA fragment, derivative DNA fragment, derivative RNA fragment, natural drug, virus particle, virus component, yeast component, blood cell, blood cell component, biological cell, non-cellular blood component, bacterium, bacterial component, natural and synthetic lipid A capsule, a synthetic and natural drug, a poison, an environmental pollutant, a polymer, a polymer particle, a glass particle, a glass surface material, a plastic particle, a plastic surface material, a polymer film, a conductor and a semiconductor. Method 2 or 3.