JPH02191674A - Method for labeling or detection of substance by using luminescent aryl sulfonate cyanine dye - Google Patents

Abstract

PURPOSE: To simply and effectively perform labeling and detecting by incorporating a specified (mero)cyanine or styryl dye into such an ingredient as an antibody and a protein in an aqueous liq.

CONSTITUTION: When an ingredient in an aqueous liq. is labeled, labeling and detection of an antibody, a protein, a peptide, etc., are performed by incorporating an emission dye (B) consisting of a (mero)cyanine or a styryl dye contg. at least one sulfonic acid group or sulfonate group bound with an arom. nucleus into an aqueous liq. (A) contg. the above described ingredients and reacting the ingredient B with the above described ingredient in the liq. so as to label the above described ingredient in the aqueous liq. with this dye.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION Cyanine and related dyes with light-absorbing properties have been used in photographic film. Such dyes require light-absorbing properties but not luminescent (fluorescent or phosphorescent) properties; hitherto cyanine dyes with luminescent properties have had very limited use. Such uses include labeling sulfhydryl groups of proteins. "
Sulfhydryl reagent dye initiates rapid Ca” release from the sarcoplasmic reticulum sac (SR) (Sulfhydryl
Reagent Dyes Trigger tha
Rapid Re! ease of Ca”
from Sacoplasmic Reticu
In a paper entitled lulIIVesicles (SR) J, Salama G.

Waggoner A, S, Abram50n J
, the cyanine chromophore having an iodoacetyl group is Ca
reported that it was used to form covalent bonds with sulfhydryl groups on sarcoplasmic reticulum proteins at pH 6.7 (Biophysical Journal).
urnal, 47.456a (19F15)), this paper also describes how fluorescent dyes can be used with proteins.

"Dynamics of conformational changes in the region of the rhodopsin molecule distant from the retinylidene binding site"
+fora+ational Changes in
a Region of the Rhodopsin
Motecule Away From thsRat
In a paper entitled Inyldene Binding 5ite) J, Waggoner^, S9, JenNn
s P, L,, Carper+ter J, P.

and Gupta R, report that the sulfhydryl group on the F1 region of bovine rhodopsin was covalently labeled with a cyanine dye with an absorbance at 660 rim.
[Biophysical Journal, 33.2
92a (1985)], which also reports the use of cyanine dyes to specifically label sulfhydryl groups of proteins, but does not disclose the use of fluorescent dyes.

``International Seminar on the Application of Fluorescent Photobleaching Techniques to Problems in Cell Biology (Inter-natjona)''
l Workshop on tt+s Applic
ation of Fluorescence pho
Tobleaching Techniques
t.

Problems in Ce1l Biolog31
) J Jacob50n K, El50n on cyanine-type fluorescent probes that can be conjugated to proteins and are excited in the deep red region of the spectrum.
E, , oppel D, and Webh 'R papers have been reported (Fed, Proc, 42 Near 2-
79 (1983)>.

The only cyanine probes described in any of the three papers mentioned above specifically bind covalently to sulfhydryl groups of proteins. The only specific cyanine compounds described are those with iodoacetyl groups, which cause cyanine dyes to covalently react with sulfhydryl groups. None of the above articles disclose the covalent reaction of cyanine dyes with substances other than proteins or any groups on protein proteins other than sulfhydryl groups.

However, many non-protein substances have sulfur l-dolyl groups, and many proteins do not have enough of these groups to be useful for fluorescence probing.

Moreover, the sulfhydryl group (-SHS) I-) is easily oxidized to disulfide (-S-3-) in the presence of air, thereby rendering it amenable to covalent attachment to a fluorescent probe.

According to the present invention, under reaction conditions suitable for fluorescence or phosphorescence detection of substances, not only sulfhydryl groups on proteins and other substances but also amines (-NH, ) and hydroxy (
Cyanine and related polymethine dyes have been developed which have substituents covalently reacting with groups such as OH) groups and other aldehyde (-C) groups. Amine and hydroxy groups are more prevalent and stable in proteins and other materials than sulfhydryl groups, which provides a considerable benefit over specific reactivity with groups. Thereby,
When using fluorescent cyanine dye to detect the presence or absence of a specific protein,
Since many dye molecules bind to the protein to be probed, a stronger fluorescence or φ light intensity signal is emitted. Moreover, amine or hydroxy groups are naturally more easily attached to the desired components of the label, such as polymer particles that do not contain sulfhydryls, amines or hydroxy groups.

The present invention also provides that the presence or absence or amount of a labeled protein or other substance is
After separation of the labeled component by chromatographic methods, a luminescent cyanine dye containing an amine or hydroxy group or a group that covalently reacts with other reactive groups can be used to detect the amine or hydroxy group that reacts with the dye in the mixture. or a method of labeling proteins or other raw materials having other groups. According to the previously cited literature, sulfhydryl groups were apparently chosen for covalent reactions because group groups play a significant role in protein function, especially when sulfhydryl groups on protein molecules are very rare. . Therefore, it was possible for the authors to attempt to identify the specific position of the sulfhydryl group on the protein structure. Furthermore, in the above-mentioned literature, sulfhydryl groups were used as probes to probe or bring about structural changes in specific proteins. Thus, in order to determine changes in absorbance due to dye or changes in free calcium ions due to dye binding, it was necessary to know where the probe was bound.

Since there are very few sulfhydryl groups on most proteins, the number of sulfhydryl groups may not be sufficient to provide sufficient total luminescence for probing. In contrast, the number of amine or hydroxyl groups on a protein molecule is significantly higher in number and widely distributed, allowing fluorescent probes to be attached to multiple locations on the molecule, thereby facilitating protein detection. Therefore, determination of light absorption or fluorescence changes is eliminated.

The present invention uses luminescent polymethine cyanine or related polymethine dyes to produce proteins, nucleic acids, DNA, drugs, toxins, blood cells, microbial substances, two particles of microbial substances, plastic or glass surface materials, polymer films, etc., on which amines or Concerning labeling at hydroxy sites. Advantageously, the dye is soluble in the aqueous or other medium containing the labeling substance.The present invention relates to a two-step labeling method in addition to a one-step labeling method. In the two-step method, a first component, such as an antibody, is labeled at a site on the component that includes an amine, hydroxy, aldehyde, or sulfhydryl moiety. The labeled component is used as a probe for a second component, such as an antigen for which the antibody is specific.

In the prior art described above, 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, cyanine or related The probe can be reacted with an amine, aldehyde, sulfhydryl, hydroxy, or other group on a second component, such as an antibody, so that in a second or staining step, the antibody has the desired specificity on a second component, such as an antigen. Achieve sex. This specificity is determined by the site of antigen binding to the antibody.

The present invention also relates to luminescent polymethine cyanine and related compounds containing groups that allow covalent attachment to amine, hydroxy, aldehyde or sulfhydryl groups on a target molecule. concerning monoclonal antibodies and other components. When the target is one type of cell, the present invention can be used to measure the amount of labeled antibody that binds to that cell. This measurement is carried out by examining the relative brightness and darkness of the luminescence of the cells.

The present invention can be used to determine the concentration of specific proteins or other components in a system. If the number of reactive groups on the protein that reacts 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.

This method can be used to quantify various proteins or other substances in a system by labeling the entire mixture of proteins in the system and then separating the labeled proteins by any means such as chromatography. The amount of luminescent protein that can be separated can be investigated using a chromatography detection system.
The dye position on the labeling substance can be confirmed.

The invention can also be used to determine the number of different cells labeled with antibodies. This number can be investigated by labeling multiple types of cells in the system and then separating the labeled cells out of the system. Furthermore, labeled cells can also be separated from unlabeled cells outside the system.

Another embodiment of the invention provides a plurality of luminescent cyanines or related dyes each coupled to a plurality of different first components, such as antibodies, each of which is specific for a different second component, such as an antigen. According to this embodiment, which involves a multiparameter method used to identify each of multiple antigens in an antigen mixture, each antibody is labeled with a dye distinct from the dye used to label the other probes. All labeled antibodies that are separately labeled with dyes that have absorption and luminescence wavelength properties are then added to the biological preparation that is analyzed to contain a second component, such as an antigen, stained with each specific labeled antibody. It will be done. Any unreacted dye material, if it interferes with the analysis, is removed from the preparation by washing. The biological preparation is then subjected to various excitation wavelengths. Each excitation wavelength used is that of a particular conjugated dye. A luminescence microscope or other luminescence microscope such as a flow cytometer or fluorescence spectrophotometer with a filter or monochromator to select the light at the excitation wavelength and to select the wavelength of the luminescence to determine the luminosity 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 to which the dye is bound, when two substances in a mixture each fluoresce at a different wavelength. A single excitation wavelength can be used to excite the luminescence from above, and the amount of each labeled species can be determined by detecting the individual fluorescence intensities at each fluorescence wavelength. If desired, an absorption detection method can be used.The two-step method of the present invention allows the first substance-dye complex to be directed to a luminescence or absorption detection system to probe for the presence of another substance coupled to the dye. For example, a dye can bind to a DNA or RNA fragment and the dye binds to the DNA or RNA fragment.
A fragment can be formed, which is then oriented to the complementary NA or RNA backbone. The same test method can be used to probe for the presence of complementary backbones of DNA.

The cyanines and related dyes of the present invention are particularly well suited for component mixture analysis requiring dyes of various excitation and emission wavelengths because specific cyanines and related dyes with a wide range of excitation and emission wavelengths have been synthesized. Specific cyanines and related dyes with specific excitation and emission wavelengths can be synthesized by varying the number of methine groups or the cyanine ring structure. In this way, it is possible to synthesize dyes with specific excitation wavelengths corresponding to specific excitation light sources, such as lasers, e.g. HeNe or diode lasers.

The present invention provides highly luminescent and highly absorbent cyanine and related dye molecules under reaction conditions for use on proteins, peptides, carbohydrates, nucleic acids, nucleic acids, lipids, and other specific biological molecules and biological cells. Covalently reacting with non-biological materials such as amines, hydroxy, aldehydes, sulfhydryls or other groups and soluble polymers, polymer particles, polymeric surfaces, polymeric films, glass surfaces and other particles or surfaces. Regarding things. Because luminescence involves highly sensitive optical techniques, it is possible to probe and quantify 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 a substance that has been absorbed. The most useful dyes have high absorbance (t=70,000-250,0000fib1 mol C
m or more), highly luminescent, at least 5
It has a quantum yield of ~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 identification reagents is the production of luminescent monoclonal antibodies. Monoclonal antibodies are protein molecules that bind very tightly and specifically to specific chemical locations or "markers" on or within cells. These antibodies are therefore specific to cell types (e.g. HLA, T cells,
It has enormous research and clinical application in identifying abnormal cells (such as bacteria and viruses) and abnormal cells. Traditionally, antibody bound to cells has been quantified by labeling the antibody by a variety of methods, including radioactive labels (radioiminoassays), enzymes (ELISA techniques), or fluorescent dyes (usually fluorescein, rhodamine, Texas Red or This has been carried out by red algae). Most manufacturers or users of a-legal antibody reagents desire to eliminate the problems associated with the use of radioactive tracers, and therefore luminescence is considered one of the most promising alternatives. In fact, many vendors today market monoclonal antibody-labeled fluorescein, Texas Red, rhodamine, and red algae.

In recent years, optical/electronic devices for detecting fluorescent antibodies on cells have become increasingly complex.
It can be measured at rates down to 0 per second, and microscopy and solution fluorescence techniques have also advanced. These devices are capable of exciting fluorescence at many wavelengths in the tJ V , visible and near-IR regions of the spectrum. Most of the useful fluorescent labeling reagents available today excite in the 400-580 nnn spectral region. An exception is some phycobiliprotein types Hf4 isolated from marine organisms, which can bind covalently to proteins and are excited at slightly longer wavelengths. Therefore, there is a large spectral window in the 580 to about 900 rim range in which new labeling reagents need to be effective in labeling biological or non-biological substances to be analyzed with today's commercially available instruments. excitation in this spectral region)
The new reagent enables the performance of multicolor luminescence analysis of cellular markers, since antibodies with different specificity and isomerism can be labeled with different colored fluorescent dyes. In this way, the presence or absence of several markers can be determined simultaneously for each cell analyzed.

The present invention also relates to luminescent (fluorescent or phosphorescent) cyanine, merocyanine and styryl dyes themselves which are covalently bound to biological or non-biological substances. Merocyanine and styryl dyes are recognized as related cyanine dyes for the purposes of this invention. The new labeling reagent itself, especially when bound to a labeling component, is initially excitable with light at a definable wavelength, for example in the 450-900 nm spectral range. Background fluorescence of cells generally occurs at lower wavelengths. Therefore,! LAM! Reagents are distinguished from background fluorescence. Especially advantageous is 63
It is a derivative that absorbs 3 nm light. This is because the derivative can be excited by a cheap, strong, stable, and long-lived HaNe laser source. Then, the light at the second defined wavelength emitted by the labeling component is fluorescent or phosphorescent. can be detected. Fluorescence or phosphorescence generally has a longer wavelength than the excitation light. The detection step can employ a luminescence microscope having a filter to absorb scattered light at the excitation wavelength and to pass the corresponding wavelength of luminescence corresponding to the particular dye label used with the analyte. Such an optical microscope is described in U.S. Pat. No. 4,621,911.

Not all cyanines and related dyes are luminescent, however, the dyes of the present invention include cyanines and related dyes that are luminescent. They are relatively photostable and many are soluble in the reaction solution, preferably an aqueous solution. The conjugated dye itself, especially when it is conjugated to a labeling component, has a molecular weight of at least 50. OOO preferably 10
It has a molecular extinction coefficient (1) of 0°000ε 1 mol cm. The extinction coefficient is a measure of the ability of a molecule to absorb light. The bound dyes of the present invention have a quantum yield of at least 2%, preferably at least 10%. In addition, the binding dye of the present invention has a molecular weight of 400-900nrn, preferably 600-900nrn.
Absorbs and emits light in a spectral range.

Ursulfone Aryl sulfonate- or arylsulfonic acid-substituted dyes as described herein above are inherently more fluorescent than similar dyes without the arylsulfonate or arylsulfonic acid group, and have improved photostability and water solubility. It was found that it has As the term arylsulfonate or arylsulfonic acid group is used herein, it refers to an interchangeable arylsulfonino acid attached to an aromatic ring structure, including a single-ring aromatic structure or a fused ring structure such as a naphthalene structure. or arylsulfonate group. Single ring aromatic structures or fused ring aromatic structures are present in polymethine, cyanine, merocyanine or styryl type dyes.

Many dyes with coplanar molecular structures, usually including cyanine dyes, are susceptible to forming dimers or more conjugates in aqueous solution, especially when inorganic salts are also present, such as in buffers or saline. , these conjugates usually have an absorption band shifted to the short wavelength side of the mercury absorption,
They are generally very weakly fluorescent species. The tendency of cyanine dyes to form conjugates in aqueous solutions is particularly well known in the photographic industry [We
stL & pierce S, J, pi B,
Cheta, 69: 1894 (+965); St
urmer D, M,, S ec, To in
) Iter.

clic Chemhair, 30 (1974
It has been found that many dye molecules, particularly cyanine dye molecules, are prone to forming conjugates in aqueous solution, however, aryl sulfonate dyes have a minimal tendency to form these conjugates. When aryl sulfonate dyes are used to form fluorescent labeling reagents, they have a reduced tendency to form conjugates when bound to proteins or other molecules, such as 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) is 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 solutions. It can be exemplified that the tendency to form a body is low.

FIG. 1 shows the monomer and dimer absorption spectra of typical dianine dyes dissolved in aqueous Ta buffer; Butylated indodicarbocyanine has an aryl sulfonate group and readily forms dimers even at concentrations below the millimolar range. Dimer spectra were calculated from dye spectra at different concentrations (
(See the method of West & Psaree, +965).

At a concentration of 3 mmolar in phosphate buffered saline solution, the absorption of the monomeric and dimeric bands was approximately equal.

Improved sulfindyloxylucyanin N.

N-diethylindodicarpobocyanine-5,5'
The spectrum of -disulfonic acid is shown in FIG. 2; this dye showed no signs of 3 mmol in saline solutions with concentrations up to 10 mmol.

It is a 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 was bis-N-hydroxysuccinimide of N,N'-dicarboxypentylindodicarbocyanine-5,5'-disulfonic acid.

Figure 3 shows that this sulfocyanine dye active ester has a pH of
A covalently labeled antibody molecule that reacts efficiently with sheep immunoglobulin in a 9,2 carbonate buffer and has a dye:antibody molar ratio ranging from less than 1 to greater than 20 depending on the relative dye to antibody concentration in the reaction solution. . The slope obtained by applying a least squares fit to the data indicates that the labeling efficiency of this dye is approximately 80% under these conditions. In a similar study, fluorescein inthiocyanate (
FITC) reacted with an efficiency of about 20%.

The reactivity of active esters of novel sulfinodicarbocyanine dyes was investigated by labeling sheep immunoglobulin (IgG). Protein (4 mg/ml was dissolved in O, 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, 2 gel permeation chromatography (Sephadex
G-50) to separate the protein from the single bound dye. The resulting dye:protein molar ratio was determined using a spectrophotometer and is shown in FIG.

At low dye:protein ratios, the absorption spectrum of the labeled protein shows bands that correspond closely to the spectrum of the free monomeric dye; either highly labeled (high dye:protein ratio) or
It has been found that antibody molecules labeled with dyes that have a high tendency to aggregate in aqueous solution often have new absorption peaks that appear at shorter wavelengths than the monomeric dye absorption band in aqueous solution. The wavelength of the new absorption peak often falls into the region exhibiting the dimeric absorption spectral characteristics of the dye (see Figure 1).

The higher the distance between the antibody labels, the higher the short wavelength:long wavelength absorption peak ratio.This shorter wavelength absorption peak is approximately 59 in Figure 4.
It can be seen at 0 nm. In Figure 4, the longer wavelength (
645 nm) peak is due to monomeric dye molecules bound to the antibody. The labeled 11i N,N'-dicarbutylindodicarbocyanine-5,5°-acetic acid bis-N-hydroxysuccinimide ester used to produce the antibody absorption spectrum of FIG. 4 has an aryl sulfonate group. Importantly, the fluorescence excitation spectra of these antibodies indicate that the excitation of the labeled antibody at short wavelength peaks, but the excitation of the labeled antibody at longer wavelength This indicates that more peak excitation does not produce fluorescence proportionally. This consideration is consistent with the concept that the 5 short wavelength absorption peaks are due to the formation of non-fluorescent dimers or conjugates on the antibody molecule.

The inventors have determined that the arylsulfonate cyanine dye used to obtain the data in Figure 3 is effective for antibody molecules, as judged by the much smaller absorption peak at the characteristic absorption wavelength of the dimer (see Figure 5). It was found that the above does not bind easily. This is important because antibodies and other "non-binding" labeling reagents should produce more fluorescently labeled proteins. In fact, arylsulfocyanine produces bright fluorescent antibodies even when the average dye/antibody ratio is relatively high (see Figure 6). Figure 4 shows sheep immunoglobulin labeled with carboxyindodicarbocyanine dye. , FIG. 5 shows a protein conjugated with a novel sulfindodicarbocyanine dye.
The presence of increased dimer in the former sample (see Figure 1) is evident.Although the dye:protein molar ratio is approximately the same in both preparations, the protein shown in Figure 5 is the same as that shown in Figure 4. The fluorescence was stronger than the sample.

In order to have a brightly 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 a protein decreases (i.e., the dye/protein ratio increases), the average quantum yield of the dye decreases. This was caused by the resulting kenching. Indeed, the formation of non-fluorescent dimers on the protein surface may contribute to this quenching. Figure 6 shows that the average quantum yield of arylsulfocyanine dyes decreases slowly as the dye/protein ratio increases (curve connecting the diamonds), in contrast to the curve connecting the circles. The curve is dye/
We show that there is a very rapid decrease in the average quantum yield for binding of a non-aryl sulfocyanine dye (N, N'-di-sulfobutylindodicarbocyanine-5-isothiocyanate) as the protein ratio increases. It shows. Therefore, the sulfocyanine dye illustrated in FIG. 6 produces more vividly fluorescent antibodies than other dyes, especially in the labeling range of 1 to 10 dye molecules per antibody molecule.

The average fluorescent plate yield of individual dye molecules on labeled proteins is a measure of the fluorescent signal obtained from these biological molecules. Data from sheep immunoglobulin (NgG) labeled with the novel sulfinodicarbocyanine dye in phosphate buffered saline is shown by the diamond-connected curve in FIG. The curve connecting the circles in Figure 6 shows a protein labeled with an indodicarbocyanine-isothiocyanate reactive dye for comparison. Values for methylamine adducts of reactive dyes (free dyes) are shown.

Luminescent probes are useful reagents for the analytical separation of molecules and cells, as well as the detection and quantification of other substances. A very small number of luminescent molecules can be detected under optimal circumstances.
Barak and Webb used a SIT camera to visualize less than 50 fluorescent lipid analogs associated with cellular LDL reception (J, Ce1l Biol.

90:595-604 (1981)], flow cytometry can be used to detect 10,000 fluorescein molecules associated with particles or specific cells [Muirhead, Horan and Po5ta,
Bi TechnoloE3:337-356 (1
9115) 1. Some specific examples of fluorescent probe applications include (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 (3) the determination of the concentration of a substance that binds to another species (e.g., an antigen-antibody reactant) by immunoassay techniques; and (3) the localization of substances in gels and other insoluble carriers by fluorescent staining techniques. For the technique of Herzenberg
g et al., [Cellular Immunology (ca1.1ular Im+au
Nology) J, 3rd edition, Chapter 22; Bla
ckwe Sc 1ant if 1cPublica
tions, 1978 (fluorescence-activated cell classification) and Go
ldman, r Fluorescent antibody method
e Antibody Methods) J, Acadeo+ic Press, New York.

+968 (Fluorescence microscopy and fluorescent staining (f Iuore
scencemicroscopy arid flu
oressence 5tajnb+g) ) and ^
pp1ficationa of Fluoresce
nce In the[1roa+ediaa[5ci
ence8, hat @Te1ar, etc. ^JanLigs I
nc,,! 966.

When using fluorescence for the above purpose, there are many restrictions on the selection of fluorescent substances. One limitation is the absorption and emission properties of fluorescent materials. Because many pastes, receptors and
This is because substances in samples such as blood, urea, and brain fluid emit fluorescence, which interferes with accurate measurement of the fluorescence of the fluorescent label.

This phenomenon is called autofluorescence or background fluorescence. Another consideration is that the fluorescent material
The binding capacity for ligands and receptors as well as other biological or non-biological substances and the effect of binding on such fluorescent substances. In many situations, binding to another molecule can result in a substantial change in the fluorescent properties of the fluorophore, in which case it substantially impairs or reduces the quantum efficiency of the fluorophore. It is also possible that binding to a fluorescent substance inactivates the function of the labeled molecule. The third consideration is the quantum efficiency of the fluorescent material, which should be high for sensitive detection. The fourth point to consider is the absorbance or absorbance of the fluorescent substance, which should be as large as possible. Also relevant is whether or not fluorescent molecules interfere with each other and cause self-quenching when they are in close proximity.

Furthermore, it is also relevant whether the fluorescent substance binds non-specifically to the container wall with other compounds, by the fluorescent substance itself, or in relation to the compound to which it is bound.

The applicability and value of the above method is closely linked to the availability of suitable fluorescent compounds, especially fluorescent substances that emit in the long wavelength visible region (yellow to near infrared). This is because excitation of these chromophores produces less autofluorescence, and when using the entire visible and near-infrared region of the spectrum, multiple chromophores emitting at different wavelengths can be analyzed simultaneously. Although the widely used fluorescent compound fluorescein is a useful emitter in the limbal region, background autofluorescence caused by excitation at fluorescein absorption wavelengths limits detection sensitivity in certain immunoassay and cell analysis systems. However, the conventional red fluorescently labeled rhodamine was found to be less effective than fluorescein. Texas Red■ is 57
It is a useful labeling reagent that excites at 8n+n and 610n
It emits maximum fluorescence at m.

Phycobiliproteins have made important contributions due to their high extinction coefficient and high quantum yield. Such chromophore-containing proteins can be covalently linked to many proteins and thus amenable to fluorescent antibody assays in microscopy and flow cytometry. Phycobiliproteins (+) have relatively complex protein labeling methods, (2) protein labeling efficiencies are usually not high (typically an average of 0.5 phycobiliprotein molecules/protein);
) Phycobiliprotein is a natural product whose preparation and purification is complex; (4) phycobiliprotein is expensive; and (5) it fluoresces further in the red region of the spectrum than allophycocyanin, which fluoresces maximally at 680 nm. m
There is currently no phycobiliprotein that is effective as a diagnostic reagent,
(6) phycobiliprotein is relatively chemically unstable; (7) it is relatively easily photobleached; (8) phycobiliprotein is a large Labeling is desirable for many proteins, including proteins, metabolites, drugs, hormones, derived nucleotides, and antibodies [1. It has the disadvantage of being larger than many substances. The latter drawback is particularly serious. Because large phycobiliprotein-labeled antibodies, avidin, DNA piperidization probes, hormones, and small molecules cannot bind to their targets due to steric limitations imposed by the dimensions of the complex, and binding to the target This is because the binding rate of substances is slower than that of low molecular weight substances.

Other techniques, including cytology, cytology, and immunoassays, also utilize fluorescent materials that exhibit high quantum efficiency, absorption and emission properties at long wavelengths, have simple means of coupling, and are virtually free of nonspecific interference. receive substantial benefits from the use of.

Day Outline The present invention uses fluorescent arylsulfonated cyanines and related dyes with relatively large absorbance and high quantum yield for assay and quantification purposes of labeled components. Fluorescent cyanine and related dyes can be used for antibodies, antigens, avidin, streptavidin, proteins, peptides, derivatized nucleoids,
It can be used to label biological substances such as carbohydrates, lipids, biological cells, bacteria, viruses, blood cells, recombinant cells, hormones, lymphokines, trace biomolecules, toxins, and drugs. Fluorescent dyes can also be used to label non-biological materials such as soluble polymers, polymeric or glass particles, drugs, conductors, semiconductors, glass or polymeric surfaces, polymeric films, and other solid particles. Can be used. The component to be labeled may be present in a mixture containing other substances. The mixture in which the labeling reaction takes place can be a liquid mixture, especially an aqueous mixture. The detection step can occur with a liquid or dry mixture such as a microscope slide.

The present invention also 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 an amine or hydroxyl site, or in the case of a sulfhydryl or aldehyde site. 5 cyanine and related dye structures in the test liquid to (1) facilitate handling in labeling reactions, (2) help prevent dye binding on the surface of the protein being labeled, and (3) ) The use or modification of cyanine and related dye structures to help prevent non-specific binding of labels to biological materials and surfaces or assay devices.

Cyanine and related dyes offer significant advantages over existing fluorescent labeling reagents. First of all, 400 to about 1l10
Cyanines and related dyes are synthesized that absorb or emit light in the spectral region of the 0rx range. Reactive derivatives of these dyes can thus be prepared for assays requiring the simultaneous measurement of multiple labeled substances. This type of multicolor (or multiparameter) analysis is simplified and
Cost effectiveness or the ratio of different labeled species on each particle in a complex mixture of particles (e.g. the ratio of antigen markers on different blood cells in a complex mixture by multiparameter flow cytometry or fluorescence microscopy) desirable for determining
Second, many cyanines and related dyes strongly absorb light and fluoresce. Third, many cyanine and related dyes are relatively photostable and do not bleach quickly under fluorescence microscopy. Fourth, cyanine and related dye derivatives are easy and effective coupling reagents to prepare. can. Fifth, many structures and synthetic methods are available, and this type of dye is versatile. Therefore, many structural modifications can be made to render the reagents more or less water soluble.

Its charge varies so as not to disturb the molecules it binds to, and to reduce nonspecific binding. Sixth, unlike phycobiliproteins, cyanine dyes are relatively small (molecular weight = i, ooo) and therefore do not appreciably sterically hinder the standard molecule's ability to rapidly reach its binding site or perform its functions. .

Thus, cyanine-type dye labeling agents offer many potential advantages. Such dyes can be used for the selective labeling of one or more components in liquids, especially aqueous liquids.The labeled components can then be detected by optical or luminescence methods. Alternatively, the labeled component is used to stain another component with which it has a strong affinity, the latter component being detected by optical or luminescent methods. In this case, the dye reacts with the amine, hydroxy, aldehyde or sulfhydryl groups of the labeled component. For example, the labeled component can be an antibody and the stained component to which it has a strong affinity is a biological cell,
It can be an antigen or hapten, or a biological cell or particle containing an antigen or hapten. In another example, the labeled component can be avidin and the component to be stained can be a biotinylated substance. Also, specific carbohydrate groups can be detected and quantified using lectins conjugated with polymethine cyanine type dyes. Over time, luminescent cyanines and related dyes can bind to DNA or RNA fragments. DNA or RNA labeled fragments are...! '
) It can be used as a fluorescent hydridization probe to examine the presence and amount of a specific complementary nucleotide sequence in a sample containing NA or RNA. The dye also binds to a hormone or ligand (eg, hormone, protein, peptide, lymphokite, metabolite), and then binds to a receptor for the hormone or ligand.

What to do with cyanine? Miraha
(U.S. Patent No. 4.337.063) and Masu et al.
da et al. (U.S. Pat. No. 4.404.289, U.S. Pat.
No. 05,711 and No. 4,414.325) are
Various cyanine dyes with N-hydroxysuccinimide active gostyl groups have been synthesized. These patents are
This shows that the above reagent can be used as a photographic sensitizer. The potential fluorescent properties of the reagent are not described in the 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 not optimal for many applications involving fluorescent detection of labeled substances.

Exekjel et al. (UK Patent No. 1,529,202)
present a number of cyanine dye derivatives that can be used as covalently reactive molecules. The reactive groups used in this reagent are azine groups to which mono- or dichlorotriazine groups belong. The British patent relates to the development and use of the above reagents as photographic film sensitizers. Fluorescence is not necessary for the process, most of the reagents described do not fluoresce, and this British patent does not relate to the development and use of reactive cyanine dyes for the purpose of detecting or quantifying labeled substances.

The present invention describes the use of luminescent cyanines and cyanine-type dyes in biological substances, in order to make the substance being labeled luminescent so that it can be detected and/or quantified by luminescence detection methods. It relates to methods of covalently bonding to non-biological molecules and macromolecules and particles.

When detecting a component in a liquid, the present invention uses a dye soluble in the liquid to covalently bond the dye to an amine or hydroxy group optionally an aldehyde or sulfhydryl group on the component. and styryl dyes so that the dyes are sensitized to the above-mentioned components. Labeled components are detected and/or quantified by luminescence or light absorption methods. a! When the labeling component is an antibody, a DNA fragment, a hormone, a lymphokite, or a drug, the labeling component can be used to test for the presence or absence of another component to which it binds, thereby detecting the other component. and/or quantified.

Any effective luminescence or absorption detection step can be used. For example, the detection step can be an optical detection step in which the liquid is first illuminated with light of a defined wavelength. The labeling component can then cause fluorescence or absorption. Light at another defined wavelength that phosphoresces is detected. Detection may also be by optical absorption. For example, the detection step may include passing light of an initial defined wavelength through the liquid and then identifying the wavelength of light transmitted by the liquid.

If desired, the detection step can include chemical analysis to chemically detect the binding of cyanine or related chromophore to component I\.

The basic structures of cyanine, merocyanine and styryl dyes that are modified to create covalently labeled reagents are shown below: and m is an integer selected from the group consisting of 1.2.3 and 4.

In the above formula, the number of methine groups partially determines the excitation color. The cyclic azine structure also partially determines the excited structure. Often, higher values of m contribute to increased luminescence or absorbance. For values of m higher than 4,
The compound becomes unstable. There, when 0m=2, luminescence is further imparted by modification in the ring structure.
The excitation wavelength is approximately 650 nm and the compound fluoresces properly. The maximum emission wavelength is approximately 15 to 15 liters longer than the maximum excitation wavelength.
100n larger.

At least one (preferably one and optionally two or more) of the RI+ R*, Rs, R4, R3, R6 and R1 groups in each molecule is a reactive group for binding the dye to the labeling component. be. At least one of the Ri, R-rich, R3, R-, R11, Ra, and Rt groups on each molecule increases the solubility of the chromophore or increases the selectivity of labeling of the labeling component or labeling with the dye. It can be a group that affects the labeling position of the component.

In the above formula, R,, R1 (when present) and Rta (
(when present) comprises at least one sulfonate group, the term sulfonate group is intended to include sulfonic acids. This is because the sulfonate group is nothing more than an ionized sulfonic acid.

Rt directly or indirectly bound to a chromophore.

Reactive groups forming R3, Rs, R-, Ra, Rs and R1 groups include - isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- to dihalogen-substituted pyridine, chino- to di-
Halogen-substituted diazine, maleimide, aziridine, sulfonyl octalide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imidoester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyldithio)propionamide,
Reactive moieties such as glyoxal and aldehyde containing groups may be included.

Rt, Rt particularly useful for labeling components with available amino, hydroxy and sulfhydryl groups.

Specific examples of R3, R-, Rs, R@ and Rt groups include: (wherein at least one of Q or W is T, Br or C
residues such as ), Rs, which are particularly useful for labeling moieties with effective sulfhydryls that can be used to label antibodies in a two-step method;
Specific examples of Rs, Rs, R4, R1, R, and R7 groups are: (where n is O or an integer).

R4, R*, which are particularly useful for labeling components by photoactivated crosslinking.
Specific examples of Rs, R-, Rs, Ra and R1 groups (where Q is a residue such as ■ or Br) include NO2.

Interaction of two or more reactive chromophores on a labeled component that increases water solubility or reduces undesired nonspecific binding of the labeled component to unsuitable components in the sample, or also results in quenching of fluorescence. In order to reduce R1, R1, R gourd,
One example is -E-F, where the R, R8, R, and R1 groups can be selected from well-known polar to charged chemical groups, where F is hydroxy, sulfonate, sulfate, carboxylate, substituted amino or Denotes quaternary amino, E is -(cOx),% (ri=o, 1.2.3
or 4), useful examples include alkylsulfonates -(cTo)s50''- and -(
cOx)4-303-.

The polymethine chains of the luminescent dyes of the present invention also contain one or more cyclic chemical groups (forming bridges between two or more carbon atoms of the polymethine chain); these bridges may affect the chemical stability of the dye or the optical It can help increase stability and can also be used to change the absorption or emission wavelength of a dye or to change the extinction coefficient of quantum yield. Improved dissolution properties can be achieved by this modification.

According to the invention, labeling components include antibodies, protein 2 peptides, enzyme substrates, hormones, lymphokines, metabolites, receptors, antigens, haptens, lectins, avidins, streptavidin, toxins, carbohydrates, oligosaccharides, polysaccharides, nucleic acids. ,
deoxynucleic acid, induced nucleic acid, induced deoxynucleic acid, DNA fragment, RNA fragment, induced DNA fragment, induced RNA fragment, natural drug, virus particle, bacterial particle, viral component, yeast component, blood cell, blood cell component, biological cell, non- Cell blood components, bacteria, bacterial components, natural or synthetic lipid groups, synthetic drugs, poisonous drugs, THL environmental pollutants, polymers, polymer particles, glass particles, glass surface materials, plastic particles, plastic surface materials, polymer membranes, Can be conductors and semiconductors.

Cyanine, nin, or related chromophores capable of absorbing light at 633 rim can be prepared upon reaction with components consisting of can. It is also possible to prepare cyanines or related dyes that absorb light maximally between 700r+m and 900nm when reacted with the components of the 5 laser diodes can be used.

The reactive groups listed above can be used with proteins, other biological agents, etc. as long as appropriate reaction conditions are used, including appropriate pH conditions. is relatively specific for labeling specific functional groups on non-biological molecules, macromolecules, surfaces, or particles.

The spectral properties of the dyes of the invention, such as reactive cyanines, merocyanines, and styryls, are not appreciably altered by the functionalization described herein, nor are the spectral properties of labeled proteins and other compounds. The dyes according to the present invention, alone or bound to a labeling substance, generally have a large extinction coefficient (ε=100.000 to 250.0), which is not significantly different from the base dye molecules not bound to the substance.
00), it has a high quantum yield of around 0.4 and emits light in the spectral range of 400-900 nm. They are thus particularly valuable as labels for luminescent detection reagents.

Any method can be used to detect the optical label or stain component. The detection method can use a light source that initially illuminates the mixture containing the labeling substance with light of a defined wavelength. Known devices are used to detect light at a second wavelength transmitted by or fluorescent or emitted by the mixture. Such detection devices include fluorescence spectrometers, absorption spectrophotometers, fluorescence microscopes, transmitted light microscopes or flow cytometers, fiber optic sensors, and immunoassay devices.

The methods of the invention can also use chemical analytical methods to detect dye binding to one or more labeling components. Chemical analysis methods include infrared spectrometry, NMR
Methods include spectrometry, absorption spectrometry, fluorescence spectrometry, mass spectrometry and chromatography.

Example - “The bond between sulfinodicarbocyanine and protein
I (sheep γ-globulin (4 mg
/m) The sample was mixed at room temperature with a 10-fold molar excess of the following sulfinodicarbocyanine active ester (m=2): Sephadex G- for an appropriate time ranging from 5 seconds to 30 minutes.
Protein samples were separated from non-covalently bound dyes by gel permeation chromatography on 50 ml. Maximum protein [knowledge is io
5.8.6, 4 and 8 for samples incubated at pH 8, 5, 89 and 9.4, respectively.
．． Resulting in a final dye/protein molar ratio of 2. The time required for a dye/protein ratio of 5 and the quantum yield of the product at different pHs are shown in the following table: ``1-pupil1LLLinterj''-LLJllta5
115 0.098.9 53
0.099.4 6.5 0.
17 The above data shows that the protein labeled with this dye was
It shows that it is better at pH 9.4 than that of unexamined. The higher the p'H of the buffer, the more rapid the binding reaction, the better the recognition efficiency, the higher the fluorescence of the product, and the higher the f1 yield is the average quantum yield per dye molecule on the labeled protein. represents the state of affairs.

Takumi-1 Combination of sulfindocarbocyanine active ester and protein
4 Sheep γ-globulin (1 mg/mρ) dissolved in phosphate-buffered saline (PBS) at pH 7.4 was adjusted to pH 9.4 using 0.1 M sodium carbonate. Cyanine dye [acid agent (structure of Example 1, m=1) was added to an aliquot of this protein solution to obtain the ratio. After 30 minutes of incubation at room temperature, separation was performed by Sephadex G-50 gel permeation chromatography using PBS as eluent. The molar ratio of dye covalently bonded to protein in the product is the initial dye/''protein ratio of 3.6.12.24
and 30 respectively 1°2.3.5.544.6.7
and 11.2.

Example - once N-aminoethyl-carboxamidomethyl (A, E
CM) Dextran was synthesized from dextran with an average molecular weight of 70,000 [Iru+ia, J, K, .
J, Immunol, lI4+ 704-709 (
197511.

A dissolved in 0.1 M carbonate buffer at pH 9.4
A portion of ECM-dextran (1mg/250uj2) was converted into sulfindodicarbocyanine active ester (Example 1).
The structure of m=2) was determined to be 0.2 mg and the dye/protein molar ratio lO was obtained. The mixture was stirred at room temperature for 30 min. Dextran was then separated from unbound dye by Sephadex G-50 gel permeation chromatography using ammonium acetate (50 nM) as the elution buffer. An average of 2.2 dye molecules were covalently attached to each dextran molecule.

Murine 1 in 1.1 M carbonate buffer (pH 9,4) of sulfindodicarbocyanine active ester of L wound-specific antibody.
Sheep gamma-globulin specific for gG (1m,/rrhI2) and sulfindocarbocyanine active ester (structure of Example 1, m=2) were mixed at a dye molecule/protein molecule ratio of 8. After incubation for 30 minutes at room temperature, the standard mixture was separated by gel filtration on Sephadex G-50 equilibrated with phosphate-buffered saline (pH 7.4). An average of 44 dye molecules covalently attached to each protein were included.

Staining of human lymphocytes with sulfindodicarbocyanine dye conjugated to sheep anti-mouse IgG antibody Freshly isolated peripheral blood lymphocytes were treated with mouse anti-62-microglobulin (0.25 μg 106 cells) for 30 min at 0°C. did. Cells were washed twice with DMEM buffer and then treated with sulfindocarbocyanine-labeled sheep anti-mouse IgG antibody (log/10@cells). 0℃
After 30 minutes of incubation, cells were centrifuged to remove excess antibody and cells were washed again with DMEM buffer. Aliquots of cells were fixed on slides for analysis by fluorescence microscopy. Under a microscope, the lymphocytes on the slide were illuminated with 610-630 nm light, and the C0T was attached to an image digitizer and a television monitor.
650-700 by U red sensing enhanced television camera
The fluorescence of nm was detected. Cells stained with this method showed fluorescence under the microscope. In a control experiment, - next mouse anti-β2-
Staining and analysis were performed as described above, except that the use of microglobulin antibodies was omitted. The control sample showed no fluorescence under the microscope, thus indicating that the sulfinocyanine-labeled sheep anti-mouse antibody did not result in significant non-specific binding to lymphocytes.

[Brief explanation of the drawing]

Figure 1 shows the monomer absorption spectrum and dimer spectrum of N,N'-disulfobutylindodicarbocyanine dye, and Figure 2 shows the N,N'-diethylindodicarbocyanine-5, Figure 3 shows the spectrum of the 5'-disulfonic dye. Labeling of sheep immunoglobulin with bis-N-hydroxysuccinimide dye of N,N'-dicarboxypentylindodicarbocyanine-5'5'-disulfonic acid. Figure 4, which shows the dye/protein molar ratio in the reaction, shows the absorption spectrum of an antibody labeled with bis-N-hydroxysuccinimide ester of N,N' dicarbutyl indodicarpocyanine-5,5-acetic acid6. Figure 5 shows the absorption spectrum of the protein bound to the novel sulfinodicarbocyanine dye. Figure 6 shows the change in quantum yield (diamond-shaped mortar) of the aryl sulfocyanine dye with increasing dye/protein ratio. Comparison with that of arylsulfocyanine dye (circle mark) is shown. Wavelength lu+i1 (dye/protein) initial

Claims (1)

[Scope of Claims] 1. When labeling a component in an aqueous liquid, (a) the liquid containing the component contains cyanine or merocyanine having at least one sulfonic acid group or sulfonate group bonded to an aromatic nucleus; and (b) reacting the dye with the component such that the dye labels the component. 2. The method according to claim 1, wherein the dye contains one or more sulfonic acid groups or sulfonate groups. 3. A claim in which the dye contains a substituent that causes the dye to covalently react with an amine, aldehyde, sulfhydryl or hydroxy group on the component, such that the dye reacts with the group on the component. The method described in paragraph 1. 4. The method according to claim 1, wherein the sulfonic acid group or sulfonate group increases the water solubility of the dye. 5. The method of claim 1, wherein the sulfonic acid or sulfonate groups reduce interactions between dyes that induce fluorescence quenching. 6. The method according to claim 1, wherein the dye is a cyanine dye. 7. The method according to claim 1, wherein the dye is a merocyanine dye. 8. Ingredients include antibodies, proteins, peptides, enzyme substrates, hormones, lymphokines, metabolites, receptors, antigens, haptens, lectins, toxins, carbohydrates, oligosaccharides, polysaccharides, nucleic acids, deoxynucleic acids, induced nucleic acids, induced deoxynucleic acids ,
DNA fragment, RNA fragment, derived DNA
Fragments, derived RNA fragments, virus particles, viral components, yeast components, blood cells, blood cell components, biological cells, non-cellular blood components, bacteria, bacterial components, natural or synthetic lipid sacs, synthetic or natural drugs,
Claim 1, which is selected from the group consisting of poisonous drugs, environmental pollutants, polymers, polymer particles, glass particles, glass surface materials, plastic particles, plastic surface materials, polymer films, conductors, and semiconductors. Method. 9. The method according to claim 1, which comprises the step of optically detecting the labeled component. 10. The method according to claim 9, wherein the detection step comprises illuminating the labeled component with light and then detecting the light emitted by the labeled component. 11. The method according to claim 9, wherein the detection step includes optical excitation with a laser. 12. The method according to claim 9, wherein the detection step uses fluorescence microscopy. 13. The method according to claim 9, wherein the detection step uses flow cytometry. 14. The method of claim 9, wherein the detected component is metered. 15. Claim 1, wherein the dye is a styryl dye
The method described in section. 16. When labeling a component in an aqueous liquid, (a) adding at least one sulfonic acid group or sulfonate group bonded to one or more rings of a heterocyclic nucleus to the liquid containing the component; (b) adding a luminescent dye that reacts with said component, selected from the group consisting of sulfoindolenine-based and naphthosulfoindolenine-based polymethine dyes, containing said dye and said component, such that said dye labels said component; (c) detecting the labeled component by an optical method. 17. The dye has a sulfonic acid group or a sulfonate group.
17. The method of claim 16, wherein the method contains more than 18. When labeling a first component and using the first component to be labeled to combine with a second component to form a labeled second component, (a) adding an aromatic nucleus to the aqueous liquid containing the first component; (b) adding a luminescent dye which reacts with said first component and is selected from the group consisting of cyanine, merocyanine and styryl dyes containing at least one sulfonic acid group or sulfonate group bonded to said first component; (c) reacting the dye with the first component to luminescently label the labeled first component to provide a labeled first component; (c) binding the labeled first component to the second component to form a labeled second component; and (d) a method comprising detecting the labeled second component by an optical method. 19. The dye has a sulfonic acid group or a sulfonate group.
19. The method of claim 18, wherein the method contains more than 20. The method according to claim 18, wherein the first component is an antibody and the second component is a substance selected from the group consisting of antigens, haptens, biological cells, viruses, and antigen- or hapten-carrying particles. . 21. Claim 18, wherein the first component is a lectin, and the second component is a substance selected from the group consisting of carbohydrates, carbohydrate-bearing macromolecules, and carbohydrate-bearing cells.
The method described in section. 22. The method according to claim 18, wherein the first component is avidin or streptavidin and the second component is a biotinylated substance. 23. The method according to claim 18, wherein the first component is a DNA fragment and the second component is a complementary strand of DNA or RNA. 24. The method according to claim 18, wherein the first component is an RNA fragment and the second component is a complementary strand of DNA or RNA. 25. Claim 1, wherein the first component is a ligand selected from the group consisting of hormones, proteins, peptides, lymphokites, and metabolites, and the second component is a receptor.
The method described in Section 8. 26. The first component is an antibody, a nucleic acid fragment, a drug, a toxin, a hormone, a metabolite, a biological cell, a bacteria,
A polymeric particle containing a substance of the group consisting of a virus particle, an antigen and a hapten, the second component being a corresponding antigen,
19. The method of claim 18, which is a complementary nucleic acid sequence, an antibody or a receptor. 27. The method according to claim 18, wherein the second component is an antibody. 28. Claim 18, wherein the first component is a substance of the group consisting of an antigen, a hapten, or a cell containing an antigen or hapten, and a particle containing an antigen or hapten, and the second component is an antibody. the method of. 29. The method of claim 18, wherein the dye reacts with amine, hydroxy or sulfhydryl groups on the first component. 30. The method of claim 18, wherein the dye reacts with the aldehyde groups on the first component. 31. Claim 18, wherein the dye is a cyanine dye and reacts with the amine or hydroxy group on the first component.
The method described in section. 32. The method of claim 18, wherein the dye is a cyanine dye and reacts with the aldehyde or sulfhydryl groups on the first component. 33. The second component is blood cells, tissue cells, bacteria,
Viruses, chromosomes, DNA, RNA, toxins, drugs,
19. The method of claim 18, wherein the method is selected from the group consisting of hormones and polymer particles. 34. A sulfindolenine base containing a component containing an amine, aldehyde, sulfhydryl or hydroxyl group and at least one substituent which causes the dye to covalently react with the amine, aldehyde, sulfhydryl or hydroxyl group on the component; A luminescent photostable reaction product with a water-soluble dye selected from the group consisting of naphthosulphoindolenine-based polymethine dyes, wherein the individual dye molecules on the reaction product have an average of at least 50,000 l/mole cm. A product having a molecular extinction coefficient and an average quantum yield of at least 2% and which absorbs or emits light in the maximum range of 400-850 nm when the labeling component is in an aqueous environment. 35. The method of claim 34, wherein the component is an antibody. 36. A group in which the components are proteins, peptides, drugs, toxins, metabolites, lymphokines, carbohydrates, lipids, blood cells, bacterial particles, virus particles, antigens, haptens, polymer particles, glass surface materials, and polymer surface materials. 35. The method of claim 34, wherein the method is selected from: 37. The method according to claim 22, wherein the second component is a DNA or RNA fragment. 38. Dyes are: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Cyanine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Merocyanine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Styryl [Here, X and Y are O, S, ▲There are mathematical formulas, chemical formulas, tables, etc.▼ is selected from the group consisting of, m is an integer selected from the group consisting of 1, 2, 3 and 4, R_1, R_2, R_3, R_4, R_5, R_6 and R_7 groups at least one of isothiocyanate,
Isocyanate, monochlorotriazine, dichlorotriazine, mono- or dihalogen-substituted pyridine, mono-
or di-halogen-substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imide ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyldithio)propionamide, glyoxal, and at least one group selected from the group consisting of aldehydes, and at least one of the R_8 and R_9 groups contains at least one arylsulfonyl or arylsulfonate group. The method described in section. 39, R_1, R_2, R_3, R_4, R_5, R_
At least one of the 6 and R_7 groups is -NCS, ▲There is a mathematical formula, chemical formula, table, etc.▼, -(C
H_2)_nHCS, -NCO, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, -(CH_2)_nNCO, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ Mathematical formulas, chemical formulas There are , tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ Mathematical formulas, chemical formulas,
There are tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲Mathematical formulas, chemical formulas,
▼, (where at least one of Q and W is selected from the group consisting of Cl, Br and I, and n is 0 or any integer) 39. The method of claim 38, comprising: 40, R_1, R_2, R_3, R_4, R_5, R_
In a patent claim, at least one of the groups 6 and R_7 contains at least one group selected from the group consisting of -N_2, ▲ has a mathematical formula, chemical formula, table, etc. ▼, ▲ has a mathematical formula, chemical formula, table, etc. The method according to scope item 38. 41, R_1, R_2, R_3, R_4, R_5, R_
Claim 38, wherein at least one of the groups 6 and R_7 also contains at least one group selected from the group consisting of hydroxy, sulfonate, sulfate, carboxylate and substituted amine to quaternary amine groups.
The method described in section. 42, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Cyanine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Merocyanine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Styryl [Here, X and Y are O, S, and ▲ Mathematical formulas, Chemical formulas, tables, etc. are selected from the group consisting of ▼, m is an integer selected from the group consisting of 1, 2, 3 and 4, and at least one of R_1, R_2, R_3, R_4, R_5, R_6 and R_7 One is isothiocyanate,
Isocyanate, monochlorotriazine, dichlorotriazine, mono- or dihalogen-substituted pyridine, mono-
or di-halogen-substituted diazine, maleimide, aziridine, sulfonyl halide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyldithio)probioliamide, glyoxal and at least one group selected from the group consisting of aldehydes, and at least one of the R_8 and R_9 groups contains at least one arylsulfonate group or an arylsulfonate group. 43. The luminescent dye according to claim 42, which is water-soluble. 44. A luminescent dye according to claim 42, having a molecular extinction coefficient of at least 50,000 l/mole cm in water and a quantum yield of at least 2%, and absorbing or emitting light in the 400-850 nm spectral range. . 45, having a molecular extinction coefficient of at least 100,000 l/mol cm in water and a quantum yield of at least 10%;
43. A luminescent dye according to claim 42, which absorbs or emits light in the 600-900 nm spectral range. 46, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Cyanine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Merocyanine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Styryl [Here, X and Y are O, S, and ▲ Mathematical formulas, Chemical formulas, tables, etc. are selected from the group consisting of ▼, Z is selected from the group consisting of O and S, m is an integer selected from the group consisting of 1, 2, 3 and 4, R_1, R_2, R_3 , R_4, R_5, R_6 and R_7, at least one of the groups is -NCS, ▲There is a mathematical formula, chemical formula, table, etc.▼, -(CH_2)_nNCS,
-NCO, ▲Mathematical formulas, chemical formulas, tables, etc.▼, -(C
H_2)_nNCO, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ Mathematical formulas ,Chemical formula,
There are tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲Mathematical formulas, chemical formulas,
There are tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (where at least one of Q and W is selected from the group consisting of I, Cl and Br, and n is 0 or an integer) containing at least one group selected from the group,
and at least one of the R_8 and R_9 groups (when present) contains at least one sulfonate group. 47, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Cyanine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Merocyanine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Styryl [Here, X and Y are O, S, and ▲ Mathematical formulas, Chemical formulas, tables, etc. are selected from the group consisting of ▼, Z is selected from the group consisting of O and S, m is an integer selected from the group consisting of 1, 2, 3 and 4, R_1, R_2, R_3 , R_4, R_5, R_6 and R_7, ▲ has a mathematical formula, chemical formula, table, etc. ▼, ▲ mathematical formula, chemical formula,
There are tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ Mathematical formulas, chemical formulas,
There are tables, etc. ▼ ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ and ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (where n is 0 or an integer, R_8, R_9, R_1_0 (if present) and R_1_
1 (when present) contains at least one group selected from the group consisting of at least one group consisting of a sulfonic acid group or a sulfonate group. 48, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Cyanine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Merocyanine ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Styryl [Here, X and Y are O, S, and ▲ Mathematical formulas, Chemical formulas, tables, etc. are selected from the group consisting of ▼, m is an integer selected from the group consisting of 1, 2, 3 and 4, and at least one of R_1, R_2, R_3, R_4, R_5, R_6 and R_7 -N_3, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, Contains at least one group selected from the group consisting of,
and at least one of the R_8 and R_9 groups contains at least one arylsulfonic acid group or an arylsulfonate group.] A luminescent dye selected from the group consisting of: 49, R_1, R_2, R_3, R_4, R_5, R_
at least one of the 6 and R_7 groups is selected from the group consisting of hydroxy, sulfonate, sulfate, carboxylate and substituted amine to quaternary amine groups;
43. The luminescent dye according to claim 42, which also contains groups for increasing water solubility and reducing non-specific binding. 50. Mixing a sulfocyanine dye labeling substance with one or more different fluorescent substances that all absorb light significantly at a single excitation wavelength but emit fluorescence at different wavelengths,
A method comprising then exciting all of these fluorophores with a single wavelength of light and quantifying each of these fluorophores by detecting their individual fluorescence signals at their different fluorescence wavelengths. 51. The method according to claim 50, wherein the sulfocyanine dye labeling substance is an antibody or a DNA hybridization probe.