JP2898264B2 - Method for labeling or detecting substances using luminescent aryl sulfonate cyanine dye - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to labeling and detection of substances using luminescent aryl sulfonate cyanine dyes.

[0002]

BACKGROUND OF THE INVENTION Cyanines and related dyes having light absorbing properties have been used in photographic films. Such dyes require light absorbing properties but do not require luminescent (fluorescent or phosphorescent) properties. Heretofore, cyanine dyes having luminescent properties have had very limited uses. Such uses include, among other things, labeling the sulfhydryl groups of the protein. "Sulfhydryl reagent dye initiates rapid Ca ²⁺ release from sarcoplasmic reticulum (SR) (Sulfhydryl Reagent Dyes)
Trigger the Rapid Release
of Ca ² from Sacoplastic
Reticulum Vesicles (SR)) in a paper by Salama G. et al. Wagoner
A. S. Abramson J. et al. Report that a cyanine chromophore bearing an iodoacetyl group was used to form a covalent bond with a sulfhydryl group on muscle reticular protein at pH 6.7 to initiate Ca ²⁺ release ( Biophysical Journal, 47, 4
56a (1985)). The article also states that fluorescent dyes have been used for labeling or isolating proteins.

[0003] The dynamics of conformational changes in the rhodopsin molecular region remote from the retinylidene binding site (Kinetics
of Informational Changes
in a Region of the Rhodo
psin MolecularAway From th
e Retinylidene BindingSit
e) ", by Wagoner A. et al. S. ,
Jenkins P. et al. L. Carpenter J. et al.
P. And Gupta R.A. Report that a sulfhydryl group on the F1 region of bovine rhodopsin was covalently labeled with a cyanine dye having absorbance at 660 nm [Biophysical Journal, 33, 2].
92a (1985)]. Again, it is reported that a cyanine dye was used to label the sulfhydryl group of the protein, but the use of a fluorescent dye was not disclosed.

"International seminar on the application of the fluorescence photobleach technique to problems in cell biology (Interna
Tional Workshop on the Ap
application of Fluorescence
photobleaching Technique
s to Problems in Cell Bio
An article entitled "Jacobson K. Logic" describes a cyanine-type fluorescent probe that can be conjugated to proteins and excited in the dark red region of the spectrum. , Els
on E. Koppel D .; And Webb W.
Has been reported (Fed. Proc. 42: 7).
2-79 (1983)).

The only cyanine probes described in any of the above three papers are those which covalently bind, especially to the sulfhydryl groups of proteins. The only specific cyanine compound described has an iodoacetyl group, which covalently reacts the 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-proteinaceous materials do not have sulfhydryl groups, and many proteins do not have enough of these groups to be useful in fluorescence probing. Moreover, the sulfhydryl groups (-SHSH-) readily oxidize to disulfides (-SS-) in the presence of air, thereby rendering them unacceptable for covalent bonding to a fluorescent probe.

[0007]

According to the present invention, there are provided the following compounds:

Embedded image Merocyanine

Embedded image Styryl

Embedded image In the above structure, X and Y are O, S and

Embedded image 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 are:
Independently hydrogen, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen 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 aldehyde Wherein at least one of the R groups is not hydrogen; the R ₈ and R ₉ groups are independently arylsulfonyl or arylsulfonate groups; Represents the carbon atom necessary to form a merocyanine and styryl dye]] and a component containing an amine, aldehyde, sulfhydryl or hydroxyl group. A photostable reaction product is provided. Preferably, the components are proteins, peptides, drugs, toxins, metabolites, lymphokines, carbohydrates, lipids, blood cells,
It is selected from the group consisting of bacterial particles, virus particles, antigens, haptens, polymer particles, glass surface materials and polymer surface materials.

[0008]

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, under appropriate reaction conditions for the fluorescence or phosphorescence detection of a substance, not only sulfhydryl groups on proteins and other substances but also amines (-NH
₂ ) and hydroxy (OH) group and other aldehydes (-
Cyanine and related polymethine dyes having substituents that covalently react with groups such as the (CHO) group 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 prevalent and stable in proteins or other substances than sulfhydryl groups. Thus, when a fluorescent cyanine dye is used for the search for the presence or absence of a specific protein, a stronger fluorescent or phosphorescent luminous intensity signal is output because many dye molecules can bind to the target protein. Moreover, the amine or hydroxy group is naturally more easily added to the desired component of the label, such as a sulfhydryl, amine or hydroxy group free polymer particle.

[0009] The present invention also provides a group covalently reactive with an amine or a hydroxyl group or another reactive group so that the presence or absence or amount of a labeled protein or other substance can be detected after the labeling component is separated by chromatography. The present invention relates to a method for labeling a protein or other raw material having an amine or a hydroxyl group or another group capable of reacting with a dye in a mixture using a luminescent cyanine dye containing the dye. According to the references cited earlier, apparently sulfhydryl groups are chosen for covalent reactions, especially because the sulfhydryl groups on protein molecules are very few and in some cases they play a significant role in the function of the protein. 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 to probe a structural change of a specific protein or to cause the change. Thus, to determine the change in absorbance due to the dye or the change in free calcium ion due to dye binding, it was necessary to know where the probe was attached.

Because the number of sulfhydryl groups on most proteins is so small, the number of sulfhydryl groups may not be sufficient to provide sufficient total luminescence for probing.
In contrast, the number of amines or hydroxy groups is significantly greater in number and widely dispersed on the protein molecule, allowing the fluorescent probe to bind to multiple locations on the molecule, thereby facilitating protein detection. Therefore, the determination of the change in absorbance or fluorescence is eliminated.

[0011] The present invention relates to the use of luminescent polymethine cyanine or related polymethine dyes for proteins and other substances, including nucleic acids, DNAs, drugs, toxins, blood cells, microbial substances, particles, plastic or glass surface materials, polymer membranes, etc.
It concerns labeling at the amine or hydroxy site on the material. Advantageously, the dye is soluble in aqueous or other media containing the label. The present invention relates to a two-stage labeling method in addition to a one-stage labeling method. In the two-step labeling method, a first component, such as an antibody, can be labeled at a location 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 specific for the antibody.

In the prior art described above, the specificity of the binding site by the cyanine probe was achieved by using a probe that covalently reacts with the sulfhydryl group. According to the two-step method of the present invention, in the first step, a cyanine or related probe is added to an amine, 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 a second component, such as an antigen. This specificity is determined by the site of antigen binding to the antibody.

[0013] The present invention also relates to luminescent polymethine cyanines 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 antigen probes. When the target is a single cell, the present invention can be used to measure the amount of a labeled antibody that binds to the cell. This measurement can be performed by examining the relative brightness 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 method can be used to quantitate various proteins or other substances in the system by labeling the entire mixture of proteins in the system and then separating the labeled proteins by any means such as chromatography. . Next, the amount of the luminescent protein to be separated can be examined, and the position of the dye on the labeling substance can be confirmed by a chromatography detection system.

The present invention can also be used to determine the number of various cells labeled with an antibody. A survey of this number 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 unlabeled cells outside the system.

Another embodiment of the present invention is directed to a plurality of different luminescent cyanines, each of which is bound to a different first component, such as an antibody, each of which is bound to a first component specific to a different second component, such as an antigen. Or related dyes include multi-parameter methods used to identify each of a plurality of antigens in an antigen mixture. According to this embodiment, each antibody is separately labeled with a dye having a different absorbance and luminescence wavelength characteristic than the dye used to label the other probe. Then
All labeled antibodies are added to a biological preparation that has been analyzed to contain a second component, such as an antigen, each of which can be stained by the particular labeled antibody. Unreacted dye material can be entirely 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 that of the particular conjugate dye. A luminescence microscope for examining the luminous intensity of the emission wavelength corresponding to the excitation wavelength, or other luminescence such as a flow cytometer or a fluorescence spectrophotometer having a filter or monochromator that selects the light of the excitation wavelength and selects the wavelength of the luminescence. A detection system is used. The luminescence intensity at a wavelength corresponding to the emission wavelength of a particular conjugate dye indicates the amount of antigen bound to the antibody to which the dye binds. In some cases, a single excitation wavelength can be used to excite luminescence from two or more substances in a mixture, each emitting fluorescence at a different wavelength, and detecting individual fluorescence intensities at each of the fluorescence wavelengths By doing so, the amount of each labeled species can be measured. If desired, absorbance detection can be used. The two-stage method of the present invention can be applied to any system that uses a first substance combined with a dye to probe for the presence of another substance to which the first substance-dye complex is directed in a luminescence or absorbance detection system. For example,
The dye binds to the DNA or RNA fragment to form a dye-bound D
An NA or RNA fragment may be formed, which is then directed to a DNA or RNA backbone where the fragment is complementary. The same test method can be used to probe for the complementary backbone of DNA.

The cyanine and related dyes of the present invention are, in particular,
Because specific cyanines and related dyes having 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. Certain cyanines and related dyes having specific excitation and emission wavelengths can be synthesized by changing the number of methine groups or the cyanine ring structure. In this way, a dye having a specific excitation wavelength corresponding to a specific excitation light source, such as a laser, for example HeNe or a diode laser, can be synthesized.

The present invention provides a method for converting cyanine and related dye molecules which are highly luminescent and have high absorbance under reaction conditions into proteins, peptides, carbohydrates, nucleic acids, derived nucleic acids, lipids, other specific biological molecules, and biological cells. Shared with non-biological substances such as amines, hydroxy, aldehydes, sulfhydryls or other groups above and soluble polymers, polymer particles, polymer facings, polymer membranes, glass facings and other particles or facings Related to reacting. Because luminescence involves sensitive optical techniques, the presence or absence of a dye `` label '' can be probed and quantified even when the label is present in very low amounts, and thus labeled using a dye-labeling reagent. Can determine the amount of the substance. The most useful dyes have high absorbance (ε = 70,000-250,000 l)
b / mol cm or more), is very luminescent and has a quantum yield of at least 5 to 80% or more. The amount applies to the dye itself and also to the dye bound to the label.

An important application of such color labeling reagents is in the production of luminescent monoclonal antibodies. Monoclonal antibodies are protein molecules that bind very tightly and specifically to specific chemical sites or "markers" on or in the cell surface. Therefore, these antibodies have enormous research and clinical applications in identifying specific cell types (eg, HLA, T cells, bacteria and viruses, etc.) and abnormal cells. Conventionally, antibody bound to cells has been quantified by labeling the antibody by various methods, and the label can be a radioactive label (radioimmunoassay), an enzyme (ELISA technique).
Or a fluorescent dye (usually fluorescein, rhodamine, Tex
as Red (registered trademark) or red algae). Most manufacturers or users of clinical antibody reagents want to eliminate the problems associated with using radioactive tracers, so luminescence is considered one of the most promising alternatives. In fact, many vendors today offer fluorescein, T-labeled with monoclonal antibodies.
exas Red®, Lodamine and Red Algain are marketed.

In recent years, optical / electronic equipment for detecting fluorescent antibodies on cells has become increasingly complex. For example, flow cytometry can be used to measure the amount of fluorescent antibody on individual cells at a rate of up to 5,000 cells / second,
Microscopy and solution fluorescence techniques have also advanced. These devices can excite 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
Can be excited in the spectral region. The exception is that they can be covalently attached to proteins and can be excited at slightly longer wavelengths. Some phycobiliprotein-type pigments isolated from marine organisms. Therefore, there is a large spectral window in the 580 to about 900 nm range where new labeling reagents need to be effective at labeling biological or non-biological materials that are analyzed on instruments commercially available today. New reagents that can be excited in this region of the spectrum allow for the performance of multicolor luminescence analysis of markers on cells since antibodies with different specificities can each be labeled with different colored fluorescent dyes. In this way, the presence or absence of several markers can be checked simultaneously for each cell analyzed.

The present invention also relates to luminescent (fluorescent or phosphorescent) cyanine, merocyanine and styryl dyes which can be covalently bound to biological or non-biological substances. Merocyanine and styryl dyes are recognized as being related to the cyanine dyes for the present invention. The new labeling reagent itself can be excited with light of a wavelength initially defined, particularly when it is bound to a labeling component, for example light in the wavelength range of 450-900 nm. Background fluorescence of the cells generally occurs at lower wavelengths. Therefore, the labeling reagent is distinguished from the background fluorescence. Particularly advantageous are derivatives that absorb light at 633 nm. Because the derivatives are inexpensive, strong, stable, and long-lived H
This is because it can be excited by an eNe laser source. The light of the second defined wavelength, which is fluorescent or phosphorescent emitted by the labeling component, can then be detected. Fluorescence or phosphorescence generally has a longer wavelength than the excitation light. The detection stage is
A luminescence microscope having a filter for absorbing the scattered light of the excitation wavelength and for passing the corresponding wavelength of luminescence corresponding to the specific dye label used with the specimen can be used. 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
Includes cyanines and related dyes that are luminescent. They are relatively stable and are often soluble in the reaction solution, preferably an aqueous solution. The binding 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 a labeling component. The extinction coefficient is a measure of the ability of a molecule to absorb light. The binding dyes of the present invention have a quantum yield of at least 2%, preferably at least 10%. In addition, the binding dyes of the present invention have a wavelength of 400 to 900 nm, preferably 600 to 900 nm.
Absorbs and generates light in the ９００900 nm spectral range.

Arylsulfonated Dyes The arylsulfonate or arylsulfonate-substituted dyes as described herein are inherently more fluorescent and have improved photostability than analogous dyes without arylsulfonate or arylsulfonate groups. And water-soluble. As used herein, the term arylsulfonate or arylsulfonate group refers to an interchangeable arylsulfonate or arylsulfonate group attached to a single ring aromatic structure or an aromatic ring structure including a fused ring structure such as a naphthalene structure. Means a group. Single ring or fused ring aromatic structures can be present in polymethine, cyanine, merocyanine or styryl type dyes.

Many dyes having a coplanar molecular structure, including cyanine dyes, usually have dimeric or higher conjugates in aqueous solution, especially when inorganic salts are also present, such as in buffers or saline. Easy to form. These conjugates usually have an absorption band shifted to the short wavelength side of monomer absorption, and are generally very weak fluorescent species. The tendency of cyanine dyes to form conjugates in aqueous solutions is particularly well known in the photographic industry [West W. et al. & Pierce S. J. P
hys. Chem . , 69; 1894 (1965); S
turmer D. M. Spec. Top in H
eterocyclic Chemistry , 30
(1974)].

Many dye molecules, especially cyanine dye molecules, tend to form conjugates in aqueous solution. Thus, aryl sulfonate dyes have been found to have a minimal tendency to form these conjugates. When an arylsulfonate dye is used in forming a fluorescent labeling reagent, it has a reduced tendency to form conjugates when it binds to other molecules, such as proteins or antibodies, at a high surface density. 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. It is therefore desirable that the dye molecules have a minimal tendency to form conjugates in aqueous salt solutions. The data shown in FIG. 2 can be used to illustrate that certain arylsulfonated dyes have a low tendency to form conjugates even at high concentrations in aqueous salt solutions.

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

Improved sulfoindodicarbocyanine N, N'-diethylindodicarbocyanine-5,5 '
FIG. 2 shows the spectrum of disulfonic acid. This dye showed no sign of conjugate in saline solution up to a concentration of 10 mM.

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 was bis-N-N, N'-dicarboxypentylindodicarbocyanine-5,5'-disulfonic acid.
-Hydroxysuccinimide. FIG. 3 shows that the sulfocyanine dye active ester efficiently reacts with sheep immunoglobulin in a pH 9.2 carbonate buffer, and ranges from less than 1 to more than 20 depending on the relative dye or antibody concentration in the reaction solution. Illustrating the formation of covalently labeled antibody molecules having a dye: antibody molar ratio over a range. The slope obtained by applying the least squares method to the data indicates that the labeling efficiency of this dye under such conditions is about 80%. In a similar study, fluorescein isothiocyanate (FITC) reacted with an efficiency of about 20%.

The reactivity of the active esters of the novel sulfoindodicarbocyanine dyes was determined by using sheep immunoglobulin (IgG).
Was determined by labeling. Protein (4mg / m
l) 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 an 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 determined with a spectrophotometer and is shown in FIG.

At low dye: protein ratios, the absorption spectrum of the labeled protein shows a band that closely corresponds to that of the free monomer dye. Antibody molecules that are highly labeled (high dye: protein ratio) or labeled with a dye that has a high tendency to aggregate in aqueous solutions often have new absorption peaks appearing at shorter wavelengths than the monomeric dye absorption bands in aqueous solutions. Has been found to have. The wavelength of the new absorption peak is often
It enters the region showing the dimer absorption spectrum characteristics 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 molecules bound to the antibody. The labeling reagent N, N'-dicarbobutylindodicarbocyanine-5,5'-acetic acid bis-N-hydroxysuccinimide ester used to produce the antibody absorption spectrum of FIG. 4 has no arylsulfonate group. In aqueous salt solutions, it also readily forms dimers on the reacted antibody. Importantly, the fluorescence excitation spectra of these antibodies indicate that excitation of the labeled antibody at the short wavelength peak does not produce fluorescence proportionally as much as at the long wavelength peak. This consideration is consistent with the notion that the short wavelength absorption peak is due to the formation of a non-fluorescent dimer or conjugate on the antibody molecule.

We have found that the arylsulfonate cyanine dye used to obtain the data of FIG. 3 is judged by a much smaller absorption peak at the characteristic absorption wavelength of the dimer (see FIG. 5). And did not readily 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 sharp fluorescent antibodies even when the average dye / antibody ratio is relatively high (see FIG. 6).

FIG. 4 shows sheep immunoglobulin labeled with a carboxyindodicarbocyanine dye. FIG. 5 shows a protein conjugated with a novel sulfoindodicarbocyanine dye. The presence of increased dimers in the former sample (see FIG. 1) is evident. Although the dye: protein molar ratio was approximately 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 clear 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 the 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 can contribute to this quenching. FIG. 6 shows that the average quantum yield of arylsulfocyanine dyes 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 dyes (N, N ′)
-Di-sulfobutylindodicarbocyanine-5-isothiocyanate), indicating a very rapid reduction in the average quantum yield. Therefore, the sulfocyanine dyes illustrated in FIG. 6 produce sharper fluorescent antibodies than other dyes, especially in the labeling range of 1-10 dye molecules per antibody molecule.

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

Background Methods Luminescent probes are useful reagents for analyzing and separating molecules and cells and for detecting and quantifying other substances. A very small number of luminescent molecules can be detected under optimal circumstances. B
arak and Webb use the SIT camera to determine L
Less than 50 fluorescent lipid analogs associated with DL acceptance were visualized (J. Cell Biol. 90: 595-60).
4 (1981)]. 1 related to particles or specific cells
Flow cytometry can be used to detect less than 0000 fluorescein molecules [Muirhead,
Horan and Poste, Bio / Technol
ogy 3: 337-356 (1985)]. Some specific examples of fluorescent probe applications include (1) the 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 The determination of the concentration of a substance (eg, an antigen-antibody reactant) that binds to another species in an immunoassay technique and (3) localization of the substance in gels and other insoluble carriers by the technique of fluorescent staining. For a description of these techniques, see Herzenberg et al., Cell Immunology (C
Cellular Immunology), 3rd edition,
Chapter 22; Blackwell Scientific
Publications, 1978 (fluorescence-activated cell classification) and Goldman, "Fluorescent antibody method (Fluo
Rescue Antibody Method
s) ", Academic Pres, New York
s, 1968 (fluorescence microscopy and fluorescent staining (fluor
esence microscopy and fl
ouresencestaining)) and A
applications of Fluorescen
ce in the Biomedical Scie
nces, Editing Taior, etc., Alan Lis I
nc. , 1986.

When using fluorescence for the above purpose, there are many restrictions on the selection of a fluorescent substance. One limitation is the absorption and emission characteristics of the phosphor. This is because many ligands, receptors and substances in analytes such as blood, urea, and cerebrospinal fluid fluoresce and interfere with the accurate measurement of the fluorescence of the fluorescent label. This phenomenon is called autofluorescence or background fluorescence. Another consideration is that
The ability of a fluorescent substance to bind ligands and receptors and other biological or non-biological substances and the effect of binding to such fluorescent substances. In many situations, binding to another molecule can result in a substantial change in the fluorescent properties of the fluorescent material, in some cases substantially impairing or reducing the quantum efficiency of the fluorescent material. It is also possible that the binding to the fluorescent substance renders the function of the label molecule inactive. A third consideration is the quantum efficiency of the fluorescent material, which should be high for sensitive detection. A fourth point to consider is the absorbance or extinction of the fluorescent material, which should be as large as possible. Also, it is related whether or not the self-quenching occurs when the fluorescent molecules interfere with each other when in the proximity state. It also concerns whether the fluorescent substance non-specifically binds to the vessel wall in relation to other compounds or to the fluorescent substance itself or to the compound to which the substance is bound.

The applicability and value of the above method is closely linked to the availability of suitable fluorescent compounds. Particularly, a fluorescent substance which emits light in a long wavelength visible region (yellow to near infrared) is necessary.
This is because the excitation of these chromophores causes less autofluorescence and multiple chromophores emitting at different wavelengths can be analyzed simultaneously when the full visible and near infrared region of the spectrum can be used. Although the widely used fluorescent compound fluorescein is a useful emitter in the green region, background autofluorescence caused by excitation at the fluorescein absorption wavelength limits detection sensitivity in certain immunoassays and cell analysis systems. However, conventional red fluorescently labeled rhodamine 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 an important contribution because of its high extinction coefficient and high quantum yield. Such chromophore-containing proteins can be covalently linked to many proteins and are thus used in microscopy and fluorescent antibody assays in flow cytometry. Phycobiliproteins are (1) relatively complex in protein labeling, and (2) protein labeling efficiencies are usually not high (typically an average of 0.5 phycobiliprotein molecules /
Protein), (3) phycobiliprotein is a natural product, its preparation and purification are complicated, (4) phycobiliprotein is expensive, (5) red region more than allophycocyanin which emits maximum fluorescence at 680 nm. There is no phycobiliprotein effective as a labeling reagent that emits fluorescence in the spectrum, (6) phycobiliprotein is relatively chemically unstable, (7) it is relatively easily photobleached, and (8) ) Phycobiliprotein is 33,000-24
It is a large protein with a molecular weight in the range of 000, and has the disadvantage that labels such as metabolites, drugs, hormones, derived nucleotides, and many proteins, including antibodies, are larger than many desirable substances. The latter disadvantage is particularly significant. Because antibodies, avidin, DNA hybridization probes, hormones and small molecules labeled with large phycobiliproteins cannot bind to the target due to the steric limitations imposed by the size of the complex complex, and will not bind to the target. This is because the binding speed of is low for low molecular weight binders.

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

Outline of the Invention The present invention uses fluorescent arylsulfonated cyanines and related dyes having relatively high extinction coefficients and high quantum yields for the purpose of assaying and quantifying labeled components. Fluorescent cyanine and related dyes include antibodies, antigens, avidin, streptavidin, proteins, peptides, derived nucleotides,
Carbohydrates, lipids, biological cells, bacteria, viruses, blood cells, tissue cells, hormones, lymphokines,
It can be used to label biological materials such as trace biomolecules, toxins and drugs. In addition, fluorescent dyes, soluble polymers, polymers or glass particles, drugs,
It can also be used to label non-biological materials such as conductors, semiconductors, glass or polymer facings, polymer films and other solid particles. The component to be labeled may be in a 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 or a dry mixture, such as a microscope slide.

The present invention requires a cyanine dye to be modified by incorporation of a reactive group covalently bonded to the target molecule, preferably at an amine or hydroxy site and, in some cases, a sulfhydryl or aldehyde site, into the cyanine molecule. The present invention also enhances the solubility of cyanine and related dye structures in test liquids, (1) facilitates handling in labeling reactions, and (2) helps prevent dye binding on the surface of the protein to be labeled. And (3) the use or modification of cyanine and related dye structures to help prevent non-specific binding of the label to biological materials and surface materials or assay devices.

Cyanines and related dyes offer important advantages over existing fluorescent labeling reagents. First of all, 400
Cyanines and related dyes that absorb or emit in the spectral range of about 1100 nm are synthesized. Thus, the reactive derivatives of these dyes can be prepared for assays that require simultaneous measurement of multiple labeling substances. This type of multicolor (or multiparameter) analysis can be simplified, cost-effective, or the ratio of different labeled species on each particle in a complex mixture of particles (e.g., multi-parameter flow cytometry or complex microscopy by fluorescence microscopy). It is desirable to determine the 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 quickly under a fluorescent microscope. Fourth, cyanine and related dye derivatives, which are simple and effective coupling reagents, can be prepared. Fifth, many structures and synthetic methods are available, and this type of dye is versatile. Therefore, many structural modifications can be made to make the reagent somewhat water soluble. The charge can vary so as not to disturb the molecule to which it binds and to reduce non-specific binding. Sixth, unlike phycobiliprotein,
Cyanine dyes are relatively small (molecular weight = 1,000),
Thus, the labeling molecule's ability to reach its binding zone quickly or perform its function is not significantly sterically hindered.

Thus, cyanine type dye labels offer a number of potential advantages. Such dyes can be used for the selective labeling of one or more components in liquids, especially aqueous liquids. The label 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, and the latter component is detected by optical or luminescent methods. In this case, the dye reacts with the amine, hydroxy, aldehyde or sulfhydryl group of the labeling component. For example, the labeling component can be an antibody, and the stained component that has 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 may be avidin and the component to be stained may be a biotinylated substance. In addition, a specific carbohydrate group can be detected and quantified using a lectin bound to a polymethine cyanine type dye. In addition, luminescent cyanines 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. The dye is a hormone or ligand (eg, hormone,
Proteins, peptides, lymphokines, metabolites) and the hormone or ligand can then 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) has synthesized various cyanine dyes having an N-hydroxysuccinimide active ester group. These patents show that the above reagents can be used as photographic photosensitizers. The potential fluorescent properties of the reagent are not described in the above patents, 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 fluorescence detection of labeled substances.

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

[0048] The embodiment described the present invention, a substance which is labeled biological substance emitting cyanine and cyanine-type dyes because it is to such luminescent be detected and / or quantified by luminescence detection methods , Non-biological and macromolecules and methods for covalent attachment to particles.

The present invention provides a dye which is soluble in the liquid when the component is detected in the liquid, and which contains a substituent which covalently bonds the dye to an amine or a hydroxy group and optionally an aldehyde or a sulfhydryl group on the component. It relates to a method of adding a dye selected from the group consisting of cyanine, merocyanine and styryl dyes so that it labels said component. The label component is detected and / or quantified by luminescence or light absorption methods. When the labeling component is an antibody, a DNA fragment, a hormone, a lymphokine or a drug, the labeling 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 effective luminescence or absorbance detection step can be used. For example, the detecting step may be an optical detecting step in which the liquid is illuminated with light of the wavelength defined initially. Next, light at another defined wavelength that is fluorescent or phosphorescent by the labeling component is detected. Detection may also be by optical absorption. For example, the detecting step involves passing light of an initially defined wavelength through the liquid and then identifying the wavelength of light transmitted by the liquid.

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

The basic structure of cyanine, merocyanine and styryl dyes that can be modified to create a covalent labeling reagent is shown below: Cyanine

Embedded image Merocyanine

Embedded image Styryl

Embedded image The following are more specific examples of polymethine type dyes: cyanine

Embedded image Merocyanine

Embedded image Styryl

Embedded image Cyanine

Embedded image Merocyanine

Embedded image Styryl

Embedded image In the above structure, X and Y are O, S and

Embedded image 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 formula, the number of methine groups partly determines the excitation color. Cyclic azine structures 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 in the ring structure. m
When = 2, the excitation wavelength is about 650 nm and the compound correctly emits fluorescence. 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 (preferably only one and optionally two or more) of the R ₆ and R ₇ groups is a reactive group for attaching the dye to the labeling component. For certain 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 will enhance the solubility of the chromophore or the label of the label component. It can be a group that affects the selectivity or the labeling position of the labeling component with a 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.

Reactive groups which can be bonded directly or indirectly to the chromophore to form R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ groups include isothiocyanates, isocyanates, monochlorotriazines, Dichlorotriazine, mono- or di-halogen 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,
Reactive moieties such as glyoxal and aldehyde containing groups may be included.

R ₁ , R ₂ , and R 3 are 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:

[0058]

Embedded image (Where at least one of Q or W is I, Br or C
l),

Embedded image Particularly useful for labeling components having effective sulfhydryls that can be used to label antibodies in a two-step method are R ₁ ,
Particular examples of R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and R ₇ groups are:

Embedded image (Where Q is a residue such as I or Br),

Embedded image and

Embedded image (Where n is 0 or an integer).

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 photoactivated crosslinking.

Embedded image Is included.

[0060] Two or more reactive chromophores on the labeled component that can increase water solubility or reduce the undesired non-specific binding of the labeled component to unsuitable components in the sample or can also result in quenching of fluorescence To reduce the interaction,
_{R 1, R 2, R 3} , R 4, and R _5, R ₆ and R ₇ groups may be selected from well-known polar to charge chemical groups. Example is-
EF wherein F is hydroxy, sulfonate,
Sulfate, carboxylate, a substituted amino or quaternary amino, E is _{- (CH 2) n (n} = 0,1,2,3
Or a spacer group as in 4). Useful examples of the alkyl sulfonate - (CH _{2) 3} SO ^3- and - (C
H _{2) 4} -SO ^3- are included.

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

According to the present invention, the labeling component may be an antibody, a protein,
Peptide, enzyme substrate, hormone, lymphokine, metabolite, receptor, antigen, hapten, lectin, avidin, streptavidin, toxin, carbohydrate, oligosaccharide,
Polysaccharide, nucleic acid, deoxynucleic acid, derived nucleic acid, derived deoxynucleic acid, DNA fragment, RNA fragment, derived DNA fragment, derived 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 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.

A cyanine or related chromophore can be prepared which can absorb light at 633 nm upon reaction with certain components;
Thus, for the detection step, a helium-neon laser emitting at this spectral wavelength can be used. Also, it is 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 step emits light in this region of the spectrum. A laser diode can be used.

Selectivity The reactive groups listed above may be proteins, other biological or non-biological molecules, macromolecules, macromolecules, surface materials, or specific groups on particles as long as appropriate reaction conditions are used, including appropriate pH conditions. Relatively specific for labeling functional groups.

[0065]Reactive cyanines, merocyanines and ste
Properties of rill dyes and their products  The spectral properties of the dyes of the present invention are determined by the governmental standards described herein.
It does not change appreciably depending on the activation. Labeled protein
The spectral properties of other compounds also affect proteins and other substances.
Not significantly different from unbound base dye molecules. Alone
Alternatively, the dye according to the present invention bound to a labeling substance is generally
A large extinction coefficient (ε = 100,000-250,0
00), and in some cases a quantum yield as high as 0.4
And emits light in the 400-900 nm spectral range.
Occur. Thus, they label the luminescence detection reagent.
Of particular value.

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

The method of the present invention can also employ a chemical analysis method to detect dye binding to one or more labeling components. Chemical analysis methods can include infrared spectrometry, NMR spectrometry, absorption spectrometry, fluorescence spectrometry, mass spectrometry and chromatographic methods.

[0068]

EXAMPLE 1 For binding of sulfoindodicarbocyanine to protein
Effect of pH Sheep γ-globulin in 0.1 M carbonate buffer (4 mg /
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:

Embedded image An appropriate time in the range of 5 seconds to 30 minutes, Sephadex G-5
Protein samples were separated from non-covalent dyes by gel permeation chromatography on HPLC. Maximum labeling of the protein occurred after 10 minutes and was 5.8, 6.4 and 8.4 for samples incubated at pH 8.5, 8.9 and 9.4, respectively.
A final dye / protein molar ratio of 2 was obtained. The time required to bring the dye / protein ratio to 5 and the quantum yield of the product at different pH are shown in the following table: pH time (sec) quantum yield 8.5 115 0.09 8.9 53 0.09 9.4 6.5 0.17 The above data show that the protein labeled with this dye is better at pH 9.4 than at pH 9 below. The higher the pH of the buffer, the faster the binding reaction, but the better the labeling efficiency and the higher the fluorescence of the product. The quantum yield indicates the average quantum yield per dye molecule on the labeled protein.

Example 2 Binding of Sulfoindocarbocyanine Active Ester to Protein
Sheep γ-globulin (1 mg / ml) dissolved in saline buffered saline (PBS) with a combined pH of 7.4 was adjusted to pH 9.4 with 0.1 M sodium carbonate. An aliquot of this protein solution was added with a cyanine dye label (the structure of Example 1, m = 1) to obtain different dye / protein molar ratios.
After incubation at room temperature for 30 minutes, separation was performed by Sephadex G-50 gel permeation chromatography using PBS as eluent. The molar ratio of dye covalently bound to protein in the product was 1.2, 3.5, 5.4, 6.7 and 11.2 for the initial dye / protein ratios of 3, 6, 12, 24 and 30, respectively. there were.

EXAMPLE 3 AECM Dext with Sulfoindodicarbocyanine
N-aminoethyl-carboxamidomethyl (AEC) containing an average of 16 amino groups per labeled dextran molecule of the orchid
M) Dextran was synthesized from dextran with an average molecular weight of 70000 [Inman, J. et al. K. , J. et al. Immu
nol. 114 : 704-709 (1975)]. pH
9.4, AECM-dissolved in 0.1 M carbonate buffer
A portion of dextran (1 mg / 250 μl) was converted to a sulfoindodicarbocyanine active ester (structure of Example 1, m
= 2) In addition to 0.2 mg, a dye / protein molar ratio of 10 was obtained. 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 elution buffer. An average of 2.2 dye molecules were covalently attached to each dextran molecule.

Example 4 Sulfoindodicarbocyanine active ester of specific antibody
Murine IgG in labeled 1.1 M carbonate buffer (pH 9.4)
(1 mg / ml) specific sheep γ-globulin and sulfoindodicarbocyanine active ester (the structure of Example 1,
m = 2) at 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 linked to each protein.

Example 5 Sulfoindodicarboconjugated to sheep anti-mouse IgG antibody
Human lymphocytes can be stained or stained with cyanine dye
Visualization Freshly isolated peripheral blood lymphocytes were treated with mouse anti-β2-microglobulin (0.25 μg 10 ⁶ cells) at 0 ° C. for 30 minutes. 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 incubation at 30 ° C. for 30 minutes, the cells were centrifuged to remove excess antibody and the 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 slides are illuminated with light at 610-630 nm and 650-700 nm by a COTU red-sensitive enhanced television camera attached to an image digitizer and television monitor.
Was detected. Cells stained by this method showed fluorescence under the microscope. In control experiments, staining and analysis were performed as described above, except that the use of the primary mouse anti-β2-microglobulin antibody was omitted. Control samples showed no fluorescence under the microscope, indicating that the sulfoindocyanine-labeled sheep anti-mouse antibody did not result in significant nonspecific binding to lymphocytes.

[Brief description of the drawings]

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

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

FIG. 3 shows the dye / protein molar ratio in the labeling reaction of sheep immunoglobulin with bis-N-hydroxysuccinimide dye of N, N′-dicarboxypentylindodicarbocyanine-5,5′-disulfonic acid. Is shown.

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 to a novel sulfoindodicarbocyanine dye.

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

Claims (2)

(57) [Claims] 1. The following compound: Cyanine Merocyanine Styryl In the above structure, X and Y are O, S and 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 are:
Independently hydrogen, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen 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 aldehyde R ₈ and R ₉ are independently an arylsulfonyl or arylsulfonate group; and the dashed line is the cyanine group, respectively, wherein at least one of the R groups is not hydrogen; Represents the carbon atom necessary to form a merocyanine and styryl dye]] and a component containing an amine, aldehyde, sulfhydryl or hydroxyl group. Stable reaction products. 2. The method according to claim 1, wherein the components are proteins, peptides, drugs, toxins, metabolites, lymphokines, carbohydrates, lipids,
The product according to claim 1, which is selected from the group consisting of blood cells, bacterial particles, virus particles, antigens, haptens, polymer particles, glass surface materials and polymer surface materials.