Method for detecting cells by repeated staining and destaining

Abstract

To provide an efficient dyeing / decoloring process of a cell.SOLUTION: There is provided a detection method of a target site in a sample of a biological specimen, by providing a composite (I), comprising: (a) bringing the sample of the biological specimen into contact with at least one composite (I), for indicating the target site recognized by an antigen recognition portion, by the composite (I); (b) exciting the indicated target site, by light having a wavelength in an absorbance spectrum of a fluorescent portion; (c) detecting fluorescence radiation discharged by the fluorescent portion, for detecting the indicated target site; and (d) over a sufficient period sufficient for supplying sufficient energy for reducing the fluorescence radiation discharged by the fluorescent portion by at least 75% of initial fluorescence radiation, radiating light having a wavelength in the absorbance spectrum of the fluorescent portion, to the composite, for decomposing the fluorescent portion of the indicated target site.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for detecting or identifying a target site or target cell from a cell sample by labeling the target site or target cell with a complex having a detection site and an antigen recognition site, wherein the detection site is decomposed by light irradiation after detection of the target site, thereby enabling subsequent labeling and detection.

[0002] For immunofluorescence analysis, fluorescent sites bound to one or more antibodies are commonly used. To enable specific detection and isolation of target cells, a vast number of improved types have been developed in the past 20 years in terms of antibodies, fluorescent sites, flow cytometers, flow sorters, and fluorescence microscopes.

[0003] It is known to utilize a fluorescent site for isolated donor detection of target cells that can be removed or destroyed after detection. For example, U.S. Patent No. 7,776,562 discloses a reversible fluorescence labeling method based on indirect non-covalent labeling of target cells with reversible peptide / MHC-multimers or Fab-streptamers.

[0004] To reduce fluorescence emission after detection, UK Patent Application Publication No. 2,372,256 discloses a process for quenching fluorescence emission by providing a complex containing a plurality of fluorescent sites bound to an antibody via a linker. The high density of fluorescent sites quenches the fluorescence signal. Further, UK Patent Application Publication No. 2,372,256 describes enzymatic degradation of the linker to cleave the fluorescent site from the complex. Since the cleaved fluorescent site does not self-quench, a stronger fluorescence signal, i.e., better resolution, is obtained.

[0005] Removal of fluorescent signals is essential for immunofluorescence techniques based on sequential staining of samples. These techniques have been shown to offer the potential for higher-order multiplexing compared to standard methods that use labeling and detection simultaneously. However, these techniques are based on the oxidative destruction of antibody-binding fluorescent sites by chemical bleaching treatment (U.S. Patent No. 7741045, European Patent No. 0810428, or German Patent Application Publication No. 10143757), or, in the case of photobleaching-based methods, the bleaching rate is slower than the methods described herein. U.S. Patent Application Publication No. 2019 / 0162721 showed increased photobleaching of dyes after multimerization on branched PEGs.

[0006] As described in U.S. Patent No. 1,0126302 or U.S. Patent No. 1,0481161, complexed polymers (CPs) bound to small molecule dyes were also used for signal amplification and as bright fluorescence sites. However, enhanced photobleaching is not mentioned.

[0007] The primary objective of conventional techniques was to provide dyes and complexes containing such dyes that emit fluorescence as strongly as possible, i.e., with the highest quantum yield. To obtain reliable and reproducible signals, the dyes are designed to be as stable as possible. While these properties are advantageous for cell detection and separation processes such as FACS, they hinder repeated staining and detection of cells.

[0008] Subject matter of the invention Therefore, there is a need to establish methods for staining and destaining target sites labeled with bright fluorescent sites, where the staining process provides the brightest possible signal that can be removed as quickly and completely as possible during destaining. It has been found that by appropriately selecting specific complexes and developing specialized methods, highly efficient staining / destaining processes can be achieved.

[0009] The object of the present invention is the general formula (I): [ka] [In the formula, Ar, MU, and L1 are repeating units of the polymer.] Ar is an aryl or heteroaryl group, MU is a polymer modification unit or bandgap correction unit that is uniformly or randomly distributed along the main chain of the polymer. L1 is an aryl or heteroaryl group that is uniformly or randomly distributed along the polymer. L2 is an aryl or heteroaryl group located at the end of the polymer. FL is the fluorescent site, G1 and G2 represent hydrogen, halogen, or antigen recognition sites, but at least one of G1 or G2 is an antigen recognition site. a is 10-100 mol%, b is 0.1 to 50 mol%, c is between 0 and 90 mol%, d is between 1 and 10,000, [Assuming a + b + c = 100 mol%] A method for detecting a target site in a biological sample by providing a complex having the following: a) Labeling a target site recognized by an antigen recognition site with complex (I) by contacting a sample of a biological specimen with at least one complex (I); b) Exciting the labeled target site with light having a wavelength within the absorbance spectrum of the fluorescent site FL; c) detecting the labeled target site by detecting the fluorescence emission emitted by the fluorescent site FL; and d) Degrading the fluorescent site FL of the labeled target site by irradiating the complex with light having wavelengths within the absorbance spectrum of the fluorescent site FL for a period of time sufficient to supply enough energy to reduce the fluorescence emission emitted by the fluorescent site FL by at least 75% of the initial fluorescence emission; This method is characterized by the following:

[0010] A further object of the present invention is the use of this method in fluorescence microscopy, flow cytometry, fluorescence spectroscopy, cell isolation, pathology, or histology.

[0011] Detailed explanation In the following, the complex according to formula (1) is called CP-FL and has a polymer backbone CP to which one or more fluorescent sites FL are bound, as defined below.

[0012] [ka]

[0013] In the method of the present invention, in order to significantly reduce the fluorescence emission emitted by the fluorescent site so that residual fluorescence emission from the first staining cycle does not interfere with subsequent staining and detection cycles, the sample is irradiated with light having a wavelength in the absorbance spectrum of the fluorescent site FL. While a reduction of at least 75% of the initial fluorescence emission is usually considered sufficient, it is preferable to reduce the fluorescence emission by at least 85%, more preferably at least 95%, and most preferably at least 99% to achieve higher quality detection, i.e., to reduce background emission not originating from the staining step of the intent. Although a 100% reduction is considered best, there is a trade-off with quenching quality and overall processing time.

[0014] In another definition, the degradation of a fluorescent site FL bound to a complexing polymer (CP) of a labeled target site is performed by irradiating the complex with light (e.g., white light) having wavelengths within the absorbance spectrum of the fluorescent site FL, CP, or both, for a time sufficient to supply enough energy to shorten the half-life of the fluorescence emission emitted by the fluorescent site. The degradation rate, indicated by the value of k from a single exponential decay fitting analysis of the fluorescent site FL, is at least 1.02 times and up to 10,000,000 times greater than the k obtained for the same fluorescent site not bound to a complexing polymer (CP).

[0015] The fluorescent site FL and the antigen recognition site can be covalently or quasi-covalently bonded to the CP. The term "covalently or quasi-covalently bonded" is defined in 10 -9 This refers to the bond between FL, CP, and Y having a dissociation constant of M or greater.

[0016] The method of the present invention can be carried out in one or more steps a) to d). After each step, the fluorescent site is degraded by irradiation with light. The terms “degraded,” “quenched,” or “bleached” are used interchangeably herein and should be understood to mean a reduction in fluorescence intensity from a labeled biological sample as a result of a change in the fluorophore due to radiation. For example, “quenched” or “bleached” of the fluorescent site FL can be achieved by oxidation initiated by radiation and / or by cleaving the fluorescent site FL from CP, followed by the removal of the unbound fluorescent site from the labeled target by washing.

[0017] The bleaching system used in the present invention can comprise two or more light sources that emit radiation of different wavelengths. For example, the bleaching system can comprise 1 to 5 light sources having a combined emission spectrum in the range of 350 to 850 nm, preferably 400 to 650 nm. The emissions of the light sources can be optically combined to irradiate the sample simultaneously or sequentially. For example, the bleaching system can comprise four light sources that emit light in the ranges of 380 to 410 (violet), 450 to 500 nm (blue), 520 to 560 nm (green), and 630 to 650 nm (red). In another embodiment, only one light source that emits light in the range of 200 to 1000 nm (white light), preferably 350 to 850 nm, most preferably 400 to 650 nm is provided. The advantage of separate light sources is that the sample is exposed only to the radiation that is necessary for the sample to bleach (remove) the fluorescent dye, thereby avoiding the sample being unnecessarily exposed to radiation of other wavelengths. The emissions of the separate light sources can be combined by a suitable device such as a mirror or an optical waveguide such as an optical fiber.

[0018] After and / or before each procedure, a washing step can be performed to remove unwanted substances such as unbound complex sites and / or unbound fluorescent sites FL from the sample.

[0019] [[ID=⑧]]The bleaching process described can be further enhanced by adding an oxidizing agent. The oxidizing agent can be, for example, O2, H2O2, peroxide, or DMSO. The added oxidizing agent can generate reactive oxygen species, which need to be present at a concentration of 0.1 to 5 ppm, preferably 2 to 5 ppm, calculated as O.

[0020] Target site Note: There seems to be a numbering error in the original text where "[[ID=⑧]]" should probably be "", and the translation has been adjusted accordingly.The target site detected using the method of the present invention may be on any biological sample such as a tissue section, cell aggregate, suspended cell, or adherent cell. The cells may be alive or dead. Preferably, the target site is an antigen expressed intracellularly or extracellularly on a biological sample such as an entire animal, an organ, a tissue section, a cell aggregate, or a single cell of an invertebrate (e.g., Caenorhabditis elegans, Drosophila melanogaster), a vertebrate (e.g., Danio rerio, Xenopus laevis), and a mammal (e.g., Mus musculus, Homo Sapiens).

[0021] Fluorescent site FL A suitable fluorescent site FL is known from the field of immunofluorescence techniques such as flow cytometry or fluorescence microscopy. In the method of the present invention, the target site labeled with the complex is detected by exciting either the CP backbone or the fluorescent site FL, or both, and detecting the luminescence (photoluminescence) obtained from FL or CP.

[0022] Useful fluorescent sites FL can be protein-based such as phycobiliprotein, organic small molecule dyes such as xanthene like fluorescein and rhodamine, cyanine, oxazine, coumarin, acridine, oxadiazole, pyrene, pyromethene, pyridyloxazole, or metal-organic complexes such as Ru, Eu, Pt complexes. In addition to the presence of single molecules, clusters of fluorescent proteins or organic small molecule dyes, as well as nanoparticles such as quantum dots, upconverting nanoparticles, gold nanoparticles, and stained polymer nanoparticles can also be used as fluorescent sites.

[0023] In another embodiment of the present invention, a target labeled with the complex is detected not by radiative emission, but by absorption of UV, visible, or NIR radiation. Suitable light-absorbing detection sites are non-fluorescent light-absorbing dyes such as N-arylrhodamine, azo dyes, and organic small molecule quencher dyes such as stilbene. In another embodiment, the light-absorbing fluorescent site FL can be irradiated with pulsed laser light to generate a photoacoustic signal.

[0024] In a modified version of the present invention, the fluorophore FL is substituted with one or more water-soluble substituents selected from the group consisting of sulfonates, phosphonates, phosphates, polyethers, sulfonamides, and carbonates. It is particularly advantageous to use fluorescent moieties having sulfonate substituents, such as the Alexa Fluor family dyes supplied by Thermo Fisher Scientific Inc. The degree of sulfonate substitution per fluorophore can be 2 or more, for example, rhodamine or cyanine dyes.

[0025] Suitable commercially available fluorescent sites can be purchased from Miltenyi Biotec BV & Co. KG's "Vio" product line, or from Thermofisher's FITC, Promofluor, or Alexa dyes and / or Bodipy dyes, or Lumiprobe's cyanines, or Dyomics GmbH's DY® fluorophores, or Abberier GmbH's Star dyes.

[0026] antigen recognition site The term "antigen recognition site" refers to any type of antibody, fragmented antibody, or fragmented antibody derivative directed toward a target site expressed on a biological sample, such as an antigen expressed inside or outside a cell. This term applies to whole antibodies, fragmented antibodies, or fragmented antibody derivatives, e.g., Fab, Fab', F(ab')2, sdAb, scFv, di-scFv, and nanobodies. Such fragmented antibody derivatives can be synthesized by recombinant procedures involving covalent and non-covalent complexes containing these types of molecules. Further examples of antigen recognition sites include peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules, and artificially engineered binding molecules, e.g., peptides or aptamers targeting cell surface molecules.

[0027] The complex used in the method of the present invention may contain up to 100, preferably 1 to 20, antigen recognition sites Y. The interaction between the antigen recognition site and the target antigen may be high affinity or low affinity. The binding interaction of a single low affinity antigen recognition site is too low to obtain stable binding to the antigen. Low affinity antigen recognition sites can be polymerized by binding to an enzymatically degradable spacer to obtain high binding activity. When the spacer is cleaved by enzyme, the low affinity antigen recognition site is monomerized and the fluorescent marker is completely removed.

[0028] Preferably, the term “antigen recognition site” refers to an antibody directed towards an antigen expressed by an intracellular biological sample (target cell) such as IL2, FoxP3, CD154, or an extracellular biological sample (target cell) such as CD19, CD3, CD14, CD4, CD25, CD34, CD56, and CD133. Antigen recognition sites G1, G2, and in particular antibodies can be bound to CP via side-chain amino groups or sulfhydryl groups. In some cases, the glycoside side chain of the antibody can be oxidized by periodate to an aldehyde functional group.

[0029] The antigen recognition site can be linked by covalent or non-covalent bonds. The methods of covalent or non-covalent linkage are known to those skilled in the art and are the same as those described for the linkage of fluorescent markers.

[0030] The method of the present invention is particularly useful for detecting and / or isolating specific cell types from complex mixtures and may comprise two or more sequential steps a) to d). The method may utilize various combinations of complexes. For example, a complex may comprise antibodies specific to two different epitopes, such as two different anti-CD34 antibodies. Different antigens can be represented by different complexes comprising different antibodies, for example, anti-CD4 and anti-CD8 to distinguish between two different T cell populations, or anti-CD4 and anti-CD25 to determine different cell subpopulations, such as regulatory T cells.

[0031] Cell detection methods A target labeled with a complex is detected by exciting either the fluorescent site (FL) or the skeletal structure (CP) and analyzing the resulting fluorescence signal. The excitation wavelength is typically selected according to the absorption maximum of the fluorescent site (FL) or CP and is provided by a laser or LED source known in the art. When multiple different detection sites (FL) are used to detect multiple colors / parameters, care must be taken to select fluorescent sites whose absorption spectra do not overlap, or at least whose absorption maxima do not overlap. In the case of a fluorescent site, the target can be detected by, for example, a fluorescence microscope, flow cytometer, spectrofluorometer, or fluorescence scanner. Light emitted by chemiluminescence can be detected by similar devices without excitation.

[0032] Method of Use The method of the present invention can be used for a variety of applications in research, diagnosis, and cell therapy, such as fluorescence microscopy, flow cytometry, fluorescence spectroscopy, cell isolation, pathology, or histology.

[0033] In a first variant of the present invention, a biological sample, such as cells, is detected for counting purposes, i.e., to determine the amount of cells from a sample having a specific set of antigens recognized by the antigen recognition site of the complex. In another variant, the biological sample detected by the complex in step d) is separated from the sample by optical means, electrostatic force, pneumatic power, mechanical separation, or acoustic means. For this purpose, the biological sample detected by the complex in step d) is separated from the sample simultaneously or before performing the subsequent step d) according to a detection signal for one or more populations by optical means, electrostatic force, pneumatic power, mechanical separation, or acoustic means.

[0034] In another variant of the present invention, the location of a target site, such as an antigen on a biological sample, is determined by the antigen recognition site of the complex. Such techniques are known as "Multi Epitope Ligand Cartography," "Chip-based Cytometry," or "Multiomyx," and are described, for example, in European Patent Publication No. 0810428, European Patent Publication No. 1181525, European Patent Publication No. 1136822, or European Patent Publication No. 1224472. In this technique, cells are immobilized and contacted with antibodies bound to fluorescent sites. The antibodies are recognized by each antigen on the biological sample (e.g., on the cell surface), and after the unbound markers are removed and fluorescence is excited, the location of the antigen is detected by the fluorescence emission of the fluorescent site. In certain variants, instead of antibodies bound to fluorescent sites, antibodies bound to sites detectable by MALDI imaging or CyTOF can be used. Those skilled in the art are aware of how to modify techniques based on fluorescence sites to make these detection sites functional.

[0035] The target site is positioned using a digital imaging device with sufficient resolution and sensitivity at the wavelength of fluorescence emission. The digital imaging device can be used, for example, with or without optical magnification, using a fluorescence microscope. The resulting images are saved to a suitable storage device, such as a hard drive, in a format such as RAW, TIF, JPEG, or HDF5.

[0036] To detect different antigens, different antibody conjugates having the same or different fluorescence sites or antigen recognition sites can be provided. Since parallel detection of fluorescence emission at different wavelengths is limited, the antibody-fluorescent dye conjugates are used sequentially individually or in small groups (2-10).

[0037] In yet another variation of the method according to the present invention, a biological sample, particularly a sample of suspended cells, is immobilized by being captured in a microcavity or by attachment.

[0038] Typically, the method of the present invention can be carried out in multiple modified forms. For example, complexes that are not recognized by the target site can be removed, for example, by washing with a buffer before the target site labeled with the complex is detected.

[0039] In a modified version of the present invention, at least two complexes are provided simultaneously or in a subsequent staining procedure, where each antigen recognition site recognizes a different antigen. In another modified version, at least two complexes can be provided to the sample simultaneously or in a subsequent staining procedure. In either case, the labeled target sites can be detected simultaneously or sequentially. [Brief explanation of the drawing]

[0040] [Figure 1] A diagram showing a schematic curve obtained regarding the photodegradation of xanthene pigments by light. [Figure 2] A diagram showing the photodegradation decay curves of a series of dyes. [Figure 3] This figure shows the photodegradation curve obtained for rhodamine dye.

[0041] Examples The absorption behavior of the following compounds was investigated. CP is, [ka] [n=0.9, m=0.1, x=11] Therefore, CP-FL is, [ka] [n=0.9, m=0.1, x=11] And FL = fluorescein, rhodamine, cyanine, or carbopyronine.

[0042] To explain the overall dynamics involved in photodegradation, Figure 1 shows a schematic curve obtained for the photodegradation of xanthene pigments by light, fitted with a single exponential decay curve such as f(x)=y0*exp(-k*x), where tau=1 / k and t 1 / 2 =tau*ln(2) is the half-life. To measure the dynamics of photolysis, organic fluorophores (i.e., coumarin, xanthene, rhodamine, cyanine, etc.) were dissolved in DMSO and then diluted with PBS, or dissolved directly in PBS, with concentrations adjusted to obtain absorbance at a maximum value of approximately 0.3 AU for each, with a path length of 1.00 cm. In this way, all solutions were normalized by absorbance for comparison. The solutions were then placed in three-way window fluorescent quartz cuvettes with low headspace and an airtight top to avoid evaporation and sample concentration. The samples in the cuvettes were then irradiated for a set period of time, and both absorbance and emission spectra were recorded. The intensity values ​​and their maximum values ​​were plotted against irradiation time, and then mathematical fitting to a single exponential decay was performed using appropriate computer software to obtain a curve in which the absorption of CP was large at approximately 400 nm and the absorption of FL was redshifted, as shown in Figure 1. To calculate the half-life, a particularly characteristic decay time (k) was used among several parameters.

[0043] Figure 2 shows the photodegradation decay curves of a series of dyes belonging to different chemical classifications according to the properties of the chromophore (i.e., fluorescein, rhodamine, cyanine, and carbopyronine), each dye covalently bound to the CP site. The data shown in Figure 2 indicates that all dye classifications are susceptible to photodegradation when bound to CP as defined below, and that the rate of photodegradation is higher in the structure for this application than in fluorescein itself.

[0044] Figure 3 shows the photodegradation curves obtained for rhodamine dyes under three different conditions: i) unbound and free in solution, ii) bound to branched PEG as described in U.S. Patent Application Publication No. 2019 / 0162721, and iii) covalently bound to linear CP (the present invention).

[0045] The results in Table 1 show that, compared to the small molecule (FL) portion, the bleaching constant K for various CP-FL structures increased by 94 times (fluorescein), 22.5 times (rhodamine), and 5.2 times (cyanine), while the half-life of the fluorophore decreased by 38 times (fluorescein), 156 times (rhodamine), and 98 times (cyanine).

[0046] As shown in Figure 3, the invention described herein (e.g., CP-rhodamine) exhibits a 46-fold increase in the breaching constant compared to the structure from U.S. Patent Application Publication 2019 / 0162721 (branched PEG rhodamine), which uses the prior art of polymerizing FL on a branched PEG. This larger breaching constant results in shorter breaching times and less background, for example, in cycling for imaging applications.

[0047] [Table 1]

Claims

1. General formula: 【Chemistry 1】 [In the formula, n is 0.9, m is 0.1, and x is 11.] FL is fluorescein, rhodamine, cyanine, or carbopyronine. d is between 1 and 10,000. Y is the antigen recognition site. A method for detecting a target site in a biological sample by providing a complex having the following: a) Labeling the target site recognized by the antigen recognition site with the complex by contacting the sample of the biological specimen with at least one complex; b) Exciting the labeled target site with light having a wavelength within the absorbance spectrum of the fluorescent site FL; c) detecting the labeled target site by detecting the fluorescence emission emitted by the fluorescent site FL; and d) Decomposing the fluorescent site FL of the labeled target site by irradiating the complex with light having wavelengths within the absorbance spectrum of the fluorescent site FL for a period of time sufficient to supply enough energy to reduce the fluorescence emission emitted by the fluorescent site FL by at least 75% of the initial fluorescence emission; A method characterized by the following.

2. The method according to claim 1, further comprising decomposing the fluorescent site FL of the labeled target site by adding an oxidizing agent.

3. Use of the method according to claim 1 or 2 in fluorescence microscopy, flow cytometry, fluorescence spectroscopy, cell isolation, pathology, or histology.

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