Novel formulations for drying polymer-dyed conjugated antibodies

Abstract

A novel dry-down buffer is provided for use in drying multiple fluorescent dye conjugates on a substrate for use in flow cytometry. The aqueous buffer comprises a water-soluble monomer; a protein stabilizer; a carbohydrate stabilizer; and a zwitterionic surfactant. When mixed with a multi-color panel comprising the fluorescent polymer-dye conjugates, dried on a substrate, and reconstituted with a biological sample, the buffer provides reduced aggregation of the fluorescent polymer-dye conjugates and reduced non-specific binding of monocytes and granulocytes when compared to the use of a buffer without the water-soluble monomer or the zwitterionic surfactant.

Description

[Technical Field]

[0001] This application was filed as a PCT international patent application on November 12, 2021. [Background technology]

[0002] background Multicolor flow cytometry is a rapidly evolving technique that uses multiple fluorescent markers to identify and characterize target cell subpopulations, enabling rapid analysis of tens of thousands of cells per second. Flow cytometry uses antibody dye conjugates to stain different biological samples, including whole blood samples, bone marrow, and other biological specimens. In a normal human whole blood sample, different cell types express different markers, for example, CD4 in T cells and CD20 in B cells. When mutually exclusive markers (markers expressed in only one specific cell type) are stained using their corresponding anti-marker antibody fluorescent dye conjugates, the signals (fluorescence) from each cell are captured and digitally converted in the flow cytometer for analysis.

[0003] Multicolor dry reagents are dried, usable cocktails containing a variety of different antibody-dye conjugates useful in flow cytometry analysis of biological samples. Dry reagent technology is used to increase the stability of biomolecules, enabling storage at room temperature, simplifying sample preparation, and minimizing user error.

[0004] A multicolor dried reagent cocktail may contain several different antibodies conjugated to low-molecular-weight (e.g., FITC), tandem (e.g., PC5.5), or large-molecular-weight (e.g., APC) dyes (e.g., CD4-FITC, CD8-PE, CD20-APC, PC5.5, etc.) and can be used to stain cells in various biological specimens and analyze them in a flow cytometer.

[0005] Compared to existing classical (monomer) dye conjugates (FITC, PC5.5, APC, etc.), polymer dye conjugates differ in structure and complexity. The structural rigidity of polymer dyes helps reduce rotational energy, resulting in brighter luminescence. Therefore, polymer dye conjugates are particularly useful in the identification and analysis of cells with poorly expressed receptors. These bright polymer dyes can enable the detection and separation (resolution) of indistinct populations. However, polymer dyes, such as blue-violet polymer dyes, tend to interact with each other and form aggregates due to their inherent hydrophobicity.

[0006] Conventional drying techniques allow for the drying down of different classical dye conjugates in a single tube without altering their functionality (affinity for antigens) or physical properties (such as brightness and leakage), while maintaining stability over a period of time. “Reagent Buffer” (RB) is a prior art formulation containing sacrificial protein, carbohydrate stabilizers, antimicrobial agents, and buffers, previously developed for drying different monomer dye conjugates in a single tube without altering their functionality (affinity for antigens) or physical properties (such as brightness and fluorescence). “Physical properties” refers to the brightness of fluorescent dye conjugates and their leakage into other channels. For example, desired flow cytometry results using CD4-PE dye conjugates, dried using conventional drying techniques and used to stain biological specimens, are shown. As shown in Figure 1A, the desired functionality, physical properties, and separation ability of CD4 PE+ monocytes and CD4 PE+ lymphocyte cell populations are demonstrated. Similarly, the desired flow cytometry results for the CD20 APC dye conjugate are shown when dried using conventional techniques and reconstituted with blood samples. As shown in Figure 1B, the desired functionality, physical properties, and flow cytometry separation ability of the CD20 APC+ cell population are demonstrated.

[0007] When two polymer dye antibody conjugates are dried down in a tube using conventional drying techniques with reagent buffer, nonspecific interactions between the polymer dye conjugates cannot be prevented, leading to aggregation. Generally, when two or more polymer dye antibody conjugates are dried using conventional drying techniques, they tend to interact with each other, leading to undesirable results. Aggregation results in false-positive populations in other channels that cannot be corrected. This challenge is illustrated in Figures 2A and 2B.

[0008] Figure 2A shows undesirable flow cytometry results for CD20-605 and CD4-786 polymer dye antibody conjugates after mixing and drying using conventional drying techniques. The inability to separate the populations on the x and y axes was assumed to be a leakage issue. However, even after correcting for leakage, the populations remained unseparated (Figure 2B).

[0009] Figure 2B shows undesirable flow cytometry results for CD20-605 and CD4-786 polymer dye antibody conjugates after mixing and drying using conventional drying techniques, along with correction for leakage. The inability to correct for populations for their individual fluorescence channels was attributed to aggregation of the polymer dye antibody conjugates when the two polymer dye antibody conjugates were dried using conventional drying techniques.

[0010] There is a need for novel buffer formulations that can keep polymer dye antibody conjugates stable, enabling improved functionality, physical properties, and flow cytometry separation capabilities, while avoiding aggregation during drying and reconstitution. [Overview of the Initiative] [Means for solving the problem]

[0011] Summary of Disclosure Novel buffer compositions are provided for use in drying multiple dye conjugates on a substrate. The dye conjugates may include fluorescent dye conjugates. Fluorescent dye conjugates may be conjugates of a fluorescent dye and a binding partner such as an antibody. Fluorescent dye conjugates may also be fluorescent polymer dyes conjugated to a binding partner such as an antibody. Fluorescent dye conjugates may be used in flow cytometry. The buffer compositions comprise water-soluble monomers; protein stabilizers; carbohydrate stabilizers; and zwitterionic surfactants. When the buffer composition is mixed with a multicolor fluorescent dye conjugate panel containing two or more fluorescent dye conjugates, dried on a substrate, and reconstituted with a biological specimen, the buffer provides reduced aggregation of the fluorescent dye conjugates compared to the use of buffers without protein stabilizers, water-soluble monomers, or zwitterionic surfactants. In some examples, the buffer compositions also provide reduced nonspecific binding of the dye to monocytes and / or granulocytes compared to the use of buffers without protein stabilizers, monomers, or zwitterionic surfactants.

[0012] In some embodiments, the disclosure provides compositions comprising multiple fluorescent dye conjugates in a panel. In the presence of a suitable buffer, multiple fluorescent dye conjugates can be used in a panel to identify subpopulations of cells. Without a suitable buffer, the fluorescent dye conjugates may interact with each other, potentially causing artificial staining phenomena that could affect data interpretation.

[0013] A buffer composition is provided for use in drying multiple dye conjugates on a substrate, comprising a water-soluble monomer; a protein stabilizer; a carbohydrate stabilizer; and an amphoteric surfactant. At least one, at least two, or at least three of the multiple dye-binding partner conjugates may contain a fluorescent polymer dye moiety.

[0014] The water-soluble monomers may be monomer units containing aryl or heteroaryl moieties, each having a water-soluble moiety bonded to them as needed. The water-soluble moieties may consist of one or more poly(ethylene glycol) moieties. The water-soluble monomers may be suitable for use in the preparation of at least one of several fluorescent polymer dyes having monomer A subunits, monomer B subunits, or combinations of monomer A and monomer B subunits. The water-soluble monomers may be dihydrophenanthrene (DHP)-based water-soluble monomers. The water-soluble monomers may also be fluorene-based water-soluble monomers.

[0015] Water-soluble monomers are given by formula (I): [ka] (In the formula, Each of G1 and G2 independently contains halogens, alkyls, PEGs, hydrogen, alkynes, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, and optionally substituted tetrahydropenes. Re Fluorine (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl groups selected from the group consisting of aryl or heteroaryl groups in which a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, and thiols is substituted on one or more terminal pendant chains; Each R 2is independently selected from the group consisting of a water-solubilizing moiety, an alkene, an alkyne, a cycloalkyl, a haloalkyl, (hetero)aryloxy, (hetero)arylamino, sulfonamide-PEG, phosphoramidate-PEG, ammonium alkyl salt, ammonium alkyl oxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamidate, [Chemical formula] selected from the group consisting of; each R 3 is a water-solubilizing moiety; each R 4 is independently selected from the group consisting of H, alkyl, PEG, a water-solubilizing moiety, a linker moiety, a chromophore, a carboxylic acid amine, an amine, a carbamate, a carboxylic acid, a carboxylic acid ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or a protecting group thereof; each R 5 is independently H, hydroxyl, C1-C 12 alkyl, C2-C 12 alkene, C2-C 12 alkyne, C3-C 12 cycloalkyl, C1-C 12 haloalkyl, C1-C 12 alkoxy, C2-C 18 (hetero)aryloxy, C2-C 18 (hetero)arylamino, C2-C 12 carboxylic acid, C2-C 12 carboxylic acid ester, and C1-C 12 alkoxy selected from the group consisting of; each Q is independently a bond, NR 4 , or -CH2; each Z is independently CH2, O, or NR 4 ; Each f is an independent integer between 0 and 50; (Each n is an independent integer between 1 and 20.) It may also be a dihydrophenanthrene (DHP) monomer having the chemical structure described above.

[0016] In some embodiments, each of G1 and G2 is independently selected from the group consisting of halo(F, Cl, Br, I), C1-C6 alkyl, and PEG.

[0017] Water-soluble monomers are given by formula (II): [ka] (In the formula, Each of G1 and G2 independently contains halogens, alkyls, PEGs, alkynes, hydrogen, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, and optionally substituted tetrahydropenes. Re Fluorine (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl groups selected from the group consisting of aryl or heteroaryl groups in which a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, and thiols is substituted on one or more terminal pendant chains; Each X is either C or Si; Each R 4These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each R 5 These are independently H, hydroxyl, and C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, C2-C 12 Carboxylic acid esters, and C1-C 12 Selected from the group consisting of alkoxys; Each Z is independently CH2, O, or NR 4 and; Each f is an independent integer between 0 and 50; (Each n is an independent integer between 1 and 20.) It may also be a fluorene monomer having the chemical structure described above.

[0018] In some embodiments, each of G1 and G2 is independently selected from the group consisting of halo(F, Cl, Br, I), C1-C6 alkyl, and PEG.

[0019] Water-soluble monomers are given by formula (III): [ka] (In the formula, each G1 and G2 is independently a halo (F, Cl, Br, I); each Z is independently selected from the group consisting of O, CH2, and NH; each R1 is independently an alkyl (C1-C3); each R2 is independently H or an alkyl (C1-C6); each n is independently 1-6; and each m is independently 5-50.) The monomer may be a dihydrophenanthrene (DHP) monomer having the chemical structure described above. In some embodiments, G1 and G2 are Br, respectively; Z is O, respectively; R1 is CH3, respectively; R2 is H, respectively; n is 2 to 4, and m is independently 5 to 20. In some embodiments, n is 3, and m is 11 or 12.

[0020] The protein stabilizer may be selected from one or more of the group consisting of casein, bovine serum albumin (BSA), and gelatin.

[0021] The carbohydrate stabilizer may be a disaccharide carbohydrate stabilizer. The disaccharide carbohydrate stabilizer may be trehalose, sucrose, maltose, cellobiose, or melibiose, or hydrates thereof. In specific embodiments, the disaccharide carbohydrate stabilizer may be trehalose or its hydrate. The carbohydrate stabilizer may be trehalose dihydrate.

[0022] Amphoteric surfactants are defined by formula (XV): [ka] (In the formula, Y = CO2- or SO3-, W = H or OH, and Z = CH3 or NHC(O)R(In the formula, R = C 1~15 (It is alkyl; independently, each p=0 or 1; q=0 to 21; and if necessary, W=H, Z=CH3, and q=11 to 15) It may include the structure described in [reference].

[0023] The amphoteric surfactants are 3-(N,N-dimethylmyristylammonium propanesulfonate (DMMA); 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonium]-propanesulfonate (DMPA); N-(alkylC 10 ~C 16 The group may be selected from the group consisting of )-N,N-dimethylglycine betaine and N,N-dimethyl-N-dodecylglycine betaine.

[0024] If necessary, the buffer composition may contain one or more, two or more, or three or more additional additives selected from the group consisting of preservatives, antioxidants, anionic surfactants, nonionic surfactants, and colorants.

[0025] The buffer composition may have a pH in the range of 6.5 to 7.5. In some examples, the aqueous buffer composition may have a pH in the range of 7.0 to 7.4.

[0026] The buffer composition may contain, per test, 200-800 μg of monomer; 2000-3000 μg of carbohydrate stabilizer; 8.4-72 μg of protein stabilizer; and 2-15 μg of amphoteric surfactant.

[0027] The buffer composition may contain, per test, 300-600 μg of monomer; 2200-2800 μg of carbohydrate stabilizer; 15-20 μg of protein stabilizer; and 8-12 μg of amphoteric surfactant.

[0028] In some embodiments, the buffer composition may contain a plurality of fluorescent dye conjugates. In some embodiments, the buffer composition may contain a plurality of fluorescent polymer dye conjugates. The fluorescent polymer dye conjugates may include structures described in formulas (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), and / or (XIV), respectively, as described herein.

[0029] In other embodiments, the aqueous buffer composition does not contain a plurality of fluorescent dye conjugates. In further embodiments, the aqueous buffer composition does not contain any fluorescent dye conjugates.

[0030] A novel method for preparing a single-reactant film is provided, comprising the steps of: dispensing a plurality of reactants together onto a substrate in a liquid phase containing a buffer composition described herein, wherein the plurality of reactants comprise a first reactant and a second reactant, the first reactant comprising a first binding partner conjugated to a first dye, the first dye comprising a fluorescent polymer dye; the second reactant comprising a second binding partner conjugated to a second dye; and drying the first and second reactants together in an aqueous buffer in the liquid phase to form a first single-reactant film on the substrate. The second dye may be a fluorescent polymer dye. The single-reactant film may be a homogeneous film comprising the plurality of reactants. The multiple reactants may consist of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more reactants, or 2 to 20, 3 to 18, or 4 to 12 different reactants, each containing a different binding partner dye conjugate. The dyes may be fluorescent dyes. The fluorescent dyes may be fluorescent polymer dyes. For example, each reactant may contain a unique fluorescent dye conjugate. The multiple reactants may contain two or more unique fluorescent polymer dye conjugates. The substrate may be a tube, well, membrane, or beads. The substrate may include the inner surface of a reaction vessel.

[0031] This disclosure provides a single-reactant film, which is a homogeneous film containing multiple reactants prepared by a novel method of this disclosure. The single-reactant film is exposed to, treated and analyzed by flow cytometry of a first aliquot of a liquid biological sample, and then provides a first flow cytometry plot showing one or more of the following when compared to a second flow cytometry plot obtained by exposing, treating and analyzing a second single-reactant film containing the first and second reactants to the second aliquot of a liquid biological sample, and the second single-reactant film containing the first and second reactants, with the second single-reactant film being prepared using a conventional liquid phase without water-soluble monomers and without amphoteric surfactants. The fluorescent dye conjugate may be a fluorescent polymer dye conjugate.

[0032] The buffer compositions described herein enable the drying of different fluorescent dye conjugates in a single tube without altering their functionality (i.e., affinity to the analyte) and physical properties (such as brightness or fluorescence), while avoiding aggregation and resulting false positive clusters to other channels, thereby providing a uniform film. [Brief explanation of the drawing]

[0033] [Figure 1A] Figure 1A shows a flow cytometry density plot of CD4-PE conjugates dried using a conventional drying technique with reagent buffer. It shows the desired functionality (affinity for antigen), physical properties (such as brightness or fluorescence), and cell population separation ability.

[0034] [Figure 1B] Figure 1B shows a flow cytometry density plot of CD20-APC conjugates dried using a conventional drying technique with reagent buffer. It demonstrates the desired functionality (affinity for antigen), physical properties (such as brightness or fluorescence), and cell population separation ability.

[0035] [Figure 2A] Figure 2A shows a two-dimensional flow cytometry dot plot of CD20-SNv605 and CD4-SNv786 polymer dye conjugates when mixed and dried using reagent buffers with conventional drying techniques without compensation. Undesirable results were observed, as well as the inability to separate the populations on the x and y axes.

[0036] [Figure 2B] Figure 2B shows flow cytometry results for CD20-SNv605 and CD4-SNv786 polymer dye conjugates when mixed and dried using reagent buffers with conventional drying techniques and compensation. The inability to compensate for the population was attributed to aggregation of the polymer dye antibody conjugate when the two polymer dyes were dried using conventional drying techniques.

[0037] [Figure 3A] Figure 3A shows a two-dimensional flow cytometry dot plot of the drydown of CD20-SNv605 and CD4-SNv786 polymer dye conjugates in DM2(DM2+S)DM buffer with no compensation stabilizer. Leakage of the 610 fluorescence channel is observed in the 780 fluorescence channel.

[0038] [Figure 3B] Figure 3B shows a two-dimensional dot plot of flow cytometry of the drydown of CD20-SNv605 and CD4-SNv786 polymer dye conjugates in the compensated DM buffer DM2+S of the present invention. The DM2+S DM buffer can separate cell populations without any nonspecific interactions.

[0039] [Figure 4] Figure 4 shows the chemical structures of representative monomers A and B.

[0040] [Figure 5] Figure 5 shows flow cytometry plots for casein dose setting in DM using CD20-SNv605 to measure mobilization percentage and demonstrate reduction of nonspecific events.

[0041] [Figure 6] Figure 6 shows two-dimensional fluorescence dot plots of the dry formulation for a three-color test at monomer A concentrations of 200 μg (upper panel) and 400 μg (lower panel) per test. A reduction in population spread was observed at monomer A concentration of 400 μg per test.

[0042] [Figure 7A] Figure 7A shows a scattering flow plot in dose optimization of DMMA. Figure 7A shows unstained (top left), dry DM alone (second from the left, top panel), DM with 0.004% dry DMMA (third from the left, top panel), DM with 0.008% dry DMMA (top right panel), DM with 0.018% dry DMMA (bottom left panel), DM with 0.021% dry DMMA (bottom center panel), and DM with 0.03% dry DMMA (bottom right panel). The 0.004% and 0.008% DMMA concentrations show less nonspecific neutrophil pull-out (indicated by arrows) compared to other DMMA concentrations.

[0043] [Figure 7B]Figure 7B shows dot plots of CD20-SNv605 for all formulations for dose optimization of DMMA in DM, including unstained (top left), dried DMMA alone (second from the left, top panel), dried DMMA 0.004% in DM (third from the left, top panel), dried DMMA 0.008% in DM (top right panel), dried DMMA 0.018% in DM (bottom left panel), dried DMMA 0.021% in DM (bottom center panel), and dried DMMA 0.03% in DM (bottom right panel). DMMA concentrations of 0.004%, 0.008%, and 0.018% show less nonspecific monocyte pullout in channel 610 (indicated by arrows) compared to DM and other DMMA concentrations.

[0044] [Figure 7C] Figure 7C shows dot plots of HLADR-786 for all formulations for dose optimization of DMMA in DM, including unstained (top left), dried DM alone (second from the left, top panel), dried DMMA 0.004% in DM (third from the left, top panel), dried DMMA 0.008% in DM (top right panel), dried DMMA 0.018% in DM (bottom left panel), dried DMMA 0.021% in DM (bottom center panel), and dried DMMA 0.03% in DM (bottom right panel).

[0045] [Figure 7D] Figure 7D shows two-dimensional dot plots of CD20-605 and HLADR-786 for dry DM alone (upper left panel), 0.004% dry DMMA in DM (upper center panel), 0.008% dry DMMA in DM (upper right panel), 0.018% dry DMMA in DM (lower left panel), 0.021% dry DMMA in DM (lower center panel), and 0.03% dry DMMA in DM (lower right panel).

[0046] [Figure 8A]Figure 8A shows flow plots of scattering for additive combinations with 0.008% DMMA, including unstained (top left), dried DM (second from the left, top panel), DMMA 0.008% (top right), DMMA 0.008% + casein 2.5× (bottom left), DMMA 0.008% + Prionex 2 dil (bottom center panel), and DMMA 0.008% + Prionex 3 dil (bottom right). The scattering appears similar for each combination, except for DM, which shows a pull-out of nonspecific granulocytes and monocytes, as indicated by the arrows.

[0047] [Figure 8B] Figure 8B shows dot plots of CD20-SNv605 for additive combinations with 0.008% DMMA, including single CD20 (top left), dried DM (second from the left, top panel), DMMA 0.008% (top right), DMMA 0.008% + casein 2.5× (bottom left), DMMA 0.002% + Prionex 2 dil (bottom center panel), and DMMA 0.008% + Prionex 3 dil (bottom right).

[0048] [Figure 8C] Figure 8C shows dot plots of HLADR-786 for additive combinations with 0.008% DMMA, including single HLADR (top left), dry DM (second from the left, top panel), DMMA 0.008% (top right), DMMA 0.008% + casein 2.5× (bottom left), DMMA 0.008% + Prionex 2 dil (bottom center panel), and DMMA 0.008% + Prionex 3 dil (bottom right). The mobilization percentage of HLADR+ was found to be similar for all combinations with 0.008% DMMA compared to the individual single liquids.

[0049] [Figure 8D]Figure 8D shows two-dimensional dot plots of CD20-SNv 605 and HLADR-786 for additive combinations with 0.008% DMMA, including dry DM (upper left panel), DMMA 0.008% (upper right), DMMA 0.002% + casein 2.5× (lower left), DMMA 0.008% + Prionex 2 dil (lower center panel), and DMMA 0.008% + Prionex 3 dil (lower right). The mobilization percentage of the double-positive population for all combinations with 0.008% DMMA was found to be similar to that for DM alone.

[0050] [Figure 9] Figure 9 shows the physical appearance and properties of the dried tubes for each of the test groups: DM2+S, trehalose + monomer, trehalose + casein, trehalose + DMMA (left to right, upper panel), DM2+S, DM2+S without monomer, DM2+S without DMMA, and DM2+S without casein (left to right, lower panel). Here, DM2+S serves as the control group. Physical observations show that in the absence of monomer, there is a change in the color of the dried film (typically, the red film turns light orange to brown). Shrinkage of the film was observed in the groups without casein and DMMA. Changes in the appearance of the dried film were not observed in the tubes without DMMA compared to DM2+S. However, the tubes without casein show minimal shrinkage of the dried film.

[0051] [Figure 10A]Figure 10A shows side-scatter SSC vs. FL plots for CD56-SNv428 in the test group DM2+S, DM2+S without DMMA, DM2+S without casein, DM2+S without monomer (left to right, upper panel), trehalose + DMMA, trehalose + casein, and trehalose + monomer (left to right, lower panel). In the absence of casein and DMMA, there is a pull-out of nonspecific monocytes (indicated by arrows). The absence of monomers leads to the spread of the negative population in lymphocytes (indicated by arrows). The spread of the negative population is mainly attributable to nonspecific interactions between SN dyes in the absence of monomers and casein.

[0052] [Figure 10B] Figure 10B shows side-scatter SSC vs. FL plots for CD20-SNv605 in the test group DM2+S, DM2+S without DMMA, DM2+S without casein, DM2+S without monomer (left to right, upper panel), trehalose + DMMA, trehalose + casein, and trehalose + monomer (left to right, lower panel). In the absence of casein, there is a pull-out of nonspecific monocytes (indicated by arrows). The absence of monomers leads to the spread of the negative population in lymphocytes (indicated by arrows). The spread of the negative population is mainly due to nonspecific interactions between SN dyes in the absence of monomers and casein.

[0053] [Figure 10C] Figure 10C shows side-scatter SSC vs. FL plots for CD4-SNv786 in the test group DM2+S, DM2+S without DMMA, DM2+S without casein, DM2+S without monomer (left to right, upper panel), trehalose + DMMA, trehalose + casein, and trehalose + monomer (left to right, lower panel). The absence of monomer leads to the spread of the negative population in lymphocytes (indicated by arrows).

[0054] [Figure 10D]Figure 10D shows two-dimensional flow plots for CD56-SNv428 versus CD20-SNv605 in the test groups DM2+S, DM2+S without DMMA, DM2+S without casein, DM2+S without monomer (left to right, upper panel), trehalose + DMMA, trehalose + casein, and trehalose + monomer (left to right, lower panel). The absence of monomers and casein causes nonspecific interactions / population spread (indicated by arrows) between SN populations. This plot shows that both monomers and casein are important to prevent nonspecific interactions.

[0055] [Figure 10E] Figure 10E shows two-dimensional flow plots for CD4-SNv786 versus CD20-SNv605 in the test groups DM2+S, DM2+S without DMMA, DM2+S without casein, DM2+S without monomer (left to right, upper panel), trehalose + DMMA, trehalose + casein, and trehalose + monomer (left to right, lower panel). The absence of monomers and casein causes nonspecific interactions / population spread (indicated by arrows) between SN populations. This plot illustrates that both monomers and casein are important in preventing nonspecific interactions.

[0056] [Figure 10F] Figure 10F shows two-dimensional flow plots of CD4-SNv786 versus CD56-SNv428 in the test group DM2+S, DM2+S without DMMA, DM2+S without casein, DM2+S without monomer (left to right, upper panel), trehalose + DMMA, trehalose + casein, and trehalose + monomer (left to right, lower panel). The absence of monomers and casein causes nonspecific interactions / population spread (indicated by arrows) between SN populations. Therefore, both monomers and casein are important to prevent nonspecific interactions.

[0057] [Figure 11A]Figure 11A shows representative SSC vs. FL superimposed flow plots for three polymer dye conjugates CD56-SNv428, CD20-SNv605, and CD4-SNv786, including gating of CD45-APC-A750, drying in a cocktail with the DM2+S drydown buffer of the present invention, and reconstitution with a blood sample. In the second group, liquid cocktails of the same antibodies were prepared using commercially available BD Horizon® Brilliant staining buffer. Compared to DM2+S dry tubes, BD Horizon® Brilliant staining buffer caused nonspecific granulocyte and monocyte pullout (indicated by arrows).

[0058] [Figure 11B] Figure 11B shows representative two-dimensional superimposed flow plots for three polymer dye antibody conjugates, CD56-SNv428, CD20-SNv605, and CD4-SNv786, dried using the DM2+S drydown buffer of the present invention and reconstituted with blood samples. In the second group, liquid cocktails of the same antibodies were prepared using commercially available BD Horizon® Brilliant staining buffer. Compared to DM2+S, BD Horizon® Brilliant staining buffer caused nonspecific lymphocyte pullout (e.g., nonspecific lymphocyte pullout in CD56 and CD20) in all combinations of SN conjugates.

[0059] [Figure 12] Figure 12 shows a photograph of a dried DM2+S buffer tube in an open pouch after 6 months.

[0060] [Figure 13A]Figure 13A shows the 6-month stability of fresh, 3-month, and 6-month aged DM2+S dry tubes. Representative two-dimensional superimposed flow plots for different combinations of SN dyes in each of the three lots are shown. The dry tubes contain 12 different binding partner dye conjugates; i.e., a 12-color (12C) panel (9C conventional conjugates + 3C SN conjugates CD56-SNv428, CD20-SNv605, CD4-SNv786). These lots were tested in single replicates on four donors in one flow cytometer instrument. The staining-dissolution-wash protocol described in Protocol and Methods was used for the study. These superimpositions show that the populations in the 3-month and 6-month aged lots completely overlap with those of the fresh lots. In addition, no nonspecific interactions or population spread were observed in the 6-month aged lots in all test donors.

[0061] [Figure 13B] Figure 13B shows representative two-dimensional superimposed flow plots for SN vs. classical combinations in all three lots, comparing the 6-month stability of DM2+S dry tubes with 3-month and fresh lots. Dry tubes containing 12-color (12C) panels (9C conventional conjugate + 3C SN conjugate CD56-SNv428, CD20-SNv605, CD4-SNv786) were tested in single replicates in one flow cytometry instrument for four donors. Superimposed flow plots for 6-month and 3-month aged lots and fresh lots for CD3 ECD-A vs. CD20 Violet610-A (left panel), CD8 KO525-A vs. CD4 Violet780-A (center panel), and CD45 APC-A750-A vs. CD56PB450-A (right panel) show that the populations in the 3-month and 6-month aged lots completely overlap with the fresh lots. In addition, no nonspecific interactions or population spread were observed in any of the test donors in the lots at 6 months post-market.

[0062] [Figure 13C] Figure 13C shows representative two-dimensional superimposed flow plots for classical vs. classical combinations in all three lots, comparing the 6-month stability of DM2+S dry tubes with that of 3-month and fresh lots. Dry tubes containing 12-color (12C) panels (9C conventional conjugate + 3C SN conjugate CD56-SNv428, CD20-SNv605, CD4-SNv786) were tested in single replicates in one flow cytometry instrument for four donors. Superimpositions of 6-month and 3-month aged lots versus fresh lots for CD16 FITC-A vs. CD25PE-A (left panel), CD8 KO525-A vs. CD45APC-A750-A (center panel), and CD3 ECD-A vs. CD10APC-A (right panel) show that the populations in the 3-month and 6-month aged lots completely overlap with those in the fresh lots. In addition, no nonspecific interactions or population spread were observed in any of the test donors in the lots at 6 months post-market. [Modes for carrying out the invention]

[0063] Detailed explanation of disclosure This disclosure provides compositions and methods for minimizing aggregation of two or more dye conjugates when they are dried down together. A dry-down buffer composition is provided that helps maintain the integrity of the fluorescent dye structure in a mixture of fluorescent dye conjugates, reduces aggregation, and minimizes nonspecific interactions and artificial staining phenomena. For example, the dry-down buffer composition may be used to reduce or prevent aggregation of two or more fluorescent polymer dye conjugates when they are dried down together or during reconstitution.

[0064] Aggregation between fluorescent dye conjugates can occur in the liquid cocktail, while drying the cocktail of fluorescent dye conjugates, or during reconstitution. The present invention solves this problem by providing a dry-down buffer composition for reducing or avoiding aggregation, for example, by preventing the fluorescent polymer dye conjugates from interacting during drying. During reconstitution, the fluorescent dye conjugates can independently bind to the target analyte in the liquid sample being analyzed, substantially reducing or eliminating erroneous results resulting from aggregation or crosslinking.

[0065] We have developed a novel drydown buffer that can reduce or eliminate nonspecific interactions of fluorescent dyes without interfering with the innate binding ability of the fluorescent dye conjugate to the target antigen.

[0066] Figures 2B and 3B show comparative flow cytometry dot plots of CD20-605 and CD4-786 polymer dye conjugates that were dried down and treated using (Figure 2B) a conventional dry-down technique and (Figure 3B) a dry-down ("DM") buffer DM2+S tube of the present invention, according to Example 1. Figure 2B shows that polymer dye conjugates dried down using a conventional dry-down technique failed to separate cell populations, while Figure 3B shows that the DM buffer DM2+S of the present invention was able to separate cell populations without any nonspecific interactions.

[0067] definition

[0068] As used herein, the following terms and their variations have the meanings set forth below unless otherwise explicitly intended by the context in which such terms are used.

[0069] The singular forms "a," "an," and "the" are intended to include plural forms unless the context explicitly indicates otherwise.

[0070] The term "and / or" refers to and encompasses any possible combination of one or more of the related enumerated items.

[0071] The term "approximately" means, when referring to measurable values ​​such as compounds, doses, time, temperature, etc., to include variations of 10%, 5%, 1%, 0.5%, or even 0.1% of the specified value, and at least industry standard variations in the test methods used to measure the value.

[0072] The terms “patient,” “subject (singular),” or “subject (plural)” include, but are not limited to, humans, and may also include other mammals, or domesticated or exotic animals, such as dogs, cats, ferrets, rabbits, pigs, horses, cattle, birds, or reptiles.

[0073] Unless otherwise specified, the term "room temperature" refers to a temperature between 18 and 27 degrees Celsius.

[0074] Unless otherwise specified, the terms “percent” or “%” refer to weight percentages.

[0075] The terms “activated ester” or “active ester,” either by themselves or as part of another substituent, refer to carboxyl-active groups used in peptide chemistry to facilitate the easy condensation of the carboxyl group of an amino acid derivative with a free amino group. Descriptions of these carboxyl-active groups can be found in general peptide chemistry textbooks, e.g., KD Kopple, “Peptides and Amino Acids”, WA Benjamin, Inc., New York, 1966, pp. 50–51 and E. Schroder and K. Lubke, “The Peptides”; Vol. 1, Academic Press, New York, 1965, pp. 77–128.

[0076] As used herein, the term "acyl" refers to a group containing a carbonyl moiety, where the group is bonded via a carbonyl carbon atom. The carbonyl carbon atom may be bonded to a hydrogen atom to form a "formyl" group, or to another carbon atom, which may be part of alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, etc. An acyl group may contain 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group may contain double or triple bonds as defined herein. An acyl group may also contain heteroatoms as it may be defined herein. Examples of acyl groups, but not limited to, include nicotinoyl (pyridyl-3-carbonyl) acetyl, bensoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acrylooxy groups. If a group containing a carbon atom bonded to a carbonyl carbon atom contains a halogen, the group is referred to as a "haloacyl" group. An example is the trifluoroacetyl group.

[0077] The term "aldehyde," either by itself or as part of another substituent, refers to a chemical compound having a -CHO group.

[0078] The terms “alkene” or “alkenyl,” either by themselves or as part of another substituent, refer to any linear, branched, or cyclic hydrocarbon having at least one double bond between two carbon atoms. Examples of alkene groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexadienyl. Alkene groups are typically monovalent, but can be divalent, for example, when two parts of an alkenyl group are linked together.

[0079] The term "alkoxy," either by itself or as part of another substituent, refers to the alkyl group defined above, having an oxygen atom connected to the alkyl group at its bonding site. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, and hexoxy. Alkoxy groups can be further substituted with the various substituents listed herein. For example, alkoxy groups can be substituted with halogens to form "halo-alkoxy" groups.

[0080] The term "alkyl," either by itself or as part of another substituent, refers to a linear or branched saturated aliphatic radical having the number of carbon atoms indicated. Alkyl alkyl groups may be substituted as needed. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and hexyl. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, and decyl. Alkyl groups may contain any number of carbon atoms, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6, and 5-6. Alkyl groups are typically monovalent, but can be divalent, for example, if two parts of an alkyl group are linked together.

[0081] The terms “alkyne” or “alkynyl,” either by themselves or as part of another substituent, refer to either linear or branched hydrocarbons having at least one triple bond between two carbon atoms. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiinyl, 1-pentynyl, 2-pentynyl, isopentinyl, 1,3-pentadinyl, 1,4-pentadinyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadinyl, 1,4-hexadinyl, 1,5-hexadinyl, 2,4-hexadinyl, or 1,3,5-hexadinyl. Alkynyl groups are typically monovalent, but can be divalent, for example, when two parts of an alkynyl group are linked together.

[0082] The term "analyte" refers to molecules, compounds, or other components in a sample. Analytes may include, but are not limited to, peptides, proteins, polynucleotides, organic molecules, sugars and other carbohydrates, and lipids.

[0083] The term “amine,” as used herein, either by itself or as part of another substituent, refers to an alkyl group as defined herein having one or more amino groups. The amino groups may be primary, secondary, or tertiary. Alkylamines may be further substituted with hydroxyl groups. Amines useful in this disclosure include, but are not limited to, ethylamine, propylamine, isopropylamine, ethylenediamine, and ethanolamine. The amino groups may link the alkylamine to a bonding site with the remainder of the compound, be located at the omega position of the alkyl group, or be linked together at at least two carbon atoms of the alkyl group. Those skilled in the art will recognize that other alkylamines may be useful in this disclosure.

[0084] The term "amino group" refers to the unprotonable -NR3 group. + Except for -NH2, -NHR, -NR2, -NR3+ (wherein each R is independently selected) refers to the form and the substituent of each protonated form. Thus, any compound substituted with an amino group can be considered an amine. In the sense of this specification, “amino group” can be a primary, secondary, tertiary, or quaternary amino group. Examples of “alkylamino” groups include monoalkylamino, dialkylamino, and trialkylamino groups.

[0085] The term "amide" refers to a functional group having a carbonyl group bonded to an amine group, having the general formula RC(=O)NR'R'' (wherein R, R', and R'' represent an organic group or a hydrogen atom). The term "amido" refers to a substituent containing an amide group.

[0086] The term "ammonium," either by itself or as part of another substituent, is used in formula NHR3 + (In the formula, each R group independently represents a cation having hydrogen, or a substituted or unsubstituted alkyl, aryl, aralkyl, or alkoxy group.) Preferably, each R group is hydrogen.

[0087] The term "antibody" refers to an immunoglobulin protein or a fragment or derivative thereof that specifically binds to an analyte. Antibodies include various classes and isotypes of immunoglobulins, such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b, IgG3, and IgM. Antibody fragments include molecules such as Fab, scFv, F(ab')2, and Fab' molecules. Antibody derivatives include antibodies or fragments thereof that have additions or substitutions, such as chimeric antibodies. Antibodies may be of human or animal origin, derived from recombinant hybridomas, or from any other method known in the art.

[0088] The term "aralkyl" refers to an alkyl group as defined herein, in which a hydrogen or carbon atom of the alkyl group is replaced by a bond to an aryl group as defined herein. Typical aralkyl groups include benzyl and phenylethyl groups, as well as condensed (cycloalkylaryl) alkyl groups, such as 4-ethyl-indanyl. An aralkenyl group is an alkenyl group as defined herein, in which a hydrogen or carbon atom of the alkyl group is replaced by a bond to an aryl group as defined herein.

[0089] The term "aryl," either by itself or as part of another substituent, refers to a cyclic aromatic hydrocarbon group that does not contain heteroatoms in the aromatic ring assembly. The "aryl" group can be a monocyclic, fused bicyclic, tricyclic, or more cyclic aromatic ring assembly containing 6 to 16 ring carbon atoms. For example, aryls may be, but are not limited to, phenyl, azlenyl, heptarenyl, biphenyl, indacenyl, fluorenyl, phenantrenyl, triphenylenyl, pyrenyl, naphthacenyl, crisenyl, biphenylenyl, anthracenyl, benzyl, or naphthyl. "Arylene" refers to a divalent radical derived from the aryl group. The aryl group may be monosubstituted, disubstituted, or trisubstituted by one, two, or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy, and oxy-C2~C3-alkylene, all of which may be further substituted as needed with, for example, those defined below herein, or with 1- or 2-naphthyl, or 1- or 2-phenantrenyl. Alkylenedioxy is a divalent substituent bonded to two adjacent carbon atoms of phenyl, e.g., methylenedioxy or ethylenedioxy. Oxy-C2~C3-alkylene is also a divalent substituent bonded to two adjacent carbon atoms of phenyl, e.g., oxyethylene or oxypropylene. An example of oxy-C2~C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0090] The term “aryloxy,” either by itself or as part of another substituent, refers to an O-aryl group, where aryl is as defined above. The aryloxy group may be unsubstituted or substituted with one or two preferred substituents. The term “phenoxy” refers to an aryloxy group, where the aryl portion is a phenyl ring. The term “(hetero)aryloxy,” as used herein, means an -O-heteroaryl group, where heteroaryl is as defined below. The term “(hetero)aryloxy” is used to indicate that the portion is either an aryloxy or a (hetero)aryloxy group.

[0091] The term "heteroaryl," either by itself or as part of another substituent, refers to a monocyclic, fused bicyclic, or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where 1 to 4 of the ring atoms are heteroatoms of N, O, or S, respectively. Examples of heteroaryls include pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radical substituted, particularly monosubstituted or disubstituted, with alkyl, nitro, or halogen, for example. Pyridyl represents 2-, 3-, or 4-pyridyl, preferably 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinyl preferably represents 2-,3-, or 4-quinolinyl. Isoquinolinyl preferably represents 1-,3-, or 4-isoquinolinyl. Benzopyranil and benzothiopyranil preferably represent 3-benzopyranil or 3-benzothiopyranil, respectively. Thiazolyl preferably represents 2- or 4-thiazolyl, with 4-thiazolyl being the most preferred. Triazolyl preferably represents 1-,2-, or 5-(1,2,4-triazolyl). Tetrazolyl preferably represents 5-tetrazolyl.

[0092] In some embodiments, the heteroaryl is any of the following: pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or a substituted, in particular, monosubstituted or disubstituted radical.

[0093] In some embodiments, substituents on aryl and heteroaryl groups are diverse, ranging in number from zero to the total number of empty valencies on the aromatic ring system, including -halogen, -OR', -OC(O)R', -NR'R'', -SR', -R', -CN, -NO2, -CO2R', -CONR'R'', -C(O)R', -OC(O)NR'R'', -NR''C(O)R', -NR''C(O)2R', -NR'-C(O)NR''R''', -NH-C(NH2)=NH, -NR'C(NH2)=NH, and -N The formulas are selected from H-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -N3, -CH(Ph)2, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, where R', R'', and R''' are independently selected from hydrogen, (C1-C5)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl.

[0094] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may, if necessary, be of the formula -TC(O)-(CH2) q -U- (wherein T and U are independently -NH-, -O-, -CH2- or a single bond, and q is an integer between 0 and 2) substituents may be replaced. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be of the formula -A-(CH2) as needed. r-B- (wherein A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer from 1 to 3) substituents may be replaced. One of the single bonds of the new ring thus formed may be replaced with a double bond, if necessary. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be replaced with substituents of the formula -(CH2) s -X-(CH2) t -(wherein s and t are independently integers from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-) substituents may be replaced by -NR'- and -S(O)2NR'- substituents R' are selected from hydrogen or unsubstituted (C1-C6) alkyl groups.

[0095] As used herein, the term "azide," either by itself or as part of another substituent, refers to structure-N3.

[0096] The term "binding partner" refers to a molecule that can specifically bind to an analyte. A binding partner can be any of many different types of molecules, including antibodies or their antigen-binding fragments, or other proteins, peptides, polysaccharides, lipids, nucleic acids, or nucleic acid analogs, such as oligonucleotides or PNA (peptide nucleic acids).

[0097] The term "boronic acid," either by itself or as part of another substituent, refers to the structure -B(OH)2. It is recognized by those skilled in the art that boronic acids can exist as boronic acid esters in various steps in the synthesis of quenchers. Boronic acid means including such esters. The terms "boronic ester" or "boronate ester," as used herein, refer to -B(Z 1 )(Z 2 ) part (where Z 1 and Z 2This refers to a chemical compound containing a portion in which the atom bonded to boron is an oxygen atom (in each case). In some embodiments, the boronic acid ester portion is a five-membered ring. In some other embodiments, the boronic acid ester portion is a six-membered ring. In some other embodiments, the boronic acid ester portion is a mixture of five-membered and six-membered rings.

[0098] The term "carbamate," either by itself or as part of another substituent, refers to a functional group having the structure -NR''CO2R' (wherein R' and R'' are independently selected from hydrogen, (C1-C8) alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4) alkyl, and (unsubstituted aryl)oxy-(C1-C4) alkyl). Examples of carbamates include t-Boc, Fmoc, benzyloxycarbonyl, alloc, methyl carbamate, ethyl carbamate, 9-(2-sulfo)fluorenyl methyl carbamate, 9-(2,7-dibromo)fluorenyl methyl carbamate, Tbfmoc, Climoc, Bimoc, DBD-Tmoc, Bsmoc, Troc, Teoc, 2-phenylethyl carbamate, Adpoc, 2-chloroethyl carbamate, 1,1-dimethyl-2-haloethyl carbamate, DB-t-BOC, TCBOC, Bpoc, t-Bumeoc, Pyoc, Bnpeoc, V-(2-pivaloylamino)-1,1-dimethylethyl carbamate, and NpSSPeoc.

[0099] The term "carboxylic acid," either by itself or as part of another substituent, refers to the structure R-COOH (where R is a carbon-containing group of an atom).

[0100] The term "carboxylate," either by itself or as part of another substituent, refers to a conjugate base of a carboxylic acid, which is generally given the formula RCOO -It can be represented by the formula RCOOR', for example, the term “magnesium carboxylate” refers to the magnesium salt of a carboxylic acid. The term “carboxylic acid ester” as used herein, either by itself or as part of another substituent, refers to a compound derived from a carboxylic acid, which can generally be represented by the formula RCOOR' (wherein R' may be an alkyl, alkene, alkyne, haloalkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, (unsubstituted aryl)alkyl, and (unsubstituted aryl)oxyalkyl or other carbon-containing group of an atom). R' may optionally contain a functional group.

[0101] The term "CD" refers to a group of differentiated antigens.

[0102] The term "chromophore" refers to a compound having a reactive group that can be covalently bonded (e.g., a carboxylate moiety, an amino moiety, a haloalkyl moiety, etc.). Examples of suitable chromophores, but not limited to, U.S. Patent Nos. 7,687,282; 7,671,214; 7,446,202; 6,972,326; 6,716,979; 6,579,718; 6,562,632; 6,399,392; 6,316,267; 6,162,931; 6,130,101; 6,005,113; 6,004,5 Examples include those described in No. 36; No. 5,863,753; No. 5,846,737; No. 5,798,276; No. 5,723,218; No. 5,696,157; ​​No. 5,658,751; No. 5,656,449; No. 5,582,977; No. 5,576,424; No. 5,573,909; and No. 5,187,288, all of which are incorporated herein by reference in their entirety.

[0103] In flow cytometry, the term "compensation" refers to a mathematical process that corrects for fluorescence leakage (the overlap of spectra in multi-parameter flow cytometry data). For example, compensation can be performed by removing the signal of any given fluorescent dye from all detectors except those dedicated to measuring that dye. Since fluorescent dyes can have a wide range of spectra, they can overlap and cause undesirable confusion during data analysis.

[0104] The term "cycloalkyl," either by itself or as part of another substituent, refers to a saturated or partially unsaturated monocyclic, condensed bicyclic, or bridging polycyclic ring assembly containing 3 to 12 ring atoms, or the number of atoms indicated in the monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Examples of bicyclic and polycyclic rings include norbornane, decahydronaphthalene, and adamantane. Examples of C3-8 cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

[0105] The term "diazonium salt" as itself or as part of another substituent is R-N2 + X - This refers to a group of organic compounds having the structure (wherein R can be any organic group (e.g., alkyl or aryl), and X can be an inorganic or organic anion (e.g., halogen).

[0106] The terms "DM-2" or "DM2" refer to DM-2. The term "S" refers to the stabilizer in reference to the "DM2+S" buffer. "DM2+S" is the DM buffer described in this disclosure, where, for example, according to Table 3, the carbohydrate stabilizer is trehalose or its hydrate, the water-soluble monomer is monomer A, the protein stabilizer is casein, and the amphoteric surfactant is DMMA.

[0107] The term "pigment conjugate" refers to a pigment that has been conjugated to a binding partner.

[0108] The term "fluorescent dye" refers to a dye containing a photoexcitable fluorophore that can re-emit light upon photoexcitation. The term "fluorescent dye" encompasses both fluorescent monomeric dyes and other conventional fluorescent dyes, as well as both fluorescent polymeric and fluorescent nonpolymeric dyes. For example, SuperNova™ ("SN") v428 (Beckman Coulter, Inc.) is a fluorescent polymeric dye that is optimally excited by a blue-violet laser (405 nm), has an excitation maximum at 414 nm, and an emission peak at 428 nm, and can be detected using a 450 / 50 bandpass filter or equivalent. SN v605 and SN v786 are tandem polymeric dyes derived from the core SN v428 polymeric dye. Both share the same absorbance characteristics, with a maximum excitation at 414 nm. These are emission peaks for SN v605 and SN v786 at 605 nm and 786 nm, respectively, which are best detected using the 610 / 2 and 780 / 60 nm bandpass filters of the flow cytometer.

[0109] The term "fluorophore" refers to a fluorescent chemical compound that can re-emit light upon photoexcitation. Fluorophores typically contain several mixed aromatic groups, or planar and cyclic molecules with several π-pi bonds.

[0110] The term "halogen," either by itself or as part of another substituent, refers to fluorine, chlorine, bromine, and iodine.

[0111] The term “(hetero)arylamino,” either by itself or as part of another substituent, refers to an amine radical substituted with an aryl group (e.g., -NH-aryl). An arylamino may also be an aryl radical substituted with an amine group (e.g., -aryl-NH2). Arylaminos can be substituted or unsubstituted.

[0112] As used herein, the term "hydrazone," whether by itself or as part of another substituent, is structural. [ka] (wherein R is, for example, a water-soluble moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or others, and may contain a carboxyl group). R may be a polymer containing six or more monomer units, a nonionic water-soluble polymer, PEG, a carboxylic acid, or a carboxylic acid ester-terminated modified PEG, or a water-soluble polymer.

[0113] The terms "hydrazine" and "hydrazide," either by themselves or as part of another substituent, refer to compounds that contain a single bonded nitrogen atom, one of which is a primary amine functional group.

[0114] The terms "hydrocarbon" or "hydrocarbyl" refer to molecules or functional groups containing carbon and hydrogen atoms. The term may also refer to molecules or functional groups that typically contain both carbon and hydrogen atoms, but where some or all of the hydrogen atoms are substituted by other functional groups. The term "hydrocarbyl" refers to functional groups derived from linear, branched, or cyclic hydrocarbons, which may be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. Hydrocarbyl groups are (C a ~C b (C1-C4) Hydrocarbyl (wherein a and b are integers, meaning that it has any number of carbon atoms from a to b). For example, (C1-C4) Hydrocarbyl means that the hydrocarbyl group can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4), and (C0-C b )Hydrocarbyl means that, in certain embodiments, the hydrocarbyl group is absent. The hydrocarbylene group is a diradical hydrocarbon, for example, a hydrocarbon bonded at two locations.

[0115] The term "labeled binding partner" refers to a binding partner that is conjugated to a dye.

[0116] The term “linker” or “link” refers to a linking portion that connects two groups and has a skeleton of 100 or fewer atoms in length. The linker or link may be a covalent bond connecting two groups, or a chain of atoms between 1 and 100 in length, e.g., a chain of carbon atoms of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, or longer, where the linker may be linear, branched, cyclic, or single-atom. In some embodiments, the linker is a branched linker, referring to a linking portion that connects three or more groups. In certain particular cases, one, two, three, four, or five, or more carbon atoms in the linker skeleton may be substituted with sulfur, nitrogen, or oxygen heteroatoms, as may be selected. In some embodiments, the linker skeleton may be a linking functional group, such as an ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester, or imine. The bonds between the skeletal atoms may be saturated or unsaturated, and in some cases, there may be no more than one, two, or three unsaturated bonds in the linker skeleton. Examples of linkers may include one or more substituents having alkyl, aryl, or alkenyl groups. Examples of linkers, but not limited to, include polyethylene glycol, ethers, thioethers, tertiary amines, and alkyl groups that may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, and 1,1-dimethylethyl (t-butyl). The linker skeleton may include cyclic groups, such as aryl, heterocyclic, or cycloalkyl groups, where two or more atoms of the cyclic group, for example, two, three, or four atoms, are included in the skeleton. The linker may be cleavable or incleavable.

[0117] The linker portion may be bonded to “L” or “A” as taught in U.S. Patent Application Publication No. 2020 / 0190253A1, which is incorporated herein by reference in its entirety. The linker portion may include sulfonamides, disulfonamides, selenamides, sulfinamides, sultams, disulfinamides, amides, selenamides, phosphonamides, phosphinamides, phosphonamides, phosphonamides, or secondary amines.

[0118] As described herein, and as each relates to a linker portion, the term "sulfonamide" refers to the partial -S(O)2NR-; the term "disulfonamide" refers to the partial -S(O)2NRS(O)2-; the term "selenamide" refers to the partial -Se(O)2NR-; the term "sulfinamide" refers to the partial -S(O)NR-; the term "disulfinamide" refers to the partial -S(O)NRS(O)-; the term "selenamide" refers to the partial - The term Se(O)NR- refers to a partial-NR-PR(O)NR-; the term "phosphonamide" refers to a partial-NR-PR(O)NR-; the term "phosphineamide" refers to a partial-PR(O)NR-; the term "phosphonamide date" refers to a partial-O-PR(O)NR-; the term "sultam" refers to a cyclic sulfonamide (for example, the R group is bonded to a sulfur atom via an alkylene moiety); where, for each term, the R group is independently H, alkyl, haloalkyl, or aryl.

[0119] As used herein, the term "N-hydroxysuccinimidyl," either by itself or as part of another substituent, is structural. [ka] (wherein R is, for example, a water-soluble moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or others, and may contain a carboxyl group). R may be, but is not limited to, a polymer containing six or more monomer units, a nonionic water-soluble polymer, PEG, a carboxylic acid, or a carboxylic acid ester-terminated modified PEG containing PEG.

[0120] The term “reactant solution” refers to a solution containing the labeled binding partner. In some embodiments, in addition to the labeled binding partner, the reactant solution further includes stabilizers, salts, buffers, surfactants, and / or other reagents. The term “maleimide” as itself or as part of another substituent refers to the structure [ka] (wherein R is, for example, a water-soluble moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or others, and may contain a carboxyl group). R may be, but is not limited to, a polymer containing six or more monomer units, a nonionic water-soluble polymer, PEG, a carboxylic acid, or a carboxylic acid ester-terminated modified PEG containing PEG.

[0121] The term "MdFl" or "MDFl" refers to the median fluorescence intensity.

[0122] The term "mobilization %" refers to the number of gated cells in the relevant population.

[0123] The term "multicolor antibody panel" refers to a cocktail containing multiple different fluorescent dye conjugates in liquid or dry format (e.g., CD4-FITC, CD8-PE, CD20-APC, CD3-PC5.5, CD16-FITC, CD25-PE, CD3-ECD, CD38-PC5.5, CD27-PC7, CD10-APC, CD14-APCA700, CD45-AA750, CD8-KRO, CD56-SNv428, CD20-SNv605, CD4-SNv786, etc.) that can be used directly to stain blood and analyze it in a flow cytometer.

[0124] The term "multicolor dried reagent" refers to a cocktail of fluorescent dye conjugates in different dried formats (such as CD4-FITC, CD8-PE, CD20-APC, CD3-PC5.5, etc.) that can be used directly to stain blood and analyze it in a flow cytometer. Multicolor dried reagent cocktails containing only conventional dyes such as FITC, PE, ECD, PC5, PC5.5, PC7, APC, AA700, AA750, PBE, and KrO can be dried using conventional drying techniques. However, conventional drying techniques have been found to be ineffective when drying multiple fluorescent polymer dye antibody conjugates in the cocktail. These polymer dye conjugates tend to interact nonspecifically, leading to difficulties in separating populations, which can present challenges in identifying desired cell populations in a given sample.

[0125] In this specification, the term "multiplexing" refers to an assay or other analytical method that allows multiple analytes to be assayed simultaneously.

[0126] The term "oligoether" refers to an oligomer containing ether-functionalized structural repeating units. As used herein, "oligomer" is understood to mean a molecule containing one or more identifiable structural repeating units of the same or different formulas.

[0127] The term "organic group" refers to any carbon-containing functional group. Examples of carbon-containing functional groups include oxygen-containing groups such as alkoxy groups, aryloxy groups, aralkyloxy groups, and oxo(carbonyl) groups; carboxyl groups, including carboxylic acids, carboxylates, and carboxylic acid esters; sulfur-containing groups such as alkyl and aryl sulfide groups; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R)2, CN, CF3, OCF3, R, C(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, O(O)R, C(O)N(R)2, O(O)N(R)2, C(S)N(R)2, (CH2) 0~2 N(R)C(O)R, (CH2) 0~2 N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N( R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, C(=NOR)R, and substituted or unsubstituted (C1~C 100 ) Hydrocarbyl is an example, where R may be a hydrogen (in examples containing other carbon atoms) or carbon-based moiety, and the carbon-based moiety may be substituted or unsubstituted.

[0128] The term "PEG" is derived from the formula -(CH2-CH2-O-) n- Refers to polyethylene glycol, or poly(ethylene glycol), a family of biocompatible, water-soluble linear polymers based on the ethylene glycol monomer unit described by or its derivatives. In some embodiments, "n" is 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, e.g., 3 to 15, or 10 to 15. The PEG polymer group may be of any convenient length, and is not limited, but is understood to include various terminal groups and / or further substituents, including alkyl, aryl, hydroxyl, amino, acyl, carboxylic acid, carboxylic acid ester, acyloxy, and amide terminal groups and / or substituents. The number after "PEG" refers to the average molecular weight, Mw refers to the weight-average molecular weight, and Mn refers to the number-average molecular weight.

[0129] The term "PBS" refers to phosphate-buffered saline, an aqueous buffer that may contain sodium chloride, disodium hydrogen phosphate, potassium chloride, and potassium dihydrogen phosphate. For example, PBS may contain milli-Q water or deionized water, and 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4. The pH may be approximately pH 7.0 to 7.4. PBS may or may not be stored with an azide such as sodium azide. PBS is an isotonic solution.

[0130] The term "phosphoramide," whether in itself or as part of another substituent, refers to structure. [ka] (wherein R is, for example, a water-soluble moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or others, and may contain a carboxyl group). R may be, but is not limited to, a polymer containing six or more monomer units, a nonionic water-soluble polymer, PEG, a carboxylic acid, or a carboxylic acid ester-terminated modified PEG containing PEG.

[0131] The term "phosphonamide" as itself or as part of another substituent is structure [ka] (wherein R is, for example, a water-soluble moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or others, and may contain a carboxyl group). R may be, but is not limited to, a polymer containing six or more monomer units, a nonionic water-soluble polymer, PEG, a carboxylic acid, or a carboxylic acid ester-terminated modified PEG containing PEG.

[0132] The term "phosphine amide," either by itself or as part of another substituent, refers to structure. [ka] (wherein R is, for example, a water-soluble moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or others, and may contain a carboxyl group). R may be, but is not limited to, a polymer containing six or more monomer units, a nonionic water-soluble polymer, PEG, a carboxylic acid, or a carboxylic acid ester-terminated modified PEG containing PEG.

[0133] The term "physical properties" refers to the properties of a fluorescent dye conjugate, including its brightness or fluorescence, and its leakage into other channels.

[0134] The term "polymer dye conjugate" refers to a polymer dye conjugated to a binding partner. For example, polymer dye conjugates may include, but are not limited to, fluorescent polymers having monomer subunits, including dihydrophenanthrene (DHP), fluorene, and combinations thereof.

[0135] The term "substantially reduced" refers to a reduction of at least 10%, at least 25%, or at least 50% of a measurable amount.

[0136] The terms “substituted” or “substituted,” as used herein in conjunction with a molecule or organic group as defined herein, refer to a state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The terms “functional group” or “substituent,” as used herein, refer to a group that may be present on or may be substituted on a molecule or organic group. Examples of substituents or functional groups include, but are not limited to, halogens (e.g., F, Cl, Br, and I); oxygen atoms in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxylic acids, carboxylates, and carboxyl groups including carboxylic acid esters; sulfur atoms in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; nitrogen atoms in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various groups. Non-limiting examples of substituents that can bond to the substituted carbon (or other) atom include F, Cl, Br, I, OR, OCO(O)N(R)2, CN, NO, NO2, ONO2, azide, CF3, OCF3, R, O(oxo), S(thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OCO(O)R, C(O)N(R)2, OCO(O)N(R)2, C(S)N(R)2, (CH2) 0~2 N(R)C(O)R, (CH2) 0~2Examples include N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, where R can be a part based on hydrogen or carbon; for example, R is hydrogen, (C1~C 100 ) may be hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or two R groups bonded to or adjacent to a nitrogen atom may, together with the nitrogen atom or atom, form a heterocyclyl.

[0137] The terms "sulfonate functional group" or "sulfonate," either by themselves or as part of another substituent, refer to both the free sulfonate anion (-S(=O)2O-) and its salts. Therefore, the term sulfonate encompasses sulfonate salts, such as sodium, lithium, potassium, and ammonium sulfonates.

[0138] The term “sulfonamide,” either by itself or as part of another substituent, refers to a group of the formula -SO2NR- (wherein R may be, for example, a water-soluble moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or others, and may contain a carboxyl group). R may be, but is not limited to, a polymer containing six or more monomer units, a nonionic water-soluble polymer, a PEG, a carboxylic acid, or a carboxylic acid ester, and may be a water-soluble polymer containing a terminally modified PEG.

[0139] The term "sulfonamide", either by itself or as part of another substituent, refers to a group of the formula -SO2NR2, where R can be, for example, a solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or others, and can contain a carboxyl group. R can be, but is not limited to, a polymer containing 6 or more monomer units, a nonionic water-soluble polymer, PEG, or a solubilizing polymer containing a modified PEG with a carboxylic acid or carboxylic acid ester at the end.

[0140] The term "sulfinamido", either by itself or as part of another substituent, refers to a group of the formula -SONR2-, where R can be, for example, a solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or others, and can contain a carboxyl group. R can be, but is not limited to, a polymer containing 6 or more monomer units, a nonionic water-soluble polymer, PEG, or a solubilizing polymer containing a modified PEG with a carboxylic acid or carboxylic acid ester at the end.

[0141] The term "silyl", either by itself or as part of another substituent, refers to Si(R z )3, where each R z is independently alkyl, aryl, or another carbon-containing group of atoms.

[0142] The term "thiol", either by itself or as part of another substituent, refers to a compound containing a functional group composed of a sulfur-hydrogen bond. The general chemical structure of the thiol functional group is R-SH, where R represents alkyl, alkene, aryl, or another carbon-containing group of atoms.

[0143] The term “water-soluble moiety,” as used herein, either by itself or as part of another substituent, refers to any hydrophilic group that can be sufficiently hydrated in an aqueous environment, such as under physiological conditions, and increase the water solubility of the molecule to which it binds. The increase in water solubility of the molecule can be highly dependent on the binding moiety. In some examples, the increase in water solubility (compared to the solubility of the molecule without the binding moiety) can be twice or more, five times or more, ten times or more, twenty-five times or more, fifty times or more, or one hundred times or more. The "water-soluble portion" includes, but is not limited to, PEG groups, carboxyl groups including carboxylic acids and carboxylates, polyvinyl alcohol, glycol, peptide, polyphosphate, polyalcohol, sulfonate, phosphonate, boronate, amine, ammonium, sulfonium, phosphonium, alcohol, amphoteric derivative, carbohydrate, nucleotide, polynucleotide, substituted PEG groups, substituted carboxyl groups including, but is not limited to, substituted carboxylic acids and substituted carboxylates, substituted glycol, substituted peptide, substituted polyphosphate, substituted polyalcohol, substituted sulfonate, substituted phosphonate, substituted boronate, substituted amine, substituted ammonium, substituted sulfonium, substituted phosphonium, alcohol, amphoteric derivative, substituted carbohydrate, substituted nucleotide, substituted polynucleotide, and combinations thereof.

[0144] The term “water-soluble polymer” (WSP), as used herein, refers to a polymer having a solubility in “water” as used herein of 1 mg / mL or higher, for example, 3 mg / mL or higher, 10 mg / mL or higher, 20 mg / mL or higher, 30 mg / mL or higher, 40 mg / mL or higher, 50 mg / mL or higher, 60 mg / mL or higher, 70 mg / mL or higher, 80 mg / mL or higher, 90 mg / mL or higher, 100 mg / mL or higher, or even higher. It is understood that, under certain conditions, water-soluble polymers can form hydrated nanoparticles in a separate water in an aqueous system and can resist aggregation. reaction vessel

[0145] The “reaction vessel” disclosed herein may be any container in which a reaction between a binding partner or its dye conjugate and a target analyte can occur. For example, a reaction vessel may be a tube, a plate, a well in a microtiter plate, a chamber, and a slide. In a preferred embodiment, the reaction vessel has a lid or cap so that the binding reaction can occur in a closed environment. Base material

[0146] The reaction vessel comprises one or more substrates. The “substrate” can be any suitable surface, including, but not limited to, plastics, nitrocellulose, cellulose acetate, quartz, and glass. Non-limiting examples of plastics may include polystyrene, polypropylene, cycloolefin, and polycarbonate. In some embodiments, the substrate is a membrane. The substrate may be the inner surface of the body of the reaction vessel, e.g., a plastic tube or a well in a microtiter plate. The substrate may also be beads. In some embodiments, at least one of the substrates that receives a labeled binding partner (e.g., a membrane) is bound to the inner surface of the body of the reaction vessel. In some embodiments, the membrane substrate is a sheet or roll, which facilitates the depositing and drying of the solution. In some embodiments, the membrane may be cut to separate individually dried reactant spots. In some embodiments, the cut membrane is easily dropped into the reaction vessel. In some preferred embodiments, the cut membrane is bound to the surface of the reaction vessel so that the membrane does not escape from the vessel when liquid is pipetteed into or out of the reaction vessel.

[0147] The acronym "SN" stands for SuperNova (trademark).

[0148] The acronym "SSC" stands for lateral scattering.

[0149] The term "WBC" refers to white blood cells. liquid sample

[0150] The reaction vessel is configured to receive a liquid sample. The liquid sample used in the present invention typically comprises a target analyte, obtained or dispersed therein, primarily as an aqueous medium. The sample may be a protein, carbohydrate, or polynucleotide, which can be obtained from any source of biomaterial, for example, directly or indirectly from an organism. Examples of samples include cells, tissues, or fluids, as well as deposits left by the organism, including viruses, mycoplasmas, and fossils. The sample may comprise, whole or in part, a target analyte prepared by synthetic means. Non-limiting examples of samples include blood, serum, plasma, urine, semen, milk, sputum, mucus, cheek swabs, vaginal swabs, rectal swabs, aspirates, needle biopsies, for example, tissue sections obtained by surgery or dissection, plasma, serum, cerebrospinal fluid, lymph, skin, exocrine secretions from the respiratory, intestinal, and urogenital tracts, tears, saliva, tumors, organs, and samples of in vitro cell culture components (including, but not limited to, conditioned media resulting from cell growth in cell culture media, putative virus-infected cells, recombinant cells, and cellular components). target analyte

[0151] The present invention is designed to detect the presence and, in some cases, the amount of a specific target analyte. The term “target analyte” refers to target molecules to be detected in a biological sample, such as peptides, proteins, polynucleotides, organic molecules, sugars and other carbohydrates, and lipids. A key aspect of the present invention is that the target analyte is contained in a liquid sample and is accessible in some respects, or becomes accessible, to bind to the analyte-specific binding partner of the present invention. The target analyte may be found in biological samples, such as blood samples, cell line development samples, tissue culture samples, etc.

[0152] The target analytes may be present on beads or on the surface of cells and accessible. Examples of useful analytes, but not limited to, include: 1) specific cell surface macromolecules and antigens (including hormones, protein complexes, and molecules recognized by cell receptors), and 2) cellular proteins, DNA, or RNA in permeabilized cells containing abnormal DNA or RNA sequences, or abnormal amounts of certain messenger RNA. Detection of these analytes is particularly useful in situations where they are present in and / or identify rare cells, such as those found in the early stages of various cancers. Integration Partners

[0153] The term "binding partner" refers to a molecule that specifically binds to the epitope of the target analyte. Many different types of binding partners can be used in this system and method. In one embodiment, the binding partner is an antibody. The antibody used to bind to a particular analyte is preferably monoclonal and therefore against a single epitope of the analyte. Monoclonal antibodies can be prepared using various techniques known in the art, typically by the creation of hybridomas using a B cell line that produces an antibody with the desired binding properties. Antibodies against a single epitope can also be produced by other methods, such as recombinant methods. In some embodiments, polyclonal antibodies can be used as specific binding partners in this system and method. For example, the binding partner may be a polyclonal antibody that arises against the epitope of the analyte. Polyclonal antibodies can be prepared by methods known in the art, for example, by immunizing a host and collecting plasma or serum from the host. Antibody fragments that lack the Fc portion of an antibody, such as Fab, Fab', and F(ab')2 fragments, and that retain their specific binding properties, can also be used as specific binding partners in the present invention. The F(ab')2 fragment can be produced by methods known in the art, for example, by cleaving a monoclonal antibody with proteolytic enzymes such as papain and pepsin. The Fab' fragment can be produced by reductive cleavage of the F(ab')2 fragment with reagents such as dithiothreitol or mercaptoethanol. Alternatively, antibody fragments can be produced using recombinant methods, for example, by using a phage display library.

[0154] Other binding partners besides antibodies, antibody fragments, or derivatives can also be used in this system and method. For example, the binding partner may be a nucleic acid or nucleic acid analog, such as an oligonucleotide or PNA probe. In one embodiment, an aptamer can be used as a specific binding partner. An aptamer is a single-stranded DNA or RNA (ssDNA or ssRNA) molecule that can bind to a pre-selected target, including proteins and peptides, with high affinity and specificity. Other binding partners that can bind to the target analyte to form receptor-ligand, enzyme-substrate, enzyme-inhibitor, and enzyme-cofactor pairs can also be used. Specific examples of such binding partner pairs include carbohydrates and lectins, biotin and avidin or streptavidin, folic acid and folate-binding proteins, vitamin B12 and intrinsic factor, protein A and immunoglobulins, and protein G and immunoglobulins. Binding partners that form a covalent bond with the target analyte are also included. pigment

[0155] A "dye" is a portion that provides a detectable signal, which can bind to or be incorporated into a binding partner, either directly or indirectly. Dyes used in the present invention can be colored, fluorescent, or luminescent and are typically detected by a detector in a flow cytometer, such as a PMT or APD. Fluorescent dyes can be monomeric or polymeric. Non-limiting examples of monomeric dyes include fluorescein, rhodamine, and cyanine. For example, commonly used monomeric fluorescent dyes include FITC (fluorescein isothiocyanate) (excitation maximum 494 nm / emission maximum 520 nm), PE (R-phycoerythrin) (excitation maximum 496 nm / emission maximum 578 nm), APC (allophycocyanin) (excitation maximum 650 nm / emission maximum 660 nm), and PerCP (carotenoid-protein complex derived from phytoplankton) (excitation maximum 482 nm / emission maximum 678 nm), and Cy5.5 (cyanine dye) (excitation maximum 675 nm / emission maximum 694 nm). Other cyanine dyes may be synthesized from 2-, 3-, 5-, or 7-methine structures and may include Cy2, Cy3, Cy3B, Cy3.5, Cy5, and Cy7.PC5.5. Fluorescent dyes may also be tandem dyes. Examples of tandem dyes include PE-Cy5.5 tandem (excitation maximum 566 nm / emission maximum 671 nm), APC-Cy5.5 tandem (excitation maximum 656 nm / emission maximum 700 nm), and PerCP-Cy5.5 tandem (excitation maximum 489 nm / emission maximum 679 nm).

[0156] The fluorescent dye may be a fluorescent polymer dye. Fluorescent polymeric dyes are particularly useful for the analysis of chemical and biological targets. Because of the multiple chromophores they contain, they are very reactive optical reporters and efficient light absorbers. Examples of fluorescent polymeric dyes include, but are not limited to, conjugated polymers having repeating units of chromophores, aggregates of conjugated molecules, luminescent dyes attached to saturated polymers via side chains, semiconductor quantum dots, and dendrimers. Fluorescent polymeric and monomeric dyes disclosed in U.S. Patent Nos. 7,214,489, 8,354,239, and 8,575,303 can also be used for the purposes of this application.

[0157] In some cases, the fluorescent dye has the formula (IV):

Chemical formula

[0158] L may be a linker moiety containing aryl or heteroaryl groups evenly or randomly distributed along the polymer backbone, and one or more of the pendant chain terminals may be substituted with functional groups selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protecting groups, for conjugation to another substrate, acceptor dye, molecule, or binder.

[0159] The fluorescent polymer dye may be conjugated to a binding partner, for example, as described in US2020 / 0190253, incorporated herein by reference, to form a fluorescent polymer dye conjugate having, for example, a monomer A subunit and a monomer B subunit. The fluorescent polymer dye conjugate is of formula V: [ka] (In the formula, Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; L 1 , L 2 , and L 3 This is the linker part; W is the water-soluble part; Each E is independently a selected chromophore, functional component, or binding partner; Each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap-modified monomers, optionally substituted ethylenes, and ethynylenes; G 1 and G 2 These are independently selected from unmodified polymer ends and modified polymer ends; The subscripts n and m are independently integers in the range of 1 to 10,000. The subscript p is an integer in the range of 0 to 10,000. The sum of the subscripts n, m, and p is in the range of 2 to 10,000; The subscript q is 1, 2, 3, or 4; The subscript r is 1, 2, 3, or 4; The subscript s is 0, 1, 2, or 3; The subscript t is either 1 or 2. The sum of the subscripts r and s is in the range of 1 to 4; A and B are distributed randomly or non-randomly within the conjugated polymer. It may have a structure.

[0160] The fluorescent dye may be a fluorescent polymer dye having a water-soluble monomer A subunit and a monomer B subunit. The polymer dye may be a water-soluble fluorescent polymer dye. For example, monomer A or monomer B may contain a dihydrophenanthrene (DHP) moiety. Monomer A or monomer B may contain a fluorene moiety. In some conjugated polymer dyes, monomer B may be used to alter the polymer's band gap. The monomer units may be water-soluble and, for example, contain one or more, or two or more, water-soluble moieties (W), such as a poly(ethylene glycol) (PEG) moiety. In some embodiments, monomer A or monomer B are each independently a water-soluble monomer molecule. Each water-soluble monomer A or monomer B may independently contain a DHP moiety and one or more, or two or more PEG moieties. Each water-soluble monomer A or monomer B may independently contain a DHP moiety together with a solubilized PEG moiety bonded via a sulfonamide group.

[0161] Water-soluble monomer A or monomer B containing the DHP moiety are each independently defined by formula (I): [ka] (In the formula, Each of G1 and G2 independently contains hydrogen, alkyl, PEG, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronic acid-substituted aryl, boronic acid ester-substituted aryl, boronic acid ester, boronic acid, and optionally substituted tetrahydrop Re Fluorine (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl groups selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protecting groups, in which a functional group selected from these groups is substituted at one or more terminal pendant chains; Each R 2 These are independently: water-soluble moieties, alkenes, alkynes, cycloalkyls, haloalkyls, (hetero)aryloxys, (hetero)arylaminos, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, sulfonamides, sulfinamides, phosphoamides, phosphineamides. [ka] Selected from the group consisting of; ; Each R 3 This is the water-soluble portion; Each R 4These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each R 5 These are independently H, hydroxyl, and C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, C2-C 12 Carboxylic acid esters, and C1-C 12 Selected from the group consisting of alkoxys; Each Q is independent, combined, NR 4 , or -CH2; Each Z is independently CH2, O, or NR 4 and; Each f is an independent integer between 0 and 50; (Each n is an independent integer between 1 and 20.) It may have the structure described above.

[0162] In some embodiments, water-soluble monomer A or monomer B containing the DHP moiety is independently derived from formula (III): [ka] (In the formula, each G1 and G2 is independently a halo (F, Cl, Br, I); each Z is independently selected from the group consisting of O, CH2, and NH; each R1 is independently an alkyl (C1-C3); each R2 is independently H or an alkyl (C1-C6); each n is independently 1-6; and each m is independently 5-50.) It may have the chemical structure described above. In some embodiments, G1 and G2 are Br, respectively; Z is O, respectively; R1 is CH3, respectively; R2 is H, respectively; n is independently 2 to 4, respectively; and m is independently 5 to 20. In some embodiments, n is 3, and m is 11.

[0163] Each water-soluble monomer A or monomer B may independently contain a fluorene moiety and one or more, or two or more, PEG moieties. Each water-soluble monomer A or monomer B containing a fluorene moiety may independently contain formula (II): [ka] (In the formula, Each of G1 and G2 independently contains hydrogen, halogen, alkyl, PEG, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronic acid-substituted aryl, boronic acid ester-substituted aryl, boronic acid ester, boronic acid, and optionally substituted tetrahydrop ReSelected from the group consisting of (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl, and aryl or heteroaryl substituted with one or more pendant chains having a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protecting groups; Each X is C or Si; Each R 4 is independently selected from the group consisting of H, alkyl, PEG, solubilizing moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​The fluorescent polymer dye may be any dye disclosed in US2019 / 0144601, which is incorporated herein by reference in its entirety. For example, the fluorescent polymer dye may be of formula VI: [ka] (In the formula, Each X is independently selected from the group consisting of C and Si; Each Y is independent, combined, and CR. 1 R 2 and SiR 1 R 2 Selected from the group consisting of; If Y is a bond, then X is directly bonded to both rings; Each R 1 These are independently polyethylene glycol (PEG), ammonium alkyl salts, ammonium alkyl oxy salts, ammonium oligo ether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligo ether salts, sulfonamide oligo ethers, and [ka] Selected from the group consisting of; Each R 2 These are independently H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, and [ka] Selected from the group consisting of; Each R 3 The following are independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG; each R4 These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each Z is independently CH2, O, or NR 4 and; Each Q independently performs as a coupled, NH, or NR. 4 Selected from the group consisting of , and CH2; Each M is an electron-rich linker unit that can independently change the polymer's band gap and is distributed evenly or randomly along the polymer backbone; Each R 4 These are nonionic side chains that, at a concentration of 10 mg / mL, can impart excessive solubility in water, and each is independently a halogen, hydroxyl, and C1-C. 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, (CH2) x’ (OCH2-CH2) y’ OCH3 (wherein each x' is an integer between 0 and 20 independently; each y' is an integer between 0 and 50 independently), and C2~C 18 Selected from the group consisting of (hetero)aryl groups; Each required linker L is an aryl or heteroaryl group evenly or randomly distributed along the polymer backbone, and may be substituted at one or more terminal pendant chains with functional groups selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protecting groups, for conjugation to another substrate, acceptor dye, molecule, or binder; Each G 1 and G 2 Each is independently selected from the group consisting of hydrogen, halogens, alkynes, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, boronic acid-substituted aryls, boronic acid ester-substituted aryls, boronic acid esters, boronic acids, optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene, amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and aryl or heteroaryls in which a functional group selected from their protecting groups is substituted at one or more terminal pendant chains; a, c, and d define the mole percent of each unit in the structure, and each unit may be repeated evenly or randomly, where a is between 10 and 100% mole percent, c is between 0 and 90% mole percent, and each d is between 0 and 25% mole percent; Each b is independently either 0 or 1; m is an integer between 1 and approximately 10,000; (Each n is an independent integer between 1 and 20.) It may also be a blue-violet fluorescent polymer dye having the structure shown.

[0165] In some cases, each M is independent, [ka] (In the formula, each R 4 These are nonionic side chains that, at a concentration of 10 mg / mL, can impart excessive solubility in water, and each is independently a halogen, hydroxyl, and C1-C. 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, (CH2) x’ (OCH2-CH2) y’ OCH3 (wherein each x' is an integer between 0 and 20 independently; each y' is an integer between 0 and 50 independently), and C2~C 18 (Selected from the group consisting of heteroaryl groups) You may choose from the group consisting of the following:

[0166] In some cases, the fluorescent polymer dye is formula VII: [ka] (In the formula, X, Y, R 2 , R 3 , G 1 , G 2 (L, M, Q, Z, a, b, c, d, m, and n are as previously defined.) It may have a structure.

[0167] In some cases, the fluorescent polymer dye is formula VIII: [ka] (In the formula, X, Y, R 2 , R4 , R 5 , G 1 , G 2 L, M, Q, Z, a, b, c, d, m, and n are as previously defined; Each f is an independent integer between 0 and 50; Each R 5 These are H, C1~C independently. 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (Hetero)arylaminos, and C1-C 12 (Selected from the group consisting of alkoxys) It may have a structure.

[0168] In some cases, the fluorescent polymer dye is formula IX: [ka] (In the formula, R 2 , R 4 , R 5 G 1 , G 2 (L, Z, a, b, d, f, m, and n are as previously defined.) It may have a structure.

[0169] In some cases, the fluorescent polymer dye is given by formula X: [ka] (In the formula, G 1 , G 2 (a, f, and n are as previously defined.) It may have a structure.

[0170] In some cases, the fluorescent polymer dye is formula XI: [ka] (In the formula, X, Y, R 1 , R 2 , R 3 , R 4 , G 1 , G 2 L, M, Q, Z, a, b, c, d, m, and n are as previously defined; (g and a together represent 10-100% moles.) It may also be a copolymer having the structure shown.

[0171] In some cases, the fluorescent polymer dye is formula XII: [ka] (In the formula, X, Y, R 2 , R 4 , R 5 , G 1 , G 2 L, M, Q, Z, a, b, c, d, m, and n are as previously defined; Each f is an independent integer between 0 and 50; (Each of g and a together is 10-100% mole%) It may also be a copolymer having the structure shown.

[0172] In some cases, the fluorescent polymer dye is formula XIII: [ka] (In the formula, X, R 2 , R 4 , R 5 , G 1 , G 2 L, Z, a, b, d, f, m, and n are as previously defined; (Each of g and a together is 10-100% mole%) It may also be a copolymer having the structure shown.

[0173] In some cases, the fluorescent polymer dye is formula XIV: [ka] (In the formula, G 1 , G 2 a, f, and n are as previously defined; (Each of g and a together is 10-100% mole%) It may also be a copolymer having the structure shown.

[0174] Fluorescent polymer dyes may be prepared by polymerization of water-soluble monomers such as monomer A and monomer B, which results in the formation of a highly conjugated fluorescent skeleton. Capping may be performed on the polymer by activation using appropriate functionalities, which results in a polymer that can be conjugated to a binding partner. Alternatively, monomer A or monomer B may be directly modified by activation using appropriate functionalities, for example, according to US2020 / 0190253, which is incorporated herein by reference in whole. The activated polymer may be conjugated to a binding partner. Any suitable binding partner, such as an antibody, may be used and subsequently purified, for example, by using standard procedures.

[0175] Polymer dyes are commercially available. For example, SuperNova™ ("SN") v428 (Beckman Coulter, Inc.) is a polymer dye that is optimally excited by a blue-violet laser (405 nm), has an excitation maximum at 414 nm, and an emission peak at 428 nm, and can be detected using a 450 / 50 bandpass filter or equivalent. SN v428 is a bright polymer dye that can be activated by an amine for tandem dyeing and subsequently for tandem conjugate. The structural rigidity of the polymer dye can help reduce rotational energy, resulting in brighter emission. This can help achieve optimized FRET (fluorescence resonance energy transfer) efficiency and increased stability.

[0176] SN v428 is one of the brightest dyes that can be excited by a blue-violet laser, and therefore it is particularly suitable for evaluating faintly expressed markers. Examples of antibodies conjugated with SN include anti-CD19 antibody-SN v428, anti-CD22 antibody-SN v428, anti-CD25 antibody-SN v428, and anti-CD38 antibody-SN v428 antibody-polymeric dye conjugates. SN v605 and SN v786 (Beckman Coulter, Inc.) are tandem polymer dyes derived from the core SN v428 polymer dye. Both share the same absorbance characteristics, with a maximum excitation at 414 nm. With emission peaks at 605 nm and 786 nm for SN v605 and SN v786, respectively, they are best detected using 610 / 2 and 780 / 60 nm bandpass filters on a flow cytometer.

[0177] The fluorescent polymer dye may be a commercially available fluorescent polymer dye from Becton Dickinson, including Brilliant® Blue, Brilliant® Violet, and Brilliant® Ultra Violet dyes. The fluorescent polymer dye may also be a commercially available fluorescent polymer dye from ThermoFisher Scientific, including Super Bright 436, Super Bright 600, Super Bright 645, Super Bright 702, and Super Bright 780 dyes. Water-soluble monomers

[0178] The "water-soluble monomers" may be monomer units containing aryl or heteroaryl moieties, each having one or more water-soluble moieties bonded to them as needed. The water-soluble moieties may consist of one or more PEG moieties. The water-soluble monomers may be suitable for use in the preparation of at least one of several fluorescent polymer dyes having monomer A subunits, monomer B subunits, or combinations of monomer A and monomer B subunits. The water-soluble monomers may be dihydrophenanthrene (DHP)-based water-soluble monomers. The water-soluble monomers may also be fluorene-based water-soluble monomers.

[0179] Water-soluble monomers are given by formula (I): [ka] (In the formula, Each of G1 and G2 independently contains halogens, alkyls, PEGs, hydrogen, alkynes, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, and optionally substituted tetrahydropenes. Re Fluorine (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl groups selected from the group consisting of aryl or heteroaryl groups in which a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, and thiols is substituted on one or more terminal pendant chains; Each R 2These are independently: water-soluble moieties, alkenes, alkynes, cycloalkyls, haloalkyls, (hetero)aryloxys, (hetero)arylaminos, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, sulfonamides, sulfinamides, phosphoamides, phosphineamides. [ka] Selected from the group consisting of; Each R 3 This is the water-soluble portion; Each R 4 These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each R 5 These are independently H, hydroxyl, and C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, C2-C 12 Carboxylic acid esters, and C1-C 12 Selected from the group consisting of alkoxys; Each Q is independent, combined, NR 4 , or -CH2; Each Z is independently CH2, O, or NR 4 and; Each f is an independent integer between 0 and 50; (Each n is an independent integer between 1 and 20.) It may also be a dihydrophenanthrene (DHP) monomer having the chemical structure described above.

[0180] Water-soluble monomers are given by formula (II): [ka] (In the formula, Each of G1 and G2 independently contains halogens, alkyls, PEGs, hydrogen, alkynes, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, and optionally substituted tetrahydropenes. Re Fluorine (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl groups selected from the group consisting of aryl or heteroaryl groups in which a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, and thiols is substituted on one or more terminal pendant chains; Each X is either C or Si; Each R 4 These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each R 5 These are independently H, hydroxyl, and C1-C 12 Alkyl, C2~C 12Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, C2-C 12 Carboxylic acid esters, and C1-C 12 Selected from the group consisting of alkoxys; Each Z is independently CH2, O, or NR 4 and; Each f is an independent integer between 0 and 50; (Each n is an independent integer between 1 and 20.) A fluorene monomer having the structure described above may also be used.

[0181] Water-soluble monomers are given by formula (III): [ka] (In the formula, each G1 and G2 is independently a halo (F, Cl, Br, I); each Z is independently selected from the group consisting of O, CH2, and NH; each R1 is independently an alkyl (C1-C3); each R2 is independently H or an alkyl (C1-C6); each n is independently 1-6; and each m is independently 5-50.) The monomer may be a dihydrophenanthrene (DHP) monomer having the chemical structure described above. In some embodiments, G1 and G2 are Br, respectively; Z is O, respectively; R1 is CH3, respectively; R2 is H, respectively; n is independently 2 to 4, and m is independently 5 to 20. In some embodiments, n is 3, and m is independently 11 or 12. Labeled binding partners

[0182] Dyes can be conjugated to their binding partners by various linking chemistry between the binding partner and the reactive pair located in the label. Reactive pairs include, but are not limited to, maleimide / thiol, succimidyl ester (NHS ester) / amine, azide chemistry, carboxy / EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) / amine, amine / sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) / thiol, and amine / BMPH (N-[ ~ Possible examples include maleimidopropionic acid hydrazide (TFA) / thiol.

[0183] Fluorescent polymer dyes are characterized by ends on a conjugated polymer chain that may contain functional groups that provide conjugation. In some cases, such functionalities are referred to as terminal linkers. These terminal linkers form covalent bonds that can bind to binding partners such as proteins, peptides, affinity ligands, antibodies, antibody fragments, polynucleotides, or aptamers. In addition, orthogonal functional groups may be inserted along the conjugated polymer chain that can be used for either the conjugation or binding of acceptor-signaling chromophores in donor-acceptor polymeric tandem dyes.

[0184] Methods for performing conjugation are well known in the art. Commercial kits for performing conjugation are also readily available, for example, from Innova Biosciences (Cambridge, UK), Novus Biologicals (Littleton, Colo.), and Thermo Fisher Scientific (Waltham, Mass.). Drydown process

[0185] Drying reagent technology can be used to increase the stability of biomolecules. The drying process can be used, for example, to create a uniform reagent layer at the bottom of a tube. Dried reagents do not require refrigeration. Dried reagents may be stored at room temperature. Antibody panels may be supplied in single-use cocktails. Antibody panels may be supplied on various substrates, including tube or plate formats. Reagents can be used to dry conjugated antibodies and to stabilize them for storage at room temperature. Reagent formats can be adapted to combine different reagents to create antibody cocktails. The antibody cocktail may contain multiple antibody-dye conjugates, for example, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more antibody conjugates, or 1 to 20, 2 to 15, or 3 to 12 different antibody-dye conjugates.

[0186] Drying reagent techniques can enable the drying of a list of beads containing cocktails. Binding partner-dye conjugates, including antibody-dye and antibody-polymer dye conjugates, can be used in flow cytometry assays. Any suitable drying process can be employed to dry the reagents to create a uniform layer at the bottom of the tube or well.

[0187] Prior art “reagent buffer” formulations (RBs) containing sacrificial proteins, carbohydrate stabilizers, antimicrobial agents, and buffers have been previously developed to dry different monomeric conjugate dyes in a single tube without altering their functionality (affinity for antigens) and physical properties (such as brightness or fluorescence). Prior art RB formulations do not contain amphoteric surfactants or water-soluble monomers. Physical properties refer to the brightness of the conjugate and its leakage into other channels.

[0188] As illustrated in Figure 1A, desired flow cytometry results using a monomeric CD4-PE dye conjugate, including desired functionality, physical properties, and separation of CD4-PE monocyte and CD4-PE+ lymphocyte cell populations, are shown when dried using conventional dry-down techniques and reconstituted in a blood sample. Similarly, as illustrated in Figure 1B, desired flow cytometry results using a monomeric CD20 APC dye conjugate, including desired functionality, physical properties, and separation of CD20 APC+ cell populations, are shown when dried using prior art techniques and reconstituted in a blood sample.

[0189] Conventional drying techniques, such as RB, have been found to alter the integrity of the polymer structure and lead to aggregation of polymer dye conjugates when used to dry down two or more polymer dye conjugates in a tube. Aggregation increases nonspecific interactions and creates an artificial phenomenon of staining. Figures 2A and 2B illustrate that conventional techniques cannot separate cell populations stained with polymer dyes when two polymer dye conjugates are dried using conventional drying techniques, due to the aggregation of polymer dye conjugates.

[0190] Figure 2A shows undesirable flow cytometry results of CD20-SNv605 and CD4-SNv786 polymer dye antibody conjugates when mixed and dried using conventional drying techniques, without compensation. This indicates nonspecific interactions, as well as the inability to separate populations on the x and y axes.

[0191] Figure 2B shows undesirable flow cytometry results for CD20-SNv605 and CD4-SNv786 polymer dye antibody conjugates when mixed and dried using conventional drying techniques with compensation. The inability to prevent nonspecific interactions was attributed to the aggregation of the polymer dye antibody conjugates when the two polymer dyes were dried using conventional drying techniques.

[0192] Therefore, there is a need for a new buffer composition to keep the fluorescent dye conjugate stable and reduce aggregation during drying.

[0193] To overcome the technical challenges described above, first, the fluorescent dye conjugates must be stable in their liquid state when mixed. While mixtures of two fluorescent dye conjugates were found to result in increased nonspecific interactions when mixed together, even in their liquid state, leakage could be compensated for. To overcome the limitations in the liquid state, commercially available buffers, including BD Biosciences (Brilliant Stain Buffer, catalog number: 563794) and Thermo Fisher (Super Bright Complete Staining Buffer, catalog number: SB-4401-42), were tested. The use of Brilliant Stain Buffer or Super Bright Complete Staining Buffer during the drying of the polymer dye conjugates did not resolve the issues of low stability and aggregation.

[0194] Other techniques were adapted for drying the fluorescent dye conjugates, which included the separate film immobilization of the fluorescent dye conjugates on a substrate, as described in US2019 / 0242882, incorporated herein by reference. From these attempts, it was concluded that the fluorescent dye conjugates could be dried separately in different spots on the substrate film, but two fluorescent dye conjugates could not be mixed and dried. The disadvantages of drying polymer dye conjugates separately include the fact that drying the fluorescent dye conjugates on a cellulose film involves a deviation from current drying techniques, the inconvenience of maintaining the cellulose film inside the DURAClone® tube substrate, and the tolerance of leakage in other channels due to high compensation values. These experiments showed that more than one fluorescent dye conjugate could be dried only in the presence of a buffer that formed a barrier during the drying process and prevented the individualization of each fluorescent dye conjugate.

[0195] Fluorescent dye conjugates may be used in multicolor dry reagents (e.g., DURAClone® tubes, Beckman Coulter, Inc.). Multicolor dry reagents are cocktails of different fluorescent dye conjugates (such as CD4-FITC, CD8-PE, CD20-APC, CD3-PC5.5, etc.) that can be used directly to stain blood and analyze it in a flow cytometer. Compared to existing monomeric conjugate dyes, polymeric dye conjugates differ in their structure and complexity.

[0196] Conventional drying techniques are used to achieve multicolor dried reagent cocktails using different conjugates. Conventional dyes, such as FITC, PE, ECD, PC5, PC5.5, PC7, APC, AA700, AA750, PBE, and KrO, can be dried using conventional drying techniques.

[0197] The introduction of polymer dye conjugates has been found to render conventional drying techniques ineffective during the drying of multiple polymer dye antibody conjugates in a cocktail. These polymer dye conjugates tend to interact nonspecifically, leading to difficulties in population separation, which can present challenges in identifying desired cell populations in a given sample. composition

[0198] To overcome the limitations of conventional drying techniques, we have developed a novel “Dry Mix” (“DM”) buffer that can be used to dry one or more fluorescent (polymer or monomer) dye conjugates in a dye cocktail. The dye cocktail may contain one or more fluorescent polymer dye conjugates. The dye cocktail may contain one or more conventional nonpolymer fluorescent dye conjugates. The dye cocktail may contain a combination of one or more fluorescent polymer dye conjugates and one or more conventional nonpolymer fluorescent dye conjugates.

[0199] The fluorescent dye conjugate may be dried together with other conventional dyes in the cocktail. Several components were evaluated for use in DM buffer formulations according to the protocol of Example 1. The experimental results for these components evaluated during the development of the DM buffer are shown in Table 1.

[0200] The DM buffer formulation of the present invention is typically an aqueous solution comprising a water-soluble monomer, a protein stabilizer, a carbohydrate stabilizer, an amphoteric surfactant, and optionally a colorant, and optionally a preservative.

[0201] Stabilizers used in the solution may include protein stabilizers (e.g., bovine serum albumin, gelatin, casein) and carbohydrate stabilizers (e.g., trehalose, dextrose, sucrose). In some embodiments, the stabilizers may facilitate the bonding of dry components to the substrate, so that when the reaction vessel is opened, the stabilizers remain at the bottom of the tube and are not blown away or stuck to the cap. The DM buffer composition may contain, per test, 200-800 μg of water-soluble monomer; 2000-3000 μg of carbohydrate stabilizer; 8.4-72 μg of protein stabilizer; and 2-15 μg of amphoteric surfactant. Diluent

[0202] The diluent for DM buffer may be selected from the group consisting of water and isotonic buffers. The water may be deionized water (DI water). The isotonic buffer may be PBS (phosphate-buffered saline) buffer. Protein stabilizer

[0203] The term "protein stabilizer" refers to a protein that helps reduce nonspecific binding, for example, by reducing intercellular interactions or by preventing nonspecific binding between antibodies and non-target molecules. Protein stabilizers may include bovine serum albumin (BSA), various gelatins, and casein. Various protein stabilizers were evaluated in DM buffer compositions, as shown in Table 1. The protein stabilizer may be casein. The protein stabilizer may be gelatin. In some embodiments, the protein stabilizer may be BSA. In some embodiments, the protein stabilizer is not BSA. The drydown buffer may contain one or more protein stabilizers.

[0204] Gelatin (or gelatin) is a protein typically derived from collagen, which is taken from parts of animal bodies. It is brittle when dry and sticky when wet. After hydrolysis, it may also be referred to as hydrolyzed collagen, collagen hydrolysate, gelatin hydrolysate, hydrolyzed gelatin, and collagen peptide. Several types of gelatin are commercially available, including type A gelatin, type B gelatin, Prionex® highly purified type A gelatin, and gelatin-coldwater fish. Each of these was evaluated as a candidate DM buffer component. As reported in Table 1, type A gelatin in dry format showed a broader negative population spread compared to the corresponding liquid cocktail. A dry mix of type B gelatin was able to prevent nonspecific interactions, but stock preparation was difficult. The effective concentration of type B gelatin was in the range of 150 to 450 micrograms per test. Prionex® highly purified type A gelatin at a concentration equivalent to that of type B gelatin was effective in preventing nonspecific interactions, although its stability was somewhat reduced. The concentration of pre-formulated solutions of Prionex® highly purified type A gelatin was quantified, and the effective concentration of the dry mix was in the range of 67 micrograms to 135 micrograms per test. Gelatin-coldwater fish was equivalent to type B gelatin in terms of performance. The effective concentration of gelatin-coldwater fish was also in the range of 150 micrograms to 450 micrograms per test.

[0205] Casein is a family of phosphoproteins (alpha-S1, alpha-S2, beta, and kappa). These proteins are found in mammalian milk and constitute approximately 80% of the proteins found in cow's milk. One common form is sodium caseinate. Casein contains a large number of proline amino acid residues, which hinders the formation of common secondary structural motifs of proteins. Casein does not contain disulfide crosslinks and therefore has a relatively small tertiary structure. Casein 10× blocking buffer (in the range of approximately 14 mg / mL to approximately 18 mg / mL) was used as one of the components of the dry mix. Casein at concentrations of 5× (2-fold dilution) and 2.5× (4-fold dilution) effectively prevented the interaction of two polymer dye antibody conjugates by adding more than 1 polymer conjugate. Carbohydrate stabilizers

[0206] "Carbohydrate stabilizers" are carbohydrate molecules used to help increase the stability of dye-antibody conjugates in solution during drying onto a substrate and / or reconstitution with a biological sample.

[0207] Candidate polysaccharides were evaluated. Carrageenan is a sulfated anionic polysaccharide. Preparation of carrageenan stocks was found to be difficult. Addition of carrageenan to DM buffer compositions increased nonspecific binding in granulocytes. Sodium alginate is the sodium salt of alginate, and it is a linear polysaccharide containing a homopolymer block of (1→4)-linked beta-D-mannuronic acid and alpha-L-guluronic acid residues. Preparation of stocks was difficult, and the overall spread of granulocytes, monocytes, and lymphocytes was higher when sodium alginate was added to DM buffer compositions than when it was added to DM buffer compositions.

[0208] The carbohydrate stabilizer may be a disaccharide. The disaccharide may be trehalose, sucrose, maltose, cellobiose, melibiose, or their hydrates or salts. In some embodiments, the disaccharide is trehalose or its hydrate. Trehalose is a non-reducing disaccharide having a 1,1-glycosidic bond between two alpha-glucose units. Trehalose may be trehalose dihydrate. The disaccharide stabilizer may be present in an aqueous buffer solution in the range of about 2000 to 3000 micrograms per test, or about 2200 to 2800 micrograms per test, or about 2500 micrograms per test, or about 400 mg / mL in the stock preparation.

[0209] Trehalose derivatives, including trehalose decanoate, trehalose tetradecanoate, and trehalose hexadecanoate, were also evaluated. For all three compounds at doses equivalent to trehalose dihydrate, the compounds completely dissolved all cells and, by reducing the dose, would not cause the tubes to dry out. surfactants

[0210] Various types of surfactants were explored to prevent nonspecific interactions in DM buffer formulations. (See Table 1.)

[0211] Anionic surfactants, including alkyl sulfates and alkyl sulfonates having at least 10-carbon alkyl groups, were evaluated. (Sarcosyl, CH3(CH2)) 10N-lauryl sarcosinate sodium salt, also known as CO-N(CH3)-CH2COONa, is an anionic surfactant. N-lauryl sarcosinate sodium salt was found to prevent nonspecific binding in monocytes and granulocytes, but it could not prevent interpolymer interactions. Lignosulfonic acid ("LSA"), 3-(2-hydroxy-3-methoxyphenyl)-2-[2-methoxy-4-(3-sulfopropyl)phenoxy]propane-1-sulfonic acid, is an anionic surfactant. Negative background in the SN v605 and SN v786 populations was increased when LSA was added compared to DM buffer.

[0212] Nonionic surfactants were evaluated for their potential use in DM buffer formulations. Polysorbate 80 was tested as a nonionic surfactant. The term "ester-linked nonionic surfactant" refers to nonionic organic compounds containing hydrophobic and hydrophilic groups that are linked or contain ester linkages. Examples of ester-linked nonionic surfactants include polyoxyethylene glycol sorbitan ester (Polysorbate, TWEEN®) and sorbitan alkyl ester (Span). Nonspecific monocyte pull-out and population spread by SN v605 conjugate were observed with 0.015% and 0.075% polysorbate 80 in the dry mix.

[0213] The potential use of amphoteric surfactants in DM buffer formulations was evaluated. The amphoteric surfactants evaluated were N,N-dimethyl-N-dodecylglycine or N-(alkylC) 10 ~C 16 Empigen® BB (Huntsman Corporation), also known as )-N,N-dimethylglycine betaine; 3-(N,N-dimethyltetradecylammonio)propanesulfonate, myristyl sulfobetaine, CH3(CH2) 13 N + (CH3)2CH2CH2CH2SO3 -) Also known as 3-(N,N-dimethylmyristylammonium propane sulfonate (DMMA); and Zwittergent® 3-16 detergent (Merck 3-[N,N-dimethyl(3-palmitoylaminopropyl) ammoniao]-propanesulfonate (DMPA), also known as KGaA (Darmstadt, Germany), was evaluated in a DM buffer composition. Empigen® BB prevented nonspecific binding in monocytes and granulocytes, but could not prevent polymer interactions. DMPA precipitated at room temperature but was functionally equivalent to Empigen® BB. The effective concentration range for DMPA was 0.002% to 0.006%. DMMA was functionally equivalent to Empigen® BB and was therefore considered for further testing. The effective concentration range for DMMA was found to be 0.002% to 0.037%, or 0.004% to 0.018%.

[0214] In some embodiments, the amphoteric surfactant has the chemical formula (XV): [ka] (In the formula, Y = CO2- or SO3-, W = H or OH, and Z = CH3 or NHC(O)R(In the formula, R = C 1~15 (It is alkyl; independently, p=0 or 1; q=0 to 21) It has the structure described above. In some embodiments, W=H, Z=CH3, and q=11-15. In some embodiments, the amphoteric surfactant is DMMA, DMPA, N-(alkylC 10 ~C 16 )-N,N-dimethylglycine betaine, lauryl hydroxysultaine, lauryl sultaine, myristyl betaine, cetyl betaine, decyl betaine, lauryl betaine, behenyl betaine, cocamidopropyl betaine, or cocamidopropyl hydroxysultaine. In some embodiments, the amphoteric surfactant may be DMMA, DMPA, N-(alkylC 10 ~C16 It may also be )-N,N-dimethylglycine betaine. Antioxidants

[0215] Antioxidant compounds were evaluated as candidate components of DM buffer formulations. Antioxidants may contain one or more, two or more, or three or more carboxylic acid or carboxylate moieties and C1-C8 or C2-C6 aliphatic moieties. The aliphatic moieties may be linear, branched, or cycloalkyl or alkenyl moieties. For example, L-ascorbic acid and citrate were evaluated as antioxidants. Nonspecific monocyte pullout with SN v605 conjugates was low with L-ascorbic acid at concentrations of 0.2 mM and 0.6 mM. 0.6 mM L-ascorbic acid had a low spread of the SN v786-positive population in the V610 channel. Citrate reduced granulocyte degranulation, and nonspecific monocyte pullout with SN v605 conjugates was low at all concentrations. However, citric acid did not have any additional effects, including population spread. monomer

[0216] To prevent nonspecific interactions between fluorescent dye conjugates, particularly between fluorescent polymer dye conjugates, various water-soluble monomer species were investigated. The monomers used were selected from synthetic monomers used in the preparation of conjugated polymer dyes. The polymer dye may be a water-soluble conjugated polymer containing a fluorescent polymer having monomer A subunit and monomer B subunit. For example, monomer A or monomer B may contain DHP. The polymer dyes and monomers are described in US2020 / 0190253, which is incorporated herein by reference in its entirety. In some embodiments, conjugated polymer dyes containing a DHP backbone may be used. In some embodiments, monomer A having a 9,10-dihydrophenanthrene DHP structure was investigated for use in DM buffer composition formulations and tested according to Example 1.

[0217] The water-soluble monomers may be monomer units containing aryl or heteroaryl moieties, each having a water-soluble moiety bonded to them as needed. The water-soluble moieties may consist of one or more PEG moieties. The water-soluble monomers may be suitable for use in the preparation of at least one of several fluorescent polymer dyes having monomer A subunits, monomer B subunits, or combinations of monomer A and monomer B subunits. The water-soluble monomers may be DHP-based water-soluble monomers. The water-soluble monomers may be fluorene-based water-soluble monomers.

[0218] Water-soluble monomers are given by formula (I): [ka] (In the formula, Each of G1 and G2 independently contains halogens, alkyls, PEGs, hydrogen, alkynes, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, and optionally substituted tetrahydropenes. Re Fluorine (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl groups selected from the group consisting of aryl or heteroaryl groups in which a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, and thiols is substituted on one or more terminal pendant chains; Each R 2These are independently: water-soluble moieties, alkenes, alkynes, cycloalkyls, haloalkyls, (hetero)aryloxys, (hetero)arylaminos, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, sulfonamides, sulfinamides, phosphoamides, phosphineamides. [ka] Selected from the group consisting of; Each R 3 This is the water-soluble portion; Each R 4 These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each R 5 These are independently H, hydroxyl, and C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, C2-C 12 Carboxylic acid esters, and C1-C 12 Selected from the group consisting of alkoxys; Each Q is independent, combined, NR 4 , or -CH2; Each Z is independently CH2, O, or NR 4 and; Each f is an independent integer between 0 and 50; (Each n is an independent integer between 1 and 20.) It may also be a DHP-based monomer having the chemical structure described above.

[0219] In some embodiments, each of G1 and G2 is independently selected from the group consisting of halo(F, Cl, Br, I), C1-C6 alkyl, and PEG.

[0220] Water-soluble monomers are given by formula (II): [ka] (In the formula, Each of G1 and G2 independently contains halogens, alkyls, PEGs, hydrogen, alkynes, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, and optionally substituted tetrahydropenes. Re Fluorine (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl groups selected from the group consisting of aryl or heteroaryl groups in which a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, and thiols is substituted on one or more terminal pendant chains; Each X is either C or Si; Each R 4These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each R5 independently consists of H, hydroxyl, and C1-C. 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, C2-C 12 Carboxylic acid esters, and C1-C 12 Selected from the group consisting of alkoxys; Each Z is independently CH2, O, or NR 4 and; Each f is an independent integer between 0 and 50; (Each n is an independent integer between 1 and 20.) A fluorene monomer having the structure described above may also be used.

[0221] In some embodiments, each of G1 and G2 is independently selected from the group consisting of halo(F, Cl, Br, I), C1-C6 alkyl, and PEG.

[0222] Water-soluble monomers are given by formula (III): [ka] (In the formula, each G1 and G2 is independently a halo (F, Cl, Br, I); each Z is independently selected from the group consisting of O, CH2, and NH; each R1 is independently an alkyl (C1-C3); each R2 is independently H or an alkyl (C1-C6); each n is independently 1-6; and each m is independently 5-50.) The DHP monomer may have the chemical structure described above. In some embodiments, G1 and R2 are Br, respectively; Z is O, respectively; R1 is CH3, respectively; R2 is H, respectively; n is independently 2 to 4, and m is independently 5 to 20. In some embodiments, n is 3, and m is 11.

[0223] The specific chemical structures of monomer A and monomer B are shown in Figure 4. Monomer A, shown in Figure 4, was found to prevent nonspecific interactions between the two SN conjugates. The effective concentration was found to be in the range of 200 to 800 micrograms (50 uL) per test. However, monomer B was unable to prevent nonspecific interactions between the two SN conjugates and also showed some nonspecific binding in negative cells.

[0224] While not bound by theory, it is assumed that monomer A interacts randomly with the polymer backbone, thus preventing interaction between the two polymer conjugates. In contrast, protein stabilizers such as gelatin or casein, due to their sticky properties, may mask these pigment conjugates, preventing them from coming into close proximity. Preservatives

[0225] The aqueous DM buffer composition may contain any suitable preservative. The preservative may be an antioxidant, biocide, or antimicrobial agent. The preservative may be an inorganic salt. The preservative may be sodium azide, 2-chloroacetamide, 2-methylisothiazolinone, salicylic acid, ProClin®, Kathon® CG, 5-chloro-2-methyl-4-isothiazolin-3-one, or 2-methyl-4-isothiazolin-3-one. Coloring agents

[0226] The aqueous DM buffer composition may contain a coloring agent. The coloring agent may be an FD&C coloring agent. For example, the coloring agent may be Allura Red (FD&C Red No. 40, 6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfonatophenyl)azo]-2-naphthalenesulfonate disodium). polymer

[0227] Fluorescent polymer dyes in different solvents were also investigated for the preparation of DM buffer compositions. However, they showed a broader negative population spread compared to liquid cocktails.

[0228] From the different reagents tested, we first formulated a DM buffer containing trehalose dihydrate, monomer A, type B gelatin, and Empigen® BB amphoteric surfactant.

[0229] Early technical solutions were developed in the form of DM buffer for drying fluorescent dye conjugates to maintain the integrity of the dye structure and reduce aggregation. In one embodiment, a DM buffer formulation having gelatin and monomer A reduced the aggregation problem during the drying process. Using the optimal protein stabilizer concentration in conventional drydown techniques, as well as optimal concentrations of monomer and gelatin, DM helps maintain the integrity of the fluorescent dye conjugate and solves the problems associated with aggregation during the drying of the fluorescent dye conjugate.

[0230] DM buffer contains a water-soluble monomer; a protein stabilizer; a carbohydrate stabilizer; and an amphoteric surfactant. DM buffer may also contain a DHP-based monomer and a water-soluble monomer containing one or more, or two or more, poly(ethylene glycol) moieties. The water-soluble monomer may have the structure described in formula (I). The protein stabilizer may be an albumin protein. The protein stabilizer may be a gelatin protein. The protein stabilizer may also contain a casein protein.

[0231] Subsequently, the DM buffer was further improved to prevent nonspecific interactions between SuperNova® conjugates and nonspecific monocyte pull-out, and this was named "DM2" (DM 2). DM2 contains trehalose dihydrate, monomer A, Prionex® type A gelatin, and DMMA surfactant.

[0232] To improve stability, DM2 was further optimized by replacing Prionex® type A gelatin with casein 10x blocking buffer. This buffer was named "DM2+S" (stabilizer). The additives present in the final formulation are provided in Table 2. The pH of the DM2+S buffer was found to be 7–7.4. Dry tubes prepared using DM2+S resulted in the prevention of nonspecific interactions of SuperNova® conjugates, as well as the prevention of nonspecific pull-out that does not impair the performance of the conjugate suspended in the cocktail (brightness and population mobilization), and the achievement of higher stability of the dry product (real-time stability for 6 months was established). A bulk formulation of SuperNova® conjugates was used in the preparation of the dry tubes. [Examples]

[0233] (Example 1A) Test Procedure The general processes and procedures used in this embodiment are shown below.

[0234] Drying: As used herein, the term drying refers to vacuum drying at a specific vacuum pressure for a certain number of hours.

[0235] Mixing two conjugates: Mixing two conjugates refers to mixing two conjugates in a 5 ml tube.

[0236] Liquid Test: The term liquid test refers to mixing conjugates together in a test tube (a liquid cocktail), and then using the mixed conjugates to stain cells.

[0237] Drying test: The term drying test refers to mixing conjugates together in a test tube, drying them using vacuum drying, and then staining cells using the dried conjugates.

[0238] When used herein, "test" refers to the following protocol: 1. Prepare the required x number of tubes (the number of tubes depends on the performance being tested). 2. Add the calculated volume of conjugated antibody (required dose) to each tube, or to the dry test tube to be used for the next step. 3. Add 100 μL of whole blood to each tube. Gently vortex the tubes for 6-8 seconds. 4. Protect from light and incubate at room temperature (18-27°C) for 15-20 minutes. 5. Add 2 ml of VersaLyse + IOTest3 Fixative mixture (2 ml Versalyse Ref. A09777 + 50 μl IOTest3 fixative 10× Ref. A07800). Immediately vortex to ensure proper mixing, protect from light, and incubate at room temperature (18-27°C) for 20 minutes. 6. Centrifuge at room temperature for 5 minutes using 300g. 7. Remove the supernatant by suction. 8. Resuspend the cell pellet in 3 mL of 1×PBS. 9. Centrifuge at room temperature for 5 minutes at 300g. 10. Remove the supernatant by aspirate and resuspend the cell pellet in 0.3 ml of 1x PBS 1X or 1x PBS + 0.1% formaldehyde (1 ml of 1x PBS + 12.5 μl of 10x IOTest3 fixative).

[0239] Compensation: In cytometry, compensation is the mathematical correction of signal overlap between channels in the emission spectra of different fluorescent dyes. Therefore, this correction factor was used to eliminate signal bleeding into other undesirable channels. Manual compensation was performed to evaluate the performance of the conjugate. (Example 1B) Flow cytometry protocol

[0240] The following protocol was used for sample processing.

[0241] Samples for flow cytometry acquisition were prepared and processed using a staining, dissolution, and washing protocol. The samples were processed and acquired using a DxFLEX / CytoFLEX flow cytometer (Beckman Coulter, Inc.) to analyze the performance of various formulations. 1. Add 100 μL of K2EDTA anticoagulant blood to a dry staining tube or a tube containing a liquid antibody cocktail and vortex for 6-8 seconds. Incubate in the dark at room temperature for 15-20 minutes. 2. Add 2 mL of Versa Fix (1 mL of Versalyse with 25 μL of IOTest 3 fixative added) dissolving reagent solution (Beckman Coulter, Inc.) and vortex briefly to ensure proper mixing. Incubate in the dark at room temperature for 20 minutes. 3. After incubation with lysis & fix buffer (Versa Fix), centrifuge at 300g at room temperature for 5 minutes to pelletize the WBCs. Aspirate and discard the supernatant, and break up the pellet by vortexing. 4. Add 3 mL of 1×PBS and centrifuge at 300 g for 5 minutes at room temperature. 5. Aspirate and discard the supernatant, resuspend the pellet in 300 μL of 1×PBS, gently vortex the tube, and retrieve the tube in a DxFLEX flow cytometer at the recommended settings.

[0242] Device settings:

[0243] Flow cytometer instrument settings were performed using CytoFLEX or DxFLEX Daily QC fluorescence spheres (Beckman Coulter, Inc.). Routine quality control management of the instrument was performed using QC beads to identify target gain values. Recommended gain settings identified by the QC protocol were used for obtaining test tubes in the flow cytometer.

[0244] Compensation was established for 12 colors using a general-purpose compensation kit (Beckman Coulter, Inc.) and a single SN polymer dye conjugate in liquid and / or dry format. (Example 2) Selection of components for the dry mix

[0245] Aqueous DM buffer formulations were developed for drying fluorescent dye conjugates. Starting with conventional drying techniques, several reagents (Table 1) were tested for prevention of nonspecific interactions and nonspecific binding of polymer dye antibody conjugates. Water or PBS was used to prepare stock solutions of these reagents. The tested reagents and their results are shown in Table 1. Reagents indicated in bold were further evaluated.

[0246] [Table 1-1] [Table 1-2] [Table 1-3]

[0247] The following DM candidate reagents were used in the dry-down of polymer dye conjugate tubes: BSA (bovine serum albumin), PEG550 (poly(ethylene glycol) methyl ether, average M n 550), BSA-ox (BSA oxide), Empigen® BB detergent (N-(alkylC 10 ~C 16)-N,N-dimethylglycine betaine), N-lauryl sarcosinate sodium salt, monomer A, monomer B, polymer in different solvents, type A gelatin, type B gelatin, lignosulfonic acid, carrageenan, sodium alginate, casein blocking buffer 10x, Prionex® highly purified type A gelatin, gelatin-coldwater fish, L-ascorbic acid, citric acid, polysorbate 80, acrylamide, trehalose decanoate, trehalose-tetradecanoate, trehalose-hexadecanoate, 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonium]-propanesulfonate (DMPA), and 3-(N,N-dimethylmyristylammonium)propanesulfonate (DMMA). The chemical structures of monomer A and monomer B are shown in Figure 4. Monomer A may also be 3,3'-((2,7-dibromo-9,10-dihydrophenanthrene-9,10-diyl)bis(oxy))bis(N-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)propane-1-sulfonamide). Drydown tubes were tested according to the protocol of Example 1. The test results are shown in Table 1. Empigen®, monomer A, type B gelatin, casein blocking buffer 10×, Prionex® highly purified type A gelatin, gelatin cold water fish, DMPA, and DMMA were selected for further testing.

[0248] From the different reagents tested, trehalose dihydrate, monomer A, type B gelatin, and Empigen BB® detergent (N,N-dimethyl-N-dodecylglycine betaine; N-(alkylC)) were selected. 10 ~C 16 We developed the first DM (DM) buffer formulation containing )-N,N-dimethylglycine betaine.

[0249] Subsequently, an improved DM buffer formulation was developed to prevent nonspecific interactions between SuperNova® conjugates and nonspecific monocyte pullout, and this was named DM2 (DM 2). DM2 contains trehalose dihydrate, monomer A, Prionex® gelatin, and DMMA. However, it was later found that dried tubes prepared using DM2 had stability issues. Finally, DM2 was further optimized by replacing Prionex with casein 10x blocking buffer. This buffer was named DM2+S (stabilizer).

[0250] The additives present in the final DM buffer formulation are shown in Table 2. The pH of the DM2+S buffer was found to be 7–7.4. Dry tubes prepared using DM buffer DM2+S resulted in the prevention of nonspecific interactions of SuperNova® conjugates, as well as the prevention of nonspecific pull-out that did not impair the performance of the conjugate suspended in the cocktail (brightness and population mobilization), and the achievement of higher stability of the dry product (real-time stability of 6 months has been established to date). Bulk formulations of SuperNova® conjugates were used in the preparation of the dry tubes.

[0251] [Table 2]

[0252] [Table 3]

[0253] Table 3 shows preferred amounts of components of the DM2+S DM buffer per test. In some embodiments, the amounts are per 28.48 microliters of buffer without the dye conjugate. In some embodiments, the amounts are per 50 microliters of buffer with the dye conjugate.

[0254] In some embodiments, the DM buffer may contain trehalose, monomer A, DMMA, and casein. In some embodiments, the dry-down buffer may contain appropriate concentrations to obtain 2000–3000 micrograms of trehalose per test; 200–800 micrograms of monomer A per test; 8.4–72 micrograms of casein per test; and 2–15 micrograms of DMMA per test. In some embodiments, the DM buffer is an aqueous buffer that may contain, without dye conjugate, 70–105 mg / mL or 80–100 mg / mL of trehalose dihydrate; 7–28 mg / mL or 10–20 mg / mL of monomer A; 0.07–0.53 mg / mL or 0.2–0.4 mg / mL of DMMA; and 0.3–2.5 mg / mL or 0.5–0.8 mg / mL of casein. The DM buffer may be prepared, for example, from stock concentrations of a carbohydrate stabilizer, a water-soluble monomer, an amphoteric surfactant, and a protein stabilizer in water or PBS buffer. For example, the stock concentration may contain about 400 mg / ml of trehalose dihydrate, about 40 mg / ml of monomer A, about 1.5 mg / ml of DMMA, and / or about 2 to 20 mg / mL of casein. (Example 3) Design of experiments for DM buffer and their results

[0255] To achieve optimal results, the concentrations of each component of the DM buffer (excluding trehalose dihydrate, sodium azide, and Allura Red) were dose-determined and optimized.

[0256] 1. Trehalose dihydrate:

[0257] Trehalose dihydrate was one of the components of the prior art. In the absence of this additive, it was difficult to dry down the components of the conjugate and other buffers.

[0258] 2. Dosage setting for casein 10 × blocking buffer:

[0259] 2.1: Experiment: 1

[0260] Objective: To find the optimal concentration of casein in a liquid format to prevent nonspecific binding.

[0261] Methods: In this experiment, pre-existing casein 10× blocking buffer solutions of different concentrations (5×, 2.5×, 1.25×, 0.625×, and 0.31×) were tested according to the protocol of Example 1.

[0262] Results and observations:

[0263] Figure 5 shows the casein dose settings and comparative CD20-SNv605 flow plots using negative control (no casein), 0.31×, 0.625×, 1.25×, 2.25×, and 5× casein. Casein at 1.25×, 2.5×, and 5× concentrations helps reduce nonspecific monocyte pullout in the V610 channel. In addition, the % of CD20+ cell recruitment was the same across all formulations compared to CD20 alone (no casein). The % of HLADR+ cell recruitment was also found to be consistent across all groups (data not shown). Therefore, casein alone was further evaluated at different concentrations in dry tubes. The casein concentration was further optimized to find the optimal concentration for dry tubes.

[0264] 2.2: Experiment: 2

[0265] Objective: To find the optimal casein concentration for preventing nonspecific binding in a dry format.

[0266] Methods: In this experiment, 12 different antibody conjugates were dried with other in-situ additives using pre-made solutions of casein 10× blocking buffer at different concentrations (4×, 2.5×, and 1×). 2.5× is the standard concentration. Therefore, the 2.5× concentration was compared to the 1× and 4× concentrations as a reference. Here, two different lots (L1 and L2) of casein 10× blocking buffer were tested.

[0267] Results and observations:

[0268] The overlap of double fluorescence plots of DM buffers at different concentrations (4×, 2.5×, and 1×) (V610 vs V780, PB450 vs V610, and PB450 vs V780) was compared (data not shown). The overlap of double fluorescence plots for SuperNova® (blue-violet channel) vs conventional channel and classical vs classical conjugate channel across different concentrations was also compared (data not shown). All of these comparisons confirmed that there was no significant variation across different concentrations of casein tested. Tables 4 and 5 show comparisons of delta recruitment absolute % and MdFI across different concentrations of casein for all specificities.

[0269] [Table 4]

[0270] [Table 5-1] [Table 5-2]

[0271] This experiment demonstrates that the superimposed plots, MdFI, and delta mobilization absolute % do not show significant variation across different concentrations (1×, 2.5×, 4×) of casein 10× blocking buffer. Delta mobilization absolute % was found to be <5% across lot-to-lot and different concentrations tested, due to minimal / no effect of different concentrations of casein 10× blocking buffer. Therefore, 1× to 4× casein blocking buffer can be used to dry SuperNova® conjugates together with other conventional dyes.

[0272] Dosage setting for monomer A: 3.1: Experiment: 1

[0273] Objective: To dose-determine the monomer A concentration for the drydown of a two-polymer dye antibody conjugate.

[0274] Methods: Monomer concentrations of 50 μg, 100 μg, 200 μg, 400 μg, and 800 μg per test were tested together with other additives kept constant in formulations consisting of CD45-FITC, CD20-SN-v605, and CD3-SN-v786 in dry format.

[0275] Results and Observations: Two-dimensional flow cytometer fluorescence plots of CD20-SN v605 and CD3-SN v786 with monomer A at various doses were analyzed (data not shown). From the different doses of monomer, 200 μg and 400 μg of monomer were sufficient to prevent interaction of polymer dye conjugates. 3.2: Experiment: 2

[0276] Objective: To dose-determine the monomer A concentration for the drydown of a three-polymer dye antibody conjugate.

[0277] Methods: Monomer A concentrations of 200 micrograms per test and 400 micrograms per test were tested together with other additives kept constant in formulations consisting of CD45-FITC, CD19-SN v428, CD20-SN v605, and CD3-SN v786 in dry format.

[0278] Results and Observations: Figure 6 shows bifluorescence plots of the dry formulations for three colors tested against monomer concentrations of 200 μg (top row) and 400 μg (bottom row) per test. With monomer A ranging from 200 μg to 400 μg, the tube containing 400 μg showed better results in terms of population spread leaking from one channel to the other (as indicated by arrows and circles in Figure 6) and was sufficient to prevent interaction of polymer dye antibody conjugates.

[0279] 4. DMMA dosage setting:

[0280] To evaluate the role of DMMA (3-(N,N-dimethylmyristylammonio)propanesulfonate) in preventing nonspecific interactions. 4.1: Experiment 1: Dose setting of the amphoteric surfactant DMMA in the presence of two SuperNova® polymer dye antibody conjugates

[0281] Objective: To find the optimal concentration of DMMA to prevent nonspecific interactions between two SuperNova® conjugates.

[0282] Methods: In this experiment, different concentrations of DMMA (Sigma, part number T7763-5G) were tested by replacing Empigen® BB with DMMA in DM version 1. DMMA is a water-soluble amphoteric surfactant. DMMA was tested to find a better alternative to Empigen® in reducing nonspecific interactions with easy manufacturability. The different concentrations of DMMA, along with details of the tested groups, are provided in Table 6.

[0283] [Table 6]

[0284] Here, different concentrations of DMMA, namely 0.03%, 0.015%, 0.0075%, and 0.00375%, were tested in liquid and dry tubes and compared to DM buffer. The DMMA concentrations were selected based on the Empigen BB® concentration (0.15%), one of the components of DM buffer. Appropriate controls were used in the experiments. As described in Table 6, 0.15% DMMA was added to the final formulation to achieve the individual concentrations of DMMA. The effects of DMMA were tested using two Supernova® conjugates, CD20-SN-v605 and HLADR-SN-v786. Samples were processed using the protocol according to Example 1B. The experiments were performed on two donors.

[0285] Results and Observations: Scattering flow plots for CD20-SN-v605, HLADR-SN-v786, and the double-positive population were compared for all tested dry DMMA formulations (data not shown). DMMA is a surfactant and can induce cell death at higher concentrations. Overlaid scatter plots of each concentration of DM versus tested DMMA were analyzed. 0.03% DMMA induced cell death and was not evaluated for other parameters. 0.0075% and 0.00375% DMMA showed comparable scattering to DM. Similarly, the mobilization percentages of CD20+ and HLADR+ were found to be similar in 0.0075% and 0.00375% DMMA compared to the individual liquid singles. In addition, 0.0075% and 0.00375% DMMA showed less spread of the 786+ population in the 610 channel compared to DM.

[0286] This experiment demonstrates that 0.0075% and 0.00375% DMMA exhibit comparable scattering properties and mobilization percentages of CD20+ and HLADR+ populations to DM. The DMMA concentration was further optimized within the range of 0.0075% to 0.00375% to obtain the optimal concentration. 4.2: Experiment 2: Dose optimization of DMMA in the presence of two Supernova(trademark) conjugates

[0287] Objective: To find the optimal concentration of DMMA to prevent nonspecific interactions between two SuperNova® conjugates.

[0288] Methods: In this experiment, the concentration of DMMA was further optimized from 0.03% to 0.004% to find the optimal concentration. Table 7 provides the different concentrations of DMMA, along with details of the groups tested.

[0289] [Table 7]

[0290] Here, different concentrations of DMMA, namely 0.03%, 0.021%, 0.018%, 0.008%, and 0.004%, were tested in dry tubes and compared to DM. Appropriate controls were used in the experiments. As described in Table 7, 0.15% DMMA was added to the final formulation to achieve the individual concentrations of DMMA. The effects of DMMA were tested using two Supernova® conjugates, CD20-SN-v605 and HLADR-SN-v786. Samples were processed using the protocol described in Example 1B. The experiments were performed on two donors.

[0291] Figures 7A–D show scattered flow plots of CD20-SN-v605, HLADR-SN-v786, and the bipositive population for all tested dry DMMA formulations. Figure 7A shows scatter plots of all concentrations of DMMA tested with unstained and DM. 0.004% and 0.008% DMMA show less nonspecific neutrophil pullout (indicated by arrows) compared to other DMMA concentrations. Similarly, 0.004%, 0.008%, and 0.018% DMMA show less nonspecific monocyte pullout in the V610 channel (indicated by arrows, Figure 7B) compared to DM and other DMMA concentrations. The mobilization percentages of CD20+ and HLADR+ were found to be similar across all DMMA concentrations compared to individual liquid singles. Similarly, the mobilization percentages of the bipositive population across all DMMA concentrations were found to be similar compared to DM. In addition, 0.004%, 0.008%, and 0.018% DMMA showed similar spread of 786+ events in the V610 channel compared to DM. This experiment demonstrates that 0.004%, 0.008%, and 0.018% DMMA exhibit better performance compared to DM. Therefore, these DMMA concentrations were further evaluated in combination with other selected additives.

[0292] 5. Evaluate the performance of selected additives at different concentrations. 5.1: Experiment 1: Performance evaluation of selected additive combinations

[0293] Objective: To evaluate the performance of selected additive combinations in preventing nonspecific binding and nonspecific interactions between SN conjugates.

[0294] Methods: This experiment evaluated selected additive combinations, e.g., DMMA + casein, DMMA + Prionex. Different concentrations of additives, along with details of the tested groups, are provided in Table 8.

[0295] [Table 8]

[0296] Here, different concentrations of DMMA, namely 0.018% and 0.008%, were tested in dry tubes with different dilutions of casein and Prionex in combination and compared to DM version 1. Appropriate controls were used in the experiments. The effects of DMMA were tested using two Supernova® conjugates, CD20-SN-v605 and HLADR-SN-v786. Samples were processed using the protocol of Example 1B. The experiments were performed on four donors.

[0297] Results and Observations: Flow plots of scattering for CD20-SN-v605, HLADR-SN-v786, and the bipositive population were compared for additive combinations with 0.008% DMMA. Figure 8A shows scatter plots of additive combinations with 0.008% DMMA compared to DM. Scattering appears similar for each concentration, except for DM. DM shows nonspecific granulocyte and monocyte pullout, as indicated by the arrows. Similarly, DM shows higher nonspecific granulocyte and monocyte pullout in the V610 channel compared to other combinations with 0.008% DMMA (indicated by the arrows, Figure 8B). The mobilization % of CD20+ and HLADR+ was found to be similar in all combinations with 0.008% DMMA compared to the individual liquids alone (Figures 8B, 8C). Similarly, the mobilization % of the bipositive population in all combinations with 0.008% DMMA was found to be similar compared to DM.

[0298] Similarly, the scatter flow plots for CD20-SN-v605, HLADR-SN-v786, and the bipositive population were compared for each additive combination with DMMA 0.018% (data not shown). Scatter plots for all additive combinations with DMMA 0.018% were compared with DM. Scattering appears similar for all combinations. DM and DMMA 0.018 + casein 1× show higher nonspecific monocyte pullout in the V610 channel compared to other combinations with 0.018% DMMA. The mobilization % of CD20+ and HLADR+ was found to be similar for each combination with 0.018% DMMA compared to the individual liquid singles. Similarly, the mobilization % of the bipositive population in all combinations with 0.018% DMMA was found to be similar compared to DM. However, the combinations of DMMA 0.018% + casein 2.5× and DMMA 0.018% with Prionex showed less spread of 786+ events in V610 compared to DM.

[0299] Flow plots of scattering for CD20-605, HLADR-786, and the double-positive population were compared for single additives of different concentrations, such as casein and Prionex (in DM version 1, gelatin was replaced with casein and Prionex) (data not shown). Scattering appears to be similar for each combination. DM alone and all singles show higher nonspecific monocyte pullout in the V610 channel compared to combinations of additives and DMMA. The mobilization percentages for CD20+ and HLADR+ were found to be similar for all singles compared to individual liquid singles. Similarly, the mobilization percentages for the double-positive population in each single were found to be similar compared to DM. Singles and Prionex dilutions show less spread of 786+ events in the V610 channel compared to DM.

[0300] Overall, among all combinations, DMMA0.008% + Prionex 2 dil and 3 dil, DMMA0.018% + Prionex 3 dil, and DMMA0.018% + Casein 2.5× provided good performance in terms of reducing nonspecific monocyte pullout in the V610 channel and reducing nonspecific interactions between SN conjugates. This experiment demonstrates that combinations including DMMA0.008% + Prionex 2 dil and 3 dil, DMMA0.018% + Prionex 3 dil, and DMMA0.018% + Casein 2.5× provided good performance in terms of reducing nonspecific binding and nonspecific interactions when tested with two SN conjugates.

[0301] These experiments showed that DM buffers containing DMMA 0.008% + Prionex 2 dilution and DMMA 0.018% + casein 2.5× dilution performed well in reducing nonspecific interactions between SN conjugates and also nonspecific pull-out. However, during development, DMMA 0.008% + Prionex 2 dilution (named DM2 formulation) was found to exhibit stability issues and was therefore not further evaluated. Subsequent experiments using DMMA 0.018% + casein 2.5× (named DM2+S) showed that this formulation has good stability: real-time stability of 6 months in dry tubes established to date (testing in progress). 6: Functional testing of additives, specifically additive minus 1 (AMO) in the final formulation (DM2+S).

[0302] Objective: To understand the effect of each additive present in the DM2+ stabilizer buffer (final formulation) on scattering and nonspecific interactions, we analyzed one of each of the buffer's negative components.

[0303] Methods: The experiment was conducted using the dried formulations described below, employing a four-color panel (CD45-AA750, CD56-SNv428, CD20-SNv605, CD4-SNv786). DM2+S served as a control group in this experiment for comparison with the other groups. 1. DM2+S 2. DM2+S monomer-free 3. DM2+S (without DMMA) 4. DM2+S Casein-free 5. DM2+S Trehalose + Monomer 6. DM2+S Trehalose + DMMA 7. DM2+S Trehalose + Casein

[0304] Previous experiments have established that trehalose is necessary for drydown; therefore, DM2+S without trehalose was not used for this experiment. The experiment was performed in single replicates on six donors. The sample preparation protocol followed the protocol and methods described. The stop gate was set to 10,000 CD45+ lymphocytes. After drydown, the dried tubes from all groups were physically observed for any obvious changes. In addition, the scattering properties of the double fluorescence plot were also observed for any nonspecific pullouts and nonspecific interactions.

[0305] Figure 9 shows the physical appearance and properties of the dried tubes for each of the test groups: DM2+S, trehalose + monomer, trehalose + casein, trehalose + DMMA (left to right, upper panel), DM2+S, DM2+S without monomer, DM2+S without DMMA, and DM2+S without casein (left to right, lower panel). Here, DM2+S serves as the control group. Physical observations show that in the absence of monomer, there is a change in the color of the dried film (typically, the red film turns light orange to brown). Shrinkage of the film was observed in the casesin and DMMA-free groups. Changes in the appearance of the dried film were not observed in the DMMA-free tubes compared to DM2+S. However, the casein-free tubes showed minimal shrinkage of the dried film.

[0306] Figures 10A-C show side-scatter SSC vs. FL plots for CD56-SNv428; CD20-SNv605; and CD4-SNv786, respectively, in the test groups DM2+S, DM2+S without DMMA, DM2+S without casein, DM2+S without monomer (left to right, upper panel), DM2+S: trehalose + DMMA, DM2+S: trehalose + casein, and DM2+S: trehalose + monomer (left to right, lower panel). The figures show that in the absence of casein and DMMA, there is a pull-out of nonspecific monocytes (indicated by arrows). The absence of monomers leads to a spread of negative populations in lymphocytes (indicated by arrows). This spread of negative populations may be primarily due to nonspecific interactions between SN dyes in the absence of monomers and casein.

[0307] Figures 10D–F show FL vs. FL double fluorescence plots for all SN combinations in all tested groups (e.g., CD56-SNv428 vs. CD20-SNv605, CD4-SNv786 vs. CD20-SNv605, and CD4-SNv786 vs. CD56-SNv428, respectively) demonstrating that the absence of monomers and casein causes nonspecific interactions / population spreading between SN conjugates within the SN population. Therefore, both monomers and casein are considered important for preventing nonspecific interactions.

[0308] This example demonstrates that monomer A and casein are important for the drydown of SN conjugates, as inefficient prevention of intercellular interactions is observed without them. In addition, casein also prevents the pull-out of nonspecific monocytes. DMMA plays a major role in preventing the pull-out of nonspecific monocytes. This experiment is important not only for providing the function of individual additives but also for troubleshooting quality control issues. 7: Performance of DM2+S dry tubes compared to BD staining buffer

[0309] Objective: To demonstrate the performance of DM2+S dry tubes compared to BD staining buffer.

[0310] Methods: This experiment was conducted to check the performance of dried tubes compared to that of BD staining buffer. Here, the performance of DM2+S dried tubes was compared to that of BD horizon brilliant buffer. The experiment was performed using a four-color panel. The group details are as follows.

[0311] Three polymer dye conjugates were evaluated with gating markers (CD45-APC-A750, CD56-SNv428, CD20-SNv605, and CD4-SNv786) using commercially available BD Horizon® Brilliant staining buffer (Becton, Dickinson and Company). The staining protocol followed the manufacturer's instructions. First, 50 μl of BD Horizon Brilliant buffer was added to a tube, followed by the addition of the four conjugates. The mixture was thoroughly mixed by vortexing. Then, 100 μl of blood sample was added. The mixture was properly mixed by vortexing, incubated at room temperature for 30 minutes, and processed by following steps 2 onward as described in the Protocol and Methods section.

[0312] The three polymer dye conjugates were dried together with gating markers (CD45-APC-A750, CD56-SNv428, CD20-SNv605, and CD4-SNv786) in the DM version 2+ stabilizer of the present invention. The sample preparation protocol was the same as that described above in terms of protocol and method.

[0313] Six donors (single replicate) were tested in all of the two described groups using the protocol described above. The stop gate was set at 10,000 CD45+ lymphocytes.

[0314] Results and observations:

[0315] Figure 11A shows representative SSC vs. FL overlay flow plots for three polymer dye conjugates CD56-SNv428, CD20-SNv605, and CD4-SNv786, either dried with one of the DM2+S drydown buffers of the present invention and reconstituted with blood samples, or using comparative BD Horizon® Brilliant staining buffer. The comparative commercially available BD Horizon® Brilliant staining buffer caused nonspecific granulocyte and monocyte pullout (indicated by arrows) compared to the DM2+S dry tube.

[0316] Figure 11B shows representative double fluorescence overlay plots for the three polymer dye conjugates along with gating markers (CD45-APC-A750, CD56-SNv428, CD20-SNv605, and CD4-SNv786). Comparative BD Horizon® Brilliant staining buffer induced nonspecific lymphocyte pullout in all SN conjugate combinations compared to DM2+S.

[0317] This example demonstrates that DM2+S and BD staining buffer show no significant differences in scattering, recruitment %, and MdFI values ​​(data not shown). However, the DM2+S dry tube shows a more robust population (no nonspecific pullouts) compared to the BD staining buffer. Therefore, from the data, it can be concluded that the DM2+S dry tube performs better than the BD staining buffer. (Example 4) Six months of provisional stability

[0318] Objective: To evaluate and check the 6-month stability of DM2+S dry tubing.

[0319] Methods: In this example, the 6-month stability of DM2+S dry tubes was evaluated. For comparison, 3-month and fresh lots were included in the study. Details of each lot are shown in Table 9. Both 3-month and 6-month dry tubes were tested in studies belonging to open pouches, i.e., the dry tubes were tested multiple times from the opened pouches. These dry tubes contained a 12-color (12C) panel (9C conventional conjugate + 3C SN conjugate CD56-SNv428, CD20-SNv605, CD4-SNv786). These lots were tested in single replicates in a single instrument for four donors. The staining-dissolution-wash protocol described in Example 1B was used for the study. The stop gate was set to 10,000 CD45+ lymphocytes. Compensation was adjusted for fresh lots. Subsequently, compensation for the fresh lot was applied to the 3-month and 6-month-old lots, and any necessary minor compensation adjustments were made to both the 3-month and 6-month-old lots. The delta mobilization absolute % and MdFI for the 3-month and 6-month-old lots were compared to those for the fresh lot (data not shown).

[0320] [Table 9]

[0321] Results and observations:

[0322] Figure 12 shows a photograph of a tube with a dry-down film after 6 months of drying. No visual deterioration was observed in the dry-down film.

[0323] Figures 13A, 13B, and 13C show representative superposition plots of all three lots for different combinations of specificities tested in the study.

[0324] Figure 13A shows representative FL vs. FL overlay plots of DM2+S dry tubes for 6-month stability compared to 3-month and fresh lots. Dry tubes containing a 12-color (12C) panel (conventional 9C conjugate + 3C SN conjugates CD56-SNv428, CD20-SNv605, CD4-SNv786) were tested in single replicates on four donors using a single flow cytometry instrument. Representative FL vs. FL overlay plots for different combinations of SN dyes in all three lots: CD56 PB450-A vs. CD20 Violet610 (left panel), CD4 Violet780 vs. CD20 Violet610 (center panel), and CD4 Violet780 vs. CD56PB450 (right panel). These overlays show that the populations in lots aged 3 months and 6 months completely overlap with the fresh lots. In addition, no nonspecific interactions or population spread were observed in any of the test donors in the lots at 6 months post-market.

[0325] Figure 13B shows representative FL vs. FL overlay plots for SN vs. classical combinations in all three lots, comparing the 6-month stability of DM2+S dry tubes with that of 3-month and fresh lots. Dry tubes containing 12-color (12C) panels (9C conventional conjugate + 3C SN conjugate CD56-SNv428, CD20-SNv605, CD4-SNv786) were tested in single replicates in a single flow cytometry instrument for four donors. Overlays for 6-month, 3-month, and fresh lots for CD3 ECD vs. CD20 Violet610 (left panel), CD8 KO525 vs. CD4 Violet780 (center panel), and CD45 APC-A750 vs. CD56PB450 (right panel) show that the populations in the 3-month and 6-month-old lots completely overlap with the fresh lots. In addition, no nonspecific interactions or population spread were observed in any of the test donors in the lots at 6 months post-market.

[0326] Figure 13C shows representative FL vs. FL overlay plots for classical vs. classical combinations in all three lots, comparing the 6-month stability of DM2+S dry tubes with that of 3-month and fresh lots. Dry tubes containing 12-color (12C) panels (9C conventional conjugate + 3C SN conjugate CD56-SNv428, CD20-SNv605, CD4-SNv786) were tested in single replicates in a single flow cytometry instrument for four donors. Overlays for 6-month, 3-month, and fresh lots for CD16 FITC vs. CD25PE (left panel), CD8 KO525 vs. CD45APC-A750 (center panel), and CD3 ECD vs. CD10APC (right panel) show that the populations in the 3-month and 6-month aged lots completely overlap with those in the fresh lots. In addition, no nonspecific interactions or population spread were observed in the 6-month aged lots in all test donors. In this embodiment, the 6-month provisional stability results indicate that the DM2+S dry tube is stable for at least 6 months. No nonspecific interactions or spreading were observed at the provisional 6-month mark. Real-time stability is planned for 24 months. (Example 5) Standardized DM Buffer Preparation

[0327] The DM buffer of the present invention can be prepared as follows. A stock preparation of the solubilized additive (component) can be prepared as follows. After stock preparation, the DM2+S buffer is prepared as shown in Table 10. The pH range of the DM2+S buffer was found to be between 7 and 7.4.

[0328] [Table 10]

[0329] Subsequently, using DM2+S DM buffer (Table 10), the panel formulations shown in Table 11 were prepared as homogeneous films of a single dried reaction product in a tube format containing three fluorescent polymer dye conjugates.

[0330] [Table 11]

[0331] The final volume per test is 50 μL. In one embodiment, for example, the following items are provided. (Item 1) A buffer composition for use in drying multiple dye conjugates on a substrate, Water-soluble monomer; Protein stabilizers; Carbohydrate stabilizers; and A buffer composition containing an amphoteric surfactant. (Item 2) The buffer composition according to item 1, wherein at least one of the plurality of dye conjugates comprises a polymer dye conjugate. (Item 3) The polymer dye conjugate is defined by formula (IV): [ka] (In the formula, Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; Each L is a linker part; Each M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap-modified monomers, optionally substituted ethylenes, and ethynylenes; G 1 and G 2 These are independently selected from unmodified polymer ends and modified polymer ends; a, c, and d independently define the mole percent of each unit in the structure, and each unit may be repeated evenly or randomly, where each a is between 10 and 100% mole percent, each c is between 0 and 90% mole percent, and each d is between 0 and 25% mole percent; Each b is independently either 0 or 1; (m is an integer between 1 and approximately 10,000) The buffer composition according to item 2, which is a binding partner conjugated to a fluorescent polymer dye having the structure described above. (Item 4) The buffer composition according to item 3, wherein the plurality of dye conjugates comprises two or more different fluorescent polymer dye conjugates, each having the structure described in formula (IV). (Item 5) The buffer composition according to item 3 or 4, wherein the water-soluble monomer is a monomer unit used in the preparation of at least one of the fluorescent polymer dye conjugates. (Item 6) The buffer composition according to any one of items 1 to 5, wherein the water-soluble monomer is a dihydrophenanthrene (DHP)-based water-soluble monomer or a fluorene-based water-soluble monomer. (Item 7) The aforementioned water-soluble monomer is of formula (I): [ka] (In the formula, Each of G1 and G2 independently contains halogens, alkyls, PEGs, hydrogen, alkynes, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, and optionally substituted tetrahydropenes. ReFluorine (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl groups selected from the group consisting of aryl or heteroaryl groups in which a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, and thiols is substituted on one or more terminal pendant chains; Each R 2 These are independently: water-soluble moieties, alkenes, alkynes, cycloalkyls, haloalkyls, (hetero)aryloxys, (hetero)arylaminos, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, sulfonamides, sulfinamides, phosphoamides, phosphineamides. [ka] Selected from the group consisting of; Each R 3 This is the water-soluble portion; Each R 4 These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each R 5 These are independently H, hydroxyl, and C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, and C2-C 12 Selected from the group consisting of carboxylic acid esters; Each Q is independent, combined, NR 4 , or -CH2; Each Z is independently CH2, O, or NR 4 and; Each f is an independent integer between 0 and 50; (Each n is an independent integer between 1 and 20.) A buffer composition according to any one of items 1 to 6, wherein the buffer is a dihydrophenanthrene (DHP) water-soluble monomer having the chemical structure described above. (Item 8) Each G1 and G2 is a halogen; each Z is O; each R2 is H; each R 5 The buffer composition according to item 7, wherein H is hydroxyl, C1-C6 alkyl, or C1-C6 alkoxy; each n is independently 2-4; and each f is independently 5-20. (Item 9) Each n is 3; each R 5 The buffer composition according to item 7 or 8, wherein the b is -OCH3; and each f is 11 to 12. (Item 10) The aforementioned water-soluble monomer is of formula (II): [ka] (In the formula, Each of G1 and G2 independently contains halogens, alkyls, PEGs, hydrogen, alkynes, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, and optionally substituted tetrahydropenes. Re Fluorine (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl groups selected from the group consisting of aryl or heteroaryl groups in which a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, and thiols is substituted on one or more terminal pendant chains; Each X is either C or Si; Each R 4 These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each R5 independently consists of H, hydroxyl, and C1-C. 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, C2-C 12 Carboxylic acid esters, and C1-C 12 Selected from the group consisting of alkoxys; Each Z is independently CH2, O, or NR 4 and; Each f is an independent integer between 0 and 50; (Each n is an independent integer between 1 and 20.) A buffer composition according to any one of items 1 to 6, wherein the fluorene-based water-soluble monomer has the chemical structure described above. (Item 11) Each G1 and G2 is a halogen; each X is C; each Z is O; each R 5 The buffer composition according to item 10, wherein H is hydroxyl, C1-C6 alkyl, or C1-C6 alkoxy; each n is independently 2-4; and each f is independently 5-20. (Item 12) The aforementioned zwitterionic surfactant is of formula (XV): [ka] (In the formula, Y = CO2- or SO3-, W = H or OH, and Z = CH3 or NHC(O)R(In the formula, R = C 1~15 (It is alkyl; independently, each p=0 or 1; q=0 to 21; and if necessary, W=H, Z=CH3, and q=11 to 15) A buffer composition according to any one of items 1 to 11, having the structure described above. (Item 13) The aforementioned amphoteric surfactants include 3-(N,N-dimethylmyristylammonium propanesulfonate (DMMA); 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonium]-propanesulfonate (DMPA); N-(alkylC 10 ~C 16 A buffer composition according to any one of items 1 to 12, selected from the group consisting of )-N,N-dimethylglycine betaine and N,N-dimethyl-N-dodecylglycine betaine. (Item 14) The buffer composition according to any one of items 1 to 13, wherein the protein stabilizer is selected from the group consisting of casein, bovine serum albumin (BSA), and gelatin. (Item 15) The buffer composition according to any one of items 1 to 14, wherein the carbohydrate stabilizer is a disaccharide carbohydrate stabilizer. (Item 16) The buffer composition according to item 15, wherein the disaccharide carbohydrate stabilizer is trehalose or its hydrate, and optionally, the trehalose or its hydrate is trehalose dihydrate. (Item 17) A buffer composition according to any one of items 1 to 16, further comprising one or more additional additives selected from the group consisting of preservatives, antioxidants, anionic surfactants, nonionic surfactants, and colorants. (Item 18) The buffer composition according to any one of items 1 to 17, wherein the composition is an aqueous composition. (Item 19) A buffer composition according to any one of items 1 to 18, having a pH in the range of pH 6.5 to 7.5, or pH 7.0 to 7.4. (Item 20) Per exam, 200 to 800 μg of the aforementioned water-soluble monomer; 2000-3000 μg of the aforementioned carbohydrate stabilizer; 8.4 to 72 μg of the protein stabilizer; and 2 to 9 μg of the aforementioned amphoteric surfactant A buffer composition according to any one of items 1 to 19, including the following: (Item 21) A method for preparing a single reactant film, wherein the method is A step of dispensing a plurality of dye conjugates together onto a substrate in a liquid phase containing an aqueous buffer composition according to any one of items 1 to 18, wherein the plurality of dye conjugates include at least one polymer dye conjugate; and The step of drying the plurality of dye conjugates together in the aqueous buffer solution of the liquid phase to form a first single reactant film on the substrate. Methods that include... (Item 22) The method according to item 21, wherein the plurality of dye conjugates comprises two or more polymer dye conjugates. (Item 23) The method according to item 20 or 21, wherein the substrate is selected from the group consisting of tubes, wells, membranes, and beads. (Item 24) The method according to any one of items 21 to 23, wherein the substrate includes the inner surface of the reaction vessel. (Item 25) The method according to any one of items 21 to 24, wherein the plurality of dye conjugates comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, or two to 20, three to 18, or four to 12 different dye conjugates, each comprising a different binding partner. (Item 26) When the first single reactant film is exposed to a first aliquot of a liquid blood sample, processed, and analyzed by flow cytometry, and compared with a second flow cytometry plot obtained by exposing a second single reactant film to a second aliquot of a liquid biological sample, processing, and analyzing it by flow cytometry, Decreased nonspecific binding of monocytes; Decreased nonspecific binding of granulocytes; Reduced nonspecific interactions of polymer dye conjugates; and Reduction in the aggregation of polymer dye conjugates The step includes obtaining a first flow cytometry plot showing one or more of the group consisting of, The second single-reactant film is prepared from a liquid phase containing the same plurality of dye conjugates that are dried together to form the first single-reactant film, as well as from a reagent buffer using a conventional drying technique without the water-soluble monomer and without the amphoteric surfactant. The method described in any one of items 21-25. (Item 27) Multiple fluorescent polymer dye conjugates; Water-soluble monomer; Protein stabilizers; Carbohydrate stabilizers; and Amphoteric surfactant A composition containing the following: (Item 28) The composition according to item 27, in the form of a single reactant film placed on a substrate. (Item 29) Per single reactant film, 200 to 800 μg of the aforementioned water-soluble monomer; 2000-3000 μg of the aforementioned carbohydrate stabilizer; 8.4 to 72 μg of the protein stabilizer; and 2 to 9 μg of the aforementioned amphoteric surfactant A composition according to item 27 or 28, including the following: (Item 30) At least one of the plurality of fluorescent polymer dye conjugates is a compound of formula (IV): [ka] (In the formula, Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; Each L is a linker part; Each M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap-modified monomers, optionally substituted ethylenes, and ethynylenes; G 1 and G 2These are independently selected from unmodified polymer ends and modified polymer ends; a, c, and d independently define the mole percent of each unit in the structure, and each unit may be repeated evenly or randomly, where each a is between 10 and 100% mole percent, each c is between 0 and 90% mole percent, and each d is between 0 and 25% mole percent; Each b is independently either 0 or 1; (m is an integer between 1 and approximately 10,000) A composition according to any one of items 27 to 29, comprising a fluorescent polymer dye portion having the structure described above. (Item 31) The composition according to any one of items 27 to 30, wherein the water-soluble monomer is a monomer unit used in the preparation of at least one of the plurality of fluorescent polymer dyes. (Item 32) The composition according to any one of items 27 to 31, wherein the water-soluble monomer is a dihydrophenanthrene (DHP)-based water-soluble monomer or a fluorene-based monomer. (Item 33) The aforementioned water-soluble monomer is of formula (I): [ka] (In the formula, Each of G1 and G2 independently contains halogens, alkyls, PEGs, hydrogen, alkynes, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, and optionally substituted tetrahydropenes. ReFluorine (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl groups selected from the group consisting of aryl or heteroaryl groups in which a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, and thiols is substituted on one or more terminal pendant chains; Each R 2 These are independently: water-soluble moieties, alkenes, alkynes, cycloalkyls, haloalkyls, (hetero)aryloxys, (hetero)arylaminos, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, sulfonamides, sulfinamides, phosphoamides, phosphineamides. [ka] Selected from the group consisting of; Each R 3 This is the water-soluble portion; Each R 4 These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each R 5 These are independently H, hydroxyl, and C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, and C2-C 12 Selected from the group consisting of carboxylic acid esters; Each Q is independent, combined, NR 4 , or -CH2; Each Z is independently CH2, O, or NR 4 and; Each f is an independent integer between 0 and 50; (Each n is an independent integer between 1 and 20.) A composition according to any one of items 27 to 32, which is a dihydrophenanthrene (DHP) water-soluble monomer having the chemical structure described above. (Item 34) The composition according to item 33, wherein G1 and G2 are halo, respectively; Z is O, respectively; R2 is H, respectively; n is independently 2-4, respectively; f is independently 5-20, respectively; n is optionally 3, and m is 11-12. (Item 35) The aforementioned water-soluble monomer is of formula (II): [ka] (In the formula, Each of G1 and G2 independently contains halogens, alkyls, PEGs, hydrogen, alkynes, optionally substituted aryls, optionally substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, and optionally substituted tetrahydropenes. ReFluorine (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl groups selected from the group consisting of aryl or heteroaryl groups in which a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, and thiols is substituted on one or more terminal pendant chains; Each X is either C or Si; Each R 4 These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each R5 independently consists of H, hydroxyl, and C1-C. 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, C2-C 12 Carboxylic acid esters, and C1-C 12 Selected from the group consisting of alkoxys; Each Z is independently CH2, O, or NR 4 and; Each f is an independent integer between 0 and 50; (Each n is an independent integer between 1 and 20.) A composition according to any one of items 27 to 32, which is a fluorene-based water-soluble monomer having the chemical structure described above. (Item 36) The aforementioned zwitterionic surfactant is of formula (XV): [ka] (In the formula, Y = CO2- or SO3-, W = H or OH, and Z = CH3 or NHC(O)R(In the formula, R = C 1~15 (It is alkyl; independently, each p=0 or 1; q=0 to 21; and if necessary, W=H, Z=CH3, and q=11 to 15) A composition according to any one of items 27 to 35, having the structure described herein. (Item 37) The aforementioned amphoteric surfactants include 3-(N,N-dimethylmyristylammonium propanesulfonate (DMMA); 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonium]-propanesulfonate (DMPA); N-(alkylC 10 ~C 16 An aqueous buffer composition according to any one of items 27 to 36, selected from the group consisting of )-N,N-dimethylglycine betaine and N,N-dimethyl-N-dodecylglycine betaine. (Item 38) The composition according to any one of items 27 to 37, wherein the protein stabilizer is selected from the group consisting of casein, bovine serum albumin (BSA), and gelatin. (Item 39) The composition according to any one of items 27 to 38, wherein the carbohydrate stabilizer is a disaccharide carbohydrate stabilizer. (Item 40) The composition according to item 39, wherein the disaccharide carbohydrate stabilizer is trehalose or its hydrate, and optionally, the trehalose or its hydrate is trehalose dihydrate. (Item 41) An aqueous buffer composition according to any one of items 27 to 40, further comprising one or more additional additives selected from the group consisting of preservatives, antioxidants, anionic surfactants, nonionic surfactants, and colorants.

Claims

1. A buffer composition for use in a method for drying multiple dye conjugates on a substrate, A water-soluble monomer containing an aryl or heteroaryl moiety, which does not contain a boronic acid-substituted aryl, a boronic acid ester-substituted aryl, a boronic acid ester, or a boronic acid, and is a dihydrophenanthrene (DHP)-based water-soluble monomer or a fluorene-based water-soluble monomer; Protein stabilizers; Carbohydrate stabilizers; and Contains an amphoteric surfactant, The aforementioned zwitterionic surfactant is of formula (XV): 【Chemistry 43】 (wherein Y = CO₂- or SO₃-, W = H or OH, Z = CH₃ or NHC(O)R (wherein R = C₁-¹⁵ alkyl); independently, p = 0 or 1 for each; q = 0-21) Having the structure described above, The plurality of dye conjugates comprises one or more fluorescent polymer dye conjugates. The method includes mixing the buffer composition with the plurality of dye conjugates and drying it on a substrate. Buffer composition.

2. One or more fluorescent polymer dye conjugates; A water-soluble monomer containing an aryl or heteroaryl moiety, which does not contain a boronic acid-substituted aryl, a boronic acid ester-substituted aryl, a boronic acid ester, or a boronic acid, and is a dihydrophenanthrene (DHP)-based water-soluble monomer or a fluorene-based water-soluble monomer; Protein stabilizers; Carbohydrate stabilizers; and Amphoteric surfactant A composition comprising, The aforementioned zwitterionic surfactant is of formula (XV): 【Chemistry 43】 (wherein Y = CO₂- or SO₃-, W = H or OH, Z = CH₃ or NHC(O)R (wherein R = C₁-¹⁵ alkyl); independently, p = 0 or 1 for each; q = 0-21) A composition having the structure described above.

3. The composition according to claim 1 or 2, wherein the water-soluble monomer comprises one or more water-soluble moieties bonded thereto.

4. The composition according to claim 1 or 2, in the form of a single reactant film disposed on a substrate.

5. The fluorescent polymer dye conjugate is of formula (IV): 【Chemistry 39】 (In the formula, Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; Each L is a linker part; Each M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap-modified monomers, unsubstituted or substituted ethylenes, and ethynylenes; G １ and G ２ These are independently selected from unmodified polymer ends and modified polymer ends; a, c, and d independently define the mole percent of each unit in the structure, and each unit may be repeated evenly or randomly, where each a is between 10 and 100% mole percent, each c is between 0 and 90% mole percent, and each d is between 0 and 25% mole percent; Each b is independently either 0 or 1; m is an integer between 1 and approximately 10,000. The composition according to claim 1 or 2, wherein the conjugated partner is a fluorescent polymer dye having the structure described above.

6. The composition according to claim 1 or 2, wherein the water-soluble monomer is a monomer unit used in the preparation of at least one of the fluorescent polymer dye conjugates.

7. The water-soluble monomer is a dihydrophenanthrene (DHP)-based water-soluble monomer having the chemical structure described in formula (I) or a fluorene-based water-soluble monomer having the chemical structure described in formula (II): 【Chemistry 40】 or 【Chemistry 42】 (In the formula, Each X is either C or Si; Each G １ G ２ This is independently selected from the group consisting of halogens, alkyls, PEGs, hydrogen, alkynes, unsubstituted or substituted aryls, unsubstituted or substituted heteroaryls, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, unsubstituted or substituted tetrahydropyrene (THP), unsubstituted or substituted fluorene, unsubstituted or substituted dihydrophenanthrene (DHP), aryls, or heteroaryls, wherein one or more pendant chains are substituted with a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, and thiols at the terminal; Each R ２ These are independently: water-soluble moieties, alkenes, alkynes, cycloalkyls, haloalkyls, (hetero)aryloxys, (hetero)arylaminos, sulfonamide-PEG, phosphoramide-PEG, ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, sulfonamides, sulfinamides, phosphoamides, phosphineamides. 【Chemistry 41】 Selected from the group consisting of; Each R ３ This is the water-soluble portion; Each R ４ These are independently selected from the group consisting of H, alkyl, PEG, water-soluble moiety, linker moiety, chromophore, carboxylic acid amine, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, or thiol, or their protecting groups; Each R ５ is independently selected from the group consisting of H, hydroxyl, C １ to C １２ alkyl, C ２ to C １２ alkene, C ２ to C １２ alkyne, C ３ to C １２ cycloalkyl, C １ to C １２ haloalkyl, C １ to C １２ alkoxy, C ２ to C １８ (hetero)aryloxy, C ２ to C １８ (hetero)arylamino, C ２ to C １２ carboxylic acid, and C ２ to C １２ carboxylic acid ester; Each Q is independent, combined, NR ４ , or -CH ２ And; Each Z is independent of CH ２ , O, or NR ４ And; Each f is an independent integer between 0 and 50; Each n is an independent integer between 1 and 20. The composition according to claim 1 or 2.

8. Each G １ G ２ However, each Z is a halogen; each R is O; ２ However, H is; each R ５ However, H, hydroxyl, C １ ~C ６ Alkyl, or C １ ~C ６ The composition according to claim 1 or 2, wherein it is an alkoxy; each n is independently 2 to 4; and each f is independently 5 to 20.

9. The aforementioned amphoteric surfactants are 3-(N,N-dimethylmyristylammonium propanesulfonate (DMMA); 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonium]-propanesulfonate (DMPA); N-(alkylC １０ ~C １６ The composition according to claim 1 or 2, selected from the group consisting of )-N,N-dimethylglycine betaine and N,N-dimethyl-N-dodecylglycine betaine.

10. The composition according to claim 1 or 2, wherein the protein stabilizer is selected from the group consisting of casein, bovine serum albumin (BSA), and gelatin.

11. The composition according to claim 1 or 2, wherein the carbohydrate stabilizer is a disaccharide carbohydrate stabilizer.

12. The composition according to claim 11, wherein the disaccharide carbohydrate stabilizer is trehalose or its hydrate.

13. The composition according to claim 1 or 2, further comprising one or more additional additives selected from the group consisting of preservatives, antioxidants, anionic surfactants, nonionic surfactants, and colorants.

14. Per exam, 200 to 800 μg of the water-soluble monomer; 2000 to 3000 μg of the carbohydrate stabilizer; 8.4 to 72 μg of the protein stabilizer; and 2 to 9 μg of the aforementioned amphoteric surfactant A composition according to claim 1 or 2, comprising:

15. A method for preparing a single reactant film, wherein the method is A step of dispensing a plurality of dye conjugates together onto a substrate in a liquid phase containing an aqueous composition according to any one of claims 1 to 14, wherein the plurality of dye conjugates include at least one fluorescent polymer dye conjugate; and A method comprising the step of drying the plurality of dye conjugates together in the aqueous buffer of the liquid phase to form a first single reactant film on the substrate.

16. When the first single-reactant film is exposed to a first aliquot of a liquid blood sample, processed, and analyzed by flow cytometry, and compared with a second flow cytometry plot obtained by exposing a second single-reactant film to a second aliquot of the liquid blood sample, processing, and analyzing it by flow cytometry, Decreased nonspecific binding of monocytes; Decreased nonspecific binding of granulocytes; Reduced nonspecific interactions of fluorescent polymer dye conjugates; and Reduction in aggregation of fluorescent polymer dye conjugates The process further includes the step of obtaining a first flow cytometry plot showing one or more of the group consisting of, The second single-reactant film is prepared from a liquid phase containing the same plurality of dye conjugates that are dried together to form the first single-reactant film, as well as from a reagent buffer using a conventional drying technique without the water-soluble monomer and without the amphoteric surfactant. The method according to claim 15.

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