Novel formulation for drying of polymer dye conjugated antibodies

Abstract

To provide a composition comprising multiple fluorescent dye conjugates in a panel.SOLUTION: A novel dry down buffer is provided for use in drying a plurality of 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 fluorescent polymer dye conjugates, dried on a substrate, and reconstituted with a biological sample, the buffer provides decreased aggregation of fluorescent polymer dye conjugates, and decreased non-specific binding of monocytes and granulocytes compared to the use of a buffer without the water-soluble monomer or zwitterionic surfactant.SELECTED DRAWING: Figure 4

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 technology that uses multiple fluorescent markers to identify and characterize cell subpopulations of interest, allowing 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 types of cells express different markers, e.g., CD4 on T cells and CD20 on B cells. When mutually exclusive markers (markers that are expressed only in one specific cell type) are stained using their counterpart anti-marker antibody-fluorochrome conjugates, the signal (fluorescence) from each cell is captured and digitally converted in the flow cytometer for analysis.

[0003] Multicolor dry reagents are dried, utilized cocktails that contain 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 to allow storage at room temperature, simplify sample preparation, and minimize user error.

[0004] Multi-color dry reagent cocktails may contain several different antibodies conjugated to small (e.g., FITC), tandem (e.g., PC5.5), or large (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 with existing classical (monomer) dye (FITC, PC5.5, APC, etc.) conjugates, polymer dye conjugates differ in structure and complexity. The rigidity of the polymer dye structure helps to reduce rotational energy resulting in brighter emission. Therefore, polymer dye conjugates are particularly useful in identifying and analyzing cells with rarely expressed receptors. These bright polymer dyes can allow for the detection and resolution of unclear 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 the drying down of different classical dye conjugates in a single tube without altering their functionality (affinity for antigen) or physical properties (such as brightness and leakage) with stability over a period of time. "Reagent Buffer" (RB) is a prior art formulation containing sacrificial proteins, carbohydrate stabilizers, antimicrobial agents and buffers that was previously developed to dry down different monomeric dye conjugates in a single tube without altering their functionality (affinity for antigen) or physical properties (such as brightness and fluorescence). "Physical properties" refer to the brightness of the fluorescent dye conjugate and its leakage into other channels. For example, the desired flow cytometry results using CD4-PE dye conjugates are shown when dried using conventional drying techniques and used to stain biological specimens. As shown in Figure 1A, the desired functionality, physical properties, and ability to separate CD4 PE+ monocyte and CD4 PE+ lymphocyte cell populations are shown. Similarly, when dried using conventional techniques and reconstituted with a blood sample, desirable flow cytometry results are exhibited for the CD20 APC dye conjugate, as shown in Figure 1B, demonstrating desirable functionality, physical properties, and flow cytometry separation ability of CD20 APC+ cell populations.

[0007] When two polymer-dye-antibody conjugates are dried down in a tube using conventional drying techniques using a reagent buffer, non-specific interactions between the polymer-dye conjugates cannot be prevented, resulting in aggregation. In general, when two or more polymer-dye-antibody conjugates are dried using conventional drying techniques, they tend to interact with each other, resulting in 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 of CD20-605 and CD4-786 polymer dye antibody conjugates when mixed and dried using conventional drying techniques. The inability to separate the populations in the x and y axes was hypothesized to be a leakage problem. However, even after correction for leakage, the populations were not separated (Figure 2B).

[0009] Figure 2B shows the undesired flow cytometry results of CD20-605 and CD4-786 polymer-dye-antibody conjugates when mixed and dried using conventional drying techniques, along with correction for leakage. The inability to correct the populations for their individual fluorescent 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] A need exists for novel buffer formulations that can keep polymer-dye-antibody conjugates stable and avoid aggregation while drying and during reconstitution, allowing for improved functionality, physical properties, and flow cytometry resolution. Summary of the Invention [Means for solving the problem]

[0011] Disclosure Summary A novel buffer composition is provided for use in drying a plurality of dye conjugates on a substrate. The dye conjugate may include a fluorescent dye conjugate. The fluorescent dye conjugate may be a conjugate of a fluorescent dye and a binding partner, such as an antibody. The fluorescent dye conjugate may be a fluorescent polymer dye conjugated to a binding partner, such as an antibody. The fluorescent dye conjugate may be used in flow cytometry. The buffer composition includes a water-soluble monomer; a protein stabilizer; a carbohydrate stabilizer; and a zwitterionic surfactant. When the buffer composition is mixed with a multi-color fluorescent dye conjugate panel including 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 when compared to the use of a buffer without a protein stabilizer, a water-soluble monomer, or a zwitterionic surfactant. In some examples, the buffer composition also provides reduced non-specific binding of the dye to monocytes and / or reduced non-specific binding of the dye to granulocytes when compared to the use of a buffer without a protein stabilizer, a monomer, or a zwitterionic surfactant.

[0012] In some embodiments, the present disclosure provides a composition comprising a plurality of fluorescent dye conjugates in a panel. In the presence of a suitable buffer, the plurality of fluorescent dye conjugates can be used in a panel to identify subpopulations of cells. Without a suitable buffer, the fluorescent dye conjugates can interact with each other, causing staining artifacts that can affect data interpretation.

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

[0014] The water-soluble monomers may be monomeric units comprising aryl or heteroaryl moieties, each having a water-soluble moiety attached thereto, as appropriate. The water-soluble moiety may be one or more poly(ethylene glycol) moieties. The water-soluble monomers may be suitable for use in the preparation of at least one of a plurality of fluorescent polymer dyes having a monomer A subunit, a monomer B subunit, or a combination of monomer A and monomer B subunits. The water-soluble monomers may be dihydrophenanthrene (DHP)-based water-soluble monomers. The water-soluble monomers may be fluorene-based water-soluble monomers.

[0015] The water soluble monomer has the formula (I): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of halogen, alkyl, PEG, hydrogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol; Each R 2are independently selected from the group consisting of water solubilizing moiety, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, sulfonamido-PEG, phosphoramido-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, [ka] 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 carboxylate amine, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protecting group thereof; Each R 5 are independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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 alkoxy; Each Q is independently a bond, NR 4 or -CH2; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. The monomer may be a dihydrophenanthrene (DHP) monomer having the chemical structure shown in

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

[0017] The water soluble monomer has the formula (II): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of halogen, alkyl, PEG, alkyne, hydrogen, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol; Each X is C or Si; Each R 4 is independently selected from the group consisting of H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylate amine, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protecting group thereof; Each R5 are independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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 alkoxy; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. The monomer may be a fluorene-based monomer having the chemical structure described in

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

[0019] The water soluble monomer has the formula (III): [ka] (wherein each G1, G2 is independently halo (F, Cl, Br, I); each Z is independently selected from the group consisting of O, CH2, and NH; each R1 is independently alkyl (C1-C3); each R2 is independently H, alkyl (C1-C6); each n is independently 1 to 6; and each m is independently 5 to 50. In some embodiments, each of G1 and G2 is Br; each of Z is O; each of R1 is CH3; each of R2 is H; each of n is 2 to 4; and each of m is independently 5 to 20. In some embodiments, each of n is 3; and each of 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 a hydrate thereof. In a specific embodiment, the disaccharide carbohydrate stabilizer may be trehalose or a hydrate thereof. The carbohydrate stabilizer may be trehalose dihydrate.

[0022] Zwitterionic surfactants have the formula (XV): [ka] (wherein Y=CO2- or SO3-, W=H or OH, and Z=CH3 or NHC(O)R, where R=C 1~15 alkyl; independently, each p=0 or 1; q=0-21; and optionally, W=H, Z=CH3, and q=11-15. The compound may include a structure as described in

[0023] Zwitterionic surfactants are 3-(N,N-dimethylmyristylammoniopropanesulfonate (DMMA); 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate (DMPA); N-(alkyl C 10 ~C 16)-N,N-dimethylglycine betaine; and N,N-dimethyl-N-dodecylglycine betaine.

[0024] Optionally, the buffer composition may contain one or more, two or more, or three or more additional additives selected from the group consisting of a preservative, an antioxidant, an anionic surfactant, a nonionic surfactant, and a coloring agent.

[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 zwitterionic detergent.

[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 zwitterionic detergent.

[0028] In some embodiments, the buffer composition may include a plurality of fluorescent dye conjugates. In some embodiments, the buffer composition may include a plurality of fluorescent polymer-dye conjugates. The fluorescent polymer-dye conjugates may include a structure according to formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X), formula (XI), formula (XII), formula (XIII), and / or formula (XIV), each of which is described herein.

[0029] In other embodiments, the aqueous buffer composition does not include more than one fluorescent dye conjugate. In further embodiments, the aqueous buffer composition does not include any fluorescent dye conjugates.

[0030] A novel method for preparing a single reactant film is provided, comprising dispensing a plurality of reactants together on a substrate in a liquid phase comprising a buffer composition as described herein, the plurality of reactants comprising 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 polymeric dye; the second reactant comprising a second binding partner conjugated to a second dye; and drying the first reactant and the second reactant together in the liquid phase aqueous buffer to form a first single reactant film on the substrate. The second dye may be a fluorescent polymeric dye. The single reactant film may be a homogeneous film comprising the plurality of reactants. The plurality of reactants may include 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 2-20, 3-18, or 4-12 different reactants, each comprising a different binding partner dye conjugate. The dye may be a fluorescent dye. The fluorescent dye may be a fluorescent polymer dye. For example, each reactant may be a unique fluorescent dye conjugate. The plurality of reactants may comprise two or more unique fluorescent polymer dye conjugates. The substrate may be a tube, a well, a membrane, or a bead. The substrate may include an inner surface of a reaction vessel.

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

[0032] The buffer compositions described in this disclosure allow for drying of different fluorescent dye conjugates in a single tube to provide a uniform film without altering their functionality (i.e., affinity for the analyte) and physical properties (such as brightness or fluorescence), while avoiding aggregation and the resulting false positive populations in other channels. [Brief description of the drawings]

[0033] [Figure 1A] Figure 1A shows flow cytometry density plots of CD4-PE conjugates when dried using conventional drying techniques using reagent buffers, demonstrating the desired functionality (affinity for antigen), physical properties (such as brightness or fluorescence), and ability to separate cell populations.

[0034] [Figure 1B] Figure IB shows flow cytometry density plots of CD20-APC conjugates when dried using conventional drying techniques using reagent buffers, exhibiting desirable functional (affinity for antigen) and physical properties (such as brightness or fluorescence) and the ability to separate cell populations.

[0035] [Figure 2A] Figure 2A shows flow cytometry two-dimensional dot plots of CD20-SNv605 and CD4-SNv786 polymer dye conjugates when mixed and dried using conventional drying technique reagent buffer without compensation, showing undesirable results and failure to separate populations in the x and y axes.

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

[0037] [Figure 3A] Figure 3A shows flow cytometry two-dimensional dot plots of the dry-down of CD20-SNv605 and CD4-SNv786 polymer-dye conjugates in DM2 with stabilizer without compensation (DM2+S) DM buffer. Leakage of the 610 fluorescence channel is observed in the 780 fluorescence channel.

[0038] [Figure 3B] Figure 3B shows two-dimensional dot plots of flow cytometry of the dry down of CD20-SNv605 and CD4-SNv786 polymer dye conjugates in the DM buffer of the present invention, DM2+S, with compensation. DM2+S DM buffer is capable of separating cell populations without any non-specific interactions.

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

[0040] [Diagram 5] FIG. 5 shows flow cytometry plots of casein titration in DM using CD20-SNv605 to measure percentage of recruitment and demonstrate reduction in non-specific events.

[0041] [Figure 6] Figure 6 shows two-dimensional fluorescence dot plots of the dried formulations for the 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 400 μg of monomer A per test.

[0042] [Figure 7A] Figure 7A shows the scatter flow plots for dose optimization of DMMA. Figure 7A shows unstained (top left), dry DM alone (second from left, top panel), 0.004% dry DMMA in DM (third from left, top panel), 0.008% dry DMMA in DM (top right panel), 0.018% dry DMMA in DM (bottom left panel), 0.021% dry DMMA in DM (bottom center panel), and 0.03% dry DMMA in DM (bottom right panel). 0.004% and 0.008% DMMA show less nonspecific neutrophil pullout (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), dry DM alone (second from left, top panel), dry DMMA 0.004% in DM (third from left, top panel), dry DMMA 0.008% in DM (top right panel), dry DMMA 0.018% in DM (bottom left panel), dry DMMA 0.021% in DM (bottom center panel), and dry DMMA 0.03% in DM (bottom right panel). DMMA at 0.004%, 0.008% and 0.018% shows less nonspecific monocyte pullout (indicated by arrows) in the 610 channel compared to DM and other DMMA concentrations.

[0044] [Figure 7C] FIG. 7C shows dot plots of HLA DR-786 for all formulations for dose optimization of DMMA in DM, including unstained (top left), dry DM alone (second from left, top panel), 0.004% dry DMMA in DM (third from left, top panel), 0.008% dry DMMA in DM (top right panel), 0.018% dry DMMA in DM (bottom left panel), 0.021% dry DMMA in DM (bottom center panel), and 0.03% dry DMMA 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 (top left panel), 0.004% dry DMMA in DM (top center panel), 0.008% dry DMMA in DM (top right panel), 0.018% dry DMMA in DM (bottom left panel), 0.021% dry DMMA in DM (bottom center panel), and 0.03% dry DMMA in DM (bottom 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 left, top panel), DMMA 0.008% (top right), DMMA 0.008% + casein 2.5x (bottom left), DMMA 0.008% + Prionex 2 dil (bottom center panel), and DMMA 0.008% + Prionex 3 dil (bottom right). Scattering appears similar for each of the combinations, with the exception of DM, which shows pullout of nonspecific granulocytes and monocytes, as indicated by the arrows.

[0047] [Figure 8B] FIG. 8B shows dot plots of CD20-SNv605 for additive combinations with 0.008% DMMA, including single CD20 (top left), dry DM (second from the left, top panel), DMMA 0.008% (top right), DMMA 0.008% + casein 2.5× (bottom left), DMMA 0.002% + 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 left, top panel), DMMA 0.008% (top right), DMMA 0.008% + casein 2.5x (bottom left), DMMA 0.008% + Prionex 2 dil (bottom center panel), and DMMA 0.008% + Prionex 3 dil (bottom right). The % recruitment of HLADR+ was found to be similar in all combinations with 0.008% DMMA compared to the individual liquid singles.

[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 (top left panel), DMMA 0.008% (top right), DMMA 0.002% + casein 2.5x (bottom left), DMMA 0.008% + Prionex 2 dil (bottom center panel), and DMMA 0.008% + Prionex 3 dil (bottom right). The % recruitment of double positive populations in all combinations with 0.008% DMMA was found to be similar compared to DM.

[0050] [Figure 9] FIG. 9 shows the physical appearance and properties of the dried tubes in each of the test groups DM2+S, trehalose+monomer, trehalose+casein, trehalose+DMMA (left to right, top panel), DM2+S, DM2+S without monomer, DM2+S without DMMA, and DM2+S without casein (left to right, bottom panel). Here, DM2+S serves as the control group. Physical observation shows that without monomer, there is a change in the color of the dried film (typically the red film becomes light orange to brown). Shrinkage of the film was observed in the groups without casein and DMMA. No change in the appearance of the dried film was observed in the tube without DMMA compared to DM2+S. However, the tube without casein shows minimal shrinkage of the dried film.

[0051] [Figure 10A]FIG. 10A shows side scatter SSC vs. FL plots for CD56-SNv428 in 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, trehalose+monomer (left to right, lower panel). In the absence of casein and DMMA, there is a non-specific monocyte pullout (indicated by arrows). The absence of monomer causes a negative population spread in lymphocytes (indicated by arrows). The negative population spread is mainly due to non-specific interactions between SN dyes in the absence of monomer and casein.

[0052] [Figure 10B] FIG. 10B shows side scatter SSC vs. FL plots for CD20-SNv605 in 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, trehalose+monomer (left to right, lower panel). In the absence of casein, there is a pullout of nonspecific monocytes (indicated by arrows). The absence of monomer causes a spread of negative population in lymphocytes (indicated by arrows). The spread of negative population is mainly due to nonspecific interactions between SN dyes in the absence of monomer and casein.

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

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

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

[0056] [Figure 10F] Figure 10F shows two-dimensional flow plots for CD4-SNv786 versus CD56-SNv428 in test groups DM2+S, DM2+S without DMMA, DM2+S without casein, DM2+S without monomer (left to right, top panel), trehalose+DMMA, trehalose+casein, trehalose+monomer (left to right, bottom panel). Absence of monomer and casein causes non-specific interactions / population spreading (indicated by arrows) between SN populations. Therefore, both monomer and casein are important to prevent non-specific interactions.

[0057] [Figure 11A]Figure 11A shows representative SSC vs. FL overlay flow plots for three polymer-dye conjugates CD56-SNv428, CD20-SNv605, and CD4-SNv786, including gating on CD45-APC-A750, drying in a cocktail with the DM2+S dry-down buffer of the present invention, and reconstitution with blood samples. In a second group, liquid cocktails of the same antibodies were prepared using commercially available BD Horizon™ Brilliant staining buffer. BD Horizon™ Brilliant staining buffer caused a pull-out of non-specific granulocytes and monocytes (indicated by arrows) compared to the DM2+S dry-down tubes.

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

[0059] [Figure 12] FIG. 12 shows a photograph of a 6-month old dried tube of DM2+S buffer in an open pouch.

[0060] [Figure 13A]FIG. 13A shows the 6-month stability of fresh, 3-month-old and 6-month-old DM2+S dried tubes. Representative two-dimensional overlay flow plots for different combinations of SN dyes in each of the three lots are shown. The dried 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 on four donors in a single replicate on one flow cytometer instrument. The stain-lyse-wash protocol described in Protocols and Methods was used for the study. These overlays show that the populations in the 3-month- and 6-month-aged lots completely overlap with the fresh lot. In addition, no non-specific interactions or population spreading were observed in the 6-month-aged lot in all tested donors.

[0061] [Figure 13B] Figure 13B shows the 6-month stability of DM2+S dried tubes compared to 3-month and fresh lots: representative two-dimensional overlay flow plots for SN vs. classical combinations in all three lots. Dried tubes containing a 12-color (12C) panel (9C conventional conjugates + 3C SN conjugates CD56-SNv428, CD20-SNv605, CD4-SNv786) were tested on four donors in a single replicate on one flow cytometry instrument. Overlay flow plots for 6-month and 3-month aged lots and fresh lots for CD3 ECD-A vs. CD20Violet610-A (left panel), CD8 KO525-A vs. CD4Violet780-A (middle 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 lot. Additionally, no non-specific interactions or population spreading were observed in the 6-month old lots across all test donors.

[0062] [Figure 13C] Figure 13C shows representative two-dimensional overlay flow plots for 6-month stability of DM2+S dried tubes compared to 3-month and fresh lots: classical vs. classical combinations in all three lots. Dried tubes containing a 12-color (12C) panel (9C conventional conjugates + 3C SN conjugates CD56-SNv428, CD20-SNv605, CD4-SNv786) were tested on four donors in a single replicate on one flow cytometry instrument. Overlays for 6-month and 3-month aged lots with fresh lots for CD16 FITC-A vs. CD25PE-A (left panel), CD8 KO525-A vs. CD45APC-A750-A (middle 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 the fresh lot. Additionally, no non-specific interactions or population spreading were observed in the 6-month old lots across all test donors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] Detailed Description of the Disclosure The present disclosure provides compositions and methods for minimizing the aggregation of two or more dye conjugates when they are dried down together. Dry-down buffer compositions are provided that help maintain the integrity of fluorescent dye structures in mixtures of fluorescent dye conjugates, reduce aggregation, and reduce non-specific interactions and staining artifacts. For example, the dry-down buffer compositions can be used to reduce or prevent the 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, or while drying the cocktail of fluorescent dye conjugates, or during reconstitution. The present invention solves the problem by providing a dry-down buffer composition to reduce or avoid aggregation, for example, so that the fluorescent polymer-dye conjugates do not interact during drying. Upon reconstitution, the fluorescent dye conjugates can independently bind to target analytes in the liquid sample being analyzed, and erroneous results resulting from aggregation or cross-linking are substantially reduced or eliminated.

[0065] A novel dry-down buffer was developed that can reduce or eliminate non-specific interactions of fluorescent dyes, and also does not interfere with the natural binding ability of the fluorescent dye conjugates to the antigen of interest.

[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 processed 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 the polymer-dye conjugates dried down using the conventional dry-down technique failed to separate the cell populations, while Figure 3B shows that the DM buffer DM2+S of the present invention was able to separate the cell populations without any non-specific interactions.

[0067] definition

[0068] As used herein, the following terms and variations thereof have the meanings indicated below, unless a different meaning is clearly intended by the context in which such term is used.

[0069] The singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0070] The term "and / or" refers to and includes every possible combination of one or more of the associated listed items.

[0071] The term "about," when referring to a measurable value such as an amount of a compound, dosage, time, temperature, etc., is meant to encompass variations of 10%, 5%, 1%, 0.5%, or even 0.1% of the stated value and at least industry standard variations in the testing method for measuring the value.

[0072] The terms "patient," "subject," or "subjects" include, but are not limited to, humans, and may also include other mammals, or domestic or exotic animals, such as dogs, cats, ferrets, rabbits, pigs, horses, cows, birds, or reptiles.

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

[0074] Unless otherwise specified, the term "percent" or "%" refers to percent by weight.

[0075] The term "activated ester" or "active ester" by itself or as part of another substituent refers to a carboxyl active group 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 textbooks of peptide chemistry, such as 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] The term "acyl" as used herein refers to a group containing a carbonyl moiety, where the group is bonded through the carbonyl carbon atom. The carbonyl carbon atom is either bonded to a hydrogen to form a "formyl" group or bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group, and the like. The acyl group can contain 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. The acyl group can contain double or triple bonds within the meaning of the present specification. The acyl group can also contain heteroatoms, as appropriate, within the meaning of the present specification. Examples of acyl groups include, but are not limited to, nicotinoyl groups (pyridyl-3-carbonyl), acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloxy groups. When the group containing the carbon atom bonded to the 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" by itself or as part of another substituent refers to a chemical compound that contains a --CHO group.

[0078] The terms "alkene" or "alkenyl" by themselves or as part of another substituent refer to either a straight-chain, branched-chain, 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-hexatrienyl. Alkene groups are typically monovalent but can be divalent, for example, when an alkenyl group links two moieties together.

[0079] The term "alkoxy" by itself or as part of another substituent refers to an alkyl group, as defined above, with an oxygen atom connecting the alkyl group to its point of attachment. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, and the like. Alkoxy groups can be further substituted with a variety of substituents described herein. For example, alkoxy groups can be substituted with halogens to form "halo-alkoxy" groups.

[0080] The term "alkyl" by itself or as part of another substituent refers to a straight or branched, saturated aliphatic radical having the number of carbon atoms indicated. The alkyl group can be an optionally substituted alkyl group. For example, C1-C6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, and the like. Alkyl can contain any number of carbons, 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, when the alkyl group links two moieties together.

[0081] The term "alkyne" or "alkynyl" by itself or as part of another substituent refers to either a straight-chain or branched hydrocarbon 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, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynyl groups are typically monovalent but can be divalent, for example, when the alkynyl group links two moieties together.

[0082] The term "analyte" refers to a molecule, compound, or other component 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" by itself or as part of another substituent, as used herein, refers to an alkyl group, as defined herein, having one or more amino groups. The amino groups may be primary, secondary, or tertiary. The alkylamines may be further substituted with hydroxy groups. Amines useful in the present disclosure include, but are not limited to, ethylamine, propylamine, isopropylamine, ethylenediamine, and ethanolamine. The amino group may link the alkylamine to the point of attachment to the remainder of the compound, may be at the omega position of the alkyl group, or may be linked together at at least two carbon atoms of the alkyl group. Those skilled in the art will recognize that other alkylamines are useful in the present disclosure.

[0084] The term "amino group" refers to the -NR3 group, which cannot be protonated. + Except for -NH2, -NHR, -NR2, -NR3+ where each R is independently selected) and each protonated form of the substituent. Thus, any compound substituted with an amino group can be considered an amine. An "amino group" within the meaning of this specification can be a primary, secondary, tertiary, or quaternary amino group. An "alkylamino" group includes monoalkylamino, dialkylamino, and trialkylamino groups.

[0085] The term "amide" refers to a functional group having a carbonyl group attached to an amine group, having the general formula RC(=O)NR'R'', where R, R', and R'' represent organic groups or hydrogen atoms. The term "amido" refers to a substituent that contains an amide group.

[0086] The term "ammonium" by itself or as part of another substituent has the formula NHR3 + where each R group is independently 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 the various classes and isotypes of immunoglobulins, e.g., 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 with additions or substitutions, such as chimeric antibodies. Antibodies may be derived from human or animal origin, from hybridomas by recombinant methods, or 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 with a bond to an aryl group, as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups, as well as fused (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 with a bond to an aryl group, as defined herein.

[0089] The term "aryl" 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. An "aryl" group can be a monocyclic, or a fused bicyclic, tricyclic or higher cyclic aromatic ring assembly containing from 6 to 16 ring carbon atoms. For example, an aryl can be, but is not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, benzyl, or naphthyl. "Arylene" means a divalent radical derived from an aryl group. The aryl group may be mono-, di- 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 are optionally further substituted, for example as defined herein below, or 1- or 2-naphthyl, or 1- or 2-phenanthrenyl. Alkylenedioxy is a divalent substituent attached to two adjacent carbon atoms of phenyl, for example methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent attached to two adjacent carbon atoms of phenyl, for example oxyethylene or oxypropylene. An example of oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0090] The term "aryloxy" by itself or as part of another substituent refers to an O-aryl group, where aryl is as defined above. An aryloxy group can be unsubstituted or substituted with one or two suitable substituents. The term "phenoxy" refers to an aryloxy group, where the aryl moiety 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 moiety is either an aryloxy or a (hetero)aryloxy group.

[0091] The term "heteroaryl" by itself or as part of another substituent refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, in which 1 to 4 of the ring atoms are heteroatoms, N, O or S, respectively. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radical, in particular mono- or di-substituted, for example, by alkyl, nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously 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. Benzopyranyl, benzothiopyranyl preferably represent 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl preferably represents 2- or 4-thiazolyl, most preferably 4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

[0092] In some embodiments, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any of the substituted, particularly mono- or di-substituted, radicals.

[0093] In some embodiments, the substituents on the aryl and heteroaryl groups are varied and include -halogen, -OR', -OC(O)R', -NR'R'', -SR', -R', -CN, -NO2, -COR', -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, -N 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 an aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CH2) q -U-, where T and U are independently -NH-, -O-, -CH2- or a single bond, and q is an integer from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2) rOptionally, one of the single bonds of the new ring thus formed may be replaced with a substituent of the formula -B-, where 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. Optionally, one of the single bonds of the new ring thus formed may be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be replaced with a substituent of the formula -(CH2) s -X-(CH2) t - (wherein s and t are independently an integer of 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-) The substituent R' in -NR'- and -S(O)2NR'- is selected from hydrogen or unsubstituted (C1-C6) alkyl.

[0095] As used herein, the term "azide" by itself or as part of another substituent refers to the 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 an antibody or an antigen-binding fragment thereof, or other protein, peptide, polysaccharide, lipid, nucleic acid, or nucleic acid analog, such as an oligonucleotide or PNA (peptide nucleic acid).

[0097] The term "boronic acid" by itself or as part of another substituent refers to the structure -B(OH). It will be recognized by those of skill in the art that boronic acids may be present as boronic esters at various steps in the synthesis of a quencher. Boronic acid is meant to include such esters. The term "boronic ester" or "boronate ester" as used herein refers to -B(Z 1 )(Z 2 ) part (in the formula, Z 1 and Z 2refers to a chemical compound containing the boronic ester moiety, where the atoms bonded to the boron in each instance together form a moiety that is an oxygen atom. In some embodiments, the boronic ester moiety is a 5-membered ring. In some other embodiments, the boronic ester moiety is a 6-membered ring. In some other embodiments, the boronic ester moiety is a mixture of 5- and 6-membered rings.

[0098] The term "carbamate" by itself or as part of another substituent refers to a functional group having the structure -NR''CO2R', where 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, benzyloxy-carbonyl, alloc, methyl carbamate, ethyl carbamate, 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl 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" by itself or as part of another substituent refers to the structure R-COOH, where R is a carbon-containing group of atoms.

[0100] The term “carboxylate” by itself or as part of another substituent refers to a conjugate base of a carboxylic acid, which generally has the formula RCOO -For example, the term "magnesium carboxylate" refers to a magnesium salt of a carboxylic acid. The term "carboxylic acid ester" as used herein 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', where R' can be an alkyl, alkene, alkyne, haloalkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, (unsubstituted aryl) alkyl, and (unsubstituted aryl) oxy-alkyl or other carbon-containing group of atoms. R' optionally contains a functional group.

[0101] The term "CD" refers to cluster of differentiation.

[0102] The term "chromophore" refers to a compound having a reactive group (e.g., a carboxylate moiety, an amino moiety, a haloalkyl moiety, etc.) that can be covalently attached. Examples of suitable chromophores include, but are not limited to, those described in U.S. Patents 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 36; 5,863,753; 5,846,737; 5,798,276; 5,723,218; 5,696,157; ​​5,658,751; 5,656,449; 5,582,977; 5,576,424; 5,573,909; and 5,187,288, which are incorporated by reference herein in their entireties.

[0103] The term "compensation" in flow cytometry is the mathematical process of correcting for fluorescence leakage (spectral overlap of multiparameter flow cytometry data). For example, compensation can be performed by removing the signal of any given fluorochrome from all detectors except those dedicated to measuring that dye. Because fluorochromes can have a wide range of spectra, they can overlap and cause undesirable confusion during data analysis.

[0104] The term "cycloalkyl" by itself or as part of another substituent refers to saturated or partially unsaturated monocyclic, fused bicyclic or bridged polycyclic ring assemblies containing 3 to 12 ring atoms, or the number of atoms indicated in the monocyclic ring, including, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and polycyclic rings include, for example, norbornane, decahydronaphthalene, and adamantane. For example, C3-8 cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

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

[0106] The term "DM-2" or "DM2" refers to DM-2. The term "S" refers to the stabilizer in reference to the "DM2+S" buffer. "DM2+S" is a DM buffer as described herein, where the carbohydrate stabilizer is trehalose or a hydrate thereof, the water-soluble monomer is monomer A, the protein stabilizer is casein, and the zwitterionic surfactant is DMMA, e.g., according to Table 3.

[0107] The term "dye conjugate" refers to a dye conjugated to a binding partner.

[0108] The term "fluorochrome" refers to a dye that contains a light-excitable fluorophore that can be re-emitted upon light excitation. The term "fluorochrome" encompasses both fluorescent polymeric and non-polymeric dyes, including both fluorescent monomeric dyes and other conventional fluorescent 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, 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 an excitation maximum at 414 nm. With emission peaks for SN v605 and SN v786 at 605 nm and 786 nm, respectively, they are optimally detected using 610 / 2 and 780 / 60 nm bandpass filters on the flow cytometer.

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

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

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

[0112] As used herein, the term "hydrazone" by itself or as part of another substituent means a compound of the structure [ka] where R may be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other, and may contain a carboxyl group. R may be a water solubilizing polymer, including but not limited to, a polymer containing six or more monomeric units, a non-ionically water soluble polymer, PEG, a modified PEG terminated with a carboxylic acid or ester.

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

[0114] The term "hydrocarbon" or "hydrocarbyl" refers to a molecule or functional group that contains carbon and hydrogen atoms. The term typically contains both carbon and hydrogen atoms, but can also refer to molecules or functional groups in which some or all of the hydrogen atoms have been replaced with other functional groups. The term "hydrocarbyl" refers to a functional group derived from a straight chain, branched or cyclic hydrocarbon, and can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. A hydrocarbyl group is one of the groups represented by the formula (C a ~C b )hydrocarbyl, where a and b are integers, meaning that the group 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, in certain embodiments, means that there are no hydrocarbyl groups present. Hydrocarbylene groups are diradical hydrocarbons, e.g., hydrocarbons bonded at two places.

[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 moiety that connects two groups and has a backbone of 100 or less atoms in length. The linker or linkage may be a covalent bond connecting two groups, or may be a chain of between 1 and 100 atoms in length, for example, a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, or more carbon atoms in length, where the linker may be linear, branched, cyclic, or a single atom. In some embodiments, the linker is a branched linker, which refers to a linking moiety that connects three or more groups. In certain cases, one, two, three, four, or five, or more carbon atoms of the linker backbone may be substituted with sulfur, nitrogen, or oxygen heteroatoms, as appropriate. In some embodiments, the linker backbone includes a linking functional group, such as ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester, or imine. The bond between backbone atoms can be saturated or unsaturated, and in some cases, there are not more than one, two, or three unsaturated bonds in the linker backbone. The linker can include one or more substituents, such as alkyl, aryl, or alkenyl groups. The linker can include, but is not limited to, polyethylene glycol, ether, thioether, tertiary amine, alkyl, which can be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, such as an aryl, heterocycle, or cycloalkyl group, where two or more atoms of the cyclic group, such as 2, 3, or 4 atoms, are included in the backbone. The linker may be cleavable or non-cleavable.

[0117] The linker moiety can be attached to "L" or "A" as taught in U.S. Patent Application Publication No. 2020 / 0190253A1, the entirety of which is incorporated herein by reference. The linker moiety can include a sulfonamide, disulfonamide, selenomide, sulfinamide, sultam, disulfinamide, amide, seleninamide, phosphonamide, phosphinamide, phosphonamidate, or secondary amine.

[0118] As described herein, and each in relation to a linker moiety, the term "sulfonamide" refers to the moiety -S(O)NR-; the term "disulfonamide" refers to the moiety -S(O)NRS(O)-; the term "selenoneamide" refers to the moiety -Se(O)NR-; the term "sulfinamide" refers to the moiety -S(O)NR-; the term "disulfinamide" refers to the moiety -S(O)NRS(O)-; and the term "seleninamide" refers to the moiety - The term "phosphonamide" refers to the moiety -NR-PR(O)NR-; the term "phosphinamide" refers to the moiety -PR(O)NR-; the term "phosphonamidate" refers to the moiety -O-PR(O)NR-; the term "sultam" refers to cyclic sulfonamides (e.g., the R group is attached to the 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" by itself or as part of another substituent means the structure [ka] where R may be, for example, a water-solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other, and may contain a carboxyl group. R may be a water-solubilizing polymer, including, but not limited to, a polymer containing six or more monomeric units, a non-ionically water soluble polymer, PEG, a modified PEG terminated with a carboxylic acid or carboxylate ester.

[0120] The term "reactant solution" refers to a solution that includes a labeled binding partner. In some embodiments, in addition to a labeled binding partner, the reactant solution further includes stabilizers, salts, buffers, detergents, and / or other reagents. The term "maleimide" by itself or as part of another substituent refers to the structure [ka] where R may be, for example, a water-solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other, and may contain a carboxyl group. R may be a water-solubilizing polymer, including, but not limited to, a polymer containing six or more monomeric units, a non-ionically water soluble polymer, PEG, a modified PEG terminated with a carboxylic acid or carboxylate ester.

[0121] The terms "MdFl" or "MDFl" refer to median fluorescence intensity.

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

[0123] The term "multicolor antibody panel" refers to a cocktail containing multiple different fluorochrome conjugates (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.) in liquid or dry format that can be used directly to stain blood and analyze it in a flow cytometer.

[0124] The term "multi-color dry reagent" refers to a cocktail of different fluorescent dye conjugates (CD4-FITC, CD8-PE, CD20-APC, CD3-PC5.5, etc.) in a dry format that can be directly used to stain blood and analyze it in a flow cytometer. Multi-color dry reagent cocktails with 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 were found to be ineffective while drying multiple fluorescent polymer-dye antibody conjugates in a cocktail. These polymer-dye conjugates tend to interact non-specifically, leading to difficulties in the separation of populations, which can lead to challenges in the identification of the desired cell population in a given sample.

[0125] As used herein, the term "multiplexed" refers to an assay or other analytical method in which multiple analytes can be assayed simultaneously.

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

[0127] The term "organic group" refers to any carbon-containing functional group. Examples of carbon-containing functional groups may include oxygen-containing groups such as alkoxy groups, aryloxy groups, aralkyloxy groups, oxo (carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylic 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), CN, CF, OCF, R, C(O), methylenedioxy, ethylenedioxy, N(R), SR, SOR, SO, SO, N(R), SO, C(O)R, C(O)C(O)R, C(O)CHC(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R), OC(O)N(R), C(S)N(R), (CH) 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, where R can be hydrogen (in instances containing other carbon atoms) or a carbon-based moiety, which can be substituted or unsubstituted.

[0128] The term "PEG" has the formula -(CH2-CH2-O-) n- 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. It is understood that the PEG polymer group can be of any convenient length and can include a variety of end groups and / or further substituents, including, but not limited to, alkyl, aryl, hydroxyl, amino, acyl, carboxylic acid, carboxylic ester, acyloxy, and amide end groups and / or substituents. The number following "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 milliQ water or deionized water, and 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4. The pH may be about pH 7.0-7.4. PBS may or may not be preserved with an azide, such as sodium azide. PBS is an isotonic solution.

[0130] The term "phosphoramide" by itself or as part of another substituent means a compound of the structure [ka] where R may be, for example, a water-solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other, and may contain a carboxyl group. R may be a water-solubilizing polymer, including, but not limited to, a polymer containing six or more monomeric units, a non-ionically water soluble polymer, PEG, a modified PEG terminated with a carboxylic acid or carboxylate ester.

[0131] The term "phosphonamidate" by itself or as part of another substituent means the structure [ka] where R may be, for example, a water-solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other, and may contain a carboxyl group. R may be a water-solubilizing polymer, including, but not limited to, a polymer containing six or more monomeric units, a non-ionically water soluble polymer, PEG, a modified PEG terminated with a carboxylic acid or carboxylate ester.

[0132] The term "phosphinamide" by itself or as part of another substituent means the structure [ka] where R may be, for example, a water-solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other, and may contain a carboxyl group. R may be a water-solubilizing polymer, including, but not limited to, a polymer containing six or more monomeric units, a non-ionically water soluble polymer, PEG, a modified PEG terminated with a carboxylic acid or carboxylate ester.

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

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

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

[0136] The term "substituted" refers to a molecule or molecule as defined herein. When used herein in conjunction with an organic group, refers to a state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term "functional group" or "substituent" as used herein refers to a group that may be on a molecule or an organic group or may be substituted thereon. 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 carboxylate 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, hydroxylamine, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various groups. Non-limiting examples of substituents which may be attached to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R), CN, NO, NO, ONO, azido, CF, OCF, R, O(oxo), S(thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R), SR, SOR, SO, SO, N(R), SO, R, C(O)R, C(O)C(O)R, C(O)CHC(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R), OC(O)N(R), C(S)N(R), (CH) 0~2 N(R)C(O)R, (CH2) 0~2 N(R)N(R), N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R), N(R)SOR, N(R)SON(R), N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R), N(R)C(S)N(R), N(COR)COR, N(OR)R, C(=NH)N(R), C(O)N(OR)R, and C(=NOR)R, where R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (C1-C 100) hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or the two R groups attached to or adjacent to the nitrogen atom can, together with the nitrogen atom or atoms, form a heterocyclyl.

[0137] The term "sulfonate functional group" or "sulfonate," either by itself or as part of another substituent, refers to both the free sulfonate anion (-S(=O)2O-) and its salts. Thus, the term sulfonate includes sulfonate salts, e.g., sodium, lithium, potassium, and ammonium sulfonates.

[0138] The term "sulfonamide" by itself or as part of another substituent refers to a group of formula -SONR-, where R can be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other, and can contain a carboxyl group. R can be a water solubilizing polymer, including, but not limited to, a polymer containing six or more monomeric units, a non-ionically water soluble polymer, a PEG, a modified PEG terminated with a carboxylic acid or carboxylate ester.

[0139] The term "sulfonamide" by itself or as part of another substituent refers to a group of formula -SO2NR2, where R can be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other, and can contain a carboxyl group. R can be a water solubilizing polymer, including, but not limited to, a polymer containing six or more monomeric units, a non-ionically water soluble polymer, PEG, a modified PEG terminated with a carboxylic acid or carboxylate ester.

[0140] The term "sulfinamide" by itself or as part of another substituent refers to a group of formula -SONR2-, where R can be, for example, a water solubilizing moiety, hydrogen, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, aryl, or other, and can contain a carboxyl group. R can be a water solubilizing polymer, including, but not limited to, a polymer containing six or more monomeric units, a non-ionically water soluble polymer, a PEG, a modified PEG terminated with a carboxylic acid or carboxylate ester.

[0141] The term “silyl” by itself or as part of another substituent means Si(R z )3 (wherein each R z are independently alkyl, aryl, or other carbon-containing groups of atoms.

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

[0143] The term "water-solubilizing moiety", as used herein 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, to increase the water solubility of the molecule to which it is attached. The increase in water solubility of a molecule can be highly dependent on the moiety to which it is attached. In some cases, the increase in water solubility (compared to the solubility of the molecule without the moiety to which it is attached) is 2-fold or more, 5-fold or more, 10-fold or more, 25-fold or more, 50-fold or more, or 100-fold or more. "Water solubilizing moieties" include moieties such as, but are not limited to, PEG groups, carboxy groups including, but not limited to, carboxylic acids and carboxylates, polyvinyl alcohols, glycols, peptides, polyphosphates, polyalcohols, sulfonates, phosphonates, boronates, amines, ammonium, sulfonium, phosphonium, alcohols, zwitterionic derivatives, carbohydrates, nucleotides, polynucleotides, substituted PEG groups, substituted carboxy groups including, but not limited to, substituted carboxylic acids and substituted carboxylates, substituted glycols, substituted peptides, substituted polyphosphates, substituted polyalcohols, substituted sulfonates, substituted phosphonates, substituted boronates, substituted amines, substituted ammonium, substituted sulfonium, substituted phosphonium, alcohols, substituted zwitterionic derivatives, substituted carbohydrates, substituted nucleotides, substituted polynucleotides, 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 more, for example, 3 mg / mL or more, 10 mg / mL or more, 20 mg / mL or more, 30 mg / mL or more, 40 mg / mL or more, 50 mg / mL or more, 60 mg / mL or more, 70 mg / mL or more, 80 mg / mL or more, 90 mg / mL or more, 100 mg / mL or more, or even more. It is understood that water-soluble polymers can form separate water-hydrated nanoparticles in aqueous systems under certain conditions and can resist aggregation. Reaction vessel

[0145] The "reaction vessel" disclosed herein can be any container in which the reaction between the binding partner or its dye conjugate and the target analyte can occur. For example, the reaction vessel can be a tube, a plate, a well of 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 includes one or more substrates. The "substrate" can be any suitable surface, including but not limited to plastic, nitrocellulose, cellulose acetate, quartz, and glass. Non-limiting examples of plastics can include polystyrene, polypropylene, cyclo-olefins, and polycarbonates. In some embodiments, the substrate is a membrane. The substrate can be the inner surface of the body of the reaction vessel, e.g., a plastic tube or a well of a microtiter plate. The substrate can also be beads. In some embodiments, at least one of the substrates that receives the labeled binding partner (e.g., a membrane) is attached to the inner surface of the body of the reaction vessel. In some embodiments, the membrane substrate is a sheet or roll, which makes it easier to deposit the solution and easier to dry. In some embodiments, the membrane can be cut to separate the 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 attached to the surface of the reaction vessel such that the membrane does not escape from the vessel when liquid is pipetted into or out of the reaction vessel.

[0147] The acronym "SN" refers to SuperNova™.

[0148] The acronym "SSC" refers to side scatter.

[0149] The term "WBC" refers to white blood cells. Liquid Samples

[0150] The reaction vessel is configured to receive a liquid sample. The liquid sample used in the present invention typically contains a target analyte obtained as or dispersed in a primarily aqueous medium. The sample can be any source of biological material, such as proteins, carbohydrates, or polynucleotides, that can be obtained directly or indirectly from an organism. Samples can include, for example, cells, tissues, or fluids, as well as deposits left by the organism, including viruses, mycoplasma, and fossils. The sample may also contain a target analyte that has been prepared in whole or in part by synthetic means. Non-limiting examples of samples include blood, serum, plasma, urine, semen, milk, sputum, mucus, buccal swabs, vaginal swabs, rectal swabs, aspirates, needle biopsies, sections of tissue obtained, e.g., by surgery or dissection, plasma, serum, cerebrospinal fluid, lymphatic fluid, external secretions of the skin, respiratory, intestinal, and genitourinary tract, tears, saliva, tumors, organs, samples of in vitro cell culture constituents (including, but not limited to, conditioned media resulting from growth of cells in cell culture media, putative virally 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 a target molecule, such as peptides, proteins, polynucleotides, organic molecules, sugars and other carbohydrates, and lipids, to be detected in a biological sample. It is an important aspect of the present invention that the target analyte is contained in a liquid sample and is accessible or becomes accessible at some point to bind to the analyte-specific binding partner of the present invention. The target analyte may be found in a biological sample, such as a blood sample, a cell line development sample, a tissue culture sample, etc.

[0152] The target analyte may be present on the bead or may be present and accessible on the surface of the cell. Examples of useful analytes include, but are not limited to: 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 that contain abnormal DNA or RNA sequences, or abnormal amounts of certain messenger RNA. The detection of these analytes is particularly useful in situations where they are contained in and / or are identifiers of rare cells, such as those found in the early stages of various cancers. Binding partners

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

[0154] Binding partners other than antibodies or antibody fragments or derivatives can also be used in the present system and method. For example, the binding partner can be a nucleic acid or a nucleic acid analog, such as an oligonucleotide or a 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 preselected 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, folate and folate binding protein, vitamin B12 and intrinsic factor, protein A and immunoglobulin, and protein G and immunoglobulin. Binding partners that form covalent bonds with the target analyte are also included. pigment

[0155] A "dye" is a moiety that provides a detectable signal, which may be bound to or incorporated into a binding partner, either directly or indirectly. The dyes used in the present invention may be colored, fluorescent, or luminescent, and are typically detected by a detector, such as a PMT or APD, in a flow cytometer. Fluorescent dyes may be monomeric or polymeric. Non-limiting examples of monomeric dyes include fluorescein, rhodamine, and cyanine. For example, commonly used monomeric dye fluorescent dyes may 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 (phytoplankton-derived carotenoid-protein complex) (excitation maximum 482 nm / emission maximum 678 nm), 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. The fluorescent dye may be a tandem dye. Tandem dyes may 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. They are highly responsive optical reporters and efficient light absorbers thanks to the multiple chromophores they contain. Examples of fluorescent polymeric dyes include, but are not limited to, conjugated polymers with repeating units of chromophores, conjugated molecular aggregates, luminescent dyes attached to saturated polymers through side chains, semiconductor quantum dots, and dendritic structures. The fluorescent polymeric and monomeric dyes disclosed in U.S. Pat. Nos. 7,214,489, 8,354,239, and 8,575,303 can also be used for this application.

[0157] In some cases, the fluorescent dye has 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 moiety; each M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene, and ethynylene; G 1 and G 2 is independently selected from an unmodified polymer end and a modified polymer end; a, c, and d independently define the mole % of each unit within the structure, and each unit may be repeated evenly or randomly, where each a is a mole % of 10-100%, each c is a mole % of 0-90%, and each d is a mole % of 0-25%; each b is independently 0 or 1; m is an integer from 1 to about 10,000. The dye may also be a fluorescent polymer dye having the structure described in

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

[0159] The fluorescent polymer dye may be conjugated to a binding partner to form, for example, a fluorescent polymer dye conjugate having a monomer A subunit and a monomer B subunit, as described, for example, in US2020 / 0190253, which is incorporated herein by reference. The fluorescent polymer dye conjugate may be represented by 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 is a linker moiety; W is a water-solubilizing moiety; each E is independently a selected chromophore, functional moiety, or binding partner; each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene, and ethynylene; G 1 and G 2 is independently selected from an unmodified polymer end and a modified polymer end; the subscripts n and m are independently integers ranging from 1 to 10,000; The subscript p is an integer ranging from 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 1 or 2, The sum of the subscripts r and s ranges from 1 to 4; A and B are randomly or non-randomly distributed in the conjugated polymer. The compound may have the structure:

[0160] The fluorescent dye may be a fluorescent polymer dye having water-soluble monomer A subunits and monomer B subunits. The polymer dye may be a water-soluble fluorescent polymer dye. For example, monomer A or monomer B may include a dihydrophenanthrene (DHP) moiety. Monomer A or monomer B may include a fluorene moiety. In some conjugated polymer dyes, monomer B may be used to modify the band gap of the polymer. The monomer units may be water-soluble, for example, including one or more, or two or more water-solubilizing moieties (W), such as poly(ethylene glycol) (PEG) moieties. In some embodiments, monomer A or monomer B is each independently a water-soluble monomer molecule. Water-soluble monomer A or monomer B may each independently include a DHP moiety and one or more, or two or more PEG moieties. Water-soluble monomer A or monomer B may each independently include a DHP moiety with a solubilizing PEG moiety attached via a sulfonamide group.

[0161] The water-soluble monomer A or monomer B containing a DHP moiety each independently has the formula (I): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of hydrogen, alkyl, PEG, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronate substituted aryl, boronate ester substituted aryl, boronate ester, boronic acid, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protecting groups thereof; Each R 2 are independently selected from the group consisting of water solubilizing moiety, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, sulfonamido-PEG, phosphoramido-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, [ka] selected from the group consisting of; ; Each R 3 is a water-solubilizing moiety; Each R 4is independently selected from the group consisting of H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylate amine, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protecting group thereof; Each R 5 are independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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 alkoxy; Each Q is independently a bond, NR 4 or -CH2; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. It may have a structure as described in

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

[0163] Water-soluble monomer A or monomer B may each independently comprise a fluorene moiety and one or more, or two or more PEG moieties. Water-soluble monomer A or monomer B comprising a fluorene moiety may each independently be represented by formula (II): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of hydrogen, halogen, alkyl, PEG, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronate-substituted aryl, boronate ester-substituted aryl, boronate ester, boronic acid, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protecting groups thereof; Each X is C or Si; Each R 4 is independently selected from the group consisting of H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylate amine, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protecting group thereof; Each R5 is independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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 alkoxy; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. It may have a structure as described in

[0164] The fluorescent polymer dye may be any dye disclosed in US2019 / 0144601, the entirety of which is incorporated herein by reference. The fluorescent polymer dye may be, for example, a compound represented by Formula VI: [ka] (In the formula, Each X is independently selected from the group consisting of C and Si; Each Y is independently a bond, CR 1 R 2 and SiR 1 R 2 selected from the group consisting of; When Y is a bond, X is directly attached to both rings; Each R 1 are independently selected from polyethylene glycol (PEG), ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, and [ka] selected from the group consisting of; Each R 2 is independently H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG, ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, and [ka] selected from the group consisting of; Each R 3 is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG; 4 is independently selected from the group consisting of H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylate amine, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protecting group thereof; Each Z is independently CH, O, or NR 4 and; Each Q is independently a bond, NH, or NR 4 and CH2; each M is independently an electron-rich linker unit capable of modifying the band gap of the polymer, evenly or randomly distributed along the polymer backbone; Each R 4 are non-ionic side chains capable of imparting excess solubility in water at 10 mg / mL, each independently being a halogen, a hydroxyl, a C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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 independently an integer from 0 to 20; each y' is independently an integer from 0 to 50), and C2 to C 18(hetero)aryl groups; Each optional linker, L, is an aryl or heteroaryl group evenly or randomly distributed along the polymer backbone, optionally substituted at one or more of its terminal pendant chains with a functional group selected from the group consisting of amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protecting groups thereof, for conjugation to another substrate, acceptor dye, molecule or binding agent; Each G 1 and G 2 are each independently an aryl or hetroaryl substituted at one or more pendant chains terminated with a functional group selected from hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronate substituted aryl, boronate ester substituted aryl, boronate ester, boronic acid, optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene, amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protecting groups thereof for conjugation to a substrate or binder. selected from the group consisting of; a, c, and d define the mole % of each unit within the structure, and each unit may be repeated evenly or randomly, where a is from 10 to 100% mole %, c is from 0 to 90% mole %, and each d is from 0 to 25% mole %; each b is independently 0 or 1; m is an integer from 1 to about 10,000; Each n is independently an integer from 1 to 20. The dye may be a blue-violet fluorescent polymer dye having the structure:

[0165] In some instances, each M can independently be: [ka] (Wherein, each R 4 are non-ionic side chains capable of imparting excess solubility in water at 10 mg / mL, each independently being a halogen, a hydroxyl, a C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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 independently an integer from 0 to 20; each y' is independently an integer from 0 to 50), and C2 to C 18 (hetero)aryl groups) may be selected from the group consisting of:

[0166] In some cases, the fluorescent polymer dye has 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). The compound may have the structure:

[0167] In some cases, the fluorescent polymer dye has Formula VIII: [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 independently an integer from 0 to 50; Each R 5 are independently H, C1 to C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (Hetero)arylamino, and C1-C 12 alkoxy) The compound may have the structure:

[0168] In some cases, the fluorescent polymer dye has 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). The compound may have the structure:

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

[0170] In some cases, the fluorescent polymer dye has 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 are in the mole percent range of 10 to 100%. It may also be a copolymer having the structure:

[0171] In some cases, the fluorescent polymer dye has 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 independently an integer from 0 to 50; Each g and a together are mol % between 10 and 100%. It may also be a copolymer having the structure:

[0172] In some cases, the fluorescent polymer dye has 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 g and a together are mol % between 10 and 100%. It may also be a copolymer having the structure:

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

[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 backbone. Capping may be performed on the polymer by activation using a suitable functionality, 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 a suitable functionality, for example, according to US2020 / 0190253, the entirety of which is incorporated herein by reference. The activated polymer may be conjugated to a binding partner. Any suitable binding partner, for example, an antibody, may be used, and may be 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) with an excitation maximum of 414 nm, an emission peak of 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 amines for tandem dyes and subsequently activated for tandem conjugates. The rigidity of the polymer dye structure 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 excitable by a blue-violet laser, so it is particularly suitable for evaluating dimly expressed markers. SN-conjugated antibodies 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 may be mentioned. SN v605 and SN v786 (Beckman Coulter, Inc.) are tandem polymeric dyes derived from the core SN v428 polymeric dye. Both share the same absorbance characteristics, with maximum excitation at 414 nm. With emission peaks for SN v605 and SN v786 at 605 nm and 786 nm, respectively, they are optimally detected using 610 / 2 and 780 / 60 nm bandpass filters on a flow cytometer.

[0177] The fluorescent polymer dyes may be fluorescent polymeric dyes available from Becton Dickinson, including Brilliant™ Blue, Brilliant™ Violet and Brilliant™ Ultra Violet dyes. The fluorescent polymeric dyes may be fluorescent polymeric dyes available 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] A "water-soluble monomer" may be a monomeric unit comprising an aryl or heteroaryl moiety, each optionally having one or more water-solubilizing moieties attached thereto. The water-soluble moieties may be one or more PEG moieties. The water-soluble monomer may be suitable for use in the preparation of at least one of a plurality of fluorescent polymer dyes having a monomer A subunit, a monomer B subunit, or a combination of a monomer A and a monomer B subunit. The water-soluble monomer may be a dihydrophenanthrene (DHP)-based water-soluble monomer. The water-soluble monomer may be a fluorene-based water-soluble monomer.

[0179] The water soluble monomer has the formula (I): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of halogen, alkyl, PEG, hydrogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol; Each R 2 are independently selected from the group consisting of water solubilizing moiety, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, sulfonamido-PEG, phosphoramido-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, [ka] 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 carboxylate amine, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protecting group thereof; Each R 5are independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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 alkoxy; Each Q is independently a bond, NR 4 or -CH2; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. The monomer may be a dihydrophenanthrene (DHP) monomer having the chemical structure shown in

[0180] The water soluble monomer has the formula (II): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of halogen, alkyl, PEG, hydrogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol; Each X is C or Si; Each R 4 is independently selected from the group consisting of H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylate amine, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protecting group thereof; Each R 5 are independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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 alkoxy; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. The fluorene monomer may have the structure shown in

[0181] The water soluble monomer has the formula (III): [ka] (wherein each G1, G2 is independently halo (F, Cl, Br, I); each Z is independently selected from the group consisting of O, CH2, and NH; each R1 is independently alkyl (C1-C3); each R2 is independently H, alkyl (C1-C6); each n is independently 1 to 6; and each m is independently 5 to 50. In some embodiments, each of G1 and G2 is Br; each of Z is O; each of R1 is CH3; each of R2 is H; each of n is independently 2 to 4; and each of m is independently 5 to 20. In some embodiments, each of n is 3; and each of m is independently 11 or 12. Labeled Binding Partners

[0182] The dyes can be conjugated to the binding partner by a variety of linking chemistries 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-[ ~ -maleimidopropionic acid]hydrazide.TFA) / thiol may be mentioned.

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

[0184] Methods for carrying out conjugation are well known in the art. Commercially available kits for carrying out conjugation are also readily available, for example, from Innova Biosciences (Cambridge, UK), Novus Biologicals (Littleton, Colo.), Thermo Fisher Scientific (Waltham, Mass.). Dry Down Process

[0185] Dry reagent technology can be used to increase the stability of biomolecules. The drying process can be used to create a uniform reagent layer, for example at the bottom of a tube. Dry reagents do not require refrigeration. Dry reagents can be stored at room temperature. The antibody panel can be supplied in a single-use cocktail. The antibody panel can be provided in a variety of substrates, including tube or plate formats. Reagents can be used to dry the conjugated antibodies and stabilize them for storage at room temperature. The reagent format can be adapted to combine different reagents to create an antibody cocktail. 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 from 1 to 20, 2 to 15, or 3 to 12 different antibody-dye conjugates.

[0186] Dry reagent technology may allow the ability to dry the beads in the list with the cocktail. Binding partner-dye conjugates, including antibody-dye and antibody-polymer dye conjugates, may be used in flow cytometry assays. Any suitable drying process may be used that dries 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 were previously developed to dry different monomeric conjugate dyes in a single tube without modifying their functionality (affinity for antigens) and physical properties (such as brightness or fluorescence). Prior art RB formulations do not contain zwitterionic detergents 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, the desired flow cytometry results using a monomeric CD4-PE dye conjugate, including the 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 with a blood sample. Similarly, as illustrated in Figure 1B, the desired flow cytometry results of a monomeric CD20 APC dye conjugate, including the desired functionality, physical properties, and separation of CD20 APC+ cell populations, are shown when dried using prior art techniques and reconstituted with a blood sample.

[0189] It has been found that conventional drying techniques such as RB, when used to dry down two or more polymer-dye conjugates in a tube, alter the integrity of the polymer structure and result in the aggregation of the polymer-dye conjugates. The aggregation increases non-specific interactions and creates staining artifacts. Figures 2A and 2B show that conventional techniques are unable to separate cell populations stained using polymer dyes due to the aggregation of the polymer-dye conjugates when the two polymer-dye conjugates are dried using conventional drying techniques.

[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, demonstrating non-specific interactions and an inability to separate populations in the x and y axes.

[0191] Figure 2B shows undesirable flow cytometry results of CD20-SNv605 and CD4-SNv786 polymer dye antibody conjugates when mixed and dried using conventional drying techniques with compensation. The failure to prevent non-specific interactions was attributed to 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 new buffer compositions to keep fluorescent dye conjugates stable and reduce aggregation during drying.

[0193] To overcome the technical challenges mentioned above, first, the fluorescent dye conjugate must be stable in its liquid state when mixed. It was found that a mixture of two fluorescent dye conjugates, even in liquid state, would result in increased non-specific interactions when mixed together, but leakage could be compensated. To overcome the limitations in the liquid state, commercially available buffers were tested, including those from BD Biosciences (Brilliant Stain Buffer, Catalog No.: 563794) and Thermo Fisher (Super Bright Complete Staining Buffer, Catalog No.: SB-4401-42). The use of Brilliant Stain Buffer or Super Bright Complete Staining Buffer during drying of the polymer-dye conjugate did not solve the problem of low stability and aggregation.

[0194] Other techniques were adapted to dry the fluorescent dye conjugates, including membrane immobilization of the fluorescent dye conjugates on a substrate separately, as described in US2019 / 0242882, which is incorporated herein by reference. From these attempts, it was concluded that the fluorescent dye conjugates could be dried separately on the substrate membrane at different spots, but the two fluorescent dye conjugates could not be mixed and dried. The disadvantages of drying the polymer dye conjugates separately include the fact that drying the fluorescent dye conjugates on a cellulose membrane involves deviations from current drying techniques, the inconvenience of maintaining the cellulose membrane inside the DURAClone™ tube substrate, and the allowance 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 multi-color dry reagents (e.g., DURAClone™ tubes, Beckman Coulter, Inc.). Multi-color dry reagents are cocktails of different fluorescent dye conjugates (CD4-FITC, CD8-PE, CD20-APC, CD3-PC5.5, etc.) that can be directly used to stain blood and analyze it in a flow cytometer. Compared with existing monomeric conjugate dyes, polymeric dye conjugates differ in their structure and complexity.

[0196] Conventional drying techniques are used to achieve multi-color dry 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] With the introduction of polymer-dye conjugates, it was found that traditional drying techniques were ineffective while drying multiple polymer-dye-antibody conjugates in a cocktail. These polymer-dye conjugates tend to interact non-specifically, leading to difficulties in separating populations, which can lead to challenges in identifying the desired cell population in a given sample. composition

[0198] To overcome the limitations of conventional drying techniques, we have developed novel "Dry Mix" ("DM") buffers that can be used to dry one or more fluorescent (polymeric or monomeric) dye conjugates in a dye cocktail. A dye cocktail may contain more than one fluorescent polymeric dye conjugate. A dye cocktail may contain more than one conventional non-polymeric fluorescent dye conjugate. A dye cocktail may contain a combination of one or more fluorescent polymeric dye conjugates and one or more conventional non-polymeric fluorescent dye conjugates.

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

[0200] The DM buffer formulations of the present invention are typically aqueous solutions containing water soluble monomers, a protein stabilizer, a carbohydrate stabilizer, a zwitterionic 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 stabilizer may facilitate binding of the dry components to the substrate, so that when the reaction vessel is opened, the stabilizer will remain at the bottom of the tube and will not blow off or stick to the cap. The DM buffer composition may contain, per test, 200-800 μg water-soluble monomer; 2000-3000 μg carbohydrate stabilizer; 8.4-72 μg protein stabilizer; and 2-15 μg zwitterionic surfactant. Diluent

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

[0203] The term "protein stabilizer" refers to a protein that serves to reduce non-specific binding, for example, to reduce cell-cell interactions or to help prevent non-specific binding between an antibody and a non-target molecule. Protein stabilizers may include bovine serum albumin (BSA), various gelatins, and casein. Various protein stabilizers were evaluated in the DM buffer composition 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 dry down buffer may include one or more protein stabilizers.

[0204] Gelatin (gelatin or gelatine) is a protein derived from collagen, usually harvested from animal body parts. It is brittle when dry and sticky when wet. After undergoing hydrolysis, it is sometimes referred to as hydrolyzed collagen, collagen hydrolysate, gelatine hydrolysate, hydrolyzed gelatin, and collagen peptides. Several types of gelatin are commercially available, including type A gelatin, type B gelatin, Prionex® highly purified type A gelatin, and gelatin-cold water 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 wider spread of negative populations compared to the corresponding liquid cocktail. Type B gelatin dry mix was able to prevent nonspecific interactions, but preparation of stocks was difficult. Effective concentrations of type B gelatin ranged from 150 micrograms to 450 micrograms per test. Prionex® Highly Purified Type A Gelatin at concentrations equivalent to Type B Gelatin was effective in preventing non-specific interactions, but with some reduced stability. The concentrations of pre-prepared solutions of Prionex® Highly Purified Type A Gelatin were quantified, and effective dry mix concentrations ranged from 67 micrograms to 135 micrograms per test. Gelatin-Cold Water Fish was equivalent to Type B Gelatin in terms of performance. Effective concentrations of Gelatin-Cold Water Fish also ranged from 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 prevents the formation of common secondary structural motifs of proteins. Casein does not contain disulfide bridges and therefore has relatively little tertiary structure. Casein 10x blocking buffer (in the range of about 14mg / mL to about 18mg / mL) was used as one of the components of the dry mix. Casein at 5x (2x dilution) and 2.5x (4x dilution) concentrations was able to effectively prevent the interaction of two polymer-dye-antibody conjugates with the addition of more than one polymer conjugate. Carbohydrate Stabilizer

[0206] A "carbohydrate stabilizer" is a carbohydrate molecule used to help increase the stability of a dye-antibody conjugate in solution upon 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 the DM buffer composition increased non-specific binding in granulocytes. Sodium alginate is the sodium salt of alginic acid, which is a linear polysaccharide containing homopolymeric blocks of (1→4)-linked beta-D-mannuronic acid and alpha-L-guluronic acid residues. Preparation of stocks was difficult and overall granulocyte, monocyte, and lymphocyte spread was higher than when sodium alginate was added to the DM buffer composition.

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

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

[0210] Various types of surfactants were explored to prevent non-specific interactions in DM buffer formulations (see Table 1).

[0211] Anionic surfactants were evaluated, including alkyl sulfates and alkyl sulfonates with alkyl groups of at least 10 carbons. Sarkosyl, CH3(CH2) 10N-Lauryl sarcosine sodium salt, also known as CO-N(CH3)-CH2COONa, is an anionic surfactant. N-Lauryl sarcosine sodium salt was found to prevent non-specific binding in monocytes and granulocytes, but was unable to prevent polymer-polymer interactions. Lignosulfonic acid ("LSA"), 3-(2-hydroxy-3-methoxyphenyl)-2-[2-methoxy-4-(3-sulfopropyl)phenoxy]propane-1-sulfonic acid, is an anionic surfactant. The negative background of the SN v605 and SN v786 populations was increased compared to DM buffer when LSA was added.

[0212] Non-ionic surfactants were evaluated for possible use in DM buffer formulations. Polysorbate 80 was tested as the non-ionic surfactant. The term "ester-linked non-ionic surfactant" refers to a non-ionic organic compound containing hydrophobic and hydrophilic groups connected by or including an ester linkage. Examples of ester-linked non-ionic surfactants include polyoxyethylene glycol sorbitan esters (Polysorbate, TWEEN®), sorbitan alkyl esters (Span). Non-specific monocyte pull-out and population spreading with SN v605 conjugates were included in the dry mix at 0.015% and 0.075% polysorbate 80.

[0213] Zwitterionic surfactants were evaluated for potential use in DM buffer formulations. The zwitterionic surfactant N,N-dimethyl-N-dodecylglycine, or N-(alkyl C 10 ~C 16 Empigen® BB (Huntsman Corporation), also known as 3-(N,N-dimethyltetradecylammonio)propanesulfonate, myristyl sulfobetaine, CH3(CH2) 13 N + (CH3)2CH2CH2CH2SO3 -3-(N,N-dimethylmyristylammoniopropanesulfonate (DMMA), also known as DMMA; and Zwittergent® 3-16 detergent (Merck 3-[N,N-Dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate (DMPA), also known as DMPA (Empigen® BB, Darmstadt, Germany), was evaluated in DM buffer compositions. Empigen® BB was able to prevent non-specific binding in monocytes and granulocytes, but was not able to prevent polymer interactions. DMPA precipitates at room temperature, but was functionally equivalent to Empigen® BB. The effective concentration range for DMPA was 0.002%-0.006%. DMMA was functionally equivalent to Empigen® BB and was therefore considered for further testing. The effective concentration of DMMA was found to be in the range of 0.002%-0.037%, or 0.004%-0.018%.

[0214] In some embodiments, the zwitterionic surfactant has formula (XV): [ka] (wherein Y=CO2- or SO3-, W=H or OH, and Z=CH3 or NHC(O)R, where R=C 1~15 alkyl; independently, p=0 or 1; and q=0 to 21. In some embodiments, W=H, Z=CH3, and q=11-15. In some embodiments, the zwitterionic surfactant is DMMA, DMPA, N-(alkyl C 10 ~C 16 )-N,N-Dimethylglycine betaine, Lauryl hydroxysultaine, Lauryl sultaine, Myristyl betaine, Cetyl betaine, Decyl betaine, Lauryl betaine, Behenyl betaine, Cocamidopropyl betaine In some embodiments, the zwitterionic surfactant may be DMMA, DMPA, N-(alkyl C10 ~C 16 )-N,N-dimethylglycine betaine. Antioxidants

[0215] Antioxidant compounds were evaluated as candidate components of the DM buffer formulation. Antioxidants may contain one or more, two or more, or three or more carboxylic acid or carboxylate moieties and a C1-C8 or C2-C6 aliphatic moiety. The aliphatic moiety may be linear, branched, or cyclo-alkyl or alkenyl moiety. For example, L-ascorbic acid and citric acid were evaluated as antioxidants. Nonspecific monocyte pull-out by SN v605 conjugates was low at 0.2 mM and 0.6 mM concentrations of L-ascorbic acid. 0.6 mM L-ascorbic acid had a low spread of SN v786 positive population in the V610 channel. Citric acid reduced granulocyte degranulation and nonspecific monocyte pull-out in SN v605 conjugates was low at all concentrations. However, citric acid had no additional effects, including population spreading. monomer

[0216] In order to prevent non-specific interactions between fluorescent dye conjugates, especially between fluorescent polymer dye conjugates, various water-soluble monomer species were examined. 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, including a fluorescent polymer having a monomer A subunit and a monomer B subunit. For example, monomer A or monomer B may include 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-based structure was examined for use in DM buffer composition formulations and tested according to Example 1.

[0217] The water-soluble monomers may be monomeric units that include an aryl or heteroaryl moiety, each having a water-soluble moiety attached thereto, as appropriate. The water-soluble moiety may be one or more PEG moieties. The water-soluble monomers may be suitable for use in the preparation of at least one of a plurality of fluorescent polymer dyes having a monomer A subunit, a monomer B subunit, or a combination of a monomer A and a monomer B subunit. The water-soluble monomers may be DHP-based water-soluble monomers. The water-soluble monomers may be fluorene-based water-soluble monomers.

[0218] The water soluble monomer has the formula (I): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of halogen, alkyl, PEG, hydrogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol; Each R 2 are independently selected from the group consisting of water solubilizing moiety, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, sulfonamido-PEG, phosphoramido-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, [ka] 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 carboxylate amine, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protecting group thereof; Each R 5are independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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 alkoxy; Each Q is independently a bond, NR 4 or -CH2; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. The monomer may be a DHP monomer having the chemical structure shown in

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

[0220] The water soluble monomer has the formula (II): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of halogen, alkyl, PEG, hydrogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol; Each X is C or Si; Each R 4 is independently selected from the group consisting of H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylate amine, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protecting group thereof; Each R5 is independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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 alkoxy; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. The fluorene monomer may have the structure shown in

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

[0222] The water soluble monomer has the formula (III): [ka] (wherein each G1, G2 is independently halo (F, Cl, Br, I); each Z is independently selected from the group consisting of O, CH2, and NH; each R1 is independently alkyl (C1-C3); each R2 is independently H, alkyl (C1-C6); each n is independently 1 to 6; and each m is independently 5 to 50. In some embodiments, each of G1 and R2 is Br; each of Z is O; each of R1 is CH3; each of R2 is H; each of n is independently 2 to 4; and each of m is independently 5 to 20. In some embodiments, each of n is 3; and each of m is 11.

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

[0224] Without being bound by theory, it is hypothesized that monomer A randomly interacts 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 nature, may mask these dye conjugates and prevent them from approaching in close proximity. Preservatives

[0225] The aqueous DM buffer composition may include any suitable preservative. The preservative may be an antioxidant, a biocide, or an 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 agent

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

[0227] Fluorescent polymeric dyes in different solvents were also explored for preparation of DM buffer compositions, however, showed a broader spread of negative populations compared to the liquid cocktail.

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

[0229] An early technical solution was 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 with gelatin and monomer A reduced the aggregation problem during the drying process. With optimal protein stabilizer concentration and optimal concentrations of monomer and gelatin in conventional dry down techniques, DM helps maintain the integrity of the fluorescent dye conjugates and solves the issues related to aggregation during drying of the fluorescent dye conjugates.

[0230] The DM buffer comprises a water-soluble monomer; a protein stabilizer; a carbohydrate stabilizer; and a zwitterionic surfactant. The DM buffer may comprise a DHP-based monomer and a water-soluble monomer comprising one or more, or two or more poly(ethylene glycol) moieties. The water-soluble monomer may comprise a structure according to formula (I). The protein stabilizer may be an albumin protein. The protein stabilizer may be a gelatin protein. The protein stabilizer may comprise a casein protein.

[0231] The DM buffer was then further improved to prevent non-specific interactions between SuperNova™ conjugates and non-specific monocyte pull-out, and 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 made using DM2+S resulted in the prevention of non-specific interactions of the SuperNova™ conjugates as well as the prevention of non-specific pull-out without interfering with the performance (brightness and population recruitment) of the conjugates suspended in the cocktail, and the achievement of higher stability of the dry product (until 6 months real-time stability was established yet). In the preparation of the dry tubes, a bulk formulation of the SuperNova™ conjugates was used. EXAMPLES

[0233] Example 1A Test procedure The general processes and procedures used in this example are set out below.

[0234] Drying: The term drying, as used herein, refers to vacuum drying at a particular vacuum pressure for a particular number of hours.

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

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

[0237] Dry test: The term dry test refers to mixing the conjugates together in a test tube and drying using vacuum drying, followed by staining the cells using the dried conjugate.

[0238] Test as used herein refers to the following protocol. 1. Prepare the required x number of tubes (x number of tubes depends on the performance to be tested). 2. Add the calculated volume of conjugated antibody (required dose) to each tube, or 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. Incubate for 15-20 minutes at room temperature (18-27°C), protected from light. 5. Add 2 ml of VersaLyse+IOTest3 Fixative mixture (2 ml of Versalyse Ref. A09777 + 50 μl of IOTest3 fixative 10× Ref. A07800). Vortex immediately to ensure proper mixing and incubate for 20 minutes at room temperature (18-27°C), protected from light. 6. Centrifuge at 300g for 5 minutes at room temperature. 7. Remove the supernatant by aspiration. 8. Resuspend the cell pellet using 3 mL of 1x PBS. 9. Centrifuge at 300g for 5 minutes at room temperature. 10. The supernatant is removed by aspiration and the cell pellet is resuspended using 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 of emission spectra of different fluorochromes. Thus, this compensation factor was used to eliminate bleeding of signals into other undesired channels. Manual compensation was performed to evaluate the performance of the conjugates. Example 1B Flow Cytometry Protocols

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

[0241] A stain-lyse-wash protocol was used to prepare and process samples for flow cytometry acquisition. Samples were processed and acquired on a DxFLEX / CytoFLEX flow cytometer (Beckman Coulter, Inc.) to analyze the performance of the various formulations. 1. Add 100 μL of K2EDTA anticoagulated blood to the dry stain tube or the tube with the 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 (for 1 ml of Versalyse, add 25 μL of IOTest 3 fixative) lysis 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), pellet the WBCs by centrifugation at 300 g for 5 minutes at room temperature. Aspirate and discard the supernatant and break up the pellet by vortexing. 4. Add 3 mL of 1x PBS and centrifuge at 300g 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 acquire the tube on a DxFLEX flow cytometer at the recommended settings.

[0242] Equipment setup:

[0243] The flow cytometer instrument settings were performed using CytoFLEX or DxFLEX Daily QC Fluorospheres (Beckman Coulter, Inc.). The QC beads are used to perform routine quality control management of the instrument and to identify target values ​​for gain. The recommended settings for gain as specified by the QC protocol were used for acquisition of the test tubes in the flow cytometer.

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

[0245] An aqueous DM buffer formulation was developed for drying fluorescent dye conjugates. Starting with conventional drying techniques, several reagents (Table 1) were tested for preventing non-specific interactions and binding of polymer-dye-antibody conjugates. Water or PBS was used in the preparation of stock solutions of these reagents. The reagents tested and the results for each reagent are shown in Table 1. Reagents in bold typeface 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 the polymer-dye conjugate tubes: BSA (bovine serum albumin), PEG550 (poly(ethylene glycol) methyl ether, average M n550), BSA-ox (oxidized BSA), Empigen® BB detergent (N-(alkyl C 10 ~C 16 )-N,N-dimethylglycine betaine), N-lauryl sarcosine sodium salt, monomer A, monomer B, polymers in different solvents, gelatin type A, gelatin type B, lignosulfonic acid, carrageenan, sodium alginate, casein blocking buffer 10x, Prionex® highly purified gelatin type A, gelatin-cold water fish, L-ascorbic acid, citric acid, polysorbate 80, acrylamide, trehalose decanoate, trehalose-tetradecanoate, trehalose-hexadecanoate, 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate (DMPA), and 3-(N,N-dimethylmyristylammonio)propanesulfonate (DMMA). The chemical structures of monomer A and monomer B are shown in FIG. 4. Monomer A may 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). The dry down tubes were tested according to the protocol of Example 1. The results of the test are shown in Table 1. Empigen®, Monomer A, Type B Gelatin, Casein Blocking Buffer 10x, 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, B gelatin, and Empigen BB® detergent (N,N-dimethyl-N-dodecylglycine betaine; N-(alkyl C 10 ~C 16 The first DM (DM) buffer formulation was developed containing N,N-dimethylglycine betaine.

[0249] An improved DM buffer formulation was then developed to prevent non-specific interactions between SuperNova™ conjugates and non-specific monocyte pull-out, which was named DM2 (DM 2). DM2 contains trehalose dihydrate, monomer A, Prionex® gelatin and DMMA. However, it was later found that dry 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 provided in Table 2. The pH of the DM2+S buffer was found to be 7-7.4. Dry tubes made using DM buffer DM2+S resulted in the prevention of non-specific interactions of the SuperNova™ conjugates as well as the prevention of non-specific pull-out without interfering with the performance (brightness and population recruitment) of the conjugates suspended in the cocktail, and the achievement of higher stability of the dry product (6 months real-time stability has been established so far). In the preparation of the dry tubes, a bulk formulation of the SuperNova™ conjugates was used.

[0251] [Table 2]

[0252] [Table 3]

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

[0254] In some embodiments, the DM buffer may include trehalose, monomer A, DMMA, and casein. In some embodiments, the dry-down buffer may include 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 include 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, without dye conjugate. DM buffer may be prepared, for example, from stock concentrations of carbohydrate stabilizer, water soluble monomer, zwitterionic detergent, and protein stabilizer in water or PBS buffer. For example, stock concentrations may include about 400 mg / ml trehalose dihydrate, about 40 mg / ml monomer A, about 1.5 mg / ml DMMA, and / or about 2-20 mg / mL casein. Example 3 Design of experiments for DM buffer and their results

[0255] The concentration of each component of the DM buffer (except for trehalose dihydrate, sodium azide and allura red) was titrated and optimized to achieve optimal results.

[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 conjugate and other buffer components.

[0258] 2. Casein 10x Blocking Buffer Titration:

[0259] 2.1:Test:1

[0260] Objective: To find the optimal concentration of casein in preventing non-specific binding in liquid format.

[0261] Methods: In this experiment, different concentrations (5x, 2.5x, 1.25x, 0.625x and 0.31x) of pre-made solutions of Casein 10x Blocking Buffer were tested following the protocol in Example 1.

[0262] Results and Observations:

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

[0264] 2.2:Experiment:2

[0265] Objective: To find the optimal concentration of casein in dry format to prevent non-specific binding.

[0266] Method: In this experiment, different concentrations (4x, 2.5x and 1x) of pre-made solution of Casein 10x Blocking Buffer were used to dry down 12 different antibody conjugates with other additives in place. 2.5x is the standard concentration. Therefore, the 2.5x concentration was compared to the 1x and 4x concentrations as a reference. Here, two different lots (L1 and L2) of Casein 10x Blocking Buffer were tested.

[0267] Results and Observations:

[0268] The overlap of the dual fluorescence plots (V610 vs. V780, PB450 vs. V610 and PB450 vs. V780) of DM buffer with different concentrations (4x, 2.5x and 1x) was compared (data not shown). The overlap of the dual 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 these comparisons confirmed that there was no significant variation across the different concentrations of casein tested. Tables 4 and 5 show the comparison across the different concentrations of casein for absolute % delta mobilization and MdFI for all specificities.

[0269] [Table 4]

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

[0271] This experiment demonstrates that the overlay plot, MdFI, and absolute % delta mobilization have no significant variation for different concentrations of Casein 10x Blocking Buffer (1x, 2.5x, 4x). Absolute % delta mobilization was found to be <5% between lots and across the different concentrations tested due to minimal / no effect of different concentrations of Casein 10x Blocking Buffer. Therefore, 1x-4x Casein Blocking Buffer can be used to dry SuperNova™ conjugates along with other conventional dyes.

[0272] Dosage setting for Monomer A: 3.1:Test:1

[0273] Objective: To titrate the monomer A concentration for the dry-down of two polymer-dye-antibody conjugates.

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

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

[0276] Objective: To titrate the monomer A concentration for the dry-down of three polymer-dye-antibody conjugates.

[0277] Methods: Monomer A concentrations of 200 micrograms per test and 400 micrograms per test were tested in formulations consisting of CD45-FITC, CD19-SN v428, CD20-SNv605 and CD3-SNv786 in dry format with other additives kept constant.

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

[0279] 4.DMMA Dosage:

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

[0281] Objective: To find the optimal concentration of DMMA in preventing non-specific 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 zwitterionic surfactant. DMMA was tested to find a better option to Empigen® in reducing non-specific interactions with easy manufacturability. Different concentrations of DMMA are provided in Table 6 along with details of the groups tested.

[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 with DM buffer. The concentration of DMMA was selected based on the Empigen BB® concentration (0.15%), which is one of the components of DM buffer. Appropriate controls were used in the experiment. 0.15% DMMA was added to the final formulation to achieve individual concentrations of DMMA, as described in Table 6. The effect of DMMA was tested using two Supernova™ conjugates CD20-SN-v605 and HLADR-SN-v786. The specimens were processed using the protocol according to Example 1B. The experiment was performed on two donors.

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

[0286] This experiment demonstrates that 0.0075% and 0.00375% DMMA show comparable % recruitment of scatter, CD20+ and HLADR+ populations as DMMA. The concentration of DMMA was further optimized within the range of 0.0075%-0.00375% to obtain the optimal concentration. 4.2: Experiment 2: Dose optimization of DMMA in the presence of two Supernova™ conjugates

[0287] Objective: To find the optimal concentration of DMMA in preventing non-specific 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. Different concentrations of DMMA are provided in Table 7 along with the 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 with DM. Appropriate controls were used in the experiments. 0.15% DMMA was added to the final formulation to achieve individual concentrations of DMMA, as described in Table 7. The effect of DMMA was tested using two Supernova™ conjugates, CD20-SN-v605 and HLADR-SN-v786. The specimens were processed using the protocol described in Example 1B. The experiments were performed on two donors.

[0291] Figures 7A-D show scatter plots of CD20-SN-v605, HLADR-SN-v786 and double positive populations for all dry DMMA formulations tested. Figure 7A shows scatter plots of all concentrations of DMMA tested along with unstained and DM. 0.004% and 0.008% DMMA show less non-specific neutrophil pull-out (indicated by arrows) compared to other DMMA concentrations. Similarly, 0.004%, 0.008% and 0.018% DMMA show less non-specific monocyte pull-out (indicated by arrows, Figure 7B) in the V610 channel compared to DM and other DMMA concentrations. The % recruitment of CD20+ and HLADR+ was found to be similar at all DMMA concentrations compared to the individual liquid singles. Similarly, the % recruitment of double positive populations at all DMMA concentrations was found to be similar compared to DM. In addition, 0.004%, 0.008% and 0.018% DMMA show similar spreading of 786+ events in the V610 channel compared to DM. This experiment demonstrates that 0.004%, 0.008% and 0.018% DMMA show better performance compared to DM. Therefore, these DMMA concentrations were further evaluated in combination with other selected additives.

[0292] 5. To 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: In this experiment, selected combinations of additives were evaluated, e.g., DMMA+Casein, DMMA+Prionex. Different concentrations of additives are provided in Table 8, along with details of the groups tested.

[0295] [Table 8]

[0296] Here, different concentrations of DMMA, i.e. 0.018% and 0.008%, were tested in combination with different dilutions of casein and Prionex in dry tubes and compared with DM version 1. Appropriate controls were used in the experiment. The effect of DMMA was tested using two Supernova™ conjugates CD20-SN-v605 and HLADR-SN-v786. The specimens were processed using the protocol of Example 1B. The experiment was performed on four donors.

[0297] Results and Observations: Flow plots of the scatter of CD20-SN-v605, HLADR-SN-v786 and double positive populations for additive combinations with DMMA 0.008% were compared. Figure 8A shows the scatter plot of additive combinations with DMMA 0.008% compared to DM. The scatter appears similar for each concentration, except for DM. DM shows pullout of non-specific granulocytes and monocytes as indicated by the arrows. Similarly, DM shows higher pullout of non-specific granulocytes and monocytes in the V610 channel compared to other combinations with DMMA 0.008% (indicated by arrows, Figure 8B). The % recruitment of CD20+ and HLADR+ was found to be similar in all combinations with DMMA 0.008% compared to the individual fluids alone (Figure 8B, Figure 8C). Similarly, the % recruitment of double positive populations in all combinations with DMMA 0.008% was found to be similar compared to DM.

[0298] Similarly, the flow plots of the scatter of CD20-SN-v605, HLADR-SN-v786 and double positive populations were compared for the additive combinations with DMMA 0.018% (data not shown). The scatter plots of all additive combinations with DMMA 0.018% were compared to DM. The scatter appears to be similar for all combinations. DM and DMMA 0.018 + casein 1x show higher non-specific monocyte pullout in the V610 channel compared to other combinations with DMMA 0.018%. The % recruitment of CD20+ and HLADR+ was found to be similar in each of the combinations with DMMA 0.018% compared to the individual fluids alone. Similarly, the % recruitment of the double positive population in all combinations with DMMA 0.018% was found to be similar compared to DM. However, the combinations of DMMA 0.018% + casein 2.5x and 0.018% DMMA with Prionex showed less prevalence of 786+ events in V610 compared to DM.

[0299] Flow plots of the scatter of CD20-605, HLADR-786 and double positive populations for different concentrations of single additives such as casein and Prionex (gelatin was replaced by casein and Prionex in DM version 1) were compared (data not shown). The scatter appears to be similar for each of the combinations. DM alone and all singles show higher non-specific monocyte pullout in the V610 channel compared to the combination of additive and DMMA. The % recruitment of CD20+ and HLADR+ was found to be similar in all singles compared to the individual fluid singles. Similarly, the % recruitment of the double positive population in each of the singles was found to be similar compared to DM. The singles and Prionex dilutions show less spreading of 786+ events in the V610 channel compared to DM.

[0300] Overall, of all the combinations, DMMA 0.008% + prioex 2 dil and 3 dil, DMMA 0.018% + Prionex 3 dil and DMMA 0.018% + Casein 2.5x provided good performance in terms of reducing non-specific monocyte pull-out in the V610 channel and reducing non-specific interactions between SN conjugates. This experiment demonstrates that combinations including DMMA 0.008% + prioex 2 dil and 3 dil, DMMA 0.018% + Prionex 3 dil and DMMA 0.018% + Casein 2.5x provided good performance in terms of reducing non-specific binding and non-specific interactions when tested with two SN conjugates.

[0301] From these experiments, DMMA 0.008% + Prionex 2 dilution and DMMA 0.018% + Casein 2.5x dilution with DM buffer showed good performance in reducing non-specific interactions between SN conjugates and also non-specific pull-out. However, during development, it was found that DMMA 0.008% + Prionex 2 dilution (named DM2 formulation) showed stability problems and was therefore not further evaluated. Subsequent experiments with DMMA 0.018% + Casein 2.5x (named DM2+S) showed that this formulation had good stability: 6 months real-time stability in dry tubes established so far (testing ongoing). 6: Functionality test of excipients, excipient 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 non-specific interactions, the buffer minus each one of the components was analyzed.

[0303] Methods: Experiments were performed using a four-color panel (CD45-AA750, CD56-SNv428, CD20-SNv605 CD4-SNv786) in the dry formulation described below. DM2+S served as a control in this experiment for comparison with the other groups. 1. DM2+S 2. DM2+S No monomer 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] It was established from previous experiments that trehalose was necessary for dry-down, therefore DM2+S without trehalose was not used for this experiment. The experiment was performed on 6 donors in a single replicate. The sample processing protocol followed that described in the Protocols and Methods. The stop gate was set at 10000 CD45+ lymphocytes. After dry-down, the dried tubes from all groups were physically observed for any obvious changes. In addition, the scattering of the dual fluorescence plots was also observed for any non-specific pull-out and non-specific interactions.

[0305] FIG. 9 shows the physical appearance and properties of the dried tubes in each of the test groups DM2+S, trehalose+monomer, trehalose+casein, trehalose+DMMA (left to right, top panel), DM2+S, DM2+S without monomer, DM2+S without DMMA, and DM2+S without casein (left to right, bottom panel). Here, DM2+S serves as the control group. Physical observations show that without monomer, there is a change in the color of the dried film (typically the red film becomes light orange to brown). Shrinkage of the film was observed in the groups without casein and DMMA. No change in the appearance of the dried film was observed in the tube without DMMA compared to DM2+S. However, the tube without casein shows minimal shrinkage of the dried film.

[0306] Figure 10A-C shows side scatter SSC vs. FL plots for CD56-SNv428; CD20-SNv605, and CD4-SNv786 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, DM2+S: trehalose + monomer (left to right, lower panel), respectively. It can be observed from the figures that in the absence of casein and DMMA, there is a pullout of nonspecific monocytes (indicated by arrows). The absence of monomer causes the spread of negative population in lymphocytes (indicated by arrows). The spread of negative population can be mainly attributed to the nonspecific interaction between SN dyes in the absence of monomer and casein.

[0307] Figure 10D-F show that FL vs. FL dual fluorescence plots for all SN combinations in all groups tested (e.g., CD56-SNv428 vs. CD20-SNv605, CD4-SNv786 vs. CD20-SNv605, and CD4-SNv786 vs. CD56-SNv428, respectively) demonstrated that the absence of monomers and casein caused non-specific interactions / population spreading between SN conjugates within the SN population. Therefore, both monomers and casein are considered important to prevent non-specific interactions.

[0308] This example demonstrates that monomer A and casein are important for the dry-down of the SN conjugate, since without them inefficient prevention of cell-cell interactions is observed. In addition, casein also prevents non-specific monocyte pull-out. DMMA has a major role in preventing non-specific monocyte pull-out. This experiment is important not only for providing the function of the individual additives, but also for troubleshooting in 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 performed to check the performance of dry tubes compared to that of BD staining buffer. Here, the performance of DM2+S dry tubes was compared to BD horizon brilliant buffer. The experiment was performed using a 4-color panel. Group details are as follows:

[0311] The three polymer-dye conjugates were evaluated together with gating markers (CD45-APC-A750, CD56-SNv428, CD20-SNv605, and CD4-SNv786) using the 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 was added to the wells of the BD Horizon Brilliant staining buffer. Brilliant buffer was added to the tube, followed by the four conjugates. Mix thoroughly by vortexing. Then 100 μl of blood sample was added. Mix properly by vortexing, incubate at room temperature for 30 minutes, and process by following step 2 onwards as mentioned in the protocol and methods section.

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

[0313] Six donors (with a single repeat) were tested using the above protocol for all two stated groups. The stop gate was set at 10000 CD45+ lymphocytes.

[0314] Results and Observations:

[0315] 11A shows representative SSC vs. FL overlay flow plots for three polymer-dye conjugates, CD56-SNv428, CD20-SNv605, and CD4-SNv786, dried with either the DM2+S dry-down buffer of the present invention and reconstituted with blood samples or with the comparative BD Horizon™ Brilliant staining buffer. The comparative commercial BD Horizon™ Brilliant staining buffer caused non-specific granulocyte and monocyte pull-out (indicated by arrows) compared to the DM2+S dry-down tubes.

[0316] Figure 11B shows representative dual fluorescence overlay plots for the three polymer-dye conjugates along with gating markers (CD45-APC-A750, CD56-SNv428, CD20-SNv605, and CD4-SNv786). The comparative BD Horizon™ Brilliant stain buffer caused non-specific lymphocyte pull-out in all combinations of SN conjugates when compared to DM2+S.

[0317] This example demonstrates that DM2+S and BD staining buffer show no significant difference in scattering, % recruitment and MdFI values ​​(data not shown). However, DM2+S dry tubes show tighter populations (without any non-specific pullout) when compared to BD staining buffer. Therefore, it can be concluded from the data that DM2+S dry tubes have better performance compared to BD staining buffer. Example 4 Interim stability at 6 months

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

[0319] Methods: In this example, the 6-month stability of DM2+S dried tubes was evaluated. For comparison, 3-month-old and fresh lots were included in the study. Details of each lot are shown in Table 9. Both 3-month-old and 6-month-old dried tubes were tested in the study belonging to open pouches, i.e., dried tubes were tested multiple times from open pouches. These dried tubes contain a 12-color (12C) panel (9C conventional conjugates + 3C SN conjugates CD56-SNv428, CD20-SNv605, CD4-SNv786). These lots were tested on 4 donors in a single replicate on a single instrument. The stain-lysis-wash protocol described in Example 1B was used for the study. The stop gate was set at 10000 CD45+ lymphocytes. Compensation was adjusted for fresh lots. The fresh lot compensation was then applied to the 3- and 6-month old lots, and any necessary minor compensation adjustments were made in both the 3- and 6-month old lots. Absolute % delta mobilization and MdFI of the 3- and 6-month old lots were compared to the fresh lots (data not shown).

[0320] [Table 9]

[0321] Results and Observations:

[0322] Figure 12 shows a photographic image of the tube with the dry down film in a 6 month old dry tube. No deterioration in appearance was visually observed in the dry down film.

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

[0324] Figure 13A shows representative FL vs. FL overlay plots of DM2+S dried tubes for 6-month stability compared to 3-month and fresh lots. Dried tubes containing a 12-color (12C) panel (9C conventional conjugates + 3C SN conjugates CD56-SNv428, CD20-SNv605, CD4-SNv786) were tested on 4 donors in a single replicate on a single flow cytometry instrument. Representative FL vs. FL overlay plots for different combinations of SN dyes in all three lots for CD56 PB450-A vs. CD20 Violet610 (left panel), CD4Violet780 vs. CD20Violet610 (middle panel), and CD4 Violet780 vs. CD56PB450 (right panel). These overlays show that the populations in the 3-month and 6-month old lots overlap completely with the fresh lots. Additionally, no non-specific interactions or population spreading were observed in the 6-month old lots across all test donors.

[0325] Figure 13B shows the 6-month stability of DM2+S dried tubes compared to 3-month and fresh lots: representative FL vs. FL overlay plots for SN vs. classical combinations in all three lots. Dried tubes containing a 12-color (12C) panel (9C traditional conjugates + 3C SN conjugates CD56-SNv428, CD20-SNv605, CD4-SNv786) were tested on four donors in a single replicate on a single flow cytometry instrument. Overlays for 6-month, 3-month, and fresh lots for CD3 ECD vs. CD20Violet610 (left panel), CD8 KO525 vs. CD4Violet780 (middle panel), and CD45 APC-A750 vs. CD56PB450 (right panel) show that the populations in the 3-month and 6-month old lots overlap completely with the fresh lot. Additionally, no non-specific interactions or population spreading were observed in the 6-month old lots across all test donors.

[0326] Figure 13C shows the 6-month stability of DM2+S dried tubes compared to 3-month and fresh lots: representative FL vs. FL overlay plots for classical vs. classical combinations in all three lots. Dried tubes containing a 12-color (12C) panel (9C of conventional conjugates + 3C of SN conjugates CD56-SNv428, CD20-SNv605, CD4-SNv786) were tested on four donors in a single replicate on a single flow cytometry instrument. CD16 FITC vs. CD25PE (left panel), CD8 KO525 vs. CD45APC-A750 (middle panel), and CD3 Overlay of 6-month, 3-month, and fresh lots for ECD vs. CD10APC (right panel) shows that the populations in the 3-month and 6-month old lots completely overlap with the fresh lot. In addition, no non-specific interactions or spreading of populations were observed in the 6-month old lots in all test donors. In this example, the 6-month interim stability results show that the DM2+S dry tubes are stable for at least 6 months. No non-specific interactions or spreading were observed at the interim 6-month time point. Real-time stability is planned for 24 months. Example 5 Standardized DM buffer formulation

[0327] The DM buffer of the present invention may be prepared as follows: A stock preparation of solubilized additives (components) may be prepared as follows: After stock preparation, DM2+S buffer is prepared as shown in Table 10. The pH range of DM2+S buffer has been found to be between 7 and 7.4.

[0328] [Table 10]

[0329] The panel formulations shown in Table 11 were then prepared as uniform films of a single dry reaction in a tube format containing the three fluorescent polymer-dye conjugates using DM2+S DM buffer (Table 10).

[0330] [Table 11]

[0331] The final volume per test is 50 uL. In one embodiment, for example, the following items are provided: (Item 1) 1. A buffer composition for use in drying a plurality of dye conjugates on a substrate, comprising: Water-soluble monomers; Protein stabilizers; Carbohydrate stabilizers; and A buffer composition comprising a zwitterionic surfactant. (Item 2) 2. The buffer composition of claim 1, wherein at least one of the plurality of dye conjugates comprises a polymer-dye conjugate. (Item 3) The polymer dye conjugate has 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 moiety; each M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene, and ethynylene; G 1 and G 2 is independently selected from an unmodified polymer end and a modified polymer end; a, c, and d independently define the mole % of each unit within the structure, and each unit may be repeated evenly or randomly, where each a is a mole % of 10-100%, each c is a mole % of 0-90%, and each d is a mole % of 0-25%; each b is independently 0 or 1; m is an integer from 1 to about 10,000. 3. The buffer composition according to claim 2, wherein the binding partner is conjugated to a fluorescent polymer dye having a structure according to (Item 4) 4. The buffer composition of claim 3, wherein the plurality of dye conjugates comprises two or more different fluorescent polymer-dye conjugates, each having a structure according to formula (IV). (Item 5) 5. The buffer composition according to item 3 or 4, wherein the water-soluble monomer is a monomeric unit used in the preparation of at least one of the fluorescent polymer-dye conjugates. (Item 6) 6. The buffer solution 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 water-soluble monomer has the formula (I): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of halogen, alkyl, PEG, hydrogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol; Each R 2are independently selected from the group consisting of water solubilizing moiety, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, sulfonamido-PEG, phosphoramido-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, [ka] selected from the group consisting of; Each R 3 is a water-solubilizing moiety; Each R 4 are independently H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylate, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, selected from the group consisting of hydrazone, azide, alkyne, aldehyde, or thiol, or a protecting group thereof; Each R 5 are independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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, and C2-C 12 carboxylic acid esters; Each Q is independently a bond, NR 4 or -CH2; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. 7. The buffer composition according to any one of items 1 to 6, wherein the water-soluble monomer is a dihydrophenanthrene (DHP)-based monomer having a chemical structure according to (Item 8) Each G1, G2 is halogen; each Z is O; each R2 is H; each R 5 is H, hydroxyl, C1-C6 alkyl, or C1-C6 alkoxy; each n is independently 2 to 4; and each f is independently 5 to 20. (Item 9) each n is 3; each R 5 is -OCH3; and each f is 11 to 12. (Item 10) The water-soluble monomer has the formula (II): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of halogen, alkyl, PEG, hydrogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol; Each X is C or Si; Each R4 are independently H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylate, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, selected from the group consisting of hydrazone, azide, alkyne, aldehyde, or thiol, or a protecting group thereof; Each R5 is independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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 alkoxy; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. 7. The buffer solution composition according to any one of items 1 to 6, wherein the water-soluble fluorene-based monomer has a chemical structure according to (Item 11) Each G1, G2 is a halogen; each X is C; each Z is O; each R 5 is H, hydroxyl, C1-C6 alkyl, or C1-C6 alkoxy; each n is independently 2 to 4; and each f is independently 5 to 20. (Item 12) The zwitterionic surfactant has formula (XV): [ka] (wherein Y=CO2- or SO3-, W=H or OH, and Z=CH3 or NHC(O)R, where R=C 1~15 alkyl; independently, each p=0 or 1; q=0-21; and optionally, W=H, Z=CH3, and q=11-15. 12. The buffer composition according to any one of items 1 to 11, having a structure according to (Item 13) The zwitterionic surfactant is 3-(N,N-dimethylmyristylammoniopropanesulfonate (DMMA); 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate (DMPA); N-(alkyl C 10 ~C 16 13. The buffer composition according to any one of items 1 to 12, wherein the glycine moiety is selected from the group consisting of N,N-dimethylglycine betaine; and N,N-dimethyl-N-dodecylglycine betaine. (Item 14) 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) 15. The buffer composition according to any one of items 1 to 14, wherein the carbohydrate stabilizer is a disaccharide carbohydrate stabilizer. (Item 16) 16. The buffer composition of claim 15, wherein the disaccharide carbohydrate stabilizer is trehalose or a hydrate thereof, and optionally the trehalose or a hydrate thereof is trehalose dihydrate. (Item 17) From preservatives, antioxidants, anionic surfactants, nonionic surfactants, and colorants 17. The buffer composition according to any one of items 1 to 16, further comprising one or more additional additives selected from the group consisting of: (Item 18) 18. The buffer composition according to any one of items 1 to 17, wherein the composition is an aqueous composition. (Item 19) 19. The 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 test, 200 to 800 μg of the water-soluble monomer; 2000-3000 μg of said carbohydrate stabilizer; 8.4 to 72 μg of the protein stabilizer; and 2 to 9 μg of the zwitterionic surfactant 20. The buffer composition according to any one of items 1 to 19, comprising: (Item 21) 1. A method for preparing a single reactant film, the method comprising: dispensing a plurality of dye conjugates together on a substrate in a liquid phase comprising the aqueous buffer composition according to any one of items 1 to 18, wherein the plurality of dye conjugates comprises at least one polymer-dye conjugate; and drying the plurality of dye conjugates together in the liquid phase aqueous buffer to form a first single reactant film on the substrate. A method comprising: (Item 22) 22. The method of claim 21, wherein the plurality of dye conjugates comprises two or more polymer-dye conjugates. (Item 23) 22. The method of claim 20 or 21, wherein the substrate is selected from the group consisting of a tube, a well, a membrane, and a bead. (Item 24) 24. The method according to any one of items 21 to 23, wherein the substrate comprises an inner surface of a reaction vessel. (Item 25) 25. The method of any one of items 21 to 24, wherein the plurality of dye conjugates comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or 2 to 20, 3 to 18, or 4 to 12 different dye conjugates each comprising a different binding partner. (Item 26) when exposed to a first aliquot of a liquid blood sample, treated, and analyzed by flow cytometry, and compared to a second flow cytometry plot obtained by exposing a second single reactant film to a second aliquot of the liquid biological sample, treated, and analyzed by flow cytometry; Decreased nonspecific binding of monocytes; Decreased nonspecific binding of granulocytes; Reduced non-specific interactions of polymer-dye conjugates; and Reduced aggregation of polymer-dye conjugates A first flow cytometry plot showing one or more of the group consisting of: obtaining a the second single reactant film is prepared from a liquid phase containing the same dye conjugates that are dried together to form the first single reactant film, and a reagent buffer of conventional drying techniques, without the water soluble monomer and without the zwitterionic surfactant; 26. The method according to any one of Items 21 to 25. (Item 27) Multiple fluorescent polymer-dye conjugates; Water-soluble monomers; Protein stabilizers; Carbohydrate stabilizers; and Zwitterionic surfactants A composition comprising: (Item 28) 28. The composition according to item 27, in the form of a single reactant film disposed on a substrate. (Item 29) Per single reactant film, 200 to 800 μg of the water-soluble monomer; 2000-3000 μg of said carbohydrate stabilizer; 8.4 to 72 μg of the protein stabilizer; and 2 to 9 μg of the zwitterionic surfactant 29. The composition according to item 27 or 28, comprising: (Item 30) At least one of the plurality of fluorescent polymer-dye conjugates has the 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 moiety; each M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene, and ethynylene; G 1 and G 2 is independently selected from an unmodified polymer end and a modified polymer end; a, c, and d independently define the mole % of each unit within the structure, and each unit may be repeated evenly or randomly, where each a is a mole % of 10-100%, each c is a mole % of 0-90%, and each d is a mole % of 0-25%; each b is independently 0 or 1; m is an integer from 1 to about 10,000. 30. The composition according to any one of items 27 to 29, comprising a fluorescent polymer dye moiety having the structure according to (Item 31) The water-soluble monomer is used in the preparation of at least one of the plurality of fluorescent polymer dyes. 31. The composition according to any one of items 27 to 30, wherein the monomer unit is used in (Item 32) 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 water-soluble monomer has the formula (I): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of halogen, alkyl, PEG, hydrogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol; Each R 2 are independently selected from the group consisting of water solubilizing moiety, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, sulfonamido-PEG, phosphoramido-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide, [ka] 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 carboxylate amine, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protecting group thereof; Each R 5 are independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, 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, and C2-C 12 carboxylic acid esters; Each Q is independently a bond, NR 4 or -CH2; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. 33. The composition according to any one of items 27 to 32, wherein the water-soluble monomer is a dihydrophenanthrene (DHP)-based monomer having a chemical structure according to the above. (Item 34) 34. The composition of claim 33, wherein G1 and G2 are each halo; each Z is O; each R2 is H; each n is independently 2 to 4; each f is independently 5 to 20; optionally each n is 3; and each m is 11 to 12. (Item 35) The water-soluble monomer has the formula (II): [ka] (In the formula, each G1, G2 is independently selected from the group consisting of halogen, alkyl, PEG, hydrogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, optionally substituted tetrahydropyran (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP), aryl or heteroaryl substituted at one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, and thiol; Each X is C or Si; Each R 4 is independently selected from the group consisting of H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylate amine, an amine, a carbamate, a carboxylic acid, a carboxylate ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protecting group thereof; Each R5 is independently H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12Cycloalkyl, 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 alkoxy; Each Z is independently CH, O, or NR 4 and; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. 33. The composition according to any one of items 27 to 32, wherein the water-soluble fluorene monomer has a chemical structure according to the formula: (Item 36) The zwitterionic surfactant has formula (XV): [ka] (wherein Y=CO2- or SO3-, W=H or OH, and Z=CH3 or NHC(O)R, where R=C 1~15 alkyl); where p=0 or 1; q=0 to 21; and optionally W=H, Z=CH3, and q=11 to 15. 36. The composition according to any one of items 27 to 35, having a structure according to (Item 37) The zwitterionic surfactant is 3-(N,N-dimethylmyristylammoniopropanesulfonate (DMMA); 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate (DMPA); N-(alkyl C 10 ~C 16 37. The aqueous buffer composition according to any one of items 27 to 36, wherein the glycine moiety is selected from the group consisting of N,N-dimethylglycine betaine; and N,N-dimethyl-N-dodecylglycine betaine. (Item 38) 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) 39. The composition according to any one of items 27 to 38, wherein the carbohydrate stabilizer is a disaccharide carbohydrate stabilizer. (Item 40) 40. The composition of claim 39, wherein the disaccharide carbohydrate stabilizer is trehalose or a hydrate thereof, and optionally the trehalose or a hydrate thereof is trehalose dihydrate. (Item 41) 41. The 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 drying a plurality of dye conjugates on a substrate, comprising: Water-soluble monomers; protein stabilizers; a carbohydrate stabilizer; and A buffer composition comprising a zwitterionic surfactant.

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