Compositions and methods for enhanced fluorescence

Abstract

This invention provides a fluorescent labeling and conjugation method that exhibits enhanced signal intensity. [Solution] This disclosure relates to the field of fluorescent dyes, and more particularly to compositions and methods for increasing fluorescence signals and decreasing fluorescence quenching.

Description

Technical Field

[0001] Field of the Invention The present disclosure relates to the field of fluorescent dyes, and specifically, to compositions and methods for increasing fluorescence signals and decreasing fluorescence quenching.

Background Art

[0002] Background Fluorescently labeled biomolecules are widely used in a variety of methods including those related to quantitative assays and cell imaging. Biomolecules such as antibodies, antigens, DNA, and RNA are fluorescently labeled and are used in applications such as immunofluorescence (IFC), flow cytometry, fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), Western blotting, drug binding assays, enzyme kinetics, imaging including HCA (immunocytochemistry ICC), in vivo imaging, etc., and in nucleic acid hybridization. Fluorescent dyes continue to be selected for these applications because they are easy to use, specific, highly sensitive, and also provide options for multiplexing. However, not all applications are suitable for fluorescent labeling. For example, in many applications, as an increased amount of fluorescent label (high dye-to-protein ratio) is added to the biomolecule, the fluorescence intensity can decrease (quenching). Additionally, fluorescence can also decrease through steric hindrance caused by the fluorescent labeling of the biomolecule. There is a need for fluorescent labels and conjugation methods that exhibit enhanced signal intensity.

Summary of the Invention

[0003] Summary Disclosed herein are compositions and methods for increasing the intensity of fluorescent dyes conjugated to biomolecules via, for example, modified linkers, chemicals, and specific protocols. Thus, one aspect of the present invention is to improve the performance of dyes. It has been found that fluorescence intensity can be increased when biomolecules are labeled with both the dye and a spacer molecule, compared to corresponding conjugates made with the dye alone. Conjugates prepared using these spacer molecules exhibit fluorescence enhancement in Western blotting, dot blot assays, plate assays, flow cytometry, and immunoassay applications (e.g., immunofluorescence imaging applications).

[0004] Furthermore, enhanced fluorescence can be observed even when fewer dye molecules are bound (covalently or noncovalently) to each biomolecule. This effect is thought to be related to the binding of spacer groups to biomolecules exhibiting enhanced fluorescence. While not theoretically bound, enhanced fluorescence is thought to result from a reduction in the quenching of dye emission. In some embodiments, the present invention relates in part to a composition comprising a first biomolecule (e.g., a first antibody) to which two or more fluorescent labels and two or more spacer molecules are covalently bound, and the fluorescent and spacer molecules are not covalently bound to each other. In some cases, the first biomolecule (e.g., a first antibody) exhibits a higher fluorescence emission level than a second biomolecule (e.g., a second antibody) prepared with an equal amount of fluorescent labels but without spacers. In further examples, the first biomolecule exhibits a higher fluorescence emission level than the second biomolecule, and the first and second biomolecules each have the same number of covalently bound fluorescent labels, while the second biomolecule does not have covalently bound spacers. In certain embodiments, the spacer molecule reduces the quenching of the fluorescent label compared to the quenching in the absence of the spacer.

[0005] In some embodiments of the present invention, spacer molecules are conjugated to biomolecules via reactive groups. Such reactive groups may be amine-reactive groups (e.g., NHS esters, where one or more amine groups may be at the amine terminus of polypeptides and / or lysine side chains), sulfhydryl groups, carboxylic acid groups, and the like. Further groups to which spacer molecules can be conjugated include cysteine ​​residues, aspartic acid residues, glutamic acid residues, and / or carboxyl terminus of polypeptides. Furthermore, fluorescent labels can be conjugated to biomolecules via conjugation arms. These fluorescent labels may be positively charged, neutral, and / or negatively charged.

[0006] In some embodiments, the fluorescent labels used in the implementation of the present invention (both compositions and methods) may be cyanine, benzorhodamine, body pea, fluorescein, benzopyrillium derivatives, and may further include ALEXA FLUOR® dyes and / or DYLIGHT® dyes. Such pigments include ALEXA FLUOR(registered trademark) 350, ALEXA FLUOR(registered trademark) 405, ALEXA FLUOR(registered trademark) 430, ALEXA FLUOR(registered trademark) 488, ALEXA FLUOR(registered trademark) 500, ALEXA FLUOR(registered trademark) 514, ALEXA FLUOR(registered trademark) 532, ALEXA FLUOR(registered trademark) 546, ALEXA FLUOR(registered trademark) 555, ALEXA FLUOR(registered trademark) 568, ALEXA FLUOR(registered trademark) 594, ALEXA FLUOR(registered trademark) 610-X, ALEXA FLUOR(registered trademark) 633, ALEXA FLUOR(registered trademark) 647, ALEXA FLUOR(registered trademark) 660, ALEXA FLUOR(registered trademark) 680, ALEXA FLUOR(registered trademark) 700, ALEXA FLUOR(registered trademark) 750, ALEXA FLUOR(registered trademark) 790, AMCA-X, BODIPY(registered trademark) 630 / 650, BODIPY(registered trademark) 650 / 665, BODIPY(registered trademark) FL, BODIPY(registered trademark) TMR, BODIPY(registered trademark) TR, BODIPY(registered trademark) TR-X, CASCADE BLUE(registered trademark), Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE(registered trademark), OREGON GREEN(registered trademark) 488, OREGON GREEN(registered trademark) 514, PACIFIC BLUE(trademark), PACIFIC ORANGE(trademark), RHODAMINE GREEN(trademark), QSY(registered trademark) 7, QSY(registered trademark) 9, QSY(registered trademark) 21, QSY(registered trademark) 35, ROX, RHODAMINE RED(trademark), TET, TAMRA, Tetramethylrhodamine, FAM, TEXAS RED (registered trademark), 7-hydroxy-9H-(1,3-dichloro-9,The following can be selected from the group consisting of 9-dimethylacridine-2-one) succinimidyl ester (DDAO-SE), DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800. Naturally, other dyes and modified forms of the above-mentioned dyes can be used in the implementation of the present invention.

[0007] In the embodiment of the present invention, the spacer may be negatively charged or neutrally charged. Furthermore, the spacer may be selected from, for example, acetate and polyethylene glycol (PEG). The spacer may contain an acetyl group and may be an acetate molecule (e.g., sulfo-NHS-acetate). Furthermore, the spacer may contain or consist of (PEG)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, and / or contain or consist of MS-(PEG)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0008] Furthermore, the spacers used in the implementation of the present invention are alkanoyl, alkenoyl, and alkinoyl (-C(O)C n H m The formula may contain or consist of one or more groups selected from ), where n is 1 to 20 atoms, m > n, and carbon atoms may be bonded to each other by single, double, and / or triple bonds. Alkyl, alkenyl, and / or alkynyl groups are -(OCH2CH2O) x -(CH2) y -It may be further substituted by OR, where x is 1 to 20, y is 1 to 6, and R is H or C 1~6It is alkyl. Furthermore, alkyl, alkenyl, and / or alkynyl groups are ammonium (-NH3 + ), quaternary ammonium (-NR3 + ) may be further substituted with a group, where R is C 1~6 It is alkyl.

[0009] Furthermore, in certain embodiments of the present invention, the fluorescent dye is one or more ALEXA FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 500, ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610-X, ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR(registered trademark) 750, ALEXA FLUOR(registered trademark) 790, AMCA-X, BODIPY(registered trademark) 630 / 650, BODIPY(registered trademark) 650 / 665, BODIPY(registered trademark) FL, BODIPY(registered trademark) TMR, BODIPY(registered trademark) TR, BODIPY(registered trademark) TR-X, CASCADE BLUE(registered trademark) dinitrophenyl, fluorescein, HEX, JOE, MARINA BLUE(registered trademark), OREGON GREEN(registered trademark) 488, OREGON GREEN(registered trademark) 514, PACIFIC BLUE(trademark), PACIFIC ORANGE(trademark), RHODAMINE GREEN(trademark), QSY(registered trademark) 7, QSY(registered trademark) 9, QSY(registered trademark) 21, QSY(registered trademark) 35, ROX, RHODAMINE RED(trademark), TET, TAMRA, tetramethyl The material may contain rhodamine, FAM, TEXAS RED®, or 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacrylidine-2-one) succinimidyl ester (DDAO-SE), and the spacer is sulfo-NHS-acetate; (PEG)n (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15);MS-(PEG)n (where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15); alkanoyl, alkenoyl, or alkynoyl (-C(O)C; n H m )(where n is 1 to 20 atoms, m > n, and the carbon atoms can be bonded to each other by single bonds, double bonds, and / or triple bonds); or -(OCH2CH2O) x -(CH2) y -OR (where x is 1 to 20, y is 1 to 6, and R is H or C 1~6 alkyl), which is further substituted by an alkyl, alkenyl, or alkynyl group, or the alkyl, alkenyl, and / or alkynyl groups are ammonium (-NH3 + ), quaternary ammonium ((-NR3 + ), where R is C 1~6 alkyl), and may include one or more of the alkyl, alkenyl, or alkynyl groups. Further, the alkyl, alkenyl, and / or alkynyl groups may be further substituted by a phosphonium group (-PQ3 1~6 ), where Q is aryl, substituted aryl, or C + alkyl).

[0010] The compositions and methods of the present invention can include or use fluorescently labeled biomolecules (e.g., antibodies), and the ratio to the fluorescently labeled biomolecule is 1 to 50, 5 to 30, or 1 to 20. The compositions and methods of the present invention can include or use fluorescently labeled biomolecules (e.g., antibodies), and the ratio of the spacer agent to the biomolecule is 1 to 50, 5 to 30, 5 to 30, or 1 to 20.

[0011] Furthermore, the compositions and methods of the present invention may include or use fluorescently labeled biomolecules (e.g., antibodies), wherein the spacer is in a molar excess of 0.1 to 25 times, 1 to 15 times, or 2.5 to 10 times the amount of the fluorescent label; wherein the spacer is in a molar excess of 2.5 times the amount of the fluorescent label; wherein the spacer is in a molar excess of 5 times the amount of the fluorescent label; wherein the spacer is in a molar excess of 7.5 times the amount of the fluorescent label; or wherein the spacer is in a molar excess of 10 times the amount of the fluorescent label.

[0012] Furthermore, the compositions and methods of the present invention may include or use fluorescently labeled biomolecules (e.g., antibodies), and the proportion of binding sites on the biomolecule (e.g., accessible amine groups) occupied by multiple fluorescent labels is between 1% and 99%.

[0013] In some embodiments, the presence of a spacer increases the detectable fluorescence of the fluorescent label by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

[0014] The present invention also relates, in part, to a method for increasing the fluorescence of a fluorescently labeled biomolecule. Such a method includes (a) conjugating a spacer molecule to a biomolecule and (b) conjugating a fluorescent label to the biomolecule, where steps (a) and (b) can be carried out simultaneously or in any order, and where the spacer and the fluorescent label are not conjugated to each other. Furthermore, the spacer molecule may reduce the quenching of the fluorescent label compared to the amount of quenching that occurs in the absence of the spacer.

[0015] The present invention also relates, in part, to a method for identifying spacer molecules that can enhance fluorescently labeled biomolecules. Such a method may include (a) conjugating a spacer molecule to a biomolecule independently of a plurality of fluorescent labels conjugated to the biomolecule; (b) testing whether the presence of the spacer agent, in addition to the plurality of fluorescent labels conjugated to the biomolecule, increases the detectable fluorescence of the plurality of fluorescent labels; and (c) identifying the spacer agent as one that reduces the quenching of fluorescent labels conjugated to a protein when the presence of the spacer agent, in addition to the plurality of fluorescent labels conjugated to the biomolecule, increases the detectable fluorescence of the plurality of fluorescent labels. In some cases, the spacer agent is conjugated to the biomolecule at the first lysine side chain present on the biomolecule. Furthermore, the biomolecule is an antibody or antibody fragment. Furthermore, the plurality of fluorescent labels may be negatively and / or positively charged. Furthermore, the spacer agent may be negatively and / or positively charged.

[0016] The present invention further relates, in part, to a method for determining the presence of a desired target in a biological sample. Such a method may include (a) contacting the biological sample with an antibody of a composition, where two or more fluorescent labels and two or more spacer molecules are covalently bound to the antibody, and the fluorescent molecules and spacer molecules are not covalently bound to each other; (b) detecting the fluorescence emitted by the multiple fluorescent labels; and (c) determining the presence of a desired target in the biological sample when the fluorescence emitted by the multiple fluorescent labels is detected. The biological sample used in the implementation of the present invention may include cell lysates, intact cells (e.g., intact cells in a fluid such as body fluid), isolated proteins, and / or recombinant proteins. Furthermore, the biological sample may be immobilized on a solid support. Furthermore, the biological sample may include living animals such as mammals.

[0017] The present invention also relates in part to a composition comprising a first nucleic acid molecule, wherein two or more fluorescent labels and two or more spacer molecules are covalently bonded to the first nucleic acid molecule, and the fluorescent and spacer molecules are not covalently bonded to each other. In some embodiments, the first nucleic acid molecule exhibits a higher fluorescence emission level than a second nucleic acid molecule prepared with an equal amount of fluorescent labels but without spacers. Furthermore, in some embodiments, the first nucleic acid molecule may exhibit a higher fluorescence emission level than the second nucleic acid molecule, and the first and second nucleic acid molecules each have an equal number of covalently bonded fluorescent labels, while the second nucleic acid molecule does not have covalently bonded spacers.

[0018] The present invention also includes a conjugated antibody comprising an antibody conjugated with a plurality of fluorescent labels (for example, an average of about 2 to about 30, about 2 to about 20, about 3 to about 30, about 2 to about 15, about 3 to about 15, about 4 to about 30, about 6 to about 20, about 7 to about 30, etc.), wherein the conjugated antibody has the following characteristics: (a) A fluorescence ratio of 0.5 or higher based on one fluorescent label, (b) Conjugate at least four fluorescent labels to the antibody. (c) The total fluorescence of the antibody is at least 20 percent greater than the fluorescence of the non-conjugate fluorescent molecule, and / or (d) Each antibody molecule contains one or more of the average 3 to 80 fluorescent labels.

[0019] Furthermore, the fluorescent labels can be conjugated to biomolecules (e.g., antibodies) by one or more multi-arm polymers (e.g., about 1 to about 15, about 2 to about 10, about 2 to about 15, about 2 to about 8, about 3 to about 10, about 3 to about 6, etc.). Furthermore, the arms of the multi-arm polymer may consist of a type of chemical selected from the group consisting of (a) polyethylene glycol, (b) polysaccharides, and (c) polypeptides, as well as other substances. In addition, the average brush distance between the fluorescent labels may be 200 to 800 angstroms (e.g., about 200 to about 700, about 300 to about 800, about 400 to about 800, about 500 to about 800, about 200 to about 600, about 500 to about 800, about 300 to about 700, about 350 to about 800, etc.). Furthermore, fluorescent labels conjugated to antibodies (or other biomolecules) can be separated from the antibody (or other biomolecule) by at least 16 covalent bonds (e.g., approximately 16 to 800, approximately 25 to 800, approximately 40 to 800, approximately 60 to 800, approximately 100 to 800, approximately 200 to 800, approximately 250 to 800, approximately 150 to 600, etc.). Furthermore, conjugate antibodies (or other biomolecules) are available for ALEXA FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 500, ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610-X, ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, and ALEXA FLUOR(registered trademark)750, ALEXAFLUOR(registered trademark) 790, AMCA-X, BODIPY(registered trademark) 630 / 650, BODIPY(registered trademark) 650 / 665, BODIPY(registered trademark) FL, BODIPY(registered trademark) TMR, BODIPY(registered trademark) TR, BODIPY(registered trademark) TR-X, CASCADE BLUE(registered trademark), Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE(registered trademark), OREGON GREEN(registered trademark) 488, OREGON GREEN(registered trademark) 514, PACIFIC BLUE(trademark), PACIFIC ORANGE(trademark), RHODAMINE GREEN(trademark), QSY(registered trademark) 7, QSY(registered trademark) 9, QSY(registered trademark) 21, QSY(registered trademark) 35, ROX, RHODAMINE RED(trademark), TET, TAMRA, Tetramethylrhodamine, FAM, TEXAS Fluorescent labels that may be one or more dyes selected from the group consisting of RED® or 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacrylidine-2-one) succinimidyl ester (DDAO-SE), and can be conjugated to fluorescent labels from the group consisting of DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800, and PEGylated DYLIGHT® dyes.

[0020] The present invention also includes a method for preparing a fluorescently labeled biomolecule, comprising (a) conjugating a reactive group and two or more fluorescent labels onto a spacer molecule to form a fluorescently labeled spacer molecule, and (b) conjugating the fluorescently labeled spacer molecule onto a biomolecule to form a fluorescently labeled biomolecule, wherein each fluorescent label of the fluorescently labeled biomolecule has a fluorescence ratio of 0.5 or more based on one fluorescent label. Furthermore, an average of 1 to 10 (e.g., about 1 to about 9, about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 2 to about 6, about 3 to about 6, about 3 to about 7, etc.) of fluorescently labeled spacer molecules can be conjugated onto each biomolecule. Furthermore, the fluorescently labeled spacer molecule may be a multi-arm polymer (e.g., a branched-chain polyethylene glycol molecule). Furthermore, the spacer molecules (e.g., multi-arm polymers) can each be conjugated to fluorescent labels with an average of 4 to 20 (e.g., about 4 to 10, about 3 to 8, about 4 to 8, about 3 to 9, etc.). In addition, the spacer molecules (e.g., multi-arm polymers) may have molecular weights of 4,000 to 80,000 Daltons (e.g., about 4,000 to 70,000, about 4,000 to 60,000, about 4,000 to 50,000, about 4,000 to 40,000, about 10,000 to 70,000, about 15,000 to 60,000, etc.).

[0021] The present invention further includes a method for detecting fluorescently labeled biomolecules. Such a method may include (a) exposing a fluorescently labeled biomolecule (e.g., an antibody) with light that excites a fluorescent label conjugated to the biomolecule, and (2) detecting the luminescence produced by the fluorescent label conjugated to the biomolecule. In some cases, a fluorescently labeled biomolecule may be conjugated to four or more fluorescent labels. Furthermore, each fluorescent label of a fluorescently labeled biomolecule may have a fluorescence ratio of 0.7 or higher based on one fluorescent label (e.g., about 0.7 to about 1.0, about 0.7 to about 0.95, about 0.7 to about 0.9, about 0.7 to about 0.85, about 0.75 to about 0.95, etc.). [Brief explanation of the drawing]

[0022] [Figure 1] Figure 1 shows a schematic diagram of one embodiment of the present invention. [Figure 2] Figure 2 shows schematic diagrams of several embodiments of the present invention. [Figure 3] Figure 3 shows the results of software analysis of dot blots acquired using imaging equipment. [Figure 4] Figure 4 shows the results of software analysis of dot blots acquired using imaging equipment. [Figure 5] Figure 5 shows the results of software analysis of dot blots acquired using imaging equipment. [Figure 6] Figure 6 shows the results of software analysis of dot blots acquired using imaging equipment. [Figure 7] Figure 7 demonstrates the effect of adding NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, and 10×) on the detectable fluorescence levels of GAM-DYLIGHT® 800-4×PEG (abbreviated as "DYLIGHT® 800" in Table 3) in a fluorescence dot blot assay. [Figure 8] Figure 8 shows a Western blot assay demonstrating the effect of adding NHS acetate (2.5×, 5×, and 10× molar excess) or MS(PEG)4 (3.75× molar excess) to the fluorescence detection level of GAR-DYLIGHT® 488 conjugated with an antibody in a molar excess ranging from 5× to 20× of dye. [Figure 9] Figure 9 shows the effect of NHS acetate (5×) or MS(PEG)4 (5×) on GAM-DYLIGHT® 650-4×PEG-GAR (with 7.5× molar excess dye) in Western blot. [Figure 10]Figure 10 shows the results of a Western blot assay testing the effect of NHS acetate (2.5×, 5×) or MS(PEG)4 on the detectable fluorescence level of GAR-DYLIGHT® 800-4×PEG (abbreviated as "DYLIGHT® 800" in Table 4). [Figure 11] Figure 11 shows the effects of adding NHS acetates (2.5×, 5×, and 10×) or MS(PEG)4 (5×) and MS(PEG)8 (5×) to GAM-DYLIGHT® 550-2×PEG (with 12.5× molar excess dye) in fluorescence Western blot and dot blot assays. [Figure 12] Figure 12 shows the effects of NHS acetate (2.5×, 5×) or MS(PEG)4 (5×) on GAM-DYLIGHT® 680-4×PEG-GAR (dye in 10x molar excess) in Western blot and dot blot assays. [Figure 13A] Figure 13A shows the IFC (Internal Fluid Factor) using DYLIGHT® 488-GAM 4 μg / ml. [Figure 13B] Figure 13B shows a similar experiment in which A549 cells were stained with pH2A × primary antibody. [Figure 14] Figure 14 shows IFC detection of PDI using DYLIGHT® 550-2×PEG (7.5×~20×) 4 μg / ml. [Figure 15] Figure 15 shows IFC detection of PDI using DYLIGHT® 650-4×PEG (7.5×~20×) 4 μg / ml. [Figure 16] Figure 16 shows the improvement ratios of IFC-DYLIGHT® 680-4×PEG compared to the basic conjugate in each molar excess with NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, and 10×). [Figure 17] Figure 17 illustrates the fluorescence levels observed for various dye / protein molar ratios of TAMRA-goat anti-mouse antibody (GAM) conjugates with and without betaine. [Figure 18]Figure 18 shows a schematic diagram of the generation of branched-chain PEG molecules containing fluorescent labeling and their binding to antibody molecules. [Figure 19] Figure 19 shows examples of single-arm connectors (such as amylose) and multi-arm connectors (such as dextran). [Figure 20] Figure 20 shows various types of star polymers, examples of some star polymer components, and examples of star polymer manufacturing methods. [Figure 21] Figure 21 shows exemplary types of polylysine molecules that can be used in carrying out the present invention. [Figure 22] Figure 22 shows the results for SK3 mouse anti-human CD4 antibody conjugated with AF647 branched PEG construct. [Figure 23] Figure 23 shows the results for SK3 mouse anti-human CD4 antibody conjugated with AF647 branched PEG construct.

[0023] (Figure 1) A schematic diagram of one embodiment of the present invention is shown. In this embodiment, the antibody is conjugated with an NHS fluorescent dye and a sulfoNHS-acetate spacer in 50 mM borate buffer (pH 8.5) in molar excess of two different dyes. The conjugation of the dye and spacer results in enhanced sensitivity and reduced quenching. (Figure 2) A schematic diagram of several embodiments of the present invention is shown. In this embodiment, the antibody is conjugated with an NHS fluorescent dye and a methyl-PEG-NHS-ester spacer in 50 mM borate buffer (pH 8.5). The spacers used in this embodiment are MS(PEG)4, MS(PEG)8 and MS(PEG) 12 That is the case. (Figure 3) This figure shows the results of software analysis of dot blots acquired using imaging equipment. The dot blots were tested using GAM antibodies co-labeled with NHS acetate or MS(PEG)4 spacer and DYLIGHT® 488 fluorescent dye (abbreviated as "DYLIGHT® 488" in Table 1). DYLIGHT® 488-GAM conjugates prepared using NHS acetate or MS(PEG)4 resulted in a 1.2 to 1.8-fold improvement in fluorescence intensity compared to the base conjugate (prepared without spacer), ranging from 1.2 to 1.8 times. The lanes in this figure are as follows: (Table 1) JPEG2026071206000002.jpg169156 (Figure 4) Dot blot - Detection of mouse IgG with DYLIGHT® 488-GAM, improvement ratios of NHS acetate (2.5×, 5×) or MS(PEG)4 (3.75×) compared to the basic conjugate with various molar excesses of dye. This figure shows the results of software analysis of dot blots acquired using imaging equipment. Dot blots were tested by assays using NHS acetate or MS(PEG)4 spacers and GAM antibodies co-labeled with DYLIGHT® 488 fluorescent dye. These data confirmed the results from Figure 3 using a different source of antibody. Both NHS acetate and MS(PEG)4 spacers resulted in significant improvements in fluorescence signal intensity (approximately a 2.6-fold increase) compared to the antibody-dye conjugate alone. (Figure 5) Dot blot - Detection of mouse IgG using DYLIGHT® 550-2×PEG-GAR. Signal / background improvement ratio from NHS acetate (2.5×, 5×) or MS(PEG)4 (3.75×) compared to the base conjugate at various molar excesses. This shows the results of software analysis of dot blots acquired using imaging equipment. Dot blots were tested with DYLIGHT® 550-GAM antibody co-labeled with NHS acetate or MS(PEG)4 spacer and DYLIGHT® 550 fluorescent dye. Mouse IgG was serially diluted 1:1 from 1000 ng / dot. All DYLIGHT® 550-2×PEG-GAR secondary antibodies were diluted to 1 / 5000 of the 1 mg / ml stock. The addition of NHS acetate or MS(PEG)4 to the conjugation mixture resulted in an improvement in signal intensity (an increase of approximately 1.6) compared to the base conjugate in molar excess of each respective dye. The improvement ranged from 1.2 to 1.6 times, and in particular, the following GAM-DYLIGHT(trademark) 550-2×PEG:10×dye+5×acetate, 15×dye+3.75×MS(PEG)4, 20×dye+5×acetate, and 20×+3.75×MS(PEG)4 showed improvements of more than 1.3 times compared to their respective base conjugates. (Figure 6) Dot blot - Detection of mouse IgG with DyLight650-4×PEG-GAM, improvement ratios of NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, 10×) compared to the basic conjugate in molar excess of each dye. This figure shows the results of software analysis of dot blots acquired using imaging equipment. Dot blots were tested with NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×) and GAM antibodies co-labeled with DYLIGHT® 650-4×PEG (abbreviated as "DYLIGHT® 650" in Table 2) (10×~20×). Mouse IgG was serially diluted 1:1 from 1000 ng / dot. All DYLIGHT® 650-4×PEG-GAR secondary antibodies were diluted to 1 / 10,000 of the 1 mg / ml stock. Conjugates with a high degree of dye substitution tend to perform better in applications such as dot blotting and Western blotting. Both NHS acetate and (MS)PEG4 resulted in significant improvements in sensitivity and signal / background (an increase of approximately 2.2) compared to the initial base conjugate. NHS acetate added to GAM-DYLIGHT® 650-4×PEG-15× in a 2.5× molar excess improved intensity by 1.7 times. The improvement provided by NHS acetate was 1.3 times better than when using conjugates prepared in the highest molar excess (20×). All MS(PEG)4 added to the GAM-DYLIGHT® 650-4×PEG-15× conjugation improved fluorescence intensity by 1.8 to 2.2 times and performed better than the corresponding best basic conjugation, GAM-DYLIGHT® 650-4×PEG-20×. The lanes in this figure are as follows: (Table 2) JPEG2026071206000003.jpg169128 (Figure 7) Dot blot - Improvement ratio of DYLIGHT® 880-4×PEG conjugate with NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, 10×) compared to the basic conjugate in molar excess. This figure demonstrates the effect of adding NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, 10×) on the detectable fluorescence level of GAM-DYLIGHT® 800-4×PEG (abbreviated as "DYLIGHT® 800" in Table 3) in a fluorescence dot blot assay. Mouse IgG was serially diluted from 1000 ng / dot to 1:2. All DYLIGHT® 800-4×PEG-GAR secondary antibodies were diluted to 1 / 20,000 of the 1 mg / ml stock. This dot blot application demonstrates that the addition of MS(PEG)4 (3.75× and 5×) and NHS acetate (5×) significantly enhanced the fluorescence intensity and sensitivity of the base DYLIGHT® 800-4×PEG conjugate by 1.5 to 6 times. The lanes in this figure are as follows: (Table 3) Figure 8 (JPEG2026071206000004.jpg169134) shows the effect of adding NHS acetate (2.5×, 5×, and 10× molar excess) or MS(PEG)4 (3.75× molar excess) to the fluorescence detection level of GAR-DYLIGHT® 488 conjugated with antibodies in molar excess of dyes ranging from 5× to 20×. A431 cell lysates were diluted 3-fold from 1 μg / well. The rabbit primary antibodies used were anti-Hsp90 diluted from 1 mg / ml to 1 / 5000 and anti-cyclophyllin B diluted from 1 mg / ml to 1 / 5000. All DYLIGHT® secondary antibodies were diluted from 1 mg / ml stock to 1 / 5000. These results demonstrate that, for Western blotting applications, DYLIGHT® 488-GAR conjugated with NHS acetate or MS(PEG)4 exhibits a significant increase in fluorescence intensity compared to the basic conjugate (prepared without spacers) at molar excesses of each dye from 7.5× to 20×. (Figure 9) Western blotting shows the effect of NHS acetate (5×) or MS(PEG)4 (5×) on GAM-DYLIGHT® 650-4×PEG-GAR (with 7.5× molar excess dye). HeLa cell lysates were diluted 4-fold from 0.5 μg / well. Primary antibody mouse anti-PDI was diluted to 1 / 5000 of 1 mg / ml. All DYLIGHT® secondary antibodies were diluted to 1 / 5000 of 1 mg / ml stock. NHS acetate added to GAM-DYLIGHT® 650-4×PEG-7.5× conjugate with a 5× molar excess improved the intensity by 1.5 times. NHS acetate added to GAM-DYLIGHT® 650-4×PEG-7.5× conjugate with a 3.75× molar excess improved the intensity by 1.4 times. (Figure 10) This figure shows the results of a Western blot assay testing the effect of NHS acetate (2.5×, 5×) or MS(PEG)4 on the detectable fluorescence level of GAR-DYLIGHT® 800-4×PEG (abbreviated as "DYLIGHT® 800" in Table 4). A431 cell lysates were serially diluted 1:1. Primary antibodies rabbit anti-Hsp90 and anti-cyclophyllin B were both diluted to 1 / 5000. All DYLIGHT® secondary antibodies were diluted to 1 / 20,000 of the 1 mg / ml stock. In this Western blotting application, the addition of MS(PEG)4 (3.75× and 5×) and NHS acetate (2.5-5×) significantly enhanced the fluorescence intensity and sensitivity of the base DYLIGHT® 800-4×PEG conjugate by 20-100% with different molar excesses of dye. The lanes in this figure are as follows: (Table 4) JPEG2026071206000005.jpg80141 (Figure 11) shows the effects of adding NHS acetates (2.5×, 5×, and 10×) or MS(PEG)4 (5×) and MS(PEG)8 (5×) to GAM-DYLIGHT® 550-2×PEG (with 12.5× molar excess dye) in fluorescence Western blot and dot blot assays. HeLa cell lysates were diluted 4-fold from 0.5 μg / well and stained with anti-PDI primary antibody diluted to 1 / 5000 of 1 mg / ml. All DYLIGHT® secondary antibodies were diluted to 1 / 5000 of 1 mg / ml stock. Western blotting and dot blot assays showed that the fluorescence intensity and sensitivity of DYLIGHT® 550-2×PEG conjugates were significantly enhanced by at least twofold upon addition of MS(PEG)4 (5×) and NHS acetate (2.5× and 5×). Conjugates prepared with long-chain MS(PEG)8 did not show significant improvement over the base conjugate. (Figure 12) Western blot and dot blot assays show the effect of NHS acetate (2.5×, 5×) or MS(PEG)4 (5×) on GAM-DYLIGHT® 680-4×PEG-GAR (dye in 10x molar excess). For Western blotting, HeLa cell lysates were diluted 4-fold from 0.5 μg / well, and anti-PDI primary antibody was diluted to 1 / 5000 of 1 mg / ml. For dot blotting, mouse IgG was serially diluted 1:2 from 1000 ng / dot. All DYLIGHT® 680-4×PEG-GAR secondary antibodies were diluted to 1 / 20000 of 1 mg / ml stock. Both Western blotting and dot blot assays showed that the fluorescence intensity and sensitivity of DYLIGHT® 680-4×PEG conjugate were significantly enhanced by 3 to 4 times upon addition of MS(PEG)4 (5×) and NHS acetate (2.5× and 5×). (Figure 13A) IFC with DYLIGHT® 488-GAM 4 μg / ml. Signal / background improvement ratio from NHS acetate (2.5×, 5×) or MS(PEG)4 (3.75×) compared to the base conjugate at various molar excesses, and Figure 13B (PDI detection with IFC-DYLIGHT® 488-GAR 4 μg / ml. Signal / background improvement ratio from NHS acetate (2.5×, 5×) or MS(PEG)4 (3.75×) compared to the base conjugate at various molar excesses). This study demonstrates the effect of adding MS(PEG)4 cells (3.75×). Figure 13A: A549 cells were stained with pH2A× primary antibody diluted to 1 / 1000 of 1 mg / ml stock. All DYLIGHT® 488 secondary antibodies were diluted to 1 / 250 of 1 mg / ml stock. NHS acetate modified conjugates, with a 15× dye molar excess, resulted in improvements in signal / background ranging from 1.4 to 1.5 times (GAM) and 1.1 to 1.6 times (GAR) compared to the basic conjugate. For GAM conjugates, the most significant improvements were observed with MS(PEG)4 for 5× NHS acetate and 3.75× GAR conjugates, and more significant improvements were observed with 2.5× NHS acetate and 3.75× MS(PEG)4. (Figure 13B) Figure 13B shows a similar experiment in which A549 cells were stained with pH2A × primary antibody. (Figure 14) IFC detection of PDI by DYLIGHT® 550-2×PEG (7.5×~20×) 4 μg / ml. Improvement ratio from the addition of NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, and 10×) to the basic conjugate with each molar excess of dye. This figure shows the effect of the addition of NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, and 10×) to GAM-DYLIGHT® 550-2×PEG-GAM (with 7.5×~20× molar excess of dye) in a fluorescence cell imaging assay. U2OS cells were stained with anti-PDI primary antibody diluted to 1 / 100 of the 1 mg / ml stock. All DYLIGHT® 550-2×PEG-GAM secondary antibodies (abbreviated as "DYLIGHT® 550" in Table 5) were diluted to 1 / 250 of the 1 mg / ml stock. For this cell imaging application, the addition of 5×NHS acetate resulted in approximately 50% improvement for the DYLIGHT® 550-2×PEG GAM conjugate with a 12.5× molal excess of dye compared to the base conjugate (made without addition), and the addition of 3.75×MS(PEG)4 with a 20-fold molal excess of dye resulted in approximately 50% improvement compared to the base conjugate. The lanes in this figure are as follows: (Table 5) JPEG2026071206000006.jpg169134 (Figure 15) IFC detection of PDI by GAM-DYLIGHT® 650-4×PEG (7.5×~20×) 4 μg / ml. Improvement ratio from the addition of NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, 10×) to the basic conjugate in each dye molar excess. This figure shows the effect of adding NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, 10×) to GAM-DYLIGHT® 650-4×PEG in a fluorescence cell imaging assay. U2OS cells were stained with anti-PDI primary antibody diluted to 1 / 100 of the 1 mg / ml stock. All DYLIGHT® 650-4×PEG-GAM secondary antibodies (abbreviated as "DYLIGHT® 650" in Table 6) were diluted to 1 / 250 of the 1 mg / ml stock. In this application, the addition of NHS acetate-5× resulted in approximately 70% improvement for the DYLIGHT® 650-4×PEG-GAM conjugate with a 20× molar excess compared to the base conjugate (made without addition), and the addition of MS(PEG)4-3.75× with a 20-fold molar excess showed approximately 90% improvement compared to the base conjugate. The lanes in this figure are as follows: (Table 6) JPEG2026071206000007.jpg169132 (Figure 16) Improvement ratios of IFC-DYLIGHT® 680-4×PEG with NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, 10×) compared to the basic conjugate in molar excess. This figure shows the effect of adding NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.3.75×, 5×, 10×) to IFC-DYLIGHT® 680-4×PEG) in a cell imaging assay. U2OS cells were stained with mouse anti-PDI primary antibody diluted to 1 / 100 of the 1 mg / ml stock. All DYLIGHT® 680-4×PEG-GAM secondary antibodies (abbreviated as "DYLIGHT® 680" in Table 7) were diluted to 1 / 250 of the 1 mg / ml stock. For this cell imaging application, the addition of NHS acetate-5× resulted in approximately 70% improvement in the dye conjugate at both 7.5× and 10× compared to the base conjugate of DYLIGHT® 680-4×PEG (prepared without addition). The GAM conjugate and MS(PEG)4-3.75× at 15× molar excess showed approximately 80% improvement over the base conjugate at 15× molar excess. The lanes in this figure are as follows: (Table 7) JPEG2026071206000008.jpg169132 (Figure 17) TAMRA-GAM co-conjugate with or without betaine. This figure illustrates the fluorescence levels observed at various dye / protein molar ratios of TAMRA-goat anti-mouse antibody (GAM) conjugates with or without betaine. Betaine levels were tested at 2.5, 5, and 10 × molar excesses compared to the number of moles of antibody. These antibodies, labeled with NHS-rhodamine (TAMRA) and conjugated with various NHS-betaine concentrations (2.5 × betaine, 5 × betaine, and 10 × betaine molar excess dyes), showed an increase in total fluorescence when the antibody was conjugated with betaine as a spacer. (Figure 18) A schematic diagram of the generation of branched chain PEG molecules containing fluorescent labels and their binding to antibody molecules is shown. In step 1, the reactive group and fluorescent label are attached to the NH2 group on the branched chain PEG molecule. In step 2, the attachment site is added to the antibody molecule. In step 3, the fluorescently labeled branched chain PEG molecule is covalently bonded to the antibody molecule. (Figure 19) This figure shows examples of single-arm connectors (such as amylose) and multi-arm connectors (such as dextran). BM represents a biomolecule, and AP represents a binding site, which means that the fluorescent label binds to that site. (Figure 20) Various types of star polymers, some examples of star polymer components, and examples of star polymer manufacturing methods. Figure 20A is a diagram of a star polymer having a core (black circle) and multiple arms (black lines). The stars represent fluorescent labels covalently bonded to the arms. Some arms are not fluorescently labeled, indicating that the labeling was incomplete. Figure 20B is a diagram of a similar star polymer where the arms are of two different types (e.g., polyethylene glycol and polyvinyl alcohol), and the different arm types are represented by solid and dashed lines. The left side of Figure 20C shows a partial view of a core (black semicircle) having reactive groups (gray bars) that can be used to bond other chemicals or act as initiators for polymerization. The core is shown in the center with adapters (black solid lines) bonded to the reactive groups. The polymer arms are shown on the right side (black dashed lines) bonded to the adapters and labeled with fluorescent molecules (stars). (Figure 21) An exemplary polylysine molecule of the type that can be used in carrying out the present invention is shown. The R1 and R2 groups are shown as unlabeled. These groups can be used as binding sites for fluorescent labeling and for conjugation to biomolecules (e.g., antibodies). (Figure 22) SK3 mouse anti-human CD4 antibody conjugated with AF647 branched-chain PEG construct. SK3 mouse anti-human CD4 antibody (5.5 mg / mL) was modified with ALEXA FLUOR® 647 succinimidyl ester in a 10-fold molar excess (dashed line). SK3 tagged with 10-fold excess azide-SE was click-conjugated with 1 mg / mL of azide-SK3 antibody to 100 μM AF647-HG20K8 PEG-sDIBO at 25°C for 20 hours, quenched with 5 mM NaN3, and purified using a Millipore AMICON® Ultra-2 100 kDa centrifugation filter (dotted line). SK3 cells tagged with azido-SE in a 20-fold excess relative to the antibody were click-conjugated with 3 mg / mL of azido-SK3 antibody in 600 μM AF647-HG20K8 PEG-sDIBO at 37°C for 3 hours, quenched with 5 mM NaN3, and purified using a Millipore AMICON® Ultra-2 100 kDa centrifugation filter (solid line). One million Ficoll-isolated PBMCs / well from a 96-well plate were stained with the SK3 conjugate using a 7-stop titration of 1 μg to 0.015 μg of antibody. Analysis of the stained cells was performed using an ATTUNE® NxT flow cytometer. (Figure 23) SK3 mouse anti-human CD4 antibody conjugated with AF647 branched-chain PEG construct. SK3 mouse anti-human CD4 antibody (5.5 mg / mL) was modified with ALEXA FLUOR® 647 succinimidyl ester in a 10-fold molar excess relative to the antibody (AF). SK3 tagged with 20-fold excess azide-SE relative to the antibody was click-conjugated with 3 mg / mL azide-SK3 antibody to 600 μM AF647-HG20K8 PEG-sDIBO at 37°C for 3 hours, quenched with 5 mM NaN3, and purified using a Millipore AMICON® Ultra-2 100 kDa centrifugation filter (B1). SK3 tagged with azide-SE in a 10-fold excess relative to the antibody was click-conjugated with 1 mg / mL of azide-SK3 antibody in 100 μM AF647-HG20K8 PEG-sDIBO at 25°C for 20 hours, quenched with 5 mM NaN3, and purified using a Millipore AMICON® Ultra-2 100 kDa centrifugation filter (B2). One million Ficoll-isolated PBMCs / well from a 96-well plate were stained with the SK3 conjugate using a 7-stop titration of 1 μg to 0.015 μg of antibody. Analysis of the stained cells was performed using an ATTUNE® NxT flow cytometer and compared with allophycocyanin (APC) (Thermo Fisher Scientific, catalog number MHCD0405). [Modes for carrying out the invention]

[0024] Detailed explanation Fluorescent labeling is widely used in imaging because it provides direct, quantitative, specific, and highly sensitive detection of biomolecules, including proteins and nucleic acids. Modified fluorescent labels that are sulfonated and / or PEG-modified are more sensitive than basic, unmodified dyes. However, even these modified fluorescent labels exhibit fluorescence quenching at specific dye-to-protein (D / P) ratios.

[0025] While increased fluorescence is observed with increasing molar excess of the labeling dye, molar excess exceeding the optimal dye-to-protein ratio (D / P) typically leads to quenching and / or precipitation of biomolecules, particularly in fluorescence imaging applications where the spatial conformation of antigen / antibody or DNA / RNA interactions can cause static quenching.

[0026] In some embodiments, the present invention includes methods for reducing quenching and / or increasing a fluorescence signal using highly labeled conjugates (e.g., biomolecules having a high dye-to-protein ratio (D / P)) that result in a decrease in fluorescence in standard conjugation. Proteins, nucleic acids, and other biomolecules (e.g., oligosaccharides) can be modified. In some embodiments, the present invention includes a composition comprising a fluorescently labeled biomolecule that exhibits an increase in fluorescence signal and / or a decrease in quenching, wherein the composition comprises the biomolecule, a spacer, and a fluorescent label, and the spacer and the fluorescent label are not directly conjugated to each other.

[0027] The present invention also relates to compositions that exhibit enhanced fluorescence based on the number of fluorescent labels, and to methods for producing and using such compositions. For example, assume that a single fluorescent label bound to a biomolecule sets a baseline of 100% fluorescence emission. Furthermore, assume that when two fluorescent labels are bound to the same biomolecule, each of the two fluorescent labels exhibits an average of 80% of the baseline fluorescence emission. The present invention relates in part to compositions and methods for increasing the average fluorescence emission beyond 80% of the baseline.

[0028] In some cases, the compositions of the present invention and the compositions used in the methods of the present invention may be defined by one or more functional properties. Examples of such properties include the number of fluorescent labels bound to the labeled molecule (e.g., biomolecule), the average distance between fluorescent labels on the labeled molecule (measured by one of a number of different methods), and / or the quantum yield of the fluorescent labels on the labeled molecule.

[0029] One method for measuring fluorescence intensity is by measuring the quantum yield. The quantum yield (Φ) for a fluorescence system is, in effect, the luminescence efficiency of a given fluorophore, and can be determined by the following equation. JPEG2026071206000009.jpg10128

[0030] As shown in Example 8 below, quantum yield can also be used to measure the quenching effect. Furthermore, instruments such as the Hamamatsu Absolute PL Quantum Yield Spectrometer (Hamamatsu Corp., Bridgewater NJ 08807, C11347-11Quantaurus-QY Absolute PL Quantum Yield Spectrometer) that can be used to measure quantum yield are commercially available.

[0031] As shown in Example 8 and Table 25, the quantum yield of fluorescently labeled molecules can be compared to the quantum yield of free fluorescent labels. If the quantum yield of a single unit of free fluorescent label under conditions where quenching does not substantially occur is set to 1, this can be used as a benchmark for comparing the fluorescence produced by each fluorescent label bound to the labeled molecule. Often, the compositions of the present invention include fluorescently labeled molecules labeled with multiple fluorescent labels, where the average amount of fluorescence emission is at least 70% (0.7 fluorescence ratio) of the fluorescence emission of free fluorescent labels, based on one fluorescent label (e.g., about 70% to about 99%, about 70% to about 90%, about 80% to about 99%, about 85% to about 99%, about 87% to about 99%, about 90% to about 99%, about 80% to about 95%, about 85% to about 96%, etc.).

[0032] As shown in Example 8 and Table 25, fluorescence intensity can be determined by measuring the total fluorescence of the fluorescently labeled molecule compared to the fluorescence of the free label. If the fluorescence of a single unit of free fluorescent label under conditions where quenching does not substantially occur is set to 1, this can be used as a benchmark for comparing the fluorescence produced by each fluorescent label bound to the labeled molecule. Often, the compositions of the present invention include fluorescently labeled molecules labeled with multiple fluorescent labels, where the average amount of fluorescence emission is at least 70% (0.7 fluorescence ratio) of the fluorescence emission of the free fluorescent label, based on one fluorescent label (e.g., about 70% to about 99%, about 70% to about 90%, about 80% to about 99%, about 85% to about 99%, about 87% to about 99%, about 90% to about 99%, about 80% to about 95%, about 85% to about 96%, etc.).

[0033] As shown in Table 25, the brightness of fluorescently labeled molecules can be determined compared to free fluorescently labeled molecules. The brightness is proportional to the product of the quantum yield (Φ), the extinction coefficient (ε), and the number of dyes per molecule (N), as given by the following equation. B = Φ × ε × N Therefore, total fluorescence enhancement can be described using the ratio of the brightness of the free fluorescent label to the brightness of the labeled molecule.

[0034] For example, the data in Table 25 are set as a benchmark for ALEXA FLUOR® 647 in deionized water. Furthermore, this sets a benchmark for the ratio of 100% quantum yield of the free dye to a brightness of 1.0. Of the samples, molecule AF647-20K8 had a quantum yield of 73% of the free dye but showed a fluorescence enhancement of 5.8 × compared to the free dye. It is also shown that sample AF647-10K4 had the highest quantum yield of the free dye (89%) but showed only a fluorescence enhancement of 3.6 × compared to the free dye. These data suggest that the degree of fluorescence enhancement observed for these molecules may directly correlate with the length of the arms. The data also show that, while keeping the arm length constant, adding more fluorescently labeled arms to the polymer tends to increase the fluorescence enhancement.

[0035] Accordingly, the present invention includes compositions and methods for linking multiple fluorescent labels to individual molecules (e.g., biomolecules) such that the fluorescent labels are spaced apart in a manner that enhances the fluorescence signal. This can be done by reducing quenching. One method for enhancing the fluorescence signal is to spatially separate the fluorescent labels present in the sample. This is particularly useful when multiple fluorescent labels are bound to the same molecule (e.g., biomolecule) to be detected.

[0036] In some embodiments, the present invention includes a method for producing an antibody conjugated with a spacer and a fluorescent label, wherein a spacer agent is used to conjugate the spacer to the antibody, and the spacer is not conjugated to the fluorescent label. The invention also includes a composition comprising a spacer, an antibody, and a fluorescent label, wherein the spacer is not conjugated to the fluorescent label.

[0037] In some embodiments, the spacer can reduce the quenching of multiple fluorescent labels conjugated to the antibody.

[0038] In some embodiments, methods for producing nucleic acids conjugated to a spacer agent, wherein the spacer agent is not directly conjugated to a fluorescent label, can reduce the quenching of the fluorescent label conjugated to the nucleic acid.

[0039] In some embodiments, the present invention includes compositions and methods relating to spatially separated fluorescent labels on molecules (e.g., biomolecules) to which they are conjugated. Often, this is accomplished by connecting one or more fluorescent labels to a spacer and connecting the spacer to a molecule (e.g., a biomolecule). Examples of such compositions and methods are shown in Figure 18.

[0040] definition This specification and exemplary embodiments should not be taken as limiting. For the purposes of this specification and the appended claims, unless otherwise indicated, all numbers representing quantities, percentages, or ratios, as well as other numerical values ​​used in the specification and claims, should in all cases be understood as being modified by the term “approximately” to the extent that they are not over-modified. Thus, unless otherwise indicated, the numerical parameters described below in the specification and the appended claims are approximations that may vary depending on the desired characteristic to be obtained. At a minimum, each numerical parameter should be interpreted by applying ordinary rounding techniques, in light of the reported number of significant figures, so as not to limit the application of the principle of equivalents to the claims.

[0041] When used herein and in the appended claims, the singular forms “a,” “an,” and “the,” as well as any singular use of any word, should be noted to include multiple referents unless explicitly and unquestionably limited to one referent. When used herein, the term “includes” and its grammatical variants are intended to be non-restrictive so as not to exclude other similar items that may be replaced by or added to the listed items.

[0042] As used herein, “biomolecules” include, but are not limited to, synthetic or naturally occurring proteins or their fragments, glycoproteins, lipoproteins, amino acids, nucleosides, nucleotides, nucleic acids, oligonucleotides, DNA, RNA, carbohydrates, sugars, lipids, fatty acids, haptens, antibodies, and the like.

[0043] The terms “protein” and “polypeptide” are used herein in a general sense to include polymers of amino acid residues of any length. Where used herein, the term “peptide” refers to a polymer in which monomers are amino acids linked to one another via amide bonds, or is called a polypeptide. When the amino acid is an α-amino acid, either the L-optical isomer or the D-optical isomer may be used. Furthermore, non-natural amino acids, such as β-alanine, phenylglycine, and homoarginine, are also included. Commonly encountered amino acids that are not genetically encoded may also be used in this invention. All amino acids used in this invention may be either D- or L-isomers. L-isomers are generally used. Furthermore, other peptide mimetic agents are also useful in this invention. For a general overview, see Spatola, AF, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983).

[0044] As used herein, the term “antibody” refers to a protein of the immunoglobulin (Ig) superfamily that non-covalently binds to a specific substance (e.g., antigen and immunogen) to form an antibody-antigen complex, including but not limited to antibodies produced by hybridoma cell lines, by immunization to induce a polyclonal antibody response, by chemosynthesis, and by recombinant host cells transformed with an expression vector encoding an antibody. In humans, immunoglobulin antibodies are classified as IgA, IgD, IgE, IgG, and IgM, and members of each class are said to have the same isotype. Human IgA and IgG isotypes are further divided into subtypes IgA1 and IgA2, as well as IgG1, IgG2, IgG3, and IgG 4に It is further subdivided. Mice generally have the same isotype as humans, but the IgG isotype is IgG1, IgG 2a IgG2b , and further subdivided into IgG3 subtypes. Therefore, when used herein, the term “antibody” will be understood to include, within its scope, (a) any various class or subclass of immunoglobulins (e.g., IgA, IgD, IgG, IgM, and IgE from any animal that produces antibodies), and (b) polyclonal and monoclonal antibodies such as mouse, chimeric, or humanized antibodies. Antibody molecules have regions of amino acid sequences (e.g., Fc region, kappa light chain, lambda light chain, hinge region, etc.) that can act as antigenic determinants. Antibodies produced against a selected region are called anti-[region] (e.g., anti-Fc, anti-kappa light chain, anti-lambda light chain, etc.). Antibodies are typically produced against antigens by immunizing an organism with a macromolecule to initiate lymphocyte activation in order to express immunoglobulin proteins. As used herein, the term “antibody” also non-limitingly encompasses any polypeptide or protein that is an antibody-binding domain comprising a single-chain Fv molecule (scFv) or has a binding domain homologous thereto, where the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site (Bird et al., Science 242:423 (1988), and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 (1988)). These may be derived from natural sources, or they may be produced synthetically, partially or entirely.

[0045] Furthermore, VHH antibodies can be used either as those obtained from antigen-stimulated cells or as genetically modified antigen-binding proteins.

[0046] As used herein, the term “antibody fragment” refers to a fragment of an antibody that retains the major selective binding properties of the entire antibody. Certain fragments, such as Fab, Fab', and F(ab')2 obtained by digestion with various proteases and lacking the Fc fragment of the intact antibody, or so-called “half-batch” fragments obtained by reductive cleavage of disulfide bonds linking the heavy chain components in the intact antibody, are well known in the art. Such fragments also include isolated fragments consisting of light chain variable regions, “Fv” fragments consisting of heavy chain and light chain variable regions, and recombinant single-chain polypeptide molecules in which the light chain variable region and heavy chain variable region are linked by a peptide linker. Other examples of binding fragments include (i) Fd fragments consisting of the VH domain and CH1 domain; (ii) dAb fragments consisting of the VH domain (Ward et al., Nature 341:544 (1989)); (iii) isolated CDR regions; and (iv) the aforementioned single-chain Fv molecules (scFv). Furthermore, arbitrary fragments can be created using recombinant technology that preserves antigen recognition properties.

[0047] An exemplary VHH antibody that can be used is a single-domain antibody, which is an antibody fragment consisting of a variable antibody domain of a single monomer. Such antibody fragments typically have a molecular weight of only 12–25 kDa, and are therefore smaller than many other antibodies (150–160 kDa) which consist of two heavy-chain protein chains and two light chains.

[0048] As used herein, “antigen” refers to a molecule, including but not limited to biological substances, that induces or can induce antibody formation, or to which antibodies selectively bind. An antigen may also refer to an “immunogen.” Antibodies selectively bind to an antigen when there is a relative lack of cross-reactivity or interference with other substances present.

[0049] As used herein, the term “reactive group” refers to a group that can react with another chemical group to form a covalent bond, i.e., a group that reacts covalently under appropriate reaction conditions, and generally represents a bond site to another substance. Reactive groups generally include nucleophilic, electrophilic, and photoactivatable groups. Exemplary reactive groups include, but are not limited to, olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazos, diazoniums, nitros, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfuric acids, isonitrile sulfenate, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids, alkynes, and azides.

[0050] As used herein, “spacer,” “spacer molecule,” or “spacer agent” refers to a compound (e.g., an organic compound) that, when conjugated directly or indirectly to a biomolecule, can enhance the fluorescence emitted from that biomolecule. This is thought to result from a reduction in the fluorescence quenching of the fluorescent label. Any number of compounds can act as spacers, and exemplary compounds include NHS acetate and various forms of polyethylene glycol (PEG). As used herein, the terms “polyethylene glycol” or “PEG” refer to oligomers or polymers of ethylene oxide. PEG polymer chain lengths can vary considerably, but tend to have high molecular weights of around 10,000,000 g / mol. PEG is also available in various forms. For example, branched-chain PEG typically has 3 to 10 PEG chains arising from a central core group. Star-chain PEG has 3 to 100 PEG chains arising from a central core group. Comb-chain PEG usually has multiple PEG chains grafted onto a polymer backbone. Most PEGs contain molecules with a molecular weight distribution (i.e., they are polydispersible). The size distribution can be statistically characterized by its weight-average molecular weight and its number-average molecular weight, and the ratio thereof is called the polydispersity index. Exemplary PEG compounds that may be used in the implementation of the present invention include MS(PEG)4, MS(PEG)8, and MS(PEG) 12 (Thermo Fisher Scientific, Waltham, MA, catalog numbers 22341, 22509B, and 22686, respectively), and (methyl-PEG) 12 This includes branched-chain PEG compounds such as 3-PEG4-NHS ester (Thermo Fisher Scientific, Waltham, MA, catalog number 22421).

[0051] As used herein, the term “direct spacer” refers to a molecule that has at least one fluorescent label bound to it and that directly binds to a biomolecule. A direct spacer may be (1) a single polymer or (2) multiple polymers bound to a core. Examples of direct spacers include single-arm polymers and multi-arm polymers.

[0052] As used herein, “polymer” refers to a molecule consisting of repeating subunits (typically at least four repeating subunits). Polymers may be synthetic or naturally occurring. The repeating units of a polymer do not need to be identical. For example, proteins are polymers composed of different amino acid subunits. Furthermore, polymers do not need to be perfectly linear molecules and may therefore be branched, such as dextran.

[0053] As used herein, the term “single-arm polymer” refers to an unbranched molecule having an unbranched chain structure to which at least one fluorescent label is attached (see Figure 19). Examples of single-arm polymers that may be used in the embodiment of the present invention are “linear” polysaccharides (e.g., amylose), polyethylene glycol, long-chain carbon molecules (e.g., Ahx), and polypeptides. In some cases, the unbranched / linear polysaccharides consist of monomers linked to each other by α1,4 bonds.

[0054] As used herein, the term “multi-arm polymer” refers to a branched-chain molecule to which at least one fluorescent label is attached and which has a non-branched-chain structure (see Figure 19). Examples of multi-arm polymers that may be used in the embodiment of the present invention are branched-chain polysaccharides (e.g., dextran, glycogegen), polyethylene glycol, branched-chain long-chain carbon molecules (e.g., Ahx), and branched-chain polypeptides.

[0055] As used herein, the terms “conjugation molecule” or “conjugation arm” refer to a linker through which a dye is bound (e.g., covalently bonded) to a molecule (e.g., a biomolecule). The conjugation molecule may be bound to a single dye molecule or to multiple dye molecules (identical or different dyes).

[0056] As used herein, the term "fluorescence" refers to the optical phenomenon in which a molecule absorbs high-energy photons and re-emits them as low-energy (long-wavelength) photons, the energy difference between the absorbed and emitted photons resulting in molecular vibrations or heat.

[0057] As used herein, the terms “fluorescent label,” “fluorescent dye,” “fluorophore,” or “fluorescent moiety” refer to a compound, chemical group, or composition that is intrinsically fluorescent. Fluorophores may include substituents that modify the solubility, spectral properties, or physical properties of the fluorophore. Numerous fluorophores are known to those skilled in the art and include, but are not limited to, coumarin, cyanine, benzofuran, quinoline, quinazolinone, indole, furan, benzazole, borapolyazindacene, and xanthenes including fluorescein, rhodamine, and rhodol, as well as other fluorophores described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9th edition, CD-ROM, September 2002). Reactive chemistry, including N-hydroxysuccinimide (NHS), maleimide, and hydrazides, as well as click chemistry (e.g., SITECLICK®), are currently used for the conjugation of fluorescent labels to biomolecules.

[0058] As used herein, the term “conjugate” means a molecule that is directly or indirectly bonded to another molecule by either a covalent or non-covalent bond.

[0059] The term "dye conjugate" refers to a dye molecule covalently or noncovalently bonded to another carrier molecule, such as an antibody, and in most cases, the dye is covalently bonded. Dye conjugates can be linked by a linker, such as a direct bond via a single covalent bond, or a series of stable covalent bonds incorporating 1 to 20 non-hydrogen atoms selected from the group consisting of C, N, O, S, and P, covalently bonding a fluorescent dye to another part or biological and non-biological component, such as an antibody or a chemically reactive group. The conjugation or linker may contain receptor-binding motifs such as biotin / avidin.

[0060] As used herein, the terms “near-infrared dye,” “near-infrared reporter molecule,” “NIR dye,” or “NIR reporter molecule” refer to dyes or reporter molecules having excitation wavelengths of approximately 580 nm to 800 nm. In most cases, NIR dyes emit light in the range of approximately 590 nm to 860 nm. In most cases, NIR dyes are excited at approximately 680 nm to 790 nm. In most cases, dyes include ALEXA FLUOR® 660 dye, ALEXA FLUOR® 680 dye, ALEXA FLUOR® 700 dye, ALEXA FLUOR® 750 dye, and ALEXA FLUOR® 790 dye. NIR dyes are particularly advantageous for in vivo imaging because they can selectively visualize endogenous substances present in living organisms without exciting them. Some NIR dyes have a large Stokes shift, so that the excitation wavelength and emission wavelength are separated by at least 20, 30, 40, 50, 60, 70, or 80 nm.

[0061] "Solid support" refers to a substrate material having a hard or semi-hard surface. Typically, at least one surface of the substrate is substantially flat, but it may be desirable to physically isolate certain areas by, for example, wells, raised regions, etched trenches, or other such topologies. Solid support materials also include spheres (including microspheres), rods (e.g., optical fibers), as well as fabricated and irregularly molded articles.

[0062] The solid support material includes, but is not limited to, any material used as an affinity matrix or support for chemical and biological molecular synthesis and analysis, such as poly(vinylidene difluoride) (PVDF), polystyrene, polycarbonate, polypropylene, nylon, glass, dextran, chitin, sand, pumice, polytetrafluoroethylene, agarose, polysaccharides, dendrimers, buckyballs, polyacrylamide, diatomaceous earth-polyacrylamide non-covalent composites, polystyrene-polyacrylamide covalent composites, polystyrene-PEG[poly(ethylene glycol)] composites, silicon, rubber, and other materials used as supports for solid-phase synthesis, affinity separation and purification, hybridization reactions, immunoassays and other such applications. The solid support material may be granular or in the form of a continuous surface such as a microtiter dish or well, a glass slide, a silicon chip, a nitrocellulose sheet, a nylon mesh, or other such material.

[0063] "Kit" refers to a packaged set of related components, typically one or more compounds or compositions.

[0064] Detectable biomolecules In some embodiments, biomolecules that are detectably labeled with multiple fluorescent labels, including spacer agents, are disclosed herein.

[0065] a. Spacer The spacer can be any molecule that, when conjugated to a biomolecule, can enhance the fluorescence emission resulting from the excitation of multiple fluorescent labels conjugated to the biomolecule independently. This is thought to be due to a reduction in the fluorescence quenching of the fluorescent labels.

[0066] In some embodiments, the spacer contains an acetyl(-C(O)CH3) group. In some embodiments, the spacer is an acetate molecule. In some embodiments, the acetate molecule is sulfo-NHS-acetate.

[0067] In some embodiments, the spacer agent comprises polyethylene glycol (PEG). In some embodiments, the spacer agent comprises (PEG)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the spacer agent comprises MS-(PEG)n.

[0068] In some embodiments, the spacer is alkanoyl, alkenoyl, and alkinoyl (-C(O)C n H m Selected from the formula, where n is 1 to 20 atoms, m > n, carbon atoms can be bonded to one another by single, double, and / or triple bonds, and alkyl, alkenyl, and / or alkynyl groups may be further substituted. In some embodiments, a specific substitution is -(OCH2CH2O) x -(CH2) y -Contains a poly(ethylene) glycol moiety such as -OR, where x is 1 to 20, y is 1 to 6, and R is H or C 1~6 It is alkyl. In some embodiments, the specific substitution is ammonium (-NH3 + ), quaternary ammonium ((-NR3 + )Base (In the formula, R is C 1~6 (It is alkyl), or a phosphonium group (-PQ3 + )(wherein Q is an aryl, a substituted aryl, or C 1~6 It contains (which is alkyl).

[0069] In some embodiments, the spacer is an alkyl, alkenyl, or alkynyl group (-C n H m Selected from the formula, where n is 1 to 20 atoms, m > n, carbon atoms can be bonded to one another by single bonds, double bonds, and / or triple bonds, and alkyl, alkenyl, and / or alkynyl groups may be further substituted. In some embodiments, specific substitutions include negatively charged sulfonate groups (-OSO3-), carboxylate groups (-CO2-), phosphate groups (-OPO3-), and / or phosphonate groups (-PO3-). In some embodiments, other substitutions include -(OCH2CH2O) x -(CH2) y -Contains a poly(ethylene) glycol moiety such as -OR, where x is 1 to 20, y is 1 to 6, and R is H or C 1~6 It is alkyl. In some embodiments, other specific substitutions include ammonium (-NH3 + ), quaternary ammonium ((-NR3 + )Base (In the formula, R is C 1~6 (It is alkyl), or a phosphonium group (-PQ3 + )(wherein Q is an aryl, a substituted aryl, or C 1~6 It contains (which is alkyl).

[0070] In some embodiments, the spacer is positively charged. In some embodiments, the spacer agent comprises betaine (i.e., trimethylglycine).

[0071] In some embodiments, the spacer material is negatively charged.

[0072] b. Fluorescent labeling The fluorescent dyes described herein function as reporter molecules that impart a detectable signal to a sample, either directly or indirectly, as a result of conjugation to functional groups on proteins, including non-limited amine or thiol groups. This generally results in the ability to detect all proteins in a sample, often in combination with the detection of a subset of all proteins in the sample. In such cases, the labeling of all proteins is distinguishable from dyes that label a subset of all proteins in the sample.

[0073] Typically, a detectable response is a change in fluorescence, such as a change in fluorescence intensity, fluorescence excitation or emission wavelength, fluorescence distribution, fluorescence lifetime, fluorescence polarization, or a combination thereof.

[0074] The fluorescent dye can be any fluorophore known to those skilled in the art. Typically, the dye comprises, but is not limited to, one or more aromatic or heteroaromatic rings that are optionally substituted once or more with various substituents, including halogens, nitro, sulfo, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring systems, benzos, or other substituents typically present on chromophores or fluorophores known in the art.

[0075] A wide variety of fluorophores suitable for total protein labeling, as described herein, are already known in the art (RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (2002)). The fluorescent dyes used in the methods and compositions described herein are any chemical moieties that exhibit an absorption maximum above 280 nm. Such chemical components include, but are not limited to, pyrene, sulfonated pyrene, sulfonated coumarin, sulfonated carbocyanine, sulfonated xanthene, anthracene, naphthalene, acridine, stilbene, indole, isoindole, indidine, benzindole, oxazole or benzoxazole, thiazole or benzothiazole, 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD), carbocyanine, carbostyryl, porphyrin, salicylate, anthranylate, azulene, perylene, pyridine, quinoline, isoquinoline, chromene, borapolyazindacene, xanthene, fluorescein, rosamine, rhodamine, rhodamine, benzo- or dibenzofluorescein, seminaphthofluorescein, naphthofluorescein, biman, oxazine or benzoxazine, carbazine, phenalenone, coumarin, benzofuran, benzphenalenone, and their derivatives. As used herein, oxazines include resolphins, aminooxazinones, diaminooxazines, and their benzo-substituted analogs.

[0076] In one embodiment, the fluorescent dye comprises one or more aromatic or heteroaromatic rings that are optionally substituted one or more times with various substituents, including halogens, nitro, sulfo, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring systems, benzos, or other substituents typically present on chromophores or fluorophores known in the art. In one embodiment, the fluorophore is a xanthene comprising one or more duroridine rings.

[0077] In exemplary embodiments, the dyes are independently substituted with substituents selected from the group consisting of hydrogen, halogens, aminos, substituted aminos, alkyls, substituted alkyls, aryls, substituted aryls, heteroaryls, substituted heteroaryls, alkoxys, sulfos, reactive groups, solid supports, and support molecules. In other embodiments, the xanthene dyes of the present invention include both substituted and unsubstituted compounds on the carbon atoms of the central ring of xanthene, with substituents typically found in xanthene dyes, such as phenyls and substituted phenyl moieties. Often, the dyes are rhodamine, fluorescein, porapolyazindacene, indole, and their derivatives.

[0078] The selection of reactive groups used to conjugate a whole protein label or expression tag label to a protein typically depends on the reactivity or functional group on the material being conjugated and the desired type or length of covalent bond. Types of functional groups typically found on organic or inorganic substrates (biomolecules or non-biomolecules) include, but are not limited to, amines, amides, thiols, alcohols, phenols, aldehydes, ketones, phosphonates, imidazoles, hydrazines, hydroxylamines, disubstituted amines, halides, epoxides, silyl halides, carboxylic acid esters, sulfonic acid esters, purines, pyrimidines, carboxylic acids, olefin bonds, or combinations of these groups. In proteins, a variety of sites can arise, including, but are not limited to, amines, thiols, alcohols, and phenols.

[0079] ALEXA FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 500, ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610-X, ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR(registered trademark) 750, ALEXA FLUOR(registered trademark) 790, AMCA-X, BODIPY(registered trademark) 630 / 650, BODIPY(registered trademark) 650 / 665, BODIPY(registered trademark) FL, BODIPY(registered trademark) TMR, BODIPY(registered trademark) TR, BODIPY(registered trademark) TR-X, CASCADE BLUE(registered trademark), Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE(registered trademark), OREGON GREEN(registered trademark) 488, OREGON GREEN(registered trademark) 514, PACIFIC BLUE(trademark), PACIFIC ORANGE(trademark), RHODAMINE GREEN(trademark), QSY(registered trademark) 7, QSY(registered trademark) 9, QSY(registered trademark) 21, QSY(registered trademark) 35, ROX, RHODAMINE Examples include, but are not limited to, RED®, TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED®, and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacrylidine-2-one) succinimidyl ester (DDAO-SE).

[0080] In some embodiments, the fluorescent reagents / dyes that bind to the protein-bound tags used in the protein labeling methods described herein may be, as a few examples, arsenic fluorophores including arsenic derivatives of fluorescein such as FlAsH-EDT2 (4'-5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(2,2-ethanedithiol)2) (LUMIO® Green, Life Technologies Corp., Carlsbad, CA), or arsenic derivatives of resolfins such as ReAsh-EDT2 (LUMIO® Red, Life Technologies Corp., Carlsbad, CA), or alternatively, oxidized derivatives such as ChoXAsH-EDT2 or HoXAsH-EDT2. Furthermore, the arsenic fluorophores may be arsenic derivatives of other known fluorophores, including, but not limited to, the ALEXA FLUOR® series described herein, such as ALEXA FLUOR® 350, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 663, and ALEXA FLUOR® 660, which are commercially available from Molecular Probes (Eugene, Oregon).

[0081] In some embodiments, the arsenic fluorophore may be present at concentrations of at least about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 50 μM, 100 μM, or higher, and at concentrations of about 500 μM, 400 μM, 300 μM, 200 μM, 100 μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM, 15 μM, 10 μM, 5 μM, 4 μM, 3 μM, 2 μM, or 1 μM or less.

[0082] In some embodiments, the tag bound to the protein to which such a fluorescent dye binds is a tetracysteine ​​peptide motif, cys-cys-Xn-cys-cys (SEQ ID NO: 1), where each X is any natural amino acid, a non-natural amino acid, or a combination thereof, and n is an integer from 2 to 100. In certain embodiments, n is an integer from 2 to 90, and in other embodiments, n is an integer from 2 to 80. In certain embodiments, n is an integer from 2 to 70, and in other embodiments, n is an integer from 2 to 60. In certain embodiments, n is an integer from 2 to 50, and in other embodiments, n is an integer from 2 to 40. In certain embodiments, n is an integer from 2 to 30, and in other embodiments, n is an integer from 2 to 20. In certain embodiments, n is an integer from 2 to 10, and in other embodiments, n is an integer from 2 to 5. In such motifs, natural amino acids include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In certain embodiments, the tetracysteine ​​tag has the sequence CCPGCC (SEQ ID NO: 2). In other embodiments, a 12-amino acid peptide containing a tetracysteine ​​motif is used, including, but is not limited to, the amino acid sequence AGGCCPGCCGGG (SEQ ID NO: 3). Furthermore, a protein can be labeled with a single tetracysteine ​​tag, or a protein can be labeled with multiple tetracysteine ​​tags, including, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 tetracysteine ​​tags. Such tags may be separated from each other within the primary amino acid sequence of the protein, or they may be directly polymerized in series as concatemers.

[0083] In certain embodiments, the tetracysteine ​​peptide has the sequence cys-cys-Xn-cys-X-cys-X (SEQ ID NO: 1), where each X is any natural amino acid, a non-natural amino acid, or a combination thereof, and n is an integer between 2 and 100. In certain embodiments, n is an integer between 2 and 90, and in other embodiments, n is an integer between 2 and 80. In certain embodiments, n is an integer between 2 and 70, and in other embodiments, n is an integer between 2 and 60. In certain embodiments, n is an integer between 2 and 50, and in other embodiments, n is an integer between 2 and 40. In certain embodiments, n is an integer between 2 and 30, and in other embodiments, n is an integer between 2 and 20. In certain embodiments, n is an integer between 2 and 10, and in other embodiments, n is an integer between 2 and 5. In such a motif, natural amino acids include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In certain embodiments, the tetracysteine ​​tag has the sequence CCGGKGNGGCGC (SEQ ID NO: 4).

[0084] Tetracysteine ​​peptide tags (one or more) can be recombinantly fused to a protein to be labeled, either at the N-terminus, C-terminus, or in-frame within the protein sequence, and expression vectors for producing tetracysteine-fused recombinant proteins can be readily constructed using techniques known to those skilled in the art. In certain embodiments, tetracysteine-tagged proteins are recombinantly expressed in host cells, including but not limited to bacterial host cells, fungal host cells, insect cells, plant cells, or mammalian cells. Such bacterial host cells include, to name just a few, genera such as Escherichia (e.g., Escherichia coli), Klebsiella, Streptomyces, Streptococcus, Shigella, Staphylococcus, Erwinia, Klebsiella, and Bacillus (e.g., Bacillus cereus). The present invention includes, but is not limited to, Gram-negative and Gram-positive bacteria of any genera, including Bacillus subtilis and Bacillus megatherium, Serratia, Pseudomonas (e.g., Pseudomonas aeruginosa and Pseudomonas syringae), and Salmonella (e.g., Salmonella typhi and Salmonella typhimurium). Suitable bacterial strains and serotypes for the present invention may include Escherichia coli serotypes K, B, C, and W. A typical bacterial host is Escherichia coli strain K-12. Fungal host cells include, as an example, Saccharomyces cerevisiae cells, while mammalian cells include, as an example, human cells. In such embodiments, the protein sample containing the protein of interest is a lysate of a host cell, which may be unpurified, partially purified, or substantially purified before labeling and analysis using the method described herein.

[0085] In other embodiments, the tetracysteine-tagged protein is expressed in vitro, and the protein sample containing the protein of interest is a cell-free extract, or a partially purified or purified fraction thereof, on which translation (and possibly transcription) takes place. In embodiments where the extract allows coupled transcription and translation in a single cell-free extract, such as the Escherichia coli-based EXPRESSWAY® or EXPRESSWAY® Plus system (Life Technologies Corp., Carlsbad, Ca), the sample is a cell-free extract, or a fraction thereof, on which transcription and translation normally occur.

[0086] Alternatively, GATEWAY® Technology (Life Technologies Corp., Carlsbad, CA) is a universal cloning technique that can be used to express target genes in E. coli.

[0087] The proteins labeled with the arsenic dyes described herein may be any protein having a tetracysteine ​​motif. The protein to which the tetracysteine ​​tag(s) are fused or conjugated may be any protein (naturally occurring or unnaturally occurring) that is desired to be labeled. Naturally occurring proteins may or may not have known biological functions, and may be known to be expressed or only predicted from their genome sequence. If the protein is naturally occurring, it may be only a full-length protein or a fragment thereof. Thus, tetracysteine-tagged proteins may be human proteins or non-human mammalian proteins, bacterial proteins including fungal proteins, eubacterial proteins and archaeal proteins, plant proteins, insect proteins, or animal proteins such as viral proteins.

[0088] In addition to tetracysteine ​​tags, other protein sequences can be recombinantly and usefully added to proteins where labeling is desired. Such additional protein sequences may include linker and / or short tags, usefully epitope tags such as FLAG tags or myc tags, or other sequences useful for purification, such as polyhistidine (e.g., 6xhis) tags. Alternatively, tetracysteine ​​tags(single or plural) can be chemically conjugated to proteins to be labeled using conjugation chemistry commonly used in the art.

[0089] In some embodiments, the fluorescent label is positively charged. In some embodiments, the fluorescent label is negatively charged.

[0090] In some embodiments, the excitation wavelength of the fluorescent label is between 350 and 850 nm. In some embodiments, the excitation wavelength of the fluorescent label is far-red. In some embodiments, the excitation wavelength of the fluorescent label is near-infrared. In some embodiments, the excitation wavelength of the fluorescent label is ultraviolet (UV).

[0091] In some embodiments, the fluorescent label comprises a DYLIGHT® phosphor. In some embodiments, the DYLIGHT® phosphor is selected from DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800. In some embodiments, the DYLIGHT® phosphor is conjugated to a PEG molecule (e.g., 2×PEG, 4×PEG, 8×PEG, or 12×PEG).

[0092] In some embodiments, the fluorescent label includes ALEXA FLUOR®. In some embodiments, ALEXA FLUOR® is used in ALEXA FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610, ALEXA FLUOR® 633, ALEXA FLUOR® 635, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR® 750, and ALEXA Select from FLUOR(registered trademark)790.

[0093] In some embodiments, the fluorescent label comprises a portion selected from xanthene, coumarin, cyanine, pyrene, oxazine, borapolyazindacene, benzopyrillilium, and carvopyronine.

[0094] In some embodiments, the fluorescent label includes fluorescein (e.g., Cy2 or FITC). In some embodiments, the fluorescent label includes rhodamine (e.g., TRITC or Cy3). In some embodiments, the fluorescent label includes MCA, coumarin, rhodamine red, Texas red, cascade blue, Cy5, Cy5.5, IR680, IR800, and Cy7.

[0095] In some embodiments, the fluorescent label is a modified fluorescent label (for example, the fluorescent label is sulfonated or conjugated with PEG).

[0096] In some embodiments, the fluorescent label is a fluorescent protein. In some embodiments, the fluorescent protein is a phycobiliprotein. Examples of phycobiliproteins useful in the present invention are allophycocyanin, phycocyanin, phycoerythrin, allophycocyanin B, β-phycoerythrin, phycoerythrocyanin, and β-phycoerythrin. The structures of phycobiliproteins have been studied, and their fluorescence spectral properties are known. See ANGlazer, "Photosynthetic Accessory Proteins with Bilin Prosthetic Groups," Biochemistry of Plants, Volume 8, MDHatch and NKBoardman, EDS., Academic Press, pp. 51-96 (1981), and ANGlazer, "Structure and Evolution of Photosynthetic Accessory Pigment Systems with Special Reference to Phycobiliproteins," The Evolution of Protein Structure and Function, BSSigman and MABrazier, EDS., Academic Press, pp. 221-244 (1980). In some embodiments, the fluorescent protein has an absorption maximum at at least about 450 nm, often at least about 500 nm, a Stokes shift of at least 15 nm, often at least about 25 nm, and a maximum fluorescence emission at at least about 500 nm, often at least about 550 nm.

[0097] In some embodiments, the fluorescent label is a dipyrometheneborone difluoride dye, such as that disclosed in US2014 / 0349,893, which is incorporated in whole herein by reference.

[0098] The amine-reactive fluorescent dyes used in the protein labeling methods described herein include, but are not limited to, reagents of the aroyl-2-quinoline-carboxyaldehyde type. Such reagents are described in U.S. Patents 5,459,272 and 5,631,374, each of which is incorporated herein by reference in whole. In some embodiments, the aroyl-2-quinoline-carboxyaldehyde reagent used is 3-(4-carboxybenzoyl)quinoline-2-carboxyaldehyde or 3-(2-froyl)quinoline-2-carboxyaldehyde. In certain embodiments, the amine-reactive fluorescent dye is 3-(2-froyl)quinoline-2-carboxyaldehyde, while in other embodiments, the amine-reactive fluorescent dye is 3-(4-carboxybenzoyl)quinoline-2-carboxyaldehyde.

[0099] c. Biomolecules The biomolecules that may be used in the compositions and methods disclosed herein include any biomolecules that are useful in molecular biological applications.

[0100] In some embodiments, the biomolecule is an antibody (e.g., a primary or secondary antibody). In some embodiments, the biomolecule is an antibody fragment. The antibody used in the implementation of the present invention may be a polyclonal antibody, a monoclonal antibody, or a genetically engineered antibody, and may come from any source (e.g., a mammal such as a shark, bird, or llama, human, mouse, rabbit, goat, or rat). Furthermore, a humanized antibody may be used.

[0101] In some embodiments, the antibody is a chimeric one.

[0102] In some embodiments, the biomolecule is a protein or polypeptide. In some embodiments, the biomolecule is a recombinant polypeptide.

[0103] In some embodiments, the biomolecule is a nucleic acid molecule. In some embodiments, the nucleic acid molecule is an oligonucleotide (e.g., 15 to 50 nucleotides long). In some embodiments, the nucleic acid molecule is longer than 50 nucleotides, longer than 100 nucleotides, longer than 500 nucleotides, longer than 1 kb, longer than 2 kb, or longer than 5 kb.

[0104] d. Conjugation of fluorescent dyes to biomolecules Typically, when the excitation wavelength is at least 580 nm, a suitable dye having the desired spectral characteristics can be selected, and then the dye can be conjugated to the target support molecule using methods well known in the art (Haugland, MOLECULAR PROBES HANDBOOK, above (2002)). In many cases, conjugation for covalent bonding consists of simply mixing the reactive compound of the present invention in a suitable solvent in which both the reactive compound and the spacer molecule to be conjugated are soluble. In many cases, the reaction proceeds spontaneously at or below room temperature without the addition of reagents. For those reactive compounds that are photoactivated, conjugation is promoted by irradiation of the reaction mixture to activate the reactive compound. Chemical modification of water-insoluble substances that can prepare the desired compound-conjugate is often carried out in aprotic solvents such as dimethylformamide, dimethyl sulfoxide, acetone, ethyl acetate, toluene, or chloroform. Similar modification of water-soluble substances is readily achieved by using the reactive compound to make them more readily soluble in organic solvents.

[0105] The preparation of biomolecular (e.g., protein) conjugates typically involves first dissolving the biomolecule to be conjugated in an aqueous buffer at a concentration of about 1–10 mg / mL at room temperature or below. For example, bicarbonate buffer (pH about 8.3), carbonate buffer and borate buffer (pH about 9) are particularly suitable for reactions with succinimidyl esters, phosphate buffer (pH about 7.2–8) is particularly suitable for reactions with thiol-reactive functional groups, and carbonate buffer and borate buffer (pH about 9) are particularly suitable for reactions with isothiocyanates and dichlorotriazines. The appropriate reactive compound is then dissolved in a non-hydroxyl solvent (usually DMSO or DMF) in an amount sufficient to give the appropriate degree of conjugation when added to the solution of the biomolecule to be conjugated. The appropriate amount of a compound for any biomolecule (e.g., a protein) or other component can be conveniently predetermined by experiments in which various amounts of the compound are added to the biomolecule, the conjugate is purified by chromatography to separate the unconjugated compound, and the compound-biomolecular conjugate is tested for the desired application.

[0106] Any number of buffers may be used for the conjugation reaction and for others as shown herein. Using Examples 1 and 5 for illustrative purposes, phosphate-buffered saline and borate buffer may and may be used for the conjugation reaction. It is also conceivable that a carbonate buffer at pH 9.5 may be used. Therefore, it is believed that conjugations at higher pH require lower molar excesses of dye and spacer molecules. Accordingly, the present invention includes compositions and methods for carrying out conjugation reactions at pH about 4.0 to about 10.0 (e.g., about 4.0 to about 10.0, about 5.0 to about 10.0, about 6.0 to about 10.0, about 7.0 to about 10.0, about 7.5 to about 10.0, about 8.0 to about 10.0, etc.). The conjugation reaction involves a fluorescent dye or a mixture of each fluorescent dye at various molar excesses and NHS-acetate, NHS-MS(PEG)4, NHS-MS(PEG)8, or NHS-MS(PEG) 12The labeling reaction can be carried out in 50 mM borate buffer, pH 8.5, along with a spacer selected from the available options. The labeling reaction can be incubated at room temperature (RT) for approximately 1 hour. The NHS activating dye and NHS activating spacer can be combined before being added to the antibody so that both reactions occur simultaneously, allowing for dye substitution and random spacing of the spacers.

[0107] Examples of buffers that may be used in carrying out the present invention include 2-(N-morpholino)ethanesulfonic acid (MES), phosphate, 3-(N-morpholino)propanesulfonic acid (MOPS), tris(hydroxymethyl)aminomethane (TRIS), borate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and carbonate buffers.

[0108] The concentration of the fluorescent reagent used in the biomolecule (e.g., protein) labeling method described herein is in the range of 50 nM to 100 mM (e.g., about 50 nM to about 25 mM, about 50 nM to about 10 mM, about 50 nM to about 5 mM, about 100 nM to about 10 mM, about 100 nM to about 5 mM, about 200 nM to about 5 mM, about 50 nM to about 100 μM, about 1 μM to about 100 μM, etc.). In certain embodiments, such concentrations are obtained by diluting a stock solution of a fluorescent dye having a concentration in the range of 100 nM to 200 mM (e.g., about 10 μM to about 500 μM, about 1 μM to about 100 μM, about 100 μM to about 200 μM, etc.). In certain embodiments, the stock solution concentration is 100 mM. In certain embodiments, the stock solution concentration is 50 mM. In a particular embodiment, the concentration of the stock solution is 20 mM. In a particular embodiment, the concentration of the stock solution is 10 mM. In a particular embodiment, the concentration of the stock solution is 1 mM. In a particular embodiment, the concentration of the stock solution is 500 μM. In a particular embodiment, the concentration of the stock solution is 10 μM.

[0109] In some embodiments, the concentration of the amine-reactive fluorescent dye used in the method for labeling biomolecules (e.g., proteins) described herein is in the range of 50 nM to about 100 mM (e.g., about 50 nM to about 50 mM, about 50 nM to about 25 mM, about 50 nM to about 10 mM, about 50 nM to about 5 mM, about 50 nM to about 5 μM, about 50 nM to about 100 μM, about 100 nM to about 5 mM, etc.). In certain embodiments, such concentrations are obtained by diluting a stock solution of the fluorescent dye having a concentration in the range of 100 nM to 200 mM. In certain embodiments, the stock solution concentration is 100 mM. In certain embodiments, the stock solution concentration is 50 mM. In certain embodiments, the stock solution concentration is 20 mM. In certain embodiments, the stock solution concentration is 10 mM. In certain embodiments, the stock solution concentration is 1 mM. In certain embodiments, the concentration of the stock solution is 500 μM. In certain embodiments, the concentration of the stock solution is 10 μM to 500 μM. In certain embodiments, the concentration of the stock solution is 1 μM to 100 μM. In certain embodiments, the concentration of the stock solution is 10 μM. In certain embodiments, the concentration of the stock solution is 100 μM to 200 μM.

[0110] In some embodiments, the concentration of the protein or protein fragment (e.g., antibody fragment) labeled using the method described herein is in the range of 0.01 mg / mL to 200 mg / mL (e.g., about 0.1 mg / mL to 100 mg / mL, about 0.1 mg / mL to about 50 mg / mL, about 0.1 mg / mL to about 10 mg / mL, about 0.2 mg / mL to about 100 mg / mL, about 0.2 mg / mL to about 50 mg / mL, about 0.2 mg / mL to about 10 mg / mL, about 0.3 mg / mL to 10 mg / mL, about 0.4 mg / mL to about 10 mg / mL, about 0.5 mg / mL to about 10 mg / mL, etc.).

[0111] In some cases, more than one dye molecule can be attached to each position on a biomolecule. One way to attach more than one dye molecule to a single position on a biomolecule is by using conjugation molecules that bind to more than one dye molecule. These conjugation molecules then bind to the biomolecule, supporting multiple dye molecules together with it. As an example, multiple fluorescent dye molecules can be used, conjugated to a single polymer backbone or core (hereinafter referred to as “dendrimer”) for the attachment of these dye molecules to the biomolecule, such as those described in U.S. Patent Application Publication No. 2012 / 0256102. These dendrimers may have a regular or irregular, branched polymer network structure that enables chemical bonding of multiple dye molecules, multiple color dyes, and / or multiple functional groups in a combinatorial manner.

[0112] Further examples of conjugation molecules that can be used include many of the same molecules used as spacers. Therefore, in some examples, the spacer molecule and the conjugation molecule will have the same structure except that the conjugation molecule has a dye molecule bound to it. Thus, various forms of PEG molecules can be used as conjugation molecules. Consequently, the spacer molecule may include a dye molecule (e.g., fluorescently labeled) (see Figure 18).

[0113] Therefore, the present invention intends to use conjugation molecules, each having an average of about 2 to about 50 bound pigment molecules (e.g., about 2 to about 45, about 2 to about 40, about 2 to about 35, about 2 to about 30, about 2 to about 20, about 5 to about 45, about 10 to about 45, etc.). In many cases, the standard deviation of the average number of pigment molecules bound to the conjugation molecule will be less than 10%, 15%, and / or 20%.

[0114] The degree of labeling can be measured for labeled biomolecules. The degree of labeling can be calculated as follows: Firstly, the molar concentration of the labeled biomolecule can be calculated using, for example, the following formula. JPEG2026071206000010.jpg 10128ε = Molar extinction coefficient of protein (for example, the molar extinction coefficient of IgG is approximately 210,000 M) -1 cm -1 (is) A 最大 = Absorbance of the dye solution measured at the maximum wavelength (λmax) for the dye molecule (A)CF = Correction factor, adjusts for the amount of absorbance at 280 nm caused by the dye (see Table 8) Dilution ratio = Protein: The degree to which the dye sample was diluted for absorbance measurement (if diluted).

[0115] Next, calculate the labeling degree using the following formula. JPEG2026071206000011.jpg 10140ε' = Molar extinction coefficient of fluorescent dye

[0116] (Table 8) Characteristics of Exemplary Dyes JPEG2026071206000012.jpg88156

[0117] In some embodiments of the present invention, enhanced fluorescence is observed for biomolecules containing spacers having a lower degree of labeling (DOL) than for biomolecules without spacers. As an example, suppose we have antibodies labeled with a fluorescent dye, with or without spacers. On an equivalent DOL basis, an antibody labeled with a dye that also has a bound spacer may show a fluorescence enhancement of between 1.5 and 3.5 times, where 1 represents the same level of fluorescence for both antibodies. Importantly, for biomolecules bound to both dye and spacers, the amount of fluorescence signal based on one dye molecule increases.

[0118] Following the addition of the reactive compound to the component solution, the mixture is incubated for an appropriate period (typically about 1 hour at room temperature to several hours on ice), and any excess compound is removed by gel filtration, dialysis, HPLC, ion exchange, adsorption to a hydrophobic polymer, or other suitable means. The compound-conjugate is then used in solution or lyophilized. In this way, suitable conjugates can be prepared from antibodies, antibody fragments, and other targeting carrier molecules.

[0119] The incubation temperatures used in the methods described herein may be room temperature, ambient temperature, or temperatures above room temperature, such as at least about 26°C, 27°C, 28°C, 29°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and even higher temperatures up to 90°C, 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C. The first and second incubation temperatures used in the methods described herein may be the same or different. In some embodiments, the first incubation temperature is between 20°C and 80°C, between 25°C and 30°C, and / or ambient temperature or room temperature. In some embodiments, the second incubation temperature is between 20°C and 80°C, between 65°C and 75°C, and / or about 70°C. In other embodiments, the second incubation temperature is ambient temperature or room temperature.

[0120] The incubation times used in the methods described herein include, but are not limited to, at least 30 seconds, at least 1 minute, at least 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, at least 1 hour, or any range as specified herein. The first and second incubation times used in the methods described herein may be the same or different. In one embodiment, the first incubation time is 0 to 60 minutes, 5 to 10 minutes, and / or 5 to 10 minutes at room temperature. In some embodiments, the second incubation time is 0 to 20 minutes and / or about 10 minutes. In a particular example, the second incubation time is about 10 minutes at about 70°C. In other embodiments, the first incubation period is 1 to 3 hours at 25°C, the second incubation period is overnight at 25°C, and the third incubation period is 2 to 3 hours at 37°C.

[0121] Conjugates of polymers, including biopolymers and other high molecular weight polymers, are typically prepared by means well known in the art (e.g., Brinkley et al., Bioconjugate Chem., 3:2 (1992)). In these embodiments, a single type of reaction site may be available, as is typical for polysaccharides, or multiple types of reaction sites may be available, as is typical for proteins (e.g., amines, thiols, alcohols, phenols). Labeling selectivity is best obtained by selecting an appropriate reactive dye. For example, modification of thiols with thiol-selective reagents such as haloacetamide or maleimide, or modification of amines with amine-reactive reagents such as active esters, acyl azides, isothiocyanates, or 3,5-dichloro-2,4,6-triazine. Partial selectivity can also be obtained by carefully controlling the reaction conditions.

[0122] When modifying polymers with compounds, an excess of the compound is typically used relative to the expected degree of compound substitution. Any residual unreacted compounds or compound hydrolysis products are usually removed by dialysis, chromatography, or precipitation. The presence of residual non-conjugated dyes can be detected by thin-layer chromatography using a solvent that elutes the dye from its conjugate. In all cases, reagents are usually concentrated to a practical degree to obtain conjugation at a suitable rate.

[0123] In certain embodiments of the methods described herein, the effectiveness of labeling can be monitored by labeling a control protein or protein either in a parallel reaction or by including it in the same reaction, if it is readily separable from the protein to be labeled.

[0124] In certain embodiments of the methods described herein, the proteins of a labeled sample can be usefully separated in parallel with a series of fluorescent molecular weight standards. Usefully, the standards spectrally match at least one fluorophore used to label the protein. Such spectral matching can be achieved, for example, by using a tetracysteine-tagged protein standard that is labeled in parallel with the same arsenic fluorophore used to label the protein sample, or by using a standard having a fluorescent moiety that spectrally matches the arsenic fluorophore or other fluorophore used to label the sample protein. Examples of standards useful in carrying out the present invention include the BENCHMARK® family of protein standards (Life Technologies Corp., Carlsbad, CA) and the MARKL2® Unstained Standard (Life Technologies Corp., Carlsbad, CA).

[0125] The methods and compositions described herein may also be used to quantify the amount of fluorescently labeled protein present in a sample. In certain embodiments, the methods described herein further include quantifying the amount of fluorescence from a diarsenic fluorophore. In certain embodiments, the methods described herein further include quantifying the amount of fluorescence from an amine-reactive fluorescent dye. In certain embodiments, the methods described herein further include quantifying the amount of fluorescence from the fluorescent moiety of an amine-reactive fluorescent dye. Quantification can be performed without separating the protein present in the protein sample, or after partially or completely separating the protein by electrophoresis such as PAGE, 2D-PAGE, or IEF, or chromatography, or a combination thereof.

[0126] e. Conjugation of spacer molecules into biomolecules In some embodiments, spacer molecules are conjugated to biomolecules using NHS-ester chemistry described in the examples herein, and SITECLICK® technology, which includes other chemistry such as maleimide, pyridyl disulfide, and hydrazide, as well as azide / alkyne, can also be used for this conjugation strategy.

[0127] In some embodiments, the spacer molecule is conjugated to a biomolecule (e.g., an antibody) at the first lysine side chain present on the protein, such as an antibody.

[0128] In some embodiments, the concentration of the protein, protein fragment, or other biomolecule labeled using the method described herein is in the range of about 0.01 mg / mL to about 200 mg / mL (e.g., about 0.1 mg / mL to about 100 mg / mL, about 0.1 mg / mL to about 50 mg / mL, about 0.1 mg / mL to about 10 mg / mL, about 0.2 mg / mL to about 100 mg / mL, about 0.2 mg / mL to about 50 mg / mL, about 0.2 mg / mL to about 10 mg / mL, about 0.3 mg / mL to about 10 mg / mL, about 0.4 mg / mL to about 10 mg / mL, about 0.5 mg / mL to about 10 mg / mL, etc.).

[0129] In some embodiments, the ratio of dye to protein in the fluorescent labeling of the antibody is 1 to 50. In some embodiments, the ratio of dye to protein in the fluorescent labeling of the antibody is 5 to 30. In some embodiments, the ratio of dye to protein in the fluorescent labeling of the antibody is 1 to 20.

[0130] In some embodiments, the ratio of spacer to protein is 1 to 50. In some embodiments, the ratio of spacer to protein is 5 to 30. In some embodiments, the ratio of spacer to protein is 1 to 20.

[0131] In some embodiments, the spacer is added in a molar excess to multiple fluorescent labels in amounts of 0.1 to 25 times, 1 to 15 times, or 2.5 to 10 times. In some embodiments, the spacer is in a molar excess to multiple fluorescent labels in an amount of 2.5 times. In some embodiments, the spacer is in a molar excess to multiple fluorescent labels in an amount of 5 times. In some embodiments, the spacer is in a molar excess to multiple fluorescent labels in an amount of 7.5 times. In some embodiments, the spacer is in a molar excess to multiple fluorescent labels in an amount of 10 times.

[0132] In some embodiments, spacers are conjugated to nucleic acid molecules. Protocols for conjugating portions (e.g., fluorescent labels) to nucleic acids have been described in the art (see, for example, Rombouts et al., Bioconjugate Chem., 27:280-207 (2016)). One method for labeling nucleic acid molecules is by using the ARES® ALEXA FLUOR® 488 DNA Labeling Kit (Thermo Fisher, catalog no. A21665). Amine-modified nucleotides can also be used to enable the labeling of nucleic acid molecules. For example, amine-modified DNA can be produced using 5-aminohexylacrylamide-dUTP (aha-dUTP) and 5-aminohexylacrylamide-dCTP (aha-dCTP) by conventional enzymatic incorporation methods such as reverse transcription, nick translation, random prime labeling, or PCR. The amine-modified DNA can then be labeled with any amine-reactive dye or hapten. This two-step technology consistently produces uniform and highly accurate DNA labeling, which is difficult to achieve by other methods.

[0133] One method for achieving high DOL using spatially separated fluorescent labels is shown in Figure 18. Figure 18 shows the preparation of fluorescently labeled branched-chain PEG molecules and the binding of the resulting PEG molecules (direct spacers) to the antibody, where the PEG molecules are covalently bound to the antibody by the CLICK-IT® reaction (see Example 7).

[0134] The PEG molecules shown in Figure 18 are each covalently bonded to seven fluorescent labels. Furthermore, the fluorescent labels can be attached to individual PEG molecules such that the labels have a specific "brush" length relative to the labeled molecule. "Brush length" refers to the extended length of the chemical group that attaches the fluorescent label to the labeled molecule. As an example, assuming that the average monomer length in a PEG molecule is approximately 3.5 angstroms, when n=1 in Figure 18, the brush length of the fluorescent labels would be in the range of approximately 5 to 10 angstroms. In many cases, n in Figure 18 will not be 1. In addition, PEG molecules typically vary in size and are described based on their average molecular weight. Therefore, using the PEG molecules shown in Figure 18 for illustrative purposes, a population of PEG molecules with an average weight of 10,000 would have approximately 30 n. A population of PEG molecules with an average weight of 40,000 would have approximately 120 n. Given that both the arms of the branched PEG molecule linking this molecule to the labeled molecule and the other arms all have repeating regions, the brush length can range from about 15 angstroms to about 800 angstroms (e.g., about 25 to about 800, about 150 to about 800, about 450 to about 800, about 600 to about 800, about 65 to about 800, about 25 to about 700, about 40 to about 700, about 28 to about 600, about 28 to about 500, about 70 to about 700, etc.). Accordingly, the present invention comprises compositions and methods for producing and using molecules covalently bound to at least one fluorescent label, the fluorescent label having a brush length of about 22 angstroms to about 800 angstroms.

[0135] Alternatively, the compositions of the present invention may be described by the number of covalent bonds between the molecules and the fluorescent labels to which they are bound. Using the branched PEG molecule shown in Figure 18 again for illustrative purposes, the number of intervening covalent bonds is about 24 to about 32, depending on where the fluorescent label is bound to the branched PEG molecule. The present invention includes compositions and methods for producing and using molecules covalently bound to at least one fluorescent label, where the fluorescent label is bound to the molecule by about 16 to about 800 intervening covalent bonds (e.g., about 16 to about 700, about 32 to about 800, about 60 to about 800, about 100 to about 800, about 150 to about 800, about 200 to about 800, about 250 to about 800, about 250 to about 700, about 250 to about 650, about 250 to about 600, about 350 to about 800, etc.).

[0136] The present invention also relates, in part, to spacing multiple fluorescent labels bound at the same position on individual labeled molecules. Similar to those described above with respect to the separation of fluorescent labels from labeled molecules, multiple fluorescent labels bound at the same position on individual labeled molecules can be separated from each other by distances of about 15 angstroms to about 800 angstroms (including the range described above) and / or by intervening covalent bonds of about 16 to about 800 (including the range described above).

[0137] As described herein, aspects of the present invention relate to the spacing of fluorescent labels. Table 9 shows the estimated properties of a series of branched PEG molecules. It should be understood that these PEG molecules, as well as other molecules, exist in multiple formats at different time points in time. For example, the arms of a branched PEG molecule can be fully extended (brushed) or fully coiled (mushroomed), and substantially all conformations in between. Furthermore, each arm can be in a different extended or coiled state independently of other arms at any given time. Thus, in Table 9, the term “brushed” refers to a fully extended PEG arm, and the term “mushroomed” refers to a fully coiled PEG arm. The mushroom state is modeled with a very large Flory radius (distance between adjacent PEG arms) of 80 angstroms to obtain the minimum number.

[0138] The FF distances in Table 9 are the distances between two fluorophores on the ends of different arms, assuming all fluorophores are equidistant from each other. The maximum distance is twice the length of the arm. Furthermore, the nearest neighbor values ​​assume a tetrahedral arrangement for four arms and a spherical / cube arrangement for eight arms. Naturally, the actual value of the FF distance at any given time will typically be somewhere between the brush distance and the mushroom distance.

[0139] (Table 9) Arm lengths of branched PEG (estimated fluorophore-to-fluorophore (FF) distance) JPEG2026071206000013.jpg90155

[0140] Dextran is another suitable substance for attaching fluorescent labels to molecules. Dextrans are hydrophilic polysaccharides characterized by their medium to high molecular weight, good water solubility, and low toxicity. Dextrans tend to be biologically inactive due to their dominant poly-(α-D-1,6-glucose) bonds. These bonds make them resistant to cleavage by most endogenous cellular glycosidases. They also typically have low immunogenicity and are generally branched-chain molecules.

[0141] Dextran is commercially available with nominal molecular weights (MW) ranging between 3,000 and 2,000,000 daltons. Suitable dextran for use in the implementation of the present invention may be any number of different molecular weights, including 3,000, 10,000, 40,000, 70,000, 500,000, and 2,000,000 daltons (e.g., about 4,000 to about 150,000, about 6,000 to about 150,000, about 8,000 to about 150,000, about 15,000 to about 150,000, about 10,000 to about 80,000, about 12,000 to about 70,000, etc.).

[0142] Dextran typically has a degree of substitution of 0.2–2 dye molecules per dextran molecule for dextran in the 10,000 MW range (e.g., DOL). Furthermore, dextran typically contains 0.2–0.7 dyes per dextran in the 3,000 MW range, 0.4–2 dyes per dextran in the 10,000 MW range, 1–4 dyes in the 40,000 MW range, and 2–6 dyes in the 70,000 MW range. Therefore, the dextran and other labeled polymers used in the implementation of the present invention may have a DOL of approximately 0.03 to 0.3 per 1,000 MW (e.g., approximately 0.03 to 0.25, approximately 0.08 to 0.3, approximately 0.09 to 0.3, approximately 0.1 to 0.3, approximately 0.05 to 0.2, approximately 0.07 to 0.25 per 1,000 MW, etc.).

[0143] Dextran and other polymers can be labeled in several ways. For example, fluorescently labeled dextran can be prepared by reacting water-soluble aminodextran with a fluorescent label having a succinimidyl ester group. Fluorescently labeled dextran can also be prepared by reacting natural dextran with an isothiocyanate derivative of a fluorescent label such as FITC. Where appropriate, once the fluorescent label is added, unreacted amines on the dextran can be capped to obtain neutral or charged dextran (i.e., negatively or positively charged). Furthermore, even if capping is not performed, charged fluorescent labels can be used to make the dextran anionic or cationic.

[0144] Another type of polysaccharide that may be useful in carrying out the present invention is amylose. Amylose is a linear polysaccharide consisting of α-D-glucose units linked by α-1,4-glycosidic bonds. Due to hydrogen bonding, amylose tends to form a helical structure containing 6 glucose units per turn. This type of molecule provides structural regularity that allows for the use of fluorescent labels arranged in a consciously designed manner. Thus, the present invention includes compositions having a fairly static structural feature that can be used to maintain a uniform distance between fluorescent labels, as well as methods for producing and using such compositions. Often, the distance between two fluorescent labels bound to such a molecule and under conditions that result in two fluorescent labels does not change by more than 30% from each other (e.g., about 5% to about 30%, about 10% to about 30%, about 15% to about 30%, about 20% to about 30%, about 10% to about 20%, etc.).

[0145] Polypeptides can also be used in the implementation of the present invention. An example of such a molecule is the branched polylysine molecule shown in Figure 21. This molecule can be prepared according to the method described in U.S. Patent Application Publication No. 2010 / 0278750. Several "R groups" representing positions to which fluorescent labels can be attached are shown in Figure 21. In such a molecule, the R groups may be the same or different (e.g., R1 and R2). Furthermore, if some R groups are the same, these groups may act as fluorescently labeled binding sites where the R groups are not fully labeled.

[0146] As an example, Figure 21 shows a polymer having 33 R groups, i.e., 32 R1 groups and 1 R2 group. We assume that all R groups are of the same type. These R groups may be partially fluorescently labeled in a semi-random manner. This means that conditions may be provided so that only a certain percentage of R groups receive fluorescent labeling. Furthermore, the labeling is semi-random because, due to their positions in the polymer, some R groups tend to receive more fluorescent labeling. Thus, the present invention includes the design of fluorescently labeled polymers in which positioning and DOL are adjusted for a desired fluorescence effect. In some cases, the average number of available binding sites based on the polymer accepting the label is in the range of 10% to 90% (e.g., about 10% to about 85%, about 15% to about 85%, about 20% to about 85%, about 20% to about 75%, about 30% to about 75%, about 30% to about 80%, about 40% to about 80%, etc.).

[0147] As mentioned above, Figure 21 shows a polymer having R1 and R2 groups. If "directed" binding of the polymer to a biomolecule (e.g., antibody) is desired, the R2 group may be different from the R1 group. If the R groups are the same, the binding will be "non-directed" in the sense that any R group at an appropriate position on the polymer can act as a conjugation site to the biomolecule. Naturally, the conditions are adjusted to achieve high levels of fluorescence while maintaining high levels of biological activity (e.g., antigen binding) to the biomolecule.

[0148] Maximizing biomolecular fluorescence may be partially independent of the number of fluorescent labels on the target biomolecule. For example, suppose a particular antibody has seven fluorescent labels when labeled under the first set of conditions, and ten fluorescent labels when labeled under the second set of conditions. Furthermore, suppose the total fluorescence of the antibody labeled under the first set of conditions is greater than the total fluorescence of the antibody labeled under the second set of conditions. In this case, fewer fluorescent labels resulted in more fluorescence. Therefore, given that other factors (e.g., functional activity of the biomolecule) are equal, the first set of conditions would be preferable to the second set of conditions.

[0149] Any number of linking groups can be used to attach a fluorescent label to a biomolecule. These bonds may be non-covalent or covalent. Furthermore, non-covalent or covalent bonds may refer to one or all of the following: (1) attachment of the fluorescent label to the polymer, (2) attachment of the polymer to the core (if present), and / or (3) attachment of the polymer or the core (if present) to the biomolecule.

[0150] In many cases, polymers (with or without a core), as well as other types of spacers described herein, can function to increase the fluorescence intensity of fluorescent labels bound to biomolecules.

[0151] One category of molecules useful in carrying out the present invention is called star polymers (see Ren et al., Star Polymers, Chemical Reviews, 116:6743-6836 (2016)). Star polymers are multi-armed molecules that consist of a core and a series of linear polymers (called "arms"). These arms typically contain terminal functional groups to facilitate the bonding of other molecules. Star polymers can be classified as homo-armed (containing arms with only one composition) or micto-armed (containing arms with more than one composition, molecular weight, or terminal functional group). The synthesis of star polymers is typically carried out using one of the following approaches: core-first, arm-first, or grafting.

[0152] The core effectively functions as a branching point in the arms. Any number of molecules can function as cores for the compositions of the present invention. Examples of suitable cores include oligoglycerols (e.g., hexaglycerol), oligoerythritols (e.g., pentaerythritol, dipentaerythritol, tripentaerythritol), sorbitol, trimethylolpropane, silanes (e.g., 1,2-bis(methylsilyl)ethane), adamantane, PAMAM (first-, second-, and third-generation (G-1-3) poly(amideamine) dendrimers, polyethyleneimine (PEI) branched-chain polymers), and peptides (e.g., polylysine, polyaspartic acid, etc.).

[0153] The core may already have functional groups for initiating polymerization or bonding to the arms, or it may require chemical modification to facilitate arm bonding, as shown in Figure 20.

[0154] For example, using hexaglycerol, this compound is commercially available and can be used for core-first synthesis of star-type PEG polymers. Star-type PEGs with a hexaglycerol core can be used for controlled drug release and wound sealing. Star-type PEGs with a hexaglycerol core can be produced by controlled polymerization of ethylene oxide from a hexaglycerol core. In this case, the core is hexaglycerol and the arms are polyethylene glycol.

[0155] The arms can consist of any number of linear polymers and will typically function as binding sites for fluorescent labeling, to space these labels from one another, and to space the labels from other fluorescent labels bound to the same labeled molecule (e.g., biomolecules). Examples of suitable arms include polyethylene glycol, poly(vinylpyrrolidone), polyglycerol, and polyvinyl alcohol, zwitterionic polymers (e.g., polysulfobetaine), and water-soluble polymers. In many cases, polymers suitable for use in this invention will be uncharged.

[0156] One region of an antibody that is particularly useful for the binding of fluorescent labels, if present, is the Fc (fragment crystallizable) region. The Fc region is located at the distal end of the antibody relative to the antigen-binding site. This region of the antibody interacts with cell surface receptors and complement system proteins called Fc receptors. Typically, the binding of chemicals in or near the Fc region has little effect on antigen binding by the antibody. However, interference with the antigen-binding event tends to increase with the size of the chemical bound to the antibody. Furthermore, large bound chemicals tend to interfere with each other in terms of their ability to bind to biomolecules due to steric hindrance. Therefore, it may be necessary to balance a set of factors to produce a biomolecule (e.g., an antibody) that has both high levels of fluorescence and high levels of functional activity (e.g., antigen-binding ability). Some of these factors are: (1) the size of the biomolecule, (2) the location of the binding site on the biomolecule for fluorescent labeling, and (3) the size and three-dimensional structure of the fluorescently labeled molecule bound to the biomolecule. With respect to antibodies, the present invention includes antibodies that have one or more of the following characteristics. - Fluorescently labeled polymers bound (e.g., covalently) at an average of 2 to 10 different positions on the antibody molecule. - An average of 3 to 80 fluorescent labels are bound to each antibody molecule. - Antigen binding affinity (K) of fluorescently labeled antibody molecules D Compared to unlabeled antibodies, the levels of labeled antibodies are reduced by more than two orders of magnitude (e.g., approximately 0.5 to 2.0, 1.0 to 2.0, 0.5 to 1.5, 0.75 to 1.5, etc.). Based on the amount of fluorescent label bound to the antibody molecule, the average fluorescence emission is at least 60% of that of the free fluorescent label (e.g., approximately 60% to 98%, 70% to 98%, 80% to 98%, 85% to 98%, 80% to 93%, etc.).

[0157] f. Combinations of biomolecules / fluorescent labels / spacers This invention is based in part on a combination of three components: a biomolecule, a fluorescent label, and a spacer. With respect to proteins (e.g., antibodies), particularly when the protein is not denatured, the groups that can be used as binding sites for the fluorescent label and spacer are not always accessible for binding. Furthermore, in many cases, it would be desirable to maintain the protein in its undenatured form.

[0158] Enhanced fluorescence emission has been found to be related to the various ratios of fluorescent labels and spacers used to label biomolecules (e.g., antibodies). Furthermore, each biomolecule may require different ratios of components in the conjugation process to produce a particular enhanced fluorescence level. This can be attributed to the different structures of biomolecules such as antibodies (e.g., primary, secondary, tertiary, and quaternary structures), as well as the properties of specific fluorescent labels and spacers.

[0159] In some cases, the ratio may be based on the individual weights of the components. In other cases, the ratio may be based on the molar ratio. Some figures and examples in this application relate to molar ratios. In some cases (e.g., when the biomolecule is large and has many conjugation sites), the use of component weights may be more appropriate.

[0160] For antibodies and other biomolecules, the ratio of biomolecules to fluorescent labels to spacers used in the conjugation process can vary considerably, but in most cases, the amount of biomolecules will be less than the amounts of both the fluorescent labels and spacers combined.

[0161] Furthermore, the density and / or spacing of conjugation sites on the biomolecule is often one factor that determines the optimal ratio of fluorescent label to spacer. This is because, assuming that enhanced fluorescence is due to reduced quenching, a lower overall or local density of conjugation sites is expected to result in less quenching. In any case, the usable ratio of biomolecule to fluorescent label to spacer is shown below:B1:FL2~30 :S 2~20 B is a biomolecule, FL is a fluorescent label, and S is a spacer. The specific range of ratios that can be used in the embodiment of the present invention includes ratios that fall within 1:2 to 25:2 to 20 (e.g., about 1:2:2 to about 1:25:20, about 1:5:2 to about 1:25:5, about 1:10:2 to about 1:25:5, about 1:5:2 to about 1:15:10, about 1:10:5 to about 1:25:20, about 1:10:5 to about 1:15:20, about 1:10:5 to about 1:20:20, etc.), where the first number is the amount of biomolecule (e.g., moles), the second number is the amount of fluorescent label, and the third number is the amount of spacer.

[0162] In some cases, the concentrations of the fluorescent label and spacer used for conjugation will be such that the available binding sites on the biomolecule are effectively saturated (e.g., at least 95% of the available binding sites bind to either the fluorescent label or the spacer). In such cases, the ratio of the fluorescent label to the spacer can be a determinant of the level of fluorescence enhancement. Often, the ratio of the fluorescent label to the spacer will be between 10:1 and 10:50 (e.g., approximately 10:1 to 10:25, 10:1 to 10:10, 10:1 to 10:5, 10:5 to 10:50, 10:5 to 10:20, 10:3 to 10:30, 10:5 to 10:30, 10:10 to 10:25, etc.).

[0163] Spacers and dyes can be conjugated simultaneously or sequentially, by first conjugating the spacer or dye to the biomolecule, and then conjugating the biomolecule. Often, when spacers and dyes bind to a biomolecule at the same site, they conjugate to the biomolecule simultaneously. However, sequential conjugation can be used when the binding site is a biomolecule and the first conjugation reaction (e.g., by the spacer) is carried out under unsaturated conditions, thus making the binding site available for a second conjugation reaction (e.g., by the dye).

[0164] g. Buffering agent In some embodiments, the compositions of the present invention include one or more spacers and one or more fluorescent labels in a buffer. Any of the fluorescent molecules and spacer molecules disclosed herein can be used with any buffer known in the art.

[0165] In some embodiments, the compositions disclosed herein include any buffer suitable for molecular biological applications.

[0166] In some embodiments, the buffer is a suitable preservation buffer (e.g., borate buffer, phosphate buffer, or carbonate buffer).

[0167] In some embodiments, the buffer is suitable for buffering biomolecules that can be detected as disclosed herein during use in a detection assay.

[0168] method The present invention has useful applications in basic research, high-throughput screening, immunohistochemistry, fluorescence in situ hybridization (FISH), microarray technology, diagnostics, and medical therapy. The present invention can be used in various assay forms for diagnostic applications in the fields of microbiology, immunology, hematology and transfusion, histopathology, forensic pathology, and veterinary pathology.

[0169] In some embodiments, the compositions described herein can be used in any molecular biological application in which a fluorescently labeled molecule is detected. For example, as disclosed herein, detectable biomolecules may be used in applications including Western blotting, ELISA, flow cytometry, and FRET. Detectable biomolecules such as those disclosed herein may also be used in fluorescence immunohistochemistry (IHC), fluorescence immunocytochemistry (ICC), and in vivo imaging applications.

[0170] In some embodiments, a method for determining the presence of a desired target in a biological sample is included, the method comprising: a) contacting the biological sample with a composition comprising one or more fluorescent labels and one or more spacer molecules, wherein the spacers and fluorescent labels are conjugated to the biomolecules but not to each other; b) detecting fluorescence emitted by the plurality of fluorescent labels; and c) determining the presence of a desired target in the biological sample when fluorescence emitted by the plurality of fluorescent labels is detected. In some embodiments, the biological sample comprises cell lysates. In some embodiments, the biological sample comprises intact cells. In some embodiments, the biological sample comprises tissue samples. Furthermore, such tissue samples may be immobilized. Furthermore, the compositions of the present invention can be used in applications such as immunohistochemistry. In some embodiments, the biological sample comprises isolated proteins. In some embodiments, the biological sample comprises recombinant proteins. In some embodiments, the biological sample is immobilized on a solid support. In some embodiments, the biological sample comprises intact cells in a fluid. In some embodiments, the biological sample is a living animal. In some embodiments, the living animal is a mammal. In some examples, the sample comprises tissues such as liver, lungs, muscles, and skin.

[0171] In some embodiments, methods for imaging target antigens in vivo are disclosed herein, comprising: a) providing antibodies conjugated to a plurality of fluorescent labels and spacers that bind to the target antigen, as disclosed herein; b) introducing the antibodies into vivo to form a contact; c) irradiating the contact with an appropriate wavelength to form an illuminator; and d) observing the illuminator on which the target antigen has been imaged. In some embodiments, these antibodies, or other target proteins or peptides, specific to the target or antigen in vivo, are conjugated with a fluorescent dye having an excitation wavelength suitable for in vivo imaging, typically about 580 nm to about 800 nm. The target-specific dye conjugates travel relatively freely in the circulating blood until their preferential sequestration occurs at a pathological or non-pathological tissue site of the target, such as a pathological or injured tissue site.

[0172] h. kit The compositions of the present invention can be incorporated into kits that facilitate the performance of various assays. The kits may be packaged with the compositions in dry form or in solution. The kits may further include one or more buffers, typically present as aqueous solutions, sample preparation reagents, additional detection reagents, organic solvents, other fluorescent detection probes, standards, microspheres, specific cell lines, antibodies, and / or instructions for performing the assay. Any additional agents may include components for testing other cellular functions in conjunction with the compounds.

[0173] In some embodiments, the kit comprises a biomolecule, a spacer, and a fluorescent label, as disclosed herein. In some embodiments, the kit further comprises a buffer. In some embodiments, the biomolecule is already conjugated to the spacer and the fluorescent label. In some embodiments, the kit may, as a bioconjugation kit, comprise a polymer conjugated to a fluorescent label and a reactive group.

[0174] The kit of the present invention may further include reagents used to prepare fluorescently labeled biomolecules (e.g., antibodies). Exemplary reagents include one or more of the following: a fluorescent dye, a spacer which is either pre-labeled or labeled according to the instructions provided in the kit, and a compound which can be used to (1) conjugate the spacer to a biomolecule and / or (2) conjugate the fluorescent label to a biomolecule.

[0175] This specification and exemplary embodiments should not be taken as limiting. For the purposes of this specification and the appended claims, unless otherwise indicated, all numbers representing quantities, percentages, or ratios, as well as other numerical values ​​used in the specification and claims, should in all cases be understood as being modified by the term “approximately” to the extent that they are not over-modified. Thus, unless otherwise indicated, the numerical parameters described below in the specification and the appended claims are approximations that may vary depending on the desired characteristic to be obtained. At a minimum, each numerical parameter should be interpreted by applying ordinary rounding techniques, in light of the reported number of significant figures, so as not to limit the application of the principle of equivalents to the claims. [Examples]

[0176] The following embodiments are provided to illustrate specific disclosed embodiments and should not be construed as limiting the scope of this disclosure.

[0177] Example 1: Fluorescent Western Blotting Method a) NHS-activating fluorescent dyes and sulfo-NHS acetate / NHS acetate and NHS-MS(PEG)4, NHS-MS(PEG)8 and NHS-MS(PEG) 12 Antibody labeling using this method. NHS-activating fluorescent dyes such as DYLIGHT® 650-4×PEG were reconstituted in dimethylformamide (DMF) at 10 mg / ml. NHS acetate was newly prepared in dimethylformamide (DMF) at 1 mg / ml. NHS-MS(PEG)4 (catalog number 22341 (Thermo Fisher Scientific)), NHS-MS(PEG)8 (catalog number 22509 (Thermo Fisher Scientific)), NHS-MS(PEG) 12 (Catalog No. 22686 (Thermo Fisher Scientific)) was reconstituted in DMF at 100 mg / ml. Immediately before use, the PEG reagent was further diluted in DMF to 1 mg / ml. 1 mg of goat anti-mouse (GAM) and goat anti-rabbit (GAR) antibodies at 7-10 mg / ml in 50 mM borate buffer (Catalog No. 28384 (Thermo Fisher Scientific), pH 8.5) were mixed with fluorescent dyes or mixtures of each fluorescent dye in various molar excesses, and NHS-acetate, NHS-MS(PEG)4, NHS-MS(PEG)8, or NHS-MS(PEG). 12Labeling was performed with a spacer selected from the available options. The labeling reaction was incubated at room temperature (RT) for approximately 1 hour. The NHS activating dye and NHS activating spacer were combined before addition to the antibody to allow both reactions to occur simultaneously, enabling dye substitution and random spacing of the spacers. 100 mM MES buffer at pH 4.7 was added to each sample to lower the pH from 8.5 to approximately 7.2. At this point, the conjugate concentration was adjusted to approximately 6 mg / ml to accommodate the final dilution in the storage buffer. Free dye was removed using Dye Removal Resin (Thermo Fisher Scientific, catalog no. 22858) and a 5 μm Harvard column (Harvard Apparatus, catalog no. 74-3820). 0.2 ml of 50% purified resin slurry was used per 1 mg of protein. The conjugates were diluted 1:50 with 0.1 M sodium phosphate buffer, pH 7.2 (PBS) and scanned using a UV Cary spectrophotometer. The concentration of the conjugate was determined using OD scanning (252nm~900nm), and the ratio of moles of dye to moles of protein (D / P) was calculated. Finally, for long-term storage, the conjugate was diluted to 1 mg / ml with STABILZYME® NOBLE Storage Buffer (Surmodics, catalog number SZ04).

[0178] Serially diluted cell lysates (500 ng to 2 ng) were mixed with SDS-PAGE sample buffer. The samples were heated at 95°C for 5 minutes and loaded onto Thermo Fisher Scientific Tris Glycine SDS-PAGE gels (Novex Gels, 4-20%, 10 wells, catalog number WT4202BX10). The gels were electrophoresed according to the manufacturer's instructions and then transferred to a nitrocellulose membrane using a semi-dry transfer method. The membranes were blocked with SEA BLOCK blocking buffer (Thermo Fisher Scientific, catalog number 0037527) for 30 minutes. Primary antibodies were prepared in Sea Block Blocking Buffer to a final concentration of 0.1-2.5 μg / ml. The blots were incubated with the antibodies prepared in Sea Block Blocking Buffer at room temperature (RT) for 1 hour with shaking. The antibody solution was decanted, and the membrane was washed twice for 10 minutes in 10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.2 (TBST). The fluorescently labeled secondary antibody conjugate was diluted to a final concentration of 20–1000 ng / mL with SEA BLOCK Blocking Buffer. The washed membrane was incubated with the appropriate secondary antibody conjugate for 30–60 minutes with agitation. The buffer was decanted, and the membrane was washed six times for 5 minutes with TBST. The membrane was imaged using a compatible fluorescence imager.

[0179] Figure 8 shows the results of experiments testing the effects of adding NHS acetate (2.5×, 5×, and 10×) and MS(PEG)4 (3.75×) spacers to GAM secondary antibodies conjugated to DYLIGHT® 488 with 5× to 20× molar excess in a Western blotting assay. A431 cell lysates were diluted 3-fold from 1 μg / well. The primary antibody rabbit used was anti-Hsp90 diluted from 1 mg / ml to 1 / 5000. All DYLIGHT® secondary antibodies were diluted from 1 mg / ml stock to 1 / 5000. For Western blotting applications, DYLIGHT® 488-GAR conjugated with NHS acetate or MS(PEG)4 showed a significant increase in fluorescence intensity compared to the basic conjugate (prepared without spacers) at each dye molar excess from 7.5× to 20×.

[0180] Figure 9 shows the results demonstrating the effect of adding NHS acetate (5X) or MS(PEG)4(5X) spacers to GAM secondary antibodies conjugated to DYLIGHT® 650-4xPEG (with 7.5× dye) in a Western blot assay. HeLa cell lysates were diluted 4-fold from 0.5 μg / well. Primary antibody mouse anti-PDI was diluted to 1 / 5000 of 1 mg / ml. All DYLIGHT® secondary antibodies were diluted to 1 / 5000 of 1 mg / ml stock. NHS acetate added to GAM-DYLIGHT® 650-4×PEG-7.5× conjugate in a 5× molar excess improved intensity by 1.5 times. NHS acetate added to GAM-DYLIGHT® 650-4×PEG-7.5× conjugate in a 3.75× molar excess improved intensity by 1.4 times.

[0181] Figure 10 and Table 10 show the results demonstrating the effects of adding NHS acetate (2.5×, 5X) and MS(PEG)4 spacers to GAR-DYLIGHT® 800-4xPEG secondary antibodies in Western blotting assays. A431 cell lysates were serially diluted 1:1. Primary antibodies rabbit anti-Hsp90 and anti-cyclophyllin B were diluted to 1 / 5000. All DYLIGHT® secondary antibodies were diluted to 1 / 20,000 of the 1 mg / ml stock. In this Western blotting application, the addition of MS(PEG)4 (3.75× and 5×) and NHS acetate (2.5-5×) significantly enhanced the fluorescence intensity and sensitivity of the base DYLIGHT® 800-4×PEG conjugate by 20-100% with different molar excesses of dye.

[0182] (Table 10) Efficacy of ±NHS acetate (2.5X, 5X) or MS(PEG)4 (5X) against GAR-DYLIGHT® 800-4xPEG in Western blot assays. JPEG2026071206000014.jpg48153NA=No additions

[0183] Figure 11 shows the results demonstrating the effects of adding NHS acetates (2.5×, 5×, and 10×) and MS(PEG)4 (5×) and MS(PEG)8 (5×) spacers to GAM-DYLIGHT® 550-2×PEG conjugates (with a 12.5× molar excess of dye) in a Western blot assay. HeLa cell lysates were diluted fourfold from 0.5 μg / well and stained with anti-PDI primary antibody diluted to 1 / 5000 of 1 mg / ml. All DYLIGHT® secondary antibodies were diluted to 1 / 5000 of 1 mg / ml stock. This experiment showed that in a Western blot assay, the addition of MS(PEG)4 (5×) and NHS acetates (2.5× and 5×) significantly enhanced the fluorescence intensity and sensitivity of the base DYLIGHT® 550-2×PEG conjugate by at least twofold. Conjugates prepared using long-chain MS(PEG)8 did not show any significant improvement over the base conjugate in this experiment.

[0184] Figure 12 shows the results demonstrating the effect of adding NHS acetate (2.5×, 5×) and MS(PEG)4 (5×) spacers to GAM-DYLIGHT® 680-4×PEG-GAR (with a 10× molar excess of dye). HeLa cell lysates were diluted fourfold from 0.5 μg / well, and the anti-PDI primary antibody was diluted to 1 / 5000 of 1 mg / ml. All DYLIGHT® 680-4×PEG-GAR secondary antibodies were diluted to 1 / 20,000 of 1 mg / ml stock. This experiment demonstrates that, in a Western blotting assay, the addition of MS(PEG)4 (5×) and NHS acetate (2.5× and 5×) significantly enhances the fluorescence intensity and sensitivity of the base DYLIGHT® 680-4×PEG conjugate by 3 to 4 times.

[0185] Example 2: Dot blot assay Serially diluted (1:1) mouse or rabbit IgG was prepared from selected stock concentrations. Using a 20 μL 12-channel multipipette, the dilutions were placed in a 96-well plate from highest to lowest concentration. One or two μL of each of the 11 serial dilutions was carefully spotted onto a nitrocellulose membrane. The membrane was dried overnight and then blocked with 2% BSA blocking buffer in TBST. The membrane was incubated at room temperature for 1 hour with agitation. The blocking solution was decanted from the container. Secondary antibody conjugates were diluted with TBS or blocking buffer. The secondary antibody conjugate dilutions varied depending on the conjugated label: 1:5,000 (DYLIGHT® 488 and 550-2×PEG conjugates); 1:10,000 (DYLIGHT® 650-4×PEG conjugate); and 1:20,000 (DYLIGHT® 680-4×PEG and DYLIGHT® 800-4×PEG conjugates). The membrane was incubated with a suitable secondary antibody conjugate for 30–60 minutes with agitation. The membrane was washed five times with TBST buffer for 5 minutes each. The membrane was imaged using a suitable imaging device, e.g., ChemiDoc MP (488, 550, 650, 680 nm) and LiCOR Odyssey CLx (650, 680, 800 nm).

[0186] The results shown in Figure 3 and Table 11 demonstrate the effects of NHS acetate (2.5×, 5×, and 10×) and MS(PEG)4 (3.75×) spacers on GAM-DYLIGHT® 488 (5× to 20× molar excess dye) in dot blot assays. For dot blot applications, DYLIGHT® 488-GAM conjugates prepared with NHS acetate and MS(PEG)4 showed a clear improvement in fluorescence intensity ranging from 1.2 to 1.8 times compared to the base conjugate (prepared without spacers) at 7.5 to 20 times molar excess of various dyes.

[0187] (Table 11) Effects of adding NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x~10x) to the conjugation of GAM-DYLIGHT™ 488 in 5x~20x molar excess in dot blots. JPEG2026071206000015.jpg57157NA=No additional information

[0188] The results shown in Figure 4 and Table 12 demonstrate the effects of adding NHS acetate (2.5× and 5×) and MS(PEG)4 (3.75×) to GAM-DYLIGHT® 488 (7.5× to 20× molar excess dye) in a dot blot assay. Different secondary antibody sources were used in this experiment. Addition of NHS acetate and MS(PEG)4 to the conjugation mixture resulted in a significant improvement in signal intensity, ranging from 1.2 to 2.6 times, at 5×, 15×, and 20× dye molar excesses compared to the base conjugation.

[0189] (Table 12) Effect of NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x) addition on conjugation of GAM-DYLIGHT® 488 in 5x-20x molar excess in dot blot assays. JPEG2026071206000016.jpg30155

[0190] The results shown in Figure 5 and Table 13 demonstrate the effects of adding NHS acetate (2.5×, 5×, and 10×) and MS(PEG)4 (3.75×) to GAM-DYLIGHT® 550-2×PEG-GAR (10× to 20× molar excess dye) in a dot blot assay. Mouse IgG was serially diluted 1:1 from 1000 ng / dot. All DYLIGHT® 550-2×PEG-GAR secondary antibodies were diluted to 1 / 5000 of the 1 mg / ml stock. Addition of NHS acetate or MS(PEG)4 to the conjugation mixture resulted in improved signal intensity compared to the basic conjugate with molar excess of each respective dye. The improvement ranged from 1.2 to 1.6 times.

[0191] (Table 13) Effect of NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x) addition on conjugation of GAM-DYLIGHT™ 550-2x PEG-GAR in 10x-20x molar excess in dot blot assays. JPEG2026071206000017.jpg48155

[0192] The results shown in Figure 6 and Table 14 [Surbhi and Marie: The data in Figure 6 and Table 14 do not completely agree. We are unsure which is correct. Please clarify this. This is not a major issue, but it needs to be done correctly.] demonstrate the effects of adding NHS acetate (2.5×, 5×, and 10×) and MS(PEG)4 (3.75×) to GAM-DYLIGHT® 650-4×PEG-GAM (10×~20× molar excess) in a dot blot assay. Mouse IgG was serially diluted 1:1 from 1000 ng / dot. All DYLIGHT® 650-4×PEG-GAR secondary antibodies were diluted to 1 / 10000 of the 1 mg / ml stock. Both NHS acetate and (MS)PEG4 resulted in significant improvements in sensitivity and signal / background compared to the initial base conjugate. NHS acetate added to GAM-DYLIGHT® 650-4×PEG-15× in a 2.5× molar excess improved the intensity by 1.7 times. The improvement brought about by the NHS acetate was 1.3 times better than when using conjugates prepared with the highest molar excess dye (20×). All MS(PEG)4 added to GAM-DYLIGHT® 650-4×PEG-15× conjugates improved the fluorescence intensity by 1.8 to 2.2 times and performed better than the corresponding highest base conjugate GAM-DYLIGHT® 650-4×PEG-20×.

[0193] (Table 14) Effect of NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x) addition on 10x-20x GAM-DYLIGHT™ 650-4x PEG-GAR conjugation in dot blot assays. JPEG2026071206000018.jpg57155NA=No additions

[0194] The results shown in Figure 7 and Table 15 demonstrate the effects of adding NHS acetate (2.5×, 5×, and 10×) and MS(PEG)4 (3.75×, 5×, and 10×) spacers to GAM-DYLIGHT® 800-4×PEG- in dot blot assays. Mouse IgG was serially diluted from 1000 ng / dot to 1:2. All DYLIGHT® 800-4×PEG-GAR secondary antibodies were diluted to 1 / 20,000 of the 1 mg / ml stock. This experiment shows that, in dot blot applications, the addition of MS(PEG)4 (3.75× and 5×) and NHS acetate (5×) significantly enhanced the fluorescence intensity and sensitivity of the base DYLIGHT® 800-4×PEG conjugate by 1.5 to 6 times.

[0195] (Table 15) Effects of adding NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x, 5x, and 10x) to GAM-DYLIGHT™ 800-4xPEG in dot blot assays. JPEG2026071206000019.jpg57152NA=No additions

[0196] The results shown in Figure 11 demonstrate the effects of adding NHS acetates (2.5×, 5×, and 10×) and MS(PEG)4 (5×) and MS(PEG)8 (5×) spacers to GAM-DYLIGHT® 550-2×PEG-GAR (12.5× molar excess dye) in dot blot assays. Mouse IgG was diluted 3-fold from 0.5 μg / well. All DYLIGHT® secondary antibodies were diluted to 1 / 5000 of the 1 mg / ml stock. These dot blot assays showed that the fluorescence intensity and sensitivity of the base DYLIGHT® 550-2×PEG conjugate were significantly enhanced by at least twofold upon addition of MS(PEG)4 (5×) and NHS acetates (2.5× and 5×). Conjugates prepared with long-chain MS(PEG)8 did not show significant improvement over the base conjugate.

[0197] The results shown in Figure 12 demonstrate the effect of adding NHS acetate (2.5×, 5×) and MS(PEG)4 (5×) spacers to GAM-DYLIGHT® 680-4×PEG-GAR (10× molar excess dye) in a dot blot assay. Mouse IgG was serially diluted from 1000 ng / dot to 1:2. All DYLIGHT® 680-4×PEG-GAR secondary antibodies were diluted to 1 / 20,000 of the 1 mg / ml stock. These dot blot assays show that the fluorescence intensity and sensitivity of the base DYLIGHT® 680-4×PEG conjugate were significantly enhanced upon addition of MS(PEG)4 (5×) and NHS acetate (2.5× and 5×).

[0198] Example 3: Plate assay method To prepare the plates, eleven (1:1) serial dilutions of mouse or rabbit IgG were prepared, starting at 10 μg / ml. Using a 300 μL 12-channel multipipette, 100 μL of each dilution was added to the corresponding wells of a 96-well plate, from highest to lowest, between 1 and 11, with PBS added to the last column (#12; negative control). This was repeated for columns A through H. After incubating the plates overnight, they were blocked and incubated with SUPERBLOCK® Blocking Buffer (Thermo Fisher, catalog no. 37515) as follows: 200 μL for 5 minutes twice, followed by 200 μL for 10 minutes once. The plates were dried and then stored dry at 4°C.

[0199] Plates coated with mouse IgG or rabbit IgG were washed twice with 200 μL of PBST 20, followed by one wash with PBS. Secondary antibody conjugates were diluted with TBS or PBS. Secondary antibody conjugates were diluted 1:100 (DYLIGHT® 488 and 550-2×PEG, DYLIGHT® 650-4×PEG, DYLIGHT® 680-4×PEG, and DYLIGHT® 800-4×PEG conjugates). 100 μL of the appropriate conjugate GAM from the mouse IgG coated plate and GAR from the rabbit IgG coated plate were added to the plate wells. Each dilution was added to a different column for each conjugate to be tested. All comparisons were performed on the same plate. The plate was incubated for 60 minutes. The plate was washed three times with 200 μL of TBST or PBST buffer. 100 μL of PBS was added to each column in each well. Fluorescence intensity was measured using a VariosKan instrument or by imaging the fluorescence signal with a suitable imaging device such as ChemiDoc MP (488, 550, 650, 680 nm) and LiCOR Odyssey CLx (650, 680, 800 nm).

[0200] Example 4: Immunofluorescence (IFC) method (i.e., cell imaging method) Method 1: Frozen U2OS cell plates stored at -80°C were thawed at 50°C for 30 minutes. The storage buffer (PBS) was removed, and the cells were permeabilized with 0.1% Triton-X100 in 1× PBS buffer for 15 minutes (100 μl / well). The plates were blocked with 2% BSA / PBS-0.1% Triton-X100 blocker for 30 minutes. Primary antibodies, mouse anti-PDI or rabbit anti-HDAC2 (10 μg / ml) (catalog number PA1-861), were diluted with 2% BSA / PBS-0.1% Triton-X100. 、Life Technologies Corp., Carlsbad, CA) was added to the plate and incubated at room temperature for 1 hour. The negative control contained only 2% BSA / PBS-0.1% Triton-X100 blocker. After incubation, the primary antibody solution was removed from the plate and the plate was washed three times with 100 μl / well PBST and once with 100 μl / well PBS. Next, GAM or GAR secondary antibodies labeled with various molar excesses of dyes were diluted to 4 μg / ml in PBS and incubated at room temperature for 1 hour. The plate was washed three times with 100 μl / well PBST and 1 × 100 μl / well PBS, and Hoechst 33342 (catalog no. 62249, Thermo Scientific, Waltham, MA) (diluted to 0.1 μg / ml in PBS) was added to each well (100 μl / well). The plate was scanned using an ARRAYSCAN™ Plate Reader VTI3 with a 20x objective lens.

[0201] Method 2: Specific experiments were performed in A549 cells in 384-well plates. Primary antibodies were used at the same concentration (1 μg / ml) while the dilution ratio of secondary antibodies was varied. B signal / noise (S / N, also called signal versus background) was measured using pH2AX measurement in cells treated with etoposide (50 μM for 3 hours, Tocris Bioscience, catalog no. 12-261-00), and brightness was compared to compare different antibodies. Secondary antibody conjugates were tested in four different dilutions (0.5, 1, 2, and 4 mg / ml). Standard procedures were used for antibody staining: fixation in 4% formaldehyde for 15 minutes. Permeabilization was performed in 0.5% Tritonx-100 for 10 minutes. Blocking was performed in 3% BSA for 30 minutes. Primary antibody incubation was performed at room temperature for 1 hour, followed by three washes with PBS. Secondary antibody conjugates were incubated at room temperature for 1 hour, followed by three washes with PBS. The cells were analyzed using ARRAYSCAN™ VTI (Thermo Fisher).

[0202] The results shown in Figure 13 and Tables 16 and 17 demonstrate the effectiveness of adding NHS acetate (2.5× and 5×) and MS(PEG)4 (3.75×) spacers to GAM-DYLIGHT® 488 (13A) and GAR-DYLIGHT® 488 ((13B) with 7.5× to 20× molar excess dye) for cell imaging applications. DYLIGHT® 488-GAM and DYLIGHT® 488-GAR. A549 cells were stained with pH2A× primary antibody diluted to 1 / 1000 of 1 mg / ml stock. All DYLIGHT® 488 secondary antibodies were diluted to 1 / 250 of 1 mg / ml stock. The addition of NHS acetate to the conjugation mixture resulted in improvements of 1.4–1.5 times (GAM) and 1.1–1.6 times (GAR) in signal / background compared to the base conjugate at a 15× molar excess of dye. For the GAM conjugate, the most significant improvements were observed with MS(PEG)4 for 5× NHS acetate and 3.75× GAR conjugates, while more significant improvements were observed with 2.5× NHS acetate and 3.75× MS(PEG)4.

[0203] (Table 16) Effects of adding NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x) to GAM-DYLIGHT® 488 conjugation at 5x-20x molar excess in cell imaging applications - DYLIGHT® 488-GAM JPEG2026071206000020.jpg27155NA=No additions

[0204] (Table 17) Effects of adding NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x) to GAR-DYLIGHT® 488 conjugation at 5x-20x molar excess in cell imaging applications - DYLIGHT® 488-GAR JPEG2026071206000021.jpg27155NA=No additions

[0205] The results shown in Figure 14 and Table 18 demonstrate the efficacy of NHS acetates (2.5×, 5×, and 10×) and MS(PEG)4 (3.75×, 5×, and 10×) on GAM-DYLIGHT® 550-2×PEG-GAM (with 7.5× to 20× molar excess dye) for cell fluorescence imaging applications. U2OS cells were stained with anti-PDI primary antibody diluted to 1 / 100 of the 1 mg / ml stock. All DYLIGHT® 550-2×PEG-GAM secondary antibodies were diluted to 1 / 250 of the 1 mg / ml stock. In this cell imaging application, the addition of 5×NHS acetate resulted in approximately 50% improvement for the DYLIGHT® 550-2×PEG GAM conjugate with a 12.5× molal excess of dye compared to the base conjugate (made without addition), and the addition of 3.75×MS(PEG)4 with a 20-fold molal excess of dye resulted in approximately 50% improvement compared to the base conjugate.

[0206] (Table 18) Effects of 2.5x-10x NHS acetate and MS(PEG)4 (3.75x-10x) on the fluorescence intensity of GAM-DYLIGHT® 550-2xPEG in cell imaging applications. JPEG2026071206000022.jpg57155NA=No additions

[0207] Figure 15 and Table 19 show the results of experiments testing the effects of NHS acetate (2.5×, 5×, and 10×) and MS(PEG)4 (3.75×, 5×, and 10×) spacers on GAM-DYLIGHT® 650-4×PEG) for cell imaging applications. U2OS cells were stained with anti-PDI primary antibody diluted to 1 / 100 of the 1 mg / ml stock. All DYLIGHT® 650-4×PEG-GAM secondary antibodies were diluted to 1 / 250 of the 1 mg / ml stock. In this cell imaging application, the addition of NHS acetate-5× resulted in approximately 70% improvement for DYLIGHT® 650-4×PEG GAM conjugates with a 20× molar excess compared to the base conjugate (made without addition), and the addition of MS(PEG)4-3.75× with a 20-fold molar excess showed approximately 90% improvement compared to the base conjugate.

[0208] (Table 19) Effects of adding NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x, 5x, 10x) to GAM-DYLIGHT® 650-4xPEG - Cell imaging applications JPEG2026071206000023.jpg57155NA=No additions

[0209] Figure 16 and Table 20 show experimental results testing the effect of adding NHS acetate (2.5×, 5×, and 10×) and MS(PEG)4 (3.3.75×, 5×, and 10×) spacers on the detectable fluorescence level of GAM-DYLIGHT® 680-4×PEG) for cell imaging applications. U2OS cells were stained with mouse anti-PDI primary antibody diluted to 1 / 100 of the 1 mg / ml stock. All DYLIGHT® 680-4×PEG-GAM secondary antibodies were diluted to 1 / 250 of the 1 mg / ml stock. For this cell imaging application, the addition of NHS acetate-5× resulted in approximately 70% improvement in the dye conjugates for both 7.5× and 10× compared to the basic DYLIGHT® 680-4×PEG conjugate (prepared without addition). GAM conjugates and MS(PEG)4-3.75× in a 15× molar excess showed approximately 80% improvement over the base conjugate in a 15× molar excess.

[0210] (Table 20) Effects of adding NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.3.75x, 5x, 10x) to GAM-DYLIGHT® 680-4xPEG - Cell imaging applications JPEG2026071206000024.jpg57155NA=No additions

[0211] result The use of spacers such as NHS-acetate, NHS-MS(PEG), and NHS-betaine increased the sensitivity and intensity of the fluorescence signal beyond the optimal D / P level that typically results in quenching. This was achieved with goat anti-mouse (GAM) and goat anti-rabbit (GAR) secondary antibodies, as well as NHS-acetate and NHS-MS(PEG)4, NHS-MS(PEG)8, and NHS-MS(PEG). 12This was demonstrated by labeling with NHS-DYLIGHT® 488, NHS-DYLIGHT® 550 2×PEG, NHS-DYLIGHT® 650 4×PEG, NHS-DYLIGHT® 680 4×PEG, and NHS-DYLIGHT® 800 4×PEG in combination with spacers including, but not limited to, the following: Our calculations of D / P values ​​after dye conjugation and purification demonstrated that the addition of spacers did not produce a significant difference in the D / P ratio, indicating that these reagents and fluorophores labeled different primary amines on the antibody (i.e., they did not compete for the same primary amine).

[0212] Conjugates prepared using different dyes and spacers were tested in various applications, including IFC, Western blotting, dot blotting, or IgG-conjugated plate-based assays. In each case, with specific spacers at certain molar excess levels relative to the dye, an increase in fluorescence intensity was observed when the spacer was used compared to a control lacking the spacer.

[0213] In addition to the experiments described above, antibodies labeled with NHS-rhodamine and conjugated with various NHS-betaine concentrations (betaine 2.5, betaine 5, and betaine 10 molar ratios) showed an increase in total fluorescence when the antibody was conjugated with betaine as a spacer modification reagent. See Figure 17 and Tables 21 and 22 below. Among the different betaine chain lengths, betaine 10 showed a positive effect on ALEXA FLUOR® 555 with a total molar excess of the dye and a D / P ratio greater than 12 (data not shown).

[0214] (Table 21) Effect of 2.5x~10x betaine on fluorescence of TAMRA-GAM conjugates JPEG2026071206000025.jpg67160MR = Mole Ratio DOL=degree of labeling

[0215] Example 5: Reaction of goat anti-mouse IgG (GAM) with and without N,N,N-trimethylglycine-N-hydroxysuccinimide bromide (betaine-SE) and 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (5(6)-TAMRA-SE) TAMRA-SE was weighed and prepared as a stock solution of 10 mg / mL in anhydrous DMSO, and betaine-SE was weighed and prepared as a stock solution of 4 mg / mL in anhydrous DMSO. Next, the DMSO solution was transferred to a reaction vial, and TAMRA-SE+ / -betaine-SE was added to the vial based on the molar ratio of the dye to IgG at 5, 10, or 20 times the amount of IgG, and equivalents of betaine-SE at a molar ratio of 0 or 10 to IgG were also added to the vial.

[0216] Separately, 0.417 mL (3.5 mg) of an 8.4 mg / mL solution of GAM in 10 mM potassium phosphate and 150 mM sodium chloride buffer (PBS) was measured and placed in a plastic tube, and the pH was raised to >8.0 with 42 μL of 1 M sodium bicarbonate, pH 9.0. The 0.5 mg GAM solution was added to a reaction vial containing SE and reacted at room temperature for 1 hour. The dye-protein conjugate was separated from the free dye and betaine by size exclusion chromatography using a 5-0.75 × 20 cm column packed with BIORAD® BIO-GEL® P-30 microparticles in PBS, and eluted with the same method. The first protein-containing band was recovered from each column.

[0217] Absorbance spectra were obtained using a Perkin-Elmer Lambda 35 UV / Vis spectrometer, and the degree of substitution (DOS) or dye moles / GAM moles was determined for each sample. Fluorescence emission spectra were obtained using a Perkin-Elmer LS 55 fluorescence spectrometer, with samples having a matched optical density at 545 nm and excited at 545 nm. Emission data were collected from 550–750 nm. Relative quantum yield (RQY) was measured as the area of ​​the sample spectrum divided by the area of ​​the sample standard spectrum. Total fluorescence was then calculated as the product of RQY*DOS.

[0218] (Table 22) Total fluorescence output of TAMRA / GAM at different molar ratios JPEG2026071206000026.jpg15156

[0219] Example 6: Reaction of goat anti-mouse IgG (GAM) with and without 1,3-propanesultone (3-hydroxy-1-propanesulfonic acid γ-sultone) with Alexa Fluor® 488 carboxylic acid, succinimidyl ester, dilithium salt (AF488-SE) AF488-SE was weighed and prepared as a stock solution of 10 mg / mL in anhydrous DMSO, and propane-sultone was weighed and prepared as a stock solution of 1 mg / mL in E-PureH2O.

[0220] 0.357 mL (4.0 mg) of an 11.2 mg / mL solution of GAM in 10 mM potassium phosphate, 150 mM sodium chloride buffer (PBS) was measured and placed in a plastic tube, and the pH was raised to >8.0 with 36 μL of 1 M sodium bicarbonate, pH 9.0. 0.5 mg of the GAM solution was transferred to a reaction vial and reacted with 0, 2, 5, or 10-fold molar excess propanesultone for 2 minutes. Then, AF488 stock was added to the mixture in an 8 or 15-fold molar excess relative to the GAM, and the mixture was reacted at room temperature for 1 hour. The dye-protein conjugate was separated from the free dye and propanesultone by size exclusion chromatography using a 5-0.75 × 20 cm column packed with BIORAD® BIO-GEL® P-30 microparticles in PBS, and eluted with the same. The first protein-containing band was recovered from each column.

[0221] Absorbance spectra were obtained using a Perkin-Elmer Lambda 35 UV / Vis spectrometer, and the degree of substitution (DOS) or dye mol / GAM mol was determined for each sample. Fluorescence emission spectra were obtained using a Perkin Elmer LS 55 fluorescence spectrometer with samples having a matched optical density at 475 nm and excited at 475 nm. Emission data were collected from 480 - 800 nm. The relative quantum yield (RQY) was measured as the area of the sample spectrum / area of the sample standard spectrum. Then, the total fluorescence was calculated as the product of RQY * DOS.

[0222] (Table 23) Total fluorescence output at different molar ratios of AF488 / GAM JPEG2026071206000027.jpg24156

[0223] Example 7: Labeling of SK3 mouse anti-human CD4 azide with 20 kDa 8-arm PEG amine (20K8PEG) modified with ALEXA FLUOR® 647 NHS ester, tris(triethylammonium salt) (AF647-SE) ALEXA FLUOR® 647 NHS / succinimidyl ester (Thermo Fisher Scientific, catalog number A37573), abbreviated as "AF647-SE", was weighed and prepared as a 32 mM stock solution in anhydrous DMSO (Thermo Fisher Scientific, D12345). CLICK-IT® SDP ester sDIBO alkyne (sDIBO) (Thermo Fisher Scientific, catalog number C20025) was prepared as a 9 mg / mL stock solution in anhydrous DMSO. An 8-arm PEG amine (hexaglycerol), HCl salt (JenKem, Plano, TX 75024, catalog number 8ARM-NH2HCl), having the following structure and designated as "20K8 PEG" JPEG2026071206000028.jpg19128 was weighed and prepared as a 40 mg / mL stock solution in anhydrous DMSO.

[0224] To the plastic tube, 300 μL of 20K8 PEG stock solution, 176 μL of sDIBO stock solution (2.4-fold molar excess / 20K8 PEG), and 6 μL of neat triethylamine (TEA) were added, and the reaction was carried out at 25 °C for 3 hours. After the reaction, 300 μL of AF647-SE stock solution (2-fold molar excess / PEG-amine) was added to the tube, and the reaction was allowed to proceed overnight at 25 °C. The AF647-20K8 PEG-sDIBO construct was purified from free dye and sDIBO by size exclusion chromatography using a 1×30 cm column packed with BioRad BIO-GEL® P-10F in PBS and eluted with the same. The first dye-containing fraction was collected and concentrated using an EMD Millipore AMICON® Ultra-4 10 kDa centrifugal filter.

[0225] Azido(PEO)4 propionic acid, succinimidyl ester (Thermo Fisher Scientific, catalog number A10280), abbreviated as "Azide-SE", was weighed and prepared as a 10 mM stock solution in anhydrous DMSO (Thermo Fisher Scientific, D12345). 266 μL of SK3 mouse anti-human CD4 antibody (2.5 mg) and 134 μL of PBS were added to a plastic tube, and the pH was raised to >8.0 with 50 μL of 1 M sodium bicarbonate, pH 9.0. 8.3 μL of azide-SE stock solution (5-fold molar excess / antibody) and 42 μL of DMSO were added to the antibody solution. The reaction was allowed to proceed at 25 °C for 2 hours. The azide-SK3 antibody was purified using a 2 mL BioRad BIO-GEL® P-30M spin column. The azide-SK3 antibody was prepared with 5- to 20-fold excess azide-SE relative to the antibody.

[0226] 24.2 μL of a 2 mM solution of AF647-20K8 PEG-sDIBO (DIBO concentration) and 116 μL of 4.3 mg / mL azide-SK3 were mixed in a plastic tube. 360 μL of PBS was added to bring the final solution concentration to 100 μM of AF647-20K8 PEG-sDIBO (DIBO concentration) and 1 mg / mL of azide-SK3 antibody. The click reaction was allowed to proceed at 37°C for 2 hours, followed by quenching with 5 mM NaN3 at room temperature for 1 hour. The AF647-20K8 PEG-SK3 conjugate was diluted to 0.5 mg / mL in PBS. Conjugation reactions were carried out using 1–3 mg / mL of azide-SK3 antibody at final DIBO concentrations of 100–600 μM, at reaction temperatures of 25°C–37°C for 2–20 hours. Table 24 shows the specific conditions for each experiment conducted.

[0227] 24.2 μL of a 2 mM solution of AF647-20K8 PEG-sDIBO (DIBO concentration) and 116 μL of 4.3 mg / mL azide-SK3 were mixed in a plastic tube. 360 μL of PBS was added to the final solution concentration of 100 μM AF647-20K8 PEG-sDIBO (DIBO concentration) and 1 mg / mL azide-SK3 antibody. The click reaction was allowed to proceed at 37°C for 2 hours, followed by quenching with 5 mM NaN3 at room temperature for 1 hour. The conjugate was purified and concentrated using an EMD Millipore AMICON® Ultra-4 100 kDa centrifuge filter. The AF647-20K8 PEG-SK3 conjugate was diluted to 0.5 mg / mL in PBS. Conjugation reactions were carried out using 1-3 mg / mL azide-SK3 antibody at a final DIBO concentration of 100-600 μM, at a reaction temperature of 25°C-37°C, for 2-20 hours. The specific conditions for each experiment are shown in Table 24.

[0228] (Table 24) Conjugation conditions JPEG2026071206000029.jpg143170

[0229] Example 8: Analysis of the quantum yield of the branched-chain PEG AF647 construct To prepare samples for quantum yield measurement, solutions of AF647 branched-chain PEG constructs (AF647-2K4, AF647-10K4, AF647-10K8, and AF647-20K8) were diluted in deionized water to a final dye concentration of 0.16 μM. Quantum yield (Φ) was measured using a Hamamatsu Absolute PL Quantum Yield Spectrometer. The quenching in the final construct was determined by comparing the quantum yield for the branched-chain PEG constructs with that of the free dye. Furthermore, brightness was determined to determine the fluorescence enhancement achieved using the branched-chain PEG spacer. The smallest construct, AF647-2K4 (a 2,000 molecular weight branched-chain PEG with four arms), showed the greatest quenching (QY of 20% free dye, fluorescence ratio of 0.2) and the lowest overall improvement in total fluorescence. The greatest fluorescence enhancement was observed for high molecular weight constructs with either four or eight arms (AF647-10K4 and AF647-20K8) in which a fluorescence quantum yield of up to 89% was maintained with the free dye (fluorescence ratio 0.9), and brightness improvements of up to 5.8 times were also observed.

[0230] (Table 25) Effect of branched-chain PEG spacers on the percent quantum yield (QY) and brightness (B) of ALEXA FLUOR® 647 JPEG2026071206000030.jpg61153

[0231] Example 9: Flow cytometry evaluation of AF647-20K8 PEG-SK3 construct Freshly collected anticoagulated whole blood (human) was lysed in ACK lysis buffer at room temperature for 20 minutes. Leukocytes were isolated by centrifugation (400 × g, 5 min) and washed twice in 1% bovine serum albumin / PBS (1% BSA / PBS). After isolation, the total number of leukocytes was determined using a COUNTESS® automated cell counter and then diluted to 10 million cells per mL. 1 million cells / well in a 96-well plate were stained with AF647-20K8 PEG-SK3 conjugate using a 7-stop titration of 1 μg to 0.015 μg of antibody. Stained cells were washed twice with 1% BSA / PBS. Stained cells were analyzed using an ATTUNE® NxT flow cytometer and compared with APC (Thermo Fisher Scientific, catalog no. MHCD0405), ALEXA FLUOR® 488 (Invitrogen, catalog no. MHCD0420), FITC (Thermo Fisher Scientific, catalog no. MA1-81103), and BRILLIANT VIOLET® 605 (BioLegend, San Diego, CA, catalog no. 300555) CD4 conjugates.

[0232] Figure 22 shows a histogram plot of CD4-positive lymphocytes as a function of fluorescence intensity in the RL1 channel of an ATTUNE® NxT flow cytometer. ALEXA FLUOR® 647 conjugated to CD4 is shown as a dashed line, and the AF647-20K8 PEG-SK3 conjugate is shown as a dotted or solid line. Compared to the AF647 conjugate alone, the Star PEG conjugate shows an increase of more than 0.5 log in brightness. Figure 23 shows the signal-to-noise ratio (S / N) and positive percentage (Positive %) plotted as a function of conjugate concentration in flow cytometry experiments. The Star PEG constructs (here B1 and B2) have an increase in S / N up to 2.5 times compared to the APC CD4 benchmark conjugate and an increase in S / N up to 2 times compared to the AF647 CD4 benchmark conjugate, while retaining the ability to accurately assess the number of several CD4-positive cells in the sample.

[0233] Example 10: Conjugation of ALEXA FLUOR® 488 to an aminodextran scaffold Preparation of a 70kD aminodextran AF488 scaffold: 10 mg of aminodextran (70,000 MW, 20 amino groups; Thermo Fisher Scientific, catalog no. D1862) was dissolved in 1.2 ml of dry DMSO containing 1.0 μl of DIEA. 0.9 mg of ALEXA FLUOR® 488 succinimimidyl lithium salt (643 MF; Thermo Fisher Scientific, catalog no. A20000) was added to the solution, and the mixture was stirred at ambient temperature for 3.5 hours. The solution was diluted with 12 mL of ethyl acetate, and the resulting suspension was centrifuged. The supernatant was discarded, and the solids were shaken with 10 mL of fresh ethyl acetate and centrifuged. This washing was repeated three more times with 10 mL of fresh ethyl acetate, and the resulting precipitate was dried under vacuum. The solid was redissolved in 0.5 ml of water, and the solution was placed in a 10 cm Spectra / Por Dialysis membrane (Spectrum Labs, MWCO12-14,000, flat, 10 mm wide) that had been cut from both ends. The dialysis membrane was slowly agitated in 1 L of water for one week. The water was changed twice a day. The dialysis membrane was opened from one end, and the solution was freeze-dried to obtain an aminodextran ALEXA FLUOR® scaffold. The measured DOL was 9.7, and the relative QY was 0.6 (referenced to the QY of ALEXA FLUOR® 488).

[0234] Bonding of thiol linkers to a 70 kD aminodextran AF488 scaffold: A 4.5 mg aminodextran AF488 scaffold was dissolved in 0.5 mL of DMSO containing 0.055 μL of N,N-diisopropylethylamine (DIEA). 20 μg of succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was added to the solution, and the mixture was left at ambient temperature overnight, then capped with succinimidyl acetate (1.0 mg, 3 hours). The solution was diluted with 10 mL of ethyl acetate. The resulting suspension was centrifuged, and the supernatant was discarded. The solid was shaken with 10 mL of fresh ethyl acetate and then centrifuged. Washing was repeated five more times. The resulting solid was vacuum-dried. The measured DOL was 0.74. The substance was redissolved in 2 mL of water, and 16 mg of DTT was added to the solution. The mixture was stirred for 5 minutes and loaded onto a G15 SEPHADEX® column. The product was eluted with DE water as a green fluorescent solution and used for conjugation to SMCC-modified streptavidin. The determined concentration was 48 μM (due to dye adsorption).

[0235] Conjugation of thiol-linker-modified aminodextran AF488 scaffold to SMCC-modified streptavidin: SMCC-modified streptavidin (35 μL aqueous solution) was treated with 1, 2, 3, and 4 equivalents of thiol-modified aminodextran AF488 scaffold (48 μM aqueous solution). The reaction was carried out at ambient temperature for 3 hours, after which the reaction mixture was maintained at 4°C overnight. The conjugate was purified using 10 nM PBS buffer with a P100 size exclusion column.

[0236] (Table 26) Streptavidin labeled with 70kD aminodextran AF488 scaffold (average 9.7 molecules of dye per scaffold) JPEG2026071206000031.jpg32170

[0237] (Table 27) Streptoavidin labeled with AF488 dye JPEG2026071206000032.jpg29128

[0238] Results: As shown in Tables 26 and 27, the conjugate made from the scaffold is brighter compared to the conjugate made from a single AF488 dye. Similarly, in contrast to the nearly constant QY for labeling with an amino dextran scaffold, the QY of the AF488 fluorophore decreases from 0.70 to 0.34 for single-dye conjugation.

[0239] The present invention is further presented by the following items.

[0240] 1. A composition comprising a first antibody, wherein two or more fluorescent labels and two or more spacer molecules are covalently bound to the first antibody, and the fluorescent labels and spacer molecules are not covalently bound to each other.

[0241] 2. The composition according to item 1, wherein the first antibody exhibits a higher fluorescence emission level than a second antibody prepared using an equal amount of fluorescent label but not containing spacer molecules.

[0242] 3. The composition according to items 1 and 2, wherein the first antibody exhibits a higher fluorescence emission level than the second antibody, the first antibody and the second antibody each have the same number of covalently bound fluorescent labels, and the second antibody does not have covalently bound spacer molecules.

[0243] 4. The composition according to items 1 to 3, wherein the spacer molecule reduces the quenching of the fluorescent label as compared to the quenching in the absence of the spacer molecule.

[0244] 5. The composition according to items 1 to 4, wherein the spacer molecule is conjugated to the antibody with respect to a reactive group.

[0245] 6. The composition according to item 5, wherein the reactive group is an amine group.

[0246] 7. The composition according to item 6, wherein the amine group is on a lysine residue.

[0247] 8. The composition according to item 1, wherein a fluorescent label is conjugated to an antibody by a conjugation molecule.

[0248] 9. The compositions according to items 1 to 5, wherein the fluorescent label is positively charged.

[0249] 10. The compositions according to items 1 to 5, wherein the fluorescent label is an ALEXA FLUOR® dye or a DYLIGHT® dye.

[0250] 11. Fluorescent labels include ALEXA FLUOR(registered trademark) 350, ALEXA FLUOR(registered trademark) 405, ALEXA FLUOR(registered trademark) 430, ALEXA FLUOR(registered trademark) 488, ALEXA FLUOR(registered trademark) 500, ALEXA FLUOR(registered trademark) 514, ALEXA FLUOR(registered trademark) 532, ALEXA FLUOR(registered trademark) 546, ALEXA FLUOR(registered trademark) 555, ALEXA FLUOR(registered trademark) 568, ALEXA FLUOR(registered trademark) 594, ALEXA FLUOR(registered trademark) 610-X, ALEXA FLUOR(registered trademark) 633, ALEXA FLUOR(registered trademark) 647, ALEXA FLUOR(registered trademark) 660, ALEXA FLUOR(registered trademark) 680, ALEXA FLUOR(registered trademark) 700, ALEXA FLUOR(registered trademark) 750, ALEXA FLUOR(registered trademark) 790, AMCA-X, BODIPY(registered trademark) 630 / 650, BODIPY(registered trademark) 650 / 665, BODIPY(registered trademark) FL, BODIPY(registered trademark) TMR, BODIPY(registered trademark) TR, BODIPY(registered trademark) TR-X, CASCADE BLUE(registered trademark), Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE(registered trademark), OREGON GREEN(registered trademark) 488, OREGON GREEN(registered trademark) 514, PACIFIC BLUE(trademark), PACIFIC ORANGE(trademark), RHODAMINE GREEN(trademark), QSY(registered trademark) 7, QSY(registered trademark) 9, QSY(registered trademark) 21, QSY(registered trademark) 35, ROX, RHODAMINE The compositions described in items 1 to 9, wherein the dye is selected from the group consisting of RED®, TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED®, or 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacrylidine-2-one) succinimidyl ester (DDAO-SE).

[0251] 12. The composition according to item 10, wherein the fluorescent label is a dye selected from the group consisting of DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800.

[0252] 13. The compositions according to items 1 to 12, wherein the spacer molecules are negatively charged or neutral.

[0253] 14. The compositions according to items 1 to 13, wherein the spacer molecule is selected from acetate and polyethylene glycol (PEG).

[0254] 15. The compositions according to items 1 to 12, wherein the spacer molecule contains an acetyl group.

[0255] 16. The compositions according to items 1 to 14, wherein the spacer molecule comprises an acetate molecule.

[0256] 17. The composition according to item 16, wherein the acetate molecule is sulfo-NHS-acetate.

[0257] 18. The composition according to claims 1 to 17, wherein the spacer molecule comprises or consists of (PEG)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0258] 19. The composition according to claims 1 to 17, wherein the spacer molecule comprises MS-(PEG)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0259] 20. Spacer molecules are alkanoyl, alkenoyl, and alkinoyl (-C(O)C n H mThe compositions according to claims 1 to 19, comprising or consisting of groups selected from, wherein n is 1 to 20 atoms, m > n, and carbon atoms can be bonded to one another by single bonds, double bonds, and / or triple bonds.

[0260] 21. Alkyl, alkenyl, and / or alkynyl groups -(OCH2CH2O) x -(CH2) y - Further substitution by OR, where x is 1 to 20, y is 1 to 6, and R is H or C 1~6 The composition described in item 20, wherein the composition is alkyl.

[0261] 22. Alkyl, alkenyl, and / or alkynyl groups are ammonium (-NH3 + ), quaternary ammonium (-NR3 + ) is further substituted with a group, where R is C 1~6 The composition described in item 21, wherein the composition is alkyl.

[0262] 23. One or more fluorescent dyes are ALEXA FLUOR(registered trademark) 350, ALEXA FLUOR(registered trademark) 405, ALEXA FLUOR(registered trademark) 430, ALEXA FLUOR(registered trademark) 488, ALEXA FLUOR(registered trademark) 500, ALEXA FLUOR(registered trademark) 514, ALEXA FLUOR(registered trademark) 532, ALEXA FLUOR(registered trademark) 546, ALEXA FLUOR(registered trademark) 555, ALEXA FLUOR(registered trademark) 568, ALEXA FLUOR(registered trademark) 594, ALEXA FLUOR(registered trademark) 610-X, ALEXA FLUOR(registered trademark) 633, ALEXA FLUOR(registered trademark) 647, ALEXA FLUOR(registered trademark) 660, ALEXA FLUOR(registered trademark) 680, ALEXA FLUOR(registered trademark) 700, ALEXA FLUOR(registered trademark) 750, ALEXA FLUOR(registered trademark) 790, AMCA-X, BODIPY(registered trademark) 630 / 650, BODIPY(registered trademark) 650 / 665, BODIPY(registered trademark) FL, BODIPY(registered trademark) TMR, BODIPY(registered trademark) TR, BODIPY(registered trademark) TR-X, CASCADE BLUE(registered trademark) dinitrophenyl, fluorescein, HEX, JOE, MARINA BLUE(registered trademark), OREGON GREEN(registered trademark) 488, OREGON GREEN(registered trademark) 514, PACIFIC BLUE(trademark), PACIFIC ORANGE(trademark), RHODAMINE GREEN(trademark), QSY(registered trademark) 7, QSY(registered trademark) 9, QSY(registered trademark) 21, QSY(registered trademark) 35, ROX, RHODAMINE RED(trademark), TET, TAMRA, tetramethyl Rhodamine, FAM, TEXAS RED®, or 7-hydroxy-9H-(1,3-dichloro-9,It comprises 9-dimethylacridine-2-one)succinimidyl ester (DDAO-SE), and is derived from the group consisting of DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 80, Spacer molecules, Sulfo-NHS-acetate; (PEG)n (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15); MS-(PEG)n (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15); Alkanoyl, alkenoyl, or alkinoyl (-C(O)C n H m )(wherein n is 1 to 20 atoms, m > n, and carbon atoms can be bonded to each other by single, double, and / or triple bonds); or -(OCH2CH2O) x -(CH2) y -OR(where x is between 1 and 20, y is between 1 and 6, and R is either H or C) 1~6 Alkyl, alkenyl, or alkynyl groups further substituted with alkyl (-NH3), or alkyl, alkenyl, and / or alkynyl groups are ammonium (-NH3 + ), quaternary ammonium ((-NR3 + ) is further substituted with a group, where R is C 1~6 Alkyl, alkenyl, or alkynyl group A composition according to items 1 to 22, comprising one or more of the following.

[0263] 24. Alkyl, alkenyl, and / or alkynyl groups are phosphonium groups (-PQ3 +) is further substituted by, where Q is an aryl, a substituted aryl, or C 1~6 The composition described in item 23, wherein the composition is alkyl.

[0264] 25. The compositions according to items 1 to 24, wherein the ratio of the fluorescent label to the antibody is 1 to 50.

[0265] 26. The composition according to item 25, wherein the ratio of the fluorescent label to the antibody is 5 to 30.

[0266] 27. The composition according to item 25, wherein the ratio of the fluorescent label to the antibody is 1 to 20.

[0267] 28. The compositions according to items 1 to 27, wherein the ratio of spacer molecules to antibody is 1 to 50.

[0268] 29. The composition according to item 28, wherein the ratio of spacer molecules to antibody is 5 to 30.

[0269] 30. The composition according to item 28, wherein the ratio of spacer molecules to antibody is 5 to 30.

[0270] 31. The composition according to item 28, wherein the ratio of spacer molecules to antibody is 1 to 20.

[0271] 32. The compositions according to items 1 to 31, wherein the spacer molecules are in molar excess relative to multiple fluorescent labels by amounts of 0.1 to 25 times, 1 to 15 times, or 2.5 to 10 times.

[0272] 33. The composition according to item 32, wherein the spacer molecule is in a molar excess of 2.5 times the amount relative to the multiple fluorescent labels.

[0273] 34. The composition according to item 33, wherein the spacer molecule is in a molar excess with respect to multiple fluorescent labels at a 5-fold amount.

[0274] 35. The composition according to item 33, wherein the spacer molecule is in a molar excess of 7.5 times the amount relative to the multiple fluorescent labels.

[0275] 36. The composition according to item 33, wherein the spacer molecule is in a molar excess with respect to multiple fluorescent labels at a 10-fold increase.

[0276] 37. The composition according to item 33, wherein the proportion of binding sites on the antibody occupied by multiple fluorescent labels is 1% to 99%.

[0277] 38. The composition according to items 1 to 37, wherein the presence of a spacer molecule increases the detectable fluorescence of the fluorescent label by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

[0278] 39. A method for increasing the fluorescence of fluorescently labeled biomolecules, (a) Conjugate spacer molecules to biomolecules, (b) Conjugating a biomolecule with a fluorescent label, Steps (a) and (b) can be performed simultaneously or in any order. The spacer and the fluorescent label are not conjugated to each other.

[0279] 40. A method for the method of item 39, wherein the spacer molecule reduces the quenching of the fluorescent label compared to the quenching in the absence of the spacer molecule.

[0280] 41. The method according to item 39, wherein a spacer molecule is conjugated to an antibody with respect to a reactive group.

[0281] 42. The method according to item 41, wherein the reactive group is an amine group.

[0282] 43. The method according to item 42, wherein the amine group is located on a lysine residue.

[0283] 44. The method described in sections 39-43, wherein the fluorescent label is positively charged.

[0284] 45. The method according to paragraphs 39-43, wherein the fluorescent dye is selected from ALEXA FLUOR® and DYLIGHT®.

[0285] 46. ​​Fluorescent labels include ALEXA FLUOR(registered trademark) 350, ALEXA FLUOR(registered trademark) 405, ALEXA FLUOR(registered trademark) 430, ALEXA FLUOR(registered trademark) 488, ALEXA FLUOR(registered trademark) 500, ALEXA FLUOR(registered trademark) 514, ALEXA FLUOR(registered trademark) 532, ALEXA FLUOR(registered trademark) 546, ALEXA FLUOR(registered trademark) 555, ALEXA FLUOR(registered trademark) 568, ALEXA FLUOR(registered trademark) 594, ALEXA FLUOR(registered trademark) 610-X, ALEXA FLUOR(registered trademark) 633, ALEXA FLUOR(registered trademark) 647, ALEXA FLUOR(registered trademark) 660, ALEXA FLUOR(registered trademark) 680, ALEXA FLUOR(registered trademark) 700, ALEXA FLUOR(registered trademark) 750, ALEXA FLUOR(registered trademark) 790, AMCA-X, BODIPY(registered trademark) 630 / 650, BODIPY(registered trademark) 650 / 665, BODIPY(registered trademark) FL, BODIPY(registered trademark) TMR, BODIPY(registered trademark) TR, BODIPY(registered trademark) TR-X, CASCADE BLUE(registered trademark), Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE(registered trademark), OREGON GREEN(registered trademark) 488, OREGON GREEN(registered trademark) 514, PACIFIC BLUE(trademark), PACIFIC ORANGE(trademark), RHODAMINE GREEN(trademark), QSY(registered trademark) 7, QSY(registered trademark) 9, QSY(registered trademark) 21, QSY(registered trademark) 35, ROX, RHODAMINE The method according to items 39 to 43, selected from the group consisting of RED®, TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED®, and 7-hydroxy-9H-(l,3-dichloro-9,9-dimethylacrylidine-2-one) succinimidyl ester (DDAO-SE).

[0286] 47. The method according to items 39-43 and 45, wherein the fluorescent label is selected from the group consisting of DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800.

[0287] 48. The method according to sections 39-43, wherein the spacer molecule is negatively charged or neutral.

[0288] 49. The method according to items 39-43, wherein the spacer molecule is selected from acetate and polyethylene glycol (PEG).

[0289] 50. The method according to items 39-48, wherein the spacer molecule contains an acetyl group.

[0290] 51. The method according to sections 39-49, wherein the spacer molecule contains an acetate molecule.

[0291] 52. The method according to item 51, wherein the acetate molecule is sulfo-NHS-acetate.

[0292] 53. The method according to items 39 to 52, wherein the spacer molecule comprises (PEG)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0293] 54. The method according to items 39 to 52, wherein the spacer molecule comprises MS-(PEG)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0294] 55. Spacer molecules are alkanoyl, alkenoyl, and alkinoyl (-C(O)C n H mThe method according to items 39-48, comprising a group selected from ), wherein n is 1 to 20 atoms, m > n, and carbon atoms can be bonded to one another by single bonds, double bonds, and / or triple bonds.

[0295] 56. Alkyl, alkenyl, and / or alkynyl groups -(OCH2CH2O) x -(CH2) y - Further substitution by OR, where x is 1 to 20, y is 1 to 6, and R is H or C 1~6 The method described in items 39-48, wherein the alkyl group is alkyl.

[0296] 57. Alkyl, alkenyl, and / or alkynyl groups are ammonium (-NH3 + ), quaternary ammonium (-NR3 + ) is further substituted with a group, where R is C 1~6 The method described in item 55, wherein the alkyl group is alkyl.

[0297] 58. Alkyl, alkenyl, and / or alkynyl groups are phosphonium groups (-PQ3 + ) is further substituted by, where Q is an aryl, a substituted aryl, or C 1~6 The method described in item 55, wherein the alkyl group is alkyl.

[0298] 59. The method described in sections 39-58, wherein the ratio of the fluorescent label to the antibody is 1-50.

[0299] 60. The method according to item 59, wherein the ratio of the fluorescently labeled substance to the antibody is 5 to 30.

[0300] 61. The method according to item 59, wherein the ratio of the fluorescent label to the antibody is 1 to 20.

[0301] 62. The method according to items 39-61, wherein the ratio of spacer molecules to proteins is 1-50.

[0302] 63. The method according to item 62, wherein the ratio of spacer molecules to proteins is 5 to 30.

[0303] 64. The method according to item 62, wherein the ratio of spacer molecules to proteins is 5 to 30.

[0304] 65. The method according to item 62, wherein the ratio of spacer molecules to proteins is 1 to 20.

[0305] 66. The method according to items 39-65, wherein the spacer molecule is in molar excess with respect to multiple fluorescent labels by an amount of 0.1-25 times, 1-15 times, or 2.5-10 times.

[0306] 67. The method according to item 66, wherein the spacer molecule is in a molar excess of 2.5 times the amount relative to multiple fluorescent labels.

[0307] 68. The method according to item 66, wherein the spacer molecule is in a molar excess for multiple fluorescent labels at a 5-fold amount.

[0308] 69. The method according to item 66, wherein the spacer molecule is in a molar excess of 7.5 times the amount relative to multiple fluorescent labels.

[0309] 70. The method according to item 66, wherein the spacer molecule is in a molar excess for multiple fluorescent labels at a 10-fold increase.

[0310] 71. The method according to items 39-70, wherein the proportion of binding sites on the antibody occupied by multiple fluorescent labels is 1% to 99%.

[0311] 72. The method according to items 39-71, wherein the presence of a spacer molecule increases the detectable fluorescence of the fluorescent label by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

[0312] 73. A method for identifying spacer molecules that can enhance the fluorescence emission of fluorescently labeled biomolecules, (a) Conjugating a spacer molecule to a biomolecule independently of multiple fluorescent labels conjugated to the biomolecule, (b) Test whether the presence of spacer molecules in addition to multiple fluorescent labels conjugated to biomolecules increases the detectable fluorescence of the multiple fluorescent labels, (c) A method comprising identifying a spacer molecule as one that reduces the quenching of a fluorescent label conjugated to a protein when the presence of the spacer molecule increases the detectable fluorescence of the multiple fluorescent labels conjugated to a biomolecule.

[0313] 74. The method according to item 73, wherein the spacer molecule is conjugated to the biomolecule by the first lysine side chain present on the biomolecule.

[0314] 75. The method according to items 73-74, wherein the biomolecule is an antibody or an antibody fragment.

[0315] 76. The method according to items 73-75, wherein multiple fluorescent labels are positively charged.

[0316] 77. The method described in sections 73-76, wherein the spacer molecule is positively charged.

[0317] 78. The method according to items 73-74, wherein multiple fluorescent labels are negatively charged.

[0318] 79. The method described in sections 73-76, wherein the spacer molecule is negatively charged.

[0319] 80. The method according to items 73-79, wherein the multiple fluorescent labels are dyes selected from ALEXA FLUOR® and DYLIGHT® molecules.

[0320] 81. Multiple fluorescent labels are ALEXA FLUOR(registered trademark) 350, ALEXA FLUOR(registered trademark) 405, ALEXA FLUOR(registered trademark) 430, ALEXA FLUOR(registered trademark) 488, ALEXA FLUOR(registered trademark) 500, ALEXA FLUOR(registered trademark) 514, ALEXA FLUOR(registered trademark) 532, ALEXA FLUOR(registered trademark) 546, ALEXA FLUOR(registered trademark) 555, ALEXA FLUOR(registered trademark) 568, ALEXA FLUOR(registered trademark) 594, ALEXA FLUOR(registered trademark) 610-X, ALEXA FLUOR(registered trademark) 633, ALEXA FLUOR(registered trademark) 647, ALEXA FLUOR(registered trademark) 660, ALEXA FLUOR(registered trademark) 680, ALEXA FLUOR(registered trademark) 700, ALEXA FLUOR(registered trademark) 750, ALEXA FLUOR(registered trademark) 790, AMCA-X, BODIPY(registered trademark) 630 / 650, BODIPY(registered trademark) 650 / 665, BODIPY(registered trademark) FL, BODIPY(registered trademark) TMR, BODIPY(registered trademark) TR, BODIPY(registered trademark) TR-X, CASCADE BLUE(registered trademark), Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE(registered trademark), OREGON GREEN(registered trademark) 488, OREGON GREEN(registered trademark) 514, PACIFIC BLUE(trademark), PACIFIC ORANGE(trademark), RHODAMINE GREEN(trademark), QSY(registered trademark) 7, QSY(registered trademark) 9, QSY(registered trademark) 21, QSY(registered trademark) 35, ROX, RHODAMINE The method according to items 73-79, selected from RED®, TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED®, and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacrylidine-2-one) succinimidyl ester (DDAO-SE).

[0321] 82. The method according to paragraphs 73-80, wherein multiple fluorescent markers are selected from DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800.

[0322] 83. The method according to sections 73-82, wherein the spacer molecule is selected from acetate and PEG.

[0323] 84. The method according to sections 73-83, wherein multiple fluorescent labels are conjugated to the antibody by a conjugation molecule.

[0324] 85. The method according to sections 73-84, wherein multiple fluorescent labels are conjugated to the antibody at the first lysine side chain.

[0325] 86. The method according to item 85, wherein a spacer molecule is conjugated to the antibody at the first lysine side chain.

[0326] 87. The method according to items 73-86, wherein multiple fluorescent labels are applied to an antibody, with a dye-to-protein ratio of 1-50.

[0327] 88. The method according to item 87, wherein multiple fluorescent labels are applied to an antibody, with a dye-to-protein ratio of 5 to 30.

[0328] 89. The method according to item 87, wherein multiple fluorescent labels are applied to the antibody, with a dye-to-protein ratio of 1 to 20.

[0329] 90. The method according to items 73-88, wherein the ratio of spacer molecules to proteins is 1-50.

[0330] 91. The method according to item 90, wherein the ratio of spacer molecules to proteins is 5 to 30.

[0331] 92. The method according to item 90, wherein the ratio of spacer molecules to proteins is 5 to 30.

[0332] 93. The method according to item 90, wherein the ratio of spacer molecules to proteins is 1 to 20.

[0333] 94. The method according to items 73-89, wherein the spacer molecule is in molar excess with respect to multiple fluorescent labels by an amount of 0.1-25 times, 1-15 times, or 2.5-10 times.

[0334] 95. The method according to item 94, wherein the spacer molecule is in a molar excess of 2.5 times the amount relative to multiple fluorescent labels.

[0335] 96. The method according to item 94, wherein the spacer molecule is in a molar excess for multiple fluorescent labels at a 5-fold amount.

[0336] 97. The method according to item 94, wherein the spacer molecule is in a molar excess of 7.5 times the amount relative to multiple fluorescent labels.

[0337] 98. The method according to item 94, wherein the spacer molecule is in a molar excess for multiple fluorescent labels at a 10-fold increase.

[0338] 99. The method according to items 73-98, wherein the proportion of binding sites on the antibody occupied by multiple fluorescent labels is 1% to 99%.

[0339] 100. The method according to paragraph 99, wherein the presence of a spacer molecule increases the detectable fluorescence of the fluorescent label by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

[0340] 101. A method for determining the presence of a desired target in a biological sample, (a) Contacting a biological sample with a composition and an antibody, wherein two or more fluorescent labels and two or more spacer molecules are covalently bonded to the antibody, and the fluorescent material and the spacer molecules are not covalently bonded to each other. (b) Detecting fluorescence emitted by multiple fluorescent labels, (c) A method comprising determining the presence of a desired target in a biological sample when fluorescence emitted by multiple fluorescent labels is detected.

[0341] 102. The method according to item 101, wherein the biological sample contains cell lysates.

[0342] 103. The method according to item 101, wherein the biological sample contains intact cells.

[0343] 104. The method according to item 101, wherein the biological sample comprises isolated proteins.

[0344] 105. The method described in item 101, wherein the biological sample contains recombinant protein.

[0345] 106. The method described in sections 101-105, wherein the biological sample is immobilized on a solid support.

[0346] 107. The method according to item 101, wherein the biological sample contains intact cells in a fluid.

[0347] 108. The method described in item 101, wherein the biological sample is a living animal.

[0348] 109. The method described in paragraph 108, wherein the living animal is a mammal.

[0349] 110. A composition comprising a first nucleic acid molecule, wherein two or more fluorescent labels and two or more spacer molecules are covalently bonded to the first nucleic acid molecule, and the fluorescent label and spacer molecules are not covalently bonded to each other.

[0350] 111. The composition according to item 110, wherein the first nucleic acid molecule exhibits a higher fluorescence emission level than the second nucleic acid molecule prepared using an equal amount of fluorescent labeling but without a spacer molecule.

[0351] 112. The composition according to item 110, wherein the first nucleic acid molecule exhibits a higher fluorescence emission level than the second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule each have the same number of covalently bonded fluorescent labels, and the second nucleic acid molecule does not have covalently bonded spacer molecules.

[0352] 113. A conjugated antibody containing an antibody conjugated with multiple fluorescent labels, having the following characteristics: (a) A fluorescence ratio of 0.5 or higher based on one fluorescent label, (b) Conjugate at least four fluorescent labels to the antibody, and / or (c) A conjugated antibody containing one or more of the following, wherein the total fluorescence of the antibody is at least 20 percent greater than the fluorescence of the non-conjugated fluorescent molecule.

[0353] 114. A conjugated antibody as described in item 113, wherein a fluorescent label is conjugated to the antibody by one or more multi-arm polymers.

[0354] 115. A conjugated antibody as described in sections 113-114, wherein a fluorescent label is conjugated to the antibody by a single multi-arm polymer.

[0355] 116. A conjugated antibody according to sections 113 and 114, wherein the antibody is conjugated with 2 to 10 multi-arm polymers that are fluorescently labeled.

[0356] 117. A conjugated antibody as described in item 116, wherein two or more fluorescent labels are conjugated to the antibody.

[0357] 118. The arms of the multi-arm polymer are (a) polyethylene glycol, (b) Polysaccharides, and (c) polypeptide A conjugate antibody as described in items 113-117, comprising a type of chemical substance selected from the group consisting of the following.

[0358] 119. A conjugate antibody as described in sections 113-118, wherein the average brush distance between fluorescent labels is 200-800 angstroms.

[0359] 120. A conjugate antibody as described in sections 113-119, wherein the fluorescent label is detached from the antibody by at least 16 covalent bonds.

[0360] 121. A conjugate antibody as described in item 120, wherein the fluorescent label is detached from the antibody by 16-800 covalent bonds.

[0361] 122. Fluorescent labels include ALEXA FLUOR(registered trademark) 350, ALEXA FLUOR(registered trademark) 405, ALEXA FLUOR(registered trademark) 430, ALEXA FLUOR(registered trademark) 488, ALEXA FLUOR(registered trademark) 500, ALEXA FLUOR(registered trademark) 514, ALEXA FLUOR(registered trademark) 532, ALEXA FLUOR(registered trademark) 546, ALEXA FLUOR(registered trademark) 555, ALEXA FLUOR(registered trademark) 568, ALEXA FLUOR(registered trademark) 594, ALEXA FLUOR(registered trademark) 610-X, ALEXA FLUOR(registered trademark) 633, ALEXA FLUOR(registered trademark) 647, ALEXA FLUOR(registered trademark) 660, ALEXA FLUOR(registered trademark) 680, ALEXA FLUOR(registered trademark) 700, ALEXA FLUOR(registered trademark) 750, ALEXA FLUOR(registered trademark) 790, AMCA-X, BODIPY(registered trademark) 630 / 650, BODIPY(registered trademark) 650 / 665, BODIPY(registered trademark) FL, BODIPY(registered trademark) TMR, BODIPY(registered trademark) TR, BODIPY(registered trademark) TR-X, CASCADE BLUE(registered trademark), Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE(registered trademark), OREGON GREEN(registered trademark) 488, OREGON GREEN(registered trademark) 514, PACIFIC BLUE(trademark), PACIFIC ORANGE(trademark), RHODAMINE GREEN(trademark), QSY(registered trademark) 7, QSY(registered trademark) 9, QSY(registered trademark) 21, QSY(registered trademark) 35, ROX, RHODAMINE RED (trademark), TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED (registered trademark), or 7-hydroxy-9H-(1,3-dichloro-9,A conjugate antibody according to item 120, comprising a dye selected from the group consisting of 9-dimethylacridine-2-one) succinimidyl ester (DDAO-SE), and a fluorescent label from the group consisting of DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800, and PEGylated DYLIGHT® dyes.

[0362] 123. A method for preparing fluorescently labeled biomolecules, (a) Conjugate a reactive group and two or more fluorescent labels to a spacer molecule, thereby forming a fluorescently labeled spacer molecule, (b) Conjugate a fluorescently labeled spacer molecule to a biomolecule, thereby forming a fluorescently labeled biomolecule, A method in which each fluorescent label of a fluorescently labeled biomolecule has a fluorescence ratio of 0.5 or higher based on the number of fluorescent labels.

[0363] 124. The method according to item 123, wherein an average of 1 to 10 fluorescently labeled spacer molecules are conjugated to each biomolecule.

[0364] 125. The method according to item 123, wherein an average of 3 to 10 fluorescently labeled spacer molecules are conjugated to each biomolecule.

[0365] 126. The method according to sections 123-125, wherein the fluorescently labeled spacer molecule is a multi-arm polymer.

[0366] 127. The method according to item 126, wherein the multi-arm polymer is a branched-chain polyethylene glycol molecule.

[0367] 128. The method according to item 126, wherein a multi-arm polymer is conjugated with 4 to 10 fluorescent labels.

[0368] 129. The method according to item 126, wherein the multi-arm polymer has a molecular weight of 4,000 to 80,000 daltons.

[0369] 130. A method for detecting fluorescently labeled biomolecules, (a) Exposing a fluorescently labeled biomolecule to light that excites a fluorescent label conjugated to the biomolecule, (b) including detecting luminescence produced by a fluorescent label conjugated to a biomolecule, Fluorescently labeled biomolecules are conjugated with four or more fluorescent labels. A method in which each fluorescent label of a fluorescently labeled biomolecule has a fluorescence ratio of 0.7 or higher based on the number of fluorescent labels.

[0370] 131. The method according to item 130, wherein the fluorescently labeled biomolecule is an antibody.

Claims

1. A composition comprising a first antibody, A composition in which two or more fluorescent labels and two or more spacer molecules are covalently bound to the first antibody, and the fluorescent labels and spacer molecules are not covalently bound to each other.

2. The composition according to claim 1, wherein the first antibody exhibits a higher fluorescence emission level than the second antibody, which is prepared using an equal amount of fluorescent labeling but does not contain the spacer molecule.

3. The composition according to claim 1, wherein the first antibody exhibits a higher fluorescence emission level than the second antibody, the first antibody and the second antibody each have the same number of covalently bonded fluorescent labels, and the second antibody does not have covalently bonded spacer molecules.

4. The composition according to claim 1, wherein the spacer molecule reduces the quenching of the fluorescent label compared to the quenching in the absence of the spacer molecule.

5. The composition according to claim 1, wherein the spacer molecule is conjugated to the antibody with respect to the reactive group.

6. The composition according to claim 5, wherein the reactive group is an amine group.

7. The composition according to claim 6, wherein the amine group is located on a lysine residue.

8. The composition according to claim 1, wherein the fluorescent label is conjugated to the antibody by a conjugation molecule.

9. The composition according to claim 1, wherein the fluorescent label is positively charged.

10. The composition according to claim 1, wherein the fluorescent label is ALEXA FLUOR® dye or DYLIGHT® dye.

11. The aforementioned fluorescent labels are ALEXA FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 500, ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610-X, ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR® 750, ALEXA FLUOR® 790, AMCA-X, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® FL, BODIPY® TMR, BODIPY® TR, BODIPY® TR-X, CASCADE BLUE®, Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE®, OREGON The composition according to claim 1, wherein the dye is selected from the group consisting of GREEN® 488, OREGON GREEN® 514, PACIFIC BLUE®, PACIFIC ORANGE®, RHODAMINE GREEN®, QSY® 7, QSY® 9, QSY® 21, QSY® 35, ROX, RHODAMINE RED®, TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED®, or 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacrylidine-2-one) succinimidyl ester (DDAO-SE).

12. The composition according to claim 1, wherein the fluorescent label is a dye selected from the group consisting of DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800.

13. The composition according to claim 1, wherein the spacer molecule is negatively charged or neutral.

14. The composition according to claim 1, wherein the spacer molecule is selected from acetate and polyethylene glycol (PEG).

15. The composition according to claim 1, wherein the spacer molecule contains an acetyl group.

16. The composition according to claim 1, wherein the spacer molecule comprises an acetate molecule.

17. The composition according to claim 1, wherein the acetate molecule is sulfo-NHS-acetate.

18. The composition according to claim 1, wherein the spacer molecule comprises or consists of (PEG)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

19. The composition according to claim 1, wherein the spacer molecule comprises MS-(PEG)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

20. The spacer molecule is an alkanoyl, alkenoyl, and alkinoyl (-C(O)C n H m The composition according to claim 1, comprising or consisting of a group selected from ), wherein n is 1 to 20 atoms, m > n, and carbon atoms may be bonded to one another by single bonds, double bonds, and / or triple bonds.

21. The alkyl, alkenyl, and / or alkynyl groups are -(OCH 2 CH 2 O) x - (CH 2 ) y Further substitution by -OR, where x is between 1 and 20, y is between 1 and 6, and R is H or C 1~6 The composition according to claim 1, wherein it is alkyl.

22. The alkyl, alkenyl, and / or alkynyl group is further substituted with an ammonium (—NH 3 + ), quaternary ammonium (—NR 3 + ), where R is C 1~6 alkyl, the composition according to claim 21.

23. The fluorescent dyes include one or more ALEXA FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 500, ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610-X, and ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR® 750, ALEXA FLUOR® 790, AMCA-X, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® FL, BODIPY® TMR, BODIPY® TR, BODIPY® TR-X, CASCADE BLUE®, Ditrophenyl, Fluorescein, HEX, JOE, MARINA BLUE (registered trademark), OREGON GREEN (registered trademark) 488, OREGON GREEN (registered trademark) 514, PACIFIC BLUE (trademark), PACIFIC ORANGE (trademark), RHODAMINE GREEN (trademark), QSY (registered trademark) 7, QSY (registered trademark) 9, QSY (registered trademark) 21, QSY (registered trademark) 35, ROX, RHODAMINE RED (trademark), TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED (registered trademark), or 7-hydroxy-9H-(1,3-dichloro-9,9-Dimethylacridine-2-one) succinimidyl ester (DDAO-SE), and those from the group consisting of DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 80, The spacer molecule, Sulfo-NHS-acetate; (PEG)n (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15); MS-(PEG)n (wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15); Alkanoyl, alkenoyl, or alkinoyl (-C(O)C) n H m ) (wherein n is 1 to 20 atoms, m > n, and carbon atoms can be bonded to each other by single bonds, double bonds, and / or triple bonds); or - (OCH 2 CH 2 O) x - (CH 2 ) y -OR (where x is between 1 and 20, y is between 1 and 6, and R is H or C) 1~6 An alkyl, alkenyl, or alkynyl group further substituted with (which is alkyl), or the alkyl, alkenyl, and / or alkynyl group is ammonium (-NH 3 + ), quaternary ammonium ((-NR 3 + ) is further substituted with a group, where R is C 1~6 Alkyl, alkenyl, or alkynyl group The composition according to claim 1, comprising one or more of the following.

24. The alkyl, alkenyl, and / or alkynyl groups are phosphonium groups (-PQ 3 + ) is further substituted by, where Q is an aryl, a substituted aryl, or C 1~6 The composition according to claim 23, wherein it is alkyl.

25. The composition according to claim 1, wherein the ratio of the fluorescent label to the antibody is 1 to 50.

26. The composition according to claim 25, wherein the ratio of the fluorescently labeled antibody to the antibody is 5 to 30.

27. The composition according to claim 25, wherein the ratio of the fluorescently labeled antibody to the antibody is 1 to 20.

28. The composition according to claim 1, wherein the ratio of the spacer molecule to the antibody is 1 to 50.

29. The composition according to claim 28, wherein the ratio of the spacer molecule to the antibody is 5 to 30.

30. The composition according to claim 28, wherein the ratio of the spacer molecule to the antibody is 5 to 30.

31. The composition according to claim 28, wherein the ratio of the spacer molecule to the antibody is 1 to 20.

32. The composition according to claim 1, wherein the spacer molecule is in a molar excess of the plurality of fluorescent labels in an amount of 0.1 to 25 times, 1 to 15 times, or 2.5 to 10 times.

33. The composition according to claim 32, wherein the spacer molecule is in a molar excess of 2.5 times the amount relative to the plurality of fluorescent labels.

34. The composition according to claim 33, wherein the spacer molecule is in a molar excess of five times the amount relative to the plurality of fluorescent labels.

35. The composition according to claim 33, wherein the spacer molecule is in a molar excess of 7.5 times the amount relative to the plurality of fluorescent labels.

36. The composition according to claim 33, wherein the spacer molecule is in a molar excess of the plurality of fluorescent labels in a 10-fold amount.

37. The composition according to claim 33, wherein the proportion of binding sites on the antibody occupied by the plurality of fluorescent labels is 1% to 99%.

38. The composition according to claim 1, wherein the presence of the spacer molecule increases the detectable fluorescence of the fluorescent label by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

39. A method for increasing the fluorescence of fluorescently labeled biomolecules, (a) Conjugate spacer molecules to biomolecules, (b) Conjugate the biomolecule with a fluorescent label, Steps (a) and (b) can be performed simultaneously or in any order. The spacer and the fluorescent label are not conjugated to each other.

40. The method according to claim 39, wherein the spacer molecule reduces the quenching of the fluorescent label compared to the quenching in the absence of the spacer molecule.

41. The method according to claim 39, wherein the spacer molecule is conjugated to the antibody with respect to the reactive group.

42. The method according to claim 41, wherein the reactive group is an amine group.

43. The method according to claim 42, wherein the amine group is located on a lysine residue.

44. The method according to claim 39, wherein the fluorescent label is positively charged.

45. The method according to claim 39, wherein the fluorescent dye is selected from ALEXA FLUOR® and DYLIGHT®.

46. The aforementioned fluorescent labels are ALEXA FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 500, ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610-X, ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR® 750, ALEXA FLUOR® 790, AMCA-X, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® FL, BODIPY® TMR, BODIPY® TR, BODIPY® TR-X, CASCADE BLUE®, Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE®, OREGON The method according to claim 39, selected from the group consisting of GREEN® 488, OREGON GREEN® 514, PACIFIC BLUE®, PACIFIC ORANGE®, RHODAMINE GREEN®, QSY® 7, QSY® 9, QSY® 21, QSY® 35, ROX, RHODAMINE RED®, TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED®, and 7-hydroxy-9H-(l,3-dichloro-9,9-dimethylacrylidine-2-one) succinimidyl ester (DDAO-SE).

47. The method according to claim 39, wherein the fluorescent label is selected from the group consisting of DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800.

48. The method according to claim 39, wherein the spacer molecule is negatively charged or neutral.

49. The method according to claim 39, wherein the spacer molecule is selected from acetate and polyethylene glycol (PEG).

50. The method according to claim 39, wherein the spacer molecule contains an acetyl group.

51. The method according to claim 39, wherein the spacer molecule includes an acetate molecule.

52. The method according to claim 39, wherein the acetate molecule is sulfo-NHS-acetate.

53. The method according to claim 39, wherein the spacer molecule comprises (PEG)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

54. The method according to claim 39, wherein the spacer molecule comprises MS-(PEG)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

55. The spacer molecule is an alkanoyl, alkenoyl, and alkinoyl (-C(O)C n H m The method according to claim 39, comprising a group selected from, wherein n is 1 to 20 atoms, m > n, and carbon atoms can be bonded to one another by single bonds, double bonds, and / or triple bonds.

56. The alkyl, alkenyl, and / or alkynyl groups are -(OCH 2 CH 2 O) x - (CH 2 ) y Further substitution by -OR, where x is between 1 and 20, y is between 1 and 6, and R is H or C 1~6 The method according to claim 55, wherein the alkyl group is alkyl.

57. The alkyl, alkenyl, and / or alkynyl groups are ammonium ((-NH 3 + ), quaternary ammonium (-NR 3 + ) is further substituted with a group, where R is C 1~6 The method according to claim 55, wherein the alkyl group is alkyl.

58. The alkyl, alkenyl, and / or alkynyl groups are phosphonium groups (-PQ 3 + ) is further substituted by, where Q is an aryl, a substituted aryl, or C 1~6 The method according to claim 55, wherein the alkyl group is alkyl.

59. The method according to claim 39, wherein the ratio of the fluorescent label to the antibody is 1 to 50.

60. The method according to claim 59, wherein the ratio of the fluorescently labeled antibody to the antibody is 5 to 30.

61. The method according to claim 59, wherein the ratio of the fluorescently labeled substance to the antibody is 1 to 20.

62. The method according to claim 39, wherein the ratio of the spacer molecule to the protein is 1 to 50.

63. The method according to claim 62, wherein the ratio of the spacer molecule to the protein is 5 to 30.

64. The method according to claim 62, wherein the ratio of the spacer molecule to the protein is 5 to 30.

65. The method according to claim 62, wherein the ratio of the spacer molecule to the protein is 1 to 20.

66. The method according to claim 39, wherein the spacer molecule is in a molar excess of the multiple fluorescent labels in an amount of 0.1 to 25 times, 1 to 15 times, or 2.5 to 10 times.

67. The method according to claim 66, wherein the spacer molecule is in a molar excess of 2.5 times the amount relative to the plurality of fluorescent labels.

68. The method according to claim 66, wherein the spacer molecule is in a molar excess of the plurality of fluorescent labels in a five-fold amount.

69. The method according to claim 66, wherein the spacer molecule is in a molar excess of 7.5 times the amount relative to the plurality of fluorescent labels.

70. The method according to claim 66, wherein the spacer molecule is in a molar excess of the plurality of fluorescent labels in a 10-fold amount.

71. The method according to claim 39, wherein the proportion of binding sites on the antibody occupied by the plurality of fluorescent labels is 1% to 99%.

72. The method according to claim 39, wherein the presence of the spacer molecule increases the detectable fluorescence of the fluorescent label by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

73. A method for identifying spacer molecules that can enhance the fluorescence emission of fluorescently labeled biomolecules, (a) Conjugating a spacer molecule to a biomolecule independently of multiple fluorescent labels conjugated to the biomolecule, (b) Test whether the presence of the spacer molecule, in addition to the plurality of fluorescent labels conjugated to the biomolecule, increases the detectable fluorescence of the plurality of fluorescent labels, (c) A method comprising identifying the spacer molecule as reducing the quenching of the fluorescent labels conjugated to the protein, in addition to the plurality of fluorescent labels conjugated to the biomolecule, when the presence of the spacer molecule increases the detectable fluorescence of the plurality of fluorescent labels.

74. The method according to claim 73, wherein the spacer molecule is conjugated to the biomolecule by the first lysine side chain present on the biomolecule.

75. The method according to claim 73, wherein the biomolecule is an antibody or an antibody fragment.

76. The method according to claim 73, wherein the plurality of fluorescent labels are positively charged.

77. The method according to claim 73, wherein the spacer molecule is positively charged.

78. The method according to claim 73, wherein the plurality of fluorescent labels are negatively charged.

79. The method according to claim 73, wherein the spacer molecule is negatively charged.

80. The method according to claim 73, wherein the plurality of fluorescent labels are dyes selected from ALEXA FLUOR® and DYLIGHT® molecules.

81. The aforementioned multiple fluorescent labels are ALEXA FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 500, ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610-X, ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR® 750, ALEXA FLUOR® 790, AMCA-X, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® FL, BODIPY® TMR, BODIPY® TR, BODIPY® TR-X, CASCADE BLUE®, Dinitrophenyl, Fluorescein, HEX, JOE, MARINA The method according to claim 73, selected from BLUE®, OREGON GREEN® 488, OREGON GREEN® 514, PACIFIC BLUE®, PACIFIC ORANGE®, RHODAMINE GREEN®, QSY® 7, QSY® 9, QSY® 21, QSY® 35, ROX, RHODAMINE RED®, TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED®, and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacrylidine-2-one) succinimidyl ester (DDAO-SE).

82. The method according to claim 73, wherein the plurality of fluorescent labels are selected from DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800.

83. The method according to claim 73, wherein the spacer molecule is selected from acetate and PEG.

84. The method according to claim 73, wherein the plurality of fluorescent labels are conjugated to the antibody by a conjugation molecule.

85. The method according to claim 73, wherein the plurality of fluorescent labels are conjugated to the antibody at the first lysine side chain.

86. The method according to claim 73, wherein the spacer molecule is conjugated to the antibody at the first lysine side chain.

87. The method according to claim 73, wherein the ratio of the dye to the protein in the plurality of fluorescent labels on the antibody is 1 to 50.

88. The method according to claim 87, wherein the ratio of the dye to the protein in the plurality of fluorescent labels on the antibody is 5 to 30.

89. The method according to claim 87, wherein the ratio of the dye to the protein in the plurality of fluorescent labels for the antibody is 1 to 20.

90. The method according to claim 73, wherein the ratio of the spacer molecule to the protein is 1 to 50.

91. The method according to claim 90, wherein the ratio of the spacer molecule to the protein is 5 to 30.

92. The method according to claim 90, wherein the ratio of the spacer molecule to the protein is 5 to 30.

93. The method according to claim 90, wherein the ratio of the spacer molecule to the protein is 1 to 20.

94. The method according to claim 73, wherein the spacer molecule is in a molar excess of the multiple fluorescent labels in an amount of 0.1 to 25 times, 1 to 15 times, or 2.5 to 10 times.

95. The method according to claim 94, wherein the spacer molecule is in a molar excess of 2.5 times the amount relative to the plurality of fluorescent labels.

96. The method according to claim 94, wherein the spacer molecule is in a molar excess of five times the amount relative to the plurality of fluorescent labels.

97. The method according to claim 94, wherein the spacer molecule is in a molar excess of 7.5 times the amount relative to the plurality of fluorescent labels.

98. The method according to claim 94, wherein the spacer molecule is in a molar excess of the plurality of fluorescent labels in a 10-fold amount.

99. The method according to claim 73, wherein the proportion of binding sites on the antibody occupied by the plurality of fluorescent labels is 1% to 99%.

100. The method according to claim 99, wherein the presence of the spacer molecule increases the detectable fluorescence of the fluorescent label by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, or at least 500%.

101. A method for determining the presence of a desired target in a biological sample, (a) Contacting the biological sample with the composition and antibody, wherein two or more fluorescent labels and two or more spacer molecules are covalently bonded to the antibody, and the fluorescent material and spacer molecules are not covalently bonded to each other. (b) Detecting fluorescence emitted by the plurality of fluorescent labels, (c) A method comprising determining the presence of the desired target in the biological sample when fluorescence emitted by the plurality of fluorescent labels is detected.

102. The method according to claim 101, wherein the biological sample comprises a cell lysate.

103. The method according to claim 101, wherein the biological sample includes intact cells.

104. The method according to claim 101, wherein the biological sample comprises isolated proteins.

105. The method according to claim 101, wherein the biological sample comprises recombinant protein.

106. The method according to claim 101, wherein the biological sample is immobilized on a solid support.

107. The method according to claim 101, wherein the biological sample includes intact cells in a fluid.

108. The method according to claim 101, wherein the biological sample is a living animal.

109. The method according to claim 108, wherein the living animal is a mammal.

110. A composition comprising a first nucleic acid molecule, Two or more fluorescent labels and two or more spacer molecules are covalently bonded to the first nucleic acid molecule. A composition in which the phosphor and spacer molecules are not covalently bonded to each other.

111. The composition according to claim 110, wherein the first nucleic acid molecule exhibits a higher fluorescence emission level than a second nucleic acid molecule prepared using an equal amount of fluorescent labeling but without the spacer molecule.

112. The composition according to claim 110, wherein the first nucleic acid molecule exhibits a higher fluorescence emission level than the second nucleic acid molecule, the first nucleic acid molecule and the second nucleic acid molecule each have the same number of covalently bonded fluorescent labels, and the second nucleic acid molecule does not have covalently bonded spacer molecules.

113. A conjugated antibody comprising an antibody conjugated with multiple fluorescent labels, having the following characteristics: (a) A fluorescence ratio of 0.5 or higher based on one fluorescent label, (b) Conjugate at least four fluorescent labels to the antibody, and / or (c) A conjugated antibody comprising one or more of the following, wherein the total fluorescence of the antibody is at least 20 percent greater than the fluorescence of a non-conjugated fluorescent molecule.

114. The conjugated antibody according to claim 113, wherein the fluorescent label is conjugated to the antibody by one or more multi-arm polymers.

115. The conjugated antibody according to claim 114, wherein the fluorescent label is conjugated to the antibody by a single multi-arm polymer.

116. The conjugated antibody according to claim 114, wherein the fluorescent label is conjugated to the antibody by 2 to 10 multi-arm polymers.

117. The conjugated antibody according to claim 116, wherein two or more fluorescent labels are conjugated to the antibody.

118. The arms of the aforementioned multi-arm polymer are (a) Polyethylene glycol, (b) Polysaccharides, and (c) polypeptide A conjugate antibody according to claim 113, comprising a type of chemical substance selected from the group consisting of the following.

119. The conjugate antibody according to claim 113, wherein the average brush distance between the fluorescent labels is 200 to 800 angstroms.

120. The conjugate antibody according to claim 113, wherein the fluorescent label is detached from the antibody by at least 16 covalent bonds.

121. The conjugate antibody according to claim 120, wherein the fluorescent label is detached from the antibody by 16 to 800 covalent bonds.

122. The aforementioned fluorescent labels are ALEXA FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 500, ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610-X, ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR® 750, ALEXA FLUOR® 790, AMCA-X, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® FL, BODIPY® TMR, BODIPY® TR, BODIPY® TR-X, CASCADE BLUE®, Dinitrophenyl, Fluorescein, HEX, JOE, MARINA BLUE®, OREGON GREEN® 488, OREGON GREEN® 514, PACIFIC BLUE®, PACIFIC ORANGE®, RHODAMINE GREEN®, QSY® 7, QSY® 9, QSY® 21, QSY® 35, ROX, RHODAMINE RED®, TET, TAMRA, Tetramethylrhodamine, FAM, TEXAS RED®, or 7-hydroxy-9H-(1,3-dichloro-9,The conjugate antibody according to claim 120, wherein the label is a dye selected from the group consisting of 9-dimethylacridine-2-one) succinimidyl ester (DDAO-SE), and a fluorescent label from the group consisting of DYLIGHT® 350, DYLIGHT® 405, DYLIGHT® 488, DYLIGHT® 550, DYLIGHT® 594, DYLIGHT® 633, DYLIGHT® 650, DYLIGHT® 680, DYLIGHT® 755, and DYLIGHT® 800, and PEG-modified DYLIGHT® dyes.

123. A method for preparing fluorescently labeled biomolecules, (a) Conjugate a reactive group and two or more fluorescent labels to a spacer molecule, thereby forming a fluorescently labeled spacer molecule, (b) Conjugate the fluorescently labeled spacer molecule to the biomolecule, thereby forming the fluorescently labeled biomolecule, A method wherein each fluorescent label of the fluorescently labeled biomolecule has a fluorescence ratio of 0.5 or more based on one fluorescent label.

124. The method according to claim 123, wherein an average of 1 to 10 fluorescently labeled spacer molecules are conjugated to each biomolecule.

125. The method according to claim 123, wherein an average of 3 to 10 fluorescently labeled spacer molecules are conjugated to each biomolecule.

126. The method according to claim 123, wherein the fluorescently labeled spacer molecule is a multi-arm polymer.

127. The method according to claim 126, wherein the multi-arm polymer is a branched-chain polyethylene glycol molecule.

128. The method according to claim 126, wherein the multi-arm polymer is conjugated with 4 to 10 fluorescent labels.

129. The method according to claim 126, wherein the multi-arm polymer has a molecular weight of 4,000 to 80,000 Daltons.

130. A method for detecting fluorescently labeled biomolecules, (a) Exposing the fluorescently labeled biomolecule to light that excites the fluorescent label conjugated to the biomolecule, (b) detecting the light emitted by the fluorescent label conjugated to the biomolecule, The fluorescently labeled biomolecule is conjugated with four or more fluorescent labels. A method wherein each fluorescent label of the fluorescently labeled biomolecule has a fluorescence ratio of 0.7 or more based on one fluorescent label.

131. The method according to claim 130, wherein the fluorescently labeled biomolecule is an antibody.

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