Composition and method for enhanced fluorescence

Abstract

To provide a fluorescent label that exhibits enhanced signal intensity and a conjugation method.SOLUTION: The present disclosure relates to a field of fluorescent dyes, in particular, to a composition and a method for increasing a fluorescent signal and reducing fluorescence quenching.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present disclosure relates to the field of fluorescent dyes, and in particular to compositions and methods for increasing fluorescent signal and reducing fluorescence quenching. [Background technology]

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

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

[0004] Furthermore, enhanced fluorescence is seen even when fewer dye molecules are attached (covalently or non-covalently) to each biomolecule. This effect is believed to be related to the attachment of spacer groups to the biomolecules that exhibit enhanced fluorescence. Without being bound by theory, it is believed that the enhanced fluorescence results from reduced 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), in which two or more fluorescent labels and two or more spacer molecules are covalently attached to the first biomolecule, and the fluorophore and spacer molecules are not covalently attached 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 label but without a spacer. In a further example, the first biomolecule exhibits a higher fluorescence emission level than the second biomolecule, in which the first biomolecule and the second biomolecule each have the same number of covalently attached fluorescent labels, and the second biomolecule does not have a covalently attached spacer. In certain embodiments, the spacer molecule reduces quenching of the fluorescent label compared to quenching in the absence of the spacer.

[0005] In some embodiments of the present invention, the spacer molecule is conjugated to the biomolecule via a reactive group. Such reactive group can be an amine reactive group (e.g., NHS ester, one or more amine groups can be at the amine terminus of the polypeptide and / or lysine side chain), a sulfhydryl group, a carboxylic acid group, etc. Additional groups to which the spacer molecule can be conjugated include cysteine ​​residues, aspartic acid residues, glutamic acid residues, and / or the carboxy terminus of the polypeptide. In addition, fluorescent labels can be conjugated to the biomolecule by a conjugation arm. These fluorescent labels can be positively charged, neutral, and / or negatively charged.

[0006] In some embodiments, fluorescent labels used in the practice of the present invention (both compositions and methods) may be cyanine, benzorhodamine, bodipy, fluorescein, benzopyrylium derivatives, and may further include ALEXA FLUOR® dyes and / or DYLIGHT™ dyes. Such dyes include 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® 760, ALEXA FLUOR® 780, ALEXA FLUOR® 790, ALEXA FLUOR® 800, ALEXA FLUOR® 810, ALEXA FLUOR® 820, ALEXA FLUOR® 830, ALEXA FLUOR® 840, ALEXA FLUOR® 850, ALEXA FLUOR® 860, ALEXA FLUOR® 870, ALEXA FLUOR® 880, ALEXA FLUOR® 890, ALEXA FLUOR® 900, ALEXA FLUOR® 950, ALEXA FLUOR® 960, ALEXA FLUOR® 970, ALEXA FLUOR® 980, ALEXA FLUOR® 990, ALEXA FLUOR® 1000, ALEXA FLUOR® 1010, ALEXA FLUOR® 1020, ALEXA FLUOR® 1030, ALEXA FLUOR® 1 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®, 7-hydroxy-9H-(1,3-dichloro-9,9-Dimethylacridin-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. Of course, other dyes and modified forms of the above dyes can be used in the practice of the invention.

[0007] In the practice of the present invention, the spacer may be negatively 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 comprise 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 may comprise 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] Additionally, the spacers used in the practice of the present invention include alkanoyl, alkenoyl, and 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, double, and / or triple bonds. The alkyl, alkenyl, and / or alkynyl groups can be selected from -(OCH2CH2O) x -(CH2) y -OR, where x is 1 to 20, y is 1 to 6, and R is H or C. 1~6Additionally, the alkyl, alkenyl, and / or alkynyl groups may be substituted with ammonium (-NH3 + ), quaternary ammonium (-NR3 + ) groups, where R is C 1~6 It is an alkyl.

[0009] Furthermore, in certain embodiments of the invention, the fluorescent dye may be one or more of 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, tetramethyl may include rhodamine, FAM, TEXAS RED®, or 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE), where the spacer is sulfo-NHS-acetate; (PEG)n, where 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 ) (wherein n is 1 to 20 atoms, m>n, and the carbon atoms may be bonded to each other by single, double, and / or triple bonds); or -(OCH2CH2O) x -(CH2) y -OR (wherein x is 1 to 20, y is 1 to 6, and R is H or C 1~6 an alkyl, alkenyl, or alkynyl group further substituted with ammonium (-NH3 + ), quaternary ammonium ((-NR3 + ) groups, wherein R is C 1~6 In addition, the alkyl, alkenyl, and / or alkynyl groups may include one or more of the following: Q is an aryl, substituted aryl, or C 1~6 The phosphonium group (-PQ3 + ) may be further substituted.

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

[0011] Additionally, compositions and methods of the invention can include or utilize fluorescently labeled biomolecules (e.g., antibodies) where the spacer agent is in molar excess over the plurality of fluorescent labels in an amount of 0.1-25x, 1-15x, or 2.5-10x; where the spacer agent is in molar excess over the plurality of fluorescent labels in an amount of 2.5x; where the spacer agent is in molar excess over the plurality of fluorescent labels in an amount of 5x; where the spacer agent is in molar excess over the plurality of fluorescent labels in an amount of 7.5x; or where the spacer agent is in molar excess over the plurality of fluorescent labels in an amount of 10x.

[0012] Additionally, compositions and methods of the invention can include or utilize fluorescently labeled biomolecules (e.g., antibodies) where the percentage of binding sites (e.g., accessible amine groups) on the biomolecule that are occupied by multiple fluorescent labels is between 1% and 99%.

[0013] In some embodiments, the presence of the 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, including those that include (a) conjugating a spacer molecule to the biomolecule and (b) conjugating a fluorescent label to the biomolecule, where steps (a) and (b) can be performed simultaneously or in any order, and where the spacer and the fluorescent label are not conjugated to each other. Furthermore, the spacer molecule can 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 a spacer molecule capable of enhancing the fluorescence emission of a fluorescently labeled biomolecule. Such a method may include (a) conjugating a spacer molecule to a biomolecule independently of the multiple fluorescent labels conjugated to the biomolecule, (b) testing whether the presence of the spacer agent in addition to the multiple fluorescent labels conjugated to the biomolecule increases the detectable fluorescence of the multiple fluorescent labels, and (c) identifying the spacer agent as one that reduces quenching of the fluorescent label conjugated to the protein when the presence of the spacer agent in addition to the multiple fluorescent labels conjugated to the biomolecule increases the detectable fluorescence of the multiple 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. Also, the multiple 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 a biological sample with an antibody of a composition, where two or more fluorescent labels and two or more spacer molecules are covalently attached to the antibody, and the fluorescent molecules and the spacer molecules are not covalently attached to each other; (b) detecting the fluorescence emitted by the multiple fluorescent labels; and (c) determining the presence of the desired target in the biological sample when the fluorescence emitted by the multiple fluorescent labels is detected. The biological sample used in the practice of the present invention may include cell lysates, intact cells (e.g., intact cells in a fluid such as a bodily fluid), isolated proteins, and / or recombinant proteins. Additionally, the biological sample may be immobilized on a solid support. Additionally, the biological sample includes 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 attached to the first nucleic acid molecule, and the fluorophores and spacer molecules are not covalently attached to each other. In some embodiments, the first nucleic acid molecule exhibits a higher level of fluorescence emission than a second nucleic acid molecule prepared with an equal amount of fluorescent labels but without a spacer. Furthermore, in some embodiments, the first nucleic acid molecule may exhibit a higher level of fluorescence emission than the second nucleic acid molecule, wherein the first nucleic acid molecule and the second nucleic acid molecule each have the same number of covalently attached fluorescent labels, and the second nucleic acid molecule does not have a covalently attached spacer.

[0018] The present invention also includes conjugated antibodies, each comprising an antibody conjugated to a plurality of fluorescent labels (e.g., 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 greater based on a single fluorescent label; (b) conjugating 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 unconjugated fluorescent molecule; and / or (d) an average of about 3 to about 80 fluorescent labels bound to each antibody molecule.

[0019] Furthermore, the fluorescent labels can be conjugated to a biomolecule (e.g., an antibody) by one or more (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.) multi-arm polymers. Furthermore, the arms of the multi-arm polymer can be composed of a type of chemical selected from the group consisting of (a) polyethylene glycol, (b) polysaccharides, and (c) polypeptides, as well as other substances. Furthermore, the average brush distance between the fluorescent labels can 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.). In addition, a fluorescent label conjugated to an antibody (or other biomolecule) can be separated from the antibody (or other biomolecule) by at least 16 (e.g., about 16 to about 800, about 25 to about 800, about 40 to about 800, about 60 to about 800, about 100 to about 800, about 200 to about 800, about 250 to about 800, about 150 to about 600, etc.) covalent bonds. Additionally, the conjugated antibody (or other biomolecule) may be used in combination with any of the following: 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, ALEXAFLUOR® 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 The fluorescent label may be one or more dyes selected from the group consisting of RED®, or 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE), as well as 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 of preparing a fluorescently labeled biomolecule, which may include (a) conjugating a reactive group and two or more fluorescent labels to a spacer molecule, thereby forming a fluorescently labeled spacer molecule, and (b) conjugating the fluorescently labeled spacer molecule to a biomolecule, thereby forming a fluorescently labeled biomolecule, wherein each fluorescent label of the fluorescently labeled biomolecule has a fluorescence ratio of 0.5 or greater on a per fluorescent label basis. Additionally, 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.) fluorescently labeled spacer molecules can be conjugated to each biomolecule. Additionally, the fluorescently labeled spacer molecule can be a multi-arm polymer (e.g., a branched polyethylene glycol molecule). Further, the spacer molecules (e.g., multi-arm polymers) may each be conjugated to an average of 4 to 20 (e.g., about 4 to about 10, about 3 to about 8, about 4 to about 8, about 3 to about 9, etc.) fluorescent labels. Further, the spacer molecules (e.g., multi-arm polymers) may have a molecular weight of 4,000 to 80,000 daltons (e.g., about 4,000 to about 70,000, about 4,000 to about 60,000, about 4,000 to about 50,000, about 4,000 to about 40,000, about 10,000 to about 70,000, about 15,000 to about 60,000, etc.).

[0021] The present invention further includes a method of detecting a fluorescently labeled biomolecule. Such a method may include (a) exposing a fluorescently labeled biomolecule (e.g., an antibody) to light that excites the fluorescent label conjugated to the biomolecule, and (2) detecting 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, the individual fluorescent labels of a fluorescently labeled biomolecule may have a fluorescence ratio of 0.7 or more (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.) on a per fluorescent label basis. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows a schematic diagram of one embodiment of the present invention. [Diagram 2] FIG. 2 shows a schematic diagram of some embodiments of the present invention. [Diagram 3] FIG. 3 shows the results of software analysis of dot blots photographed using an imaging device. [Figure 4] FIG. 4 shows the results of software analysis of dot blots photographed using an imaging device. [Diagram 5] FIG. 5 shows the results of software analysis of dot blots photographed using the imaging device. [Figure 6] FIG. 6 shows the results of software analysis of dot blots photographed using the imaging device. [Figure 7] FIG. 7 demonstrates the effect of addition of NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, 10×) on the detectable fluorescence levels of GAM-DYLIGHT™ 800-4×PEG (abbreviated as “DYLIGHT™ 800” in Table 3) in a fluorescent dot blot assay. [Figure 8] FIG. 8 shows a Western blot assay demonstrating the effect of addition of NHS acetate (2.5×, 5×, and 10× molar excess) or MS(PEG)4 (3.75× molar excess) on the fluorescence detection levels of GAR-DYLIGHT™ 488 conjugated to antibody at molar excesses of the dye ranging from 5× to 20×. [Figure 9] FIG. 9 shows the effect of NHS acetate (5×) or MS(PEG)4 (5×) on GAM-DYLIGHT™ 650-4×PEG-GAR (at 7.5× molar excess of dye) on a Western blot. [Figure 10]FIG. 10 shows the results of a Western blot assay examining the effect of NHS acetate (2.5×, 5×) or MS(PEG)4 on the detectable fluorescence levels of GAR-DYLIGHT™ 800-4×PEG (abbreviated as “DYLIGHT™ 800” in Table 4). [Figure 11] FIG. 11 shows the effect of adding NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (5×) and MS(PEG)8 (5×) to GAM-DYLIGHT™ 550-2×PEG (at 12.5× molar excess of dye) on fluorescent Western blot and dot blot assays. [Figure 12] FIG. 12 shows the effect of NHS acetate (2.5×, 5×) or MS(PEG)4 (5×) on GAM-DYLIGHT™ 680-4×PEG-GAR (dye at 10-fold molar excess) in Western blot and dot blot assays. [Figure 13A] FIG. 13A shows IFC with DYLIGHT™ 488-GAM 4 μg / ml. [Figure 13B] FIG. 13B shows a similar experiment in which A549 cells were stained with pH2A×primary antibody. [Figure 14] FIG. 14 shows IFC detection of PDI with DYLIGHT™ 550-2×PEG (7.5× to 20×) 4 μg / ml. [Figure 15] FIG. 15 shows IFC detection of PDI with DYLIGHT™ 650-4×PEG (7.5× to 20×) 4 μg / ml. [Figure 16] FIG. 16 shows the fold improvement with IFC-DYLIGHT™ 680-4×PEG, NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, 10×) over the base conjugate at a molar excess of each. [Figure 17] FIG. 17 illustrates the fluorescence levels observed at various dye / protein molar ratios of TAMRA-goat anti-mouse antibody (GAM) conjugates with and without betaine. [Figure 18]FIG. 18 shows a schematic diagram of the generation and attachment of branched PEG molecules containing fluorescent labels to antibody molecules. [Figure 19] FIG. 19 shows an example of a single-arm connector (such as amylose) and a multi-arm connector (such as dextran). [Figure 20] FIG. 20 shows examples of various types of star polymers, some of the components of star polymers, and examples of methods for producing star polymers. [Figure 21] FIG. 21 shows exemplary polylysine molecules of the type that can be used in the practice of the present invention. [Figure 22] FIG. 22 shows results for the SK3 mouse anti-human CD4 antibody conjugated to the AF647 branched PEG construct. [Diagram 23] FIG. 23 shows results for the SK3 mouse anti-human CD4 antibody conjugated to the AF647 branched PEG construct.

[0023] (Figure 1) Schematic diagram of one embodiment of the present invention. In this embodiment, an antibody is conjugated with an NHS fluorescent dye and a sulfo-NHS-acetate spacer at two different dye molar excesses in 50 mM borate buffer (pH 8.5). The conjugation of the dye and spacer results in enhanced sensitivity and reduced quenching. (FIG. 2) Schematic diagram of some embodiments of the present invention. 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 It is. (Figure 3) shows the results of software analysis of dot blots photographed with an imaging instrument. The dot blots were tested with GAM antibodies co-labeled with NHS acetate or MS(PEG)4 spacers and DYLIGHT™ 488 fluorescent dye (abbreviated "DYLIGHT™ 488" in Table 1). DYLIGHT™ 488-GAM conjugates made with NHS acetate or MS(PEG)4 resulted in a 1.2- to 1.8-fold improvement in fluorescence intensity over the base conjugate (made without a spacer). The lanes in this figure are as follows: (Table 1) JPEG2025028883000002.jpg169156 (Figure 4) Dot blot - DYLIGHT™ 488-GAM detection of mouse IgG, fold improvement of NHS acetate (2.5x, 5x) or MS(PEG)4 (3.75x) over the base conjugate at various molar excesses of dye. This figure shows the results of software analysis of dot blots taken with an imaging instrument. The dot blots were tested by assay using GAM antibody co-labeled with NHS acetate or MS(PEG)4 spacer and DYLIGHT™ 488 fluorescent dye. These data confirmed the results from Figure 3 using another source of antibody. Both NHS acetate and MS(PEG)4 spacer provided a significant improvement in fluorescent signal intensity (approximately 2.6-fold increase) over the antibody-dye conjugate alone. (FIG. 5) Dot blot - Detection of mouse IgG with DYLIGHT™ 550-2×PEG-GAR. Fold improvement in signal / background from NHS acetate (2.5×, 5×) or MS(PEG)4 (3.75×) over base conjugate at various molar excesses. This shows the results of software analysis of dot blots taken with an imaging instrument. 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 1 / 5000 of the 1 mg / ml stock. Adding NHS acetate or MS(PEG)4 to the conjugation mixture resulted in an improvement in signal intensity (an increase of about 1.6) compared to the base conjugate at a molar excess of each respective dye. The improvement ranged from 1.2-1.6-fold, with the following in particular showing an improvement of over 1.3-fold over the respective base conjugates: GAM-DYLIGHT™ 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. (Figure 6) Dot blot - DyLight650-4xPEG-GAM detection of mouse IgG, improvement factor with NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x, 5x, 10x) over the base conjugate at molar excess of each dye. This figure shows the results of software analysis of dot blots taken with an imaging instrument. Dot blots were tested with GAM antibody co-labeled with NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x) and DYLIGHT™ 650-4xPEG (abbreviated as "DYLIGHT™ 650" in Table 2) (10x to 20x). Mouse IgG was serially diluted 1:1 from 1000 ng / dot. All DYLIGHT™ 650-4xPEG-GAR secondary antibodies were diluted 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 blots and Western blotting. Both NHS acetate and (MS)PEG4 provide significant improvements in sensitivity and signal / background (an increase of about 2.2) over the initial base conjugate. NHS acetate added at 2.5x molar excess to GAM-DYLIGHT™ 650-4xPEG-15x improved intensity by 1.7x. The improvement provided by NHS acetate represented a 1.3x better performance than with the conjugate prepared at the highest molar excess (20x). All MS(PEG)4 appended to the GAM-DYLIGHT™ 650-4xPEG-15x conjugation improved fluorescence intensity by 1.8-2.2 fold, performing better than the corresponding highest base conjugate GAM-DYLIGHT™ 650-4xPEG-20x. The lanes in this figure are as follows: (Table 2) JPEG2025028883000003.jpg169128 (Figure 7) Dot blot - fold improvement of DYLIGHT™ 880-4xPEG conjugate with NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x, 5x, 10x) over the base conjugate at molar excess of each. This figure demonstrates the effect of addition of NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x, 5x, 10x) on the detectable fluorescence level of GAM-DYLIGHT™ 800-4xPEG (abbreviated as "DYLIGHT™ 800" in Table 3) in a fluorescent dot blot assay. Mouse IgG was serially diluted 1:2 from 1000 ng / dot. All DYLIGHT™ 800-4xPEG-GAR secondary antibodies were diluted 1 / 20,000 of the 1 mg / ml stock. This dot blot application shows that the addition of MS(PEG)4 (3.75x and 5x) and NHS acetate (5x) significantly enhanced the fluorescence intensity and sensitivity of the base DYLIGHT™ 800-4xPEG conjugate by 1.5-6 fold. The lanes in this figure are as follows: (Table 3) JPEG2025028883000004.jpg169134 (Figure 8) Western blot assay showing the effect of addition of NHS acetate (2.5x, 5x, and 10x molar excess) or MS(PEG)4 (3.75x molar excess) on the fluorescence detection level of GAR-DYLIGHT™ 488 conjugated to antibody at molar excess of dye ranging from 5x to 20x. A431 cell lysates were diluted 3-fold from 1 μg / well. Rabbit primary antibodies used were anti-Hsp90 diluted 1 / 5000 from 1 mg / ml and anti-cyclophilin B diluted 1 / 5000 from 1 mg / ml. All DYLIGHT™ secondary antibodies were diluted 1 / 5000 from 1 mg / ml stocks. These results show that in Western blot applications, there is a significant increase in fluorescence intensity over the base conjugate (made without a spacer) for DYLIGHT™ 488-GAR conjugated with NHS acetate or MS(PEG)4 addition at 7.5× to 20× molar excess of each dye. (FIG. 9) Effect of NHS acetate (5×) or MS(PEG)4 (5×) on GAM-DYLIGHT™ 650-4×PEG-GAR (at 7.5× molar excess of dye) on Western blot. HeLa cell lysates were diluted 4-fold from 0.5 μg / well. Primary antibody mouse anti-PDI was diluted 1 / 5000 of 1 mg / ml. All DYLIGHT™ secondary antibodies were diluted 1 / 5000 of 1 mg / ml stock. NHS acetate added at 5× molar excess to GAM-DYLIGHT™ 650-4×PEG-7.5× conjugate improved intensity by 1.5-fold. NHS acetate added at 3.75× molar excess to GAM-DYLIGHT™ 650-4×PEG-7.5× conjugate improved intensity by 1.4-fold. (FIG. 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 levels 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-cyclophilin B were both diluted 1 / 5000. All DYLIGHT™ secondary antibodies were diluted 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%, at different molar excesses of the dye. The lanes in this figure are as follows: (Table 4) JPEG2025028883000005.jpg80141 (FIG. 11) Shows the effect of adding NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (5×) and MS(PEG)8 (5×) to GAM-DYLIGHT™ 550-2×PEG (at 12.5× molar excess of dye) on fluorescent 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 1 / 5000 of 1 mg / ml. All DYLIGHT™ secondary antibodies were diluted 1 / 5000 of 1 mg / ml stock. Western blotting and dot blot assays showed that the addition of MS(PEG)4 (5x) and NHS acetate (2.5x and 5x) significantly enhanced the fluorescence intensity and sensitivity of the DYLIGHT™ 550-2xPEG conjugate by at least 2-fold. Conjugates prepared with the long chain MS(PEG)8 did not show any significant improvement over the base conjugate. (FIG. 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 at 10-fold molar excess). For Western blots, HeLa cell lysates were diluted 4-fold from 0.5 μg / well and anti-PDI primary antibodies were diluted 1 / 5000 of 1 mg / ml. For dot blots, mouse IgG was serially diluted 1:2 from 1000 ng / dot. All DYLIGHT™ 680-4×PEG-GAR secondary antibodies were diluted 1 / 20000 of 1 mg / ml stock. Both Western blotting and dot blot assays showed that the addition of MS(PEG)4 (5×) and NHS acetate (2.5× and 5×) significantly enhanced the fluorescence intensity and sensitivity of the DYLIGHT™ 680-4×PEG conjugate by 3-4 fold. (FIG. 13A) (IFC with DYLIGHT™ 488-GAM 4 μg / ml. Fold improvement in signal / background from NHS acetate (2.5×, 5×) or MS(PEG)4 (3.75×) over base conjugate at various molar excesses, and FIG. 13B (IFC-DYLIGHT™ 488-GAR 4 μg / ml. Fold improvement in signal / background from NHS acetate (2.5×, 5×) or MS(PEG)4 (3.75×) over base conjugate at various molar excesses) show the effect of NHS acetate (2.5× and 5×) on the fluorescence of GAM-DYLIGHT™ 488 (13A) and GAR-DYLIGHT™ 488 (13B) at 7.5× to 20× molar excess of the dye in a cell imaging assay. Figure 13A: A549 cells were stained with pH2Ax primary antibodies diluted 1 / 1000 of 1 mg / ml stock. All DYLIGHT™ 488 secondary antibodies were diluted 1 / 250 of 1 mg / ml stock. NHS acetate modified conjugates provided improvements in signal / background in the range of 1.4-1.5 fold (GAM) and 1.1-1.6 fold (GAR) compared to base conjugates at 15x dye molar excess. For GAM conjugates, the most significant improvements were observed with NHS acetate at 5x and MS(PEG)4 at 3.75x, and for GAR conjugates, more significant improvements were observed with NHS acetate at 2.5x and MS(PEG)4 at 3.75x. (Figure 13B) Figure 13B shows a similar experiment in which A549 cells were stained with pH2A x primary antibody. (FIG. 14) IFC detection of PDI with DYLIGHT™ 550-2×PEG (7.5×-20×) 4 μg / ml. Fold improvement from addition of NHS acetate (2.5×, 5×, and 10×) or MS(PEG)4 (3.75×, 5×, and 10×) to the base conjugate at molar excess of each dye. This figure shows the effect of 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 (at 7.5×-20× molar excess of dye) in a fluorescent cell imaging assay. U2OS cells were stained with anti-PDI primary antibody diluted 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 1 / 250 of a 1 mg / ml stock. In this cell imaging application, the addition of 5×NHS acetate provided approximately a 50% improvement over the base conjugate (made without addition) for the DYLIGHT™ 550-2×PEG GAM conjugate at 12.5× dye molar excess, and the addition of 3.75×MS(PEG)4 provided approximately a 50% improvement over the base conjugate at 20× dye molar excess. The lanes in this figure are as follows: (Table 5) JPEG2025028883000006.jpg169134 (Figure 15) IFC detection of PDI with DYLIGHT™ 650-4xPEG (7.5x-20x) 4 μg / ml. Fold improvement from addition of NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x, 5x, 10x) to the base conjugate at molar excess of each dye. This figure shows the effect of addition of NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x, 5x, 10x) to GAM-DYLIGHT™ 650-4xPEG in a fluorescent cell imaging assay. U2OS cells were stained with anti-PDI primary antibody diluted 1 / 100 of the 1 mg / ml stock. All DYLIGHT™ 650-4xPEG-GAM secondary antibodies (abbreviated as "DYLIGHT™ 650" in Table 6) were diluted 1 / 250 of a 1 mg / ml stock. In this application, the addition of NHS acetate-5x resulted in approximately 70% improvement over the base conjugate (made without addition) for the DYLIGHT™ 650-4xPEG GAM conjugate at a 20x molar excess, and the addition of MS(PEG)4-3.75x at a 20-fold molar excess showed approximately 90% improvement over the base conjugate. The lanes in this figure are as follows: (Table 6) JPEG2025028883000007.jpg169132 (Figure 16) Fold improvement with NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x, 5x, 10x) over the base conjugate IFC-DYLIGHT™ 680-4xPEG, each at molar excess. This figure shows the effect of adding NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.3.75x, 5x, 10x) to GAM-DYLIGHT™ 680-4xPEG) in a cell imaging assay. U2OS cells were stained with mouse anti-PDI primary antibody diluted 1 / 100 of a 1 mg / ml stock. All DYLIGHT™ 680-4xPEG-GAM secondary antibodies (abbreviated as "DYLIGHT™ 680" in Table 7) were diluted 1 / 250 of the 1 mg / ml stock, and in this cell imaging application, the addition of NHS acetate-5x resulted in approximately 70% improvement of the dye conjugates at both 7.5x and 10x compared to the base conjugate of DYLIGHT™ 680-4xPEG (made without addition). The GAM conjugate at 15x molar excess and MS(PEG)4-3.75x showed approximately 80% improvement over the base conjugate at 15x molar excess. The lanes in this figure are as follows: (Table 7) JPEG2025028883000008.jpg169132 (Figure 17) TAMRA-GAM co-conjugates 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. Levels of betaine were tested at 2.5, 5, and 10x molar excess compared to the molar number of antibody. These antibodies labeled with NHS-rhodamine (TAMRA) and conjugated with various NHS-betaine concentrations (2.5x betaine, 5x betaine, 10x betaine molar excess of dye) showed an increase in total fluorescence when the antibody was conjugated with betaine as a spacer agent. (Figure 18) Schematic diagram of the generation of branched PEG molecules containing fluorescent labels and their conjugation to antibody molecules. In step 1, a reactive group and a fluorescent label are attached to an NH2 group on the branched PEG molecule. In step 2, an attachment site is added to the antibody molecule. In step 3, the fluorescently labeled branched PEG molecule is covalently attached to the antibody molecule. FIG. 19 shows an example of a single-arm connector (such as amylose) and a multi-arm connector (such as dextran). BM represents the biomolecule and AP represents the attachment point, which means that the fluorescent label is attached to that point. (Figure 20) Examples of various types of star polymers and some star polymer components and methods for manufacturing star polymers. Figure 20A is a diagram of a star polymer with a core (black circle) and multiple arms (black lines). The star represents a fluorescent label covalently attached to the arm. Some arms are not fluorescently labeled, indicating that the labeling was not completed. Figure 20B is a diagram of a similar star polymer in which 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 the core (black semicircle) with reactive groups (gray bars) that can be used to attach other chemicals or act as initiators for polymerization. The core is represented in the center with an adaptor (solid black line) attached to the reactive group. The polymer arms are shown on the right (dashed black line) attached to the adaptor and labeled with a fluorescent molecule (star). (FIG. 21) Exemplary polylysine molecules of the type that can be used in the practice of the present invention are shown. The R1 and R2 groups are shown without labels. These groups can be used as attachment points for fluorescent labels and for conjugation to biomolecules (e.g., antibodies). (FIG. 22) SK3 mouse anti-human CD4 antibody conjugated with AF647 branched PEG construct. SK3 mouse anti-human CD4 antibody (5.5 mg / mL) was modified with ALEXA FLUOR® 647 succinimidyl ester at 10-fold molar excess of ester over antibody (dashed line). SK3 tagged with 10-fold excess of azido-SE over antibody was click conjugated to 100 μM AF647-HG20K8 PEG-sDIBO with 1 mg / mL azido-SK3 antibody for 20 hours at 25° C., quenched with 5 mM NaN3, and the conjugate was purified with Millipore AMICON™ Ultra-2 100 kDa centrifugal filters (dotted line). SK3 tagged with azide-SE in 20-fold excess over antibody was click-conjugated to 600 μM AF647-HG20K8 PEG-sDIBO with 3 mg / mL azide-SK3 antibody for 3 h at 37 °C, quenched with 5 mM NaN3, and the conjugate was purified with Millipore AMICON™ Ultra-2 100 kDa centrifugal filters (solid line). One million Ficoll-isolated PBMCs / well in a 96-well plate were stained with SK3 conjugate using a 7-point titration of 1 μg to 0.015 μg of antibody. Analysis of stained cells was performed using an ATTUNE™ NxT flow cytometer. (FIG. 23) SK3 mouse anti-human CD4 antibody conjugated with AF647 branched PEG construct. SK3 mouse anti-human CD4 antibody (5.5 mg / mL) was modified with ALEXA FLUOR® 647 succinimidyl ester at a 10-fold molar excess of ester over antibody (AF). SK3 tagged with 20-fold excess of azido-SE over antibody was click conjugated to 600 μM AF647-HG20K8 PEG-sDIBO at 3 mg / mL azido-SK3 antibody for 3 hours at 37° C., quenched with 5 mM NaN3, and the conjugate was purified with Millipore AMICON™ Ultra-2 100 kDa centrifugal filters (B1). SK3 tagged with azide-SE in 10-fold excess over antibody was click-conjugated to 100 μM AF647-HG20K8 PEG-sDIBO with 1 mg / mL azide-SK3 antibody for 20 h at 25 °C, quenched with 5 mM NaN3, and the conjugate was purified with Millipore AMICON™ Ultra-2 100 kDa centrifugal filters (B2). One million Ficoll-isolated PBMCs / well in a 96-well plate were stained with SK3 conjugate using a 7-point titration of 1 μg to 0.015 μg of antibody. Analysis of stained cells was performed using an ATTUNE™ NxT flow cytometer and compared to allophycocyanin (APC) (Thermo Fisher Scientific, catalog no. MHCD0405). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description Fluorescent labels are widely used in imaging because they provide direct, quantitative, specific, and sensitive detection of biomolecules, including proteins and nucleic acids. Modified fluorescent labels that are sulfonated and / or PEG-modified have higher sensitivity compared to basic unmodified dyes. However, even these modified fluorescent labels exhibit fluorescence quenching at certain dye-to-protein (D / P) ratios.

[0025] Although an increase in fluorescence is seen with increasing molar excess of the labeling dye, molar excesses above the optimal dye to protein ratio (D / P) typically result in quenching and / or precipitation of biomolecules, especially in fluorescence imaging applications where the spatial conformation of antigen / antibody or DNA / RNA interactions can cause static quenching.

[0026] In some embodiments, the invention includes methods to reduce quenching and / or increase fluorescent signal using highly labeled conjugates (e.g., biomolecules with high dye-to-protein ratios (D / P)) that result in reduced fluorescence upon standard conjugation. Modifications can be made to proteins, nucleic acids and other biomolecules (e.g., oligosaccharides). In some embodiments, the invention includes compositions comprising fluorescently labeled biomolecules that exhibit increased fluorescent signal and / or reduced quenching, where the composition includes a biomolecule, a spacer, and a fluorescent label, and where 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 on a per fluorescent label basis, as well as methods of making and using such compositions.As an example, assume that a single fluorescent label bound to a biomolecule sets a baseline of 100% fluorescent emission.Further 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 fluorescent emission.The present invention relates, in part, to compositions and methods for increasing the average fluorescent emission above 80% of the baseline.

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

[0029] One means of measuring fluorescence intensity is by measuring the quantum yield. The quantum yield (Φ) for a fluorescent system is effectively the efficiency of light emission of a given fluorophore and can be determined by the following equation: JPEG2025028883000009.jpg10128

[0030] Quantum yield can also be used to measure quenching efficiency, as shown below in Example 8. Additionally, 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 a fluorescently labeled molecule can be compared to the quantum yield of a free fluorescent label. If the quantum yield of a single unit of a free fluorescent label under conditions where quenching is not substantially present is set to 1, this can be used as a benchmark for comparing the fluorescence generated by each fluorescent label bound to the labeled molecule. In many cases, 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) (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.) of the fluorescence emission of the free fluorescent label on a per fluorescent label basis.

[0032] As shown in Example 8 and Table 25, the fluorescence intensity can be determined by measuring the total fluorescence of the fluorescently labeled molecule compared to the fluorescence of the free label. The fluorescence of a single unit of free fluorescent label under conditions where there is substantially no quenching is set to 1, which can be used as a benchmark for comparing the fluorescence generated by each fluorescent label bound to the labeled molecule. In many cases, the compositions of the 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) (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.) of the fluorescence emission of the free fluorescent label on a per fluorescent label basis.

[0033] The brightness of the fluorescently labeled molecule compared to the free fluorescent label can be determined as shown in Table 25. 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 formula: B = Φ × ε × N Therefore, the ratio of the brightness of the free fluorescent label to the brightness of the labeled molecule can be used to describe the total fluorescence enhancement.

[0034] As an example, the data in Table 25 is set as a benchmark for ALEXA FLUOR® 647 in deionized water. Moreover, it sets a benchmark for 100% quantum yield of free dye and a brightness ratio of 1.0. Among the samples, molecule AF647-20K8 had a quantum yield of 73% of the free dye, but showed a 5.8x fluorescence enhancement over the free dye. It is also shown that sample AF647-10K4 has the highest quantum yield of the free dye (89%), but only a 3.6x fluorescence enhancement over the free dye. These data show that the degree of fluorescence enhancement seen for these molecules can be directly correlated to the arm length. The data also show that keeping the arm length constant and adding more fluorescently labeled arms to the polymer tends to increase the fluorescence enhancement.

[0035] Therefore, the present invention includes compositions and methods for linking multiple fluorescent labels to individual molecules (e.g., biomolecules) so that the fluorescent labels are spaced apart in a manner that enhances the fluorescent signal. This can be done by reducing quenching. One way to enhance the fluorescent 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 invention includes methods of producing an antibody conjugated to a spacer and a fluorescent label, where the spacer is conjugated to the antibody using a spacer agent, and the spacer is not conjugated to the fluorescent label. Also included are compositions comprising a spacer, an antibody, and a fluorescent label, where the spacer is not conjugated to the fluorescent label.

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

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

[0039] In some embodiments, the present invention includes compositions and methods for spatially separating fluorescent labels from points on the 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 the molecule (e.g., biomolecule). Examples of such compositions and methods are shown in FIG.

[0040] definition The present 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 expressing quantities, proportions, or ratios, and other numerical values ​​used in the specification and claims should be understood in all cases as modified by the term "about" to the extent that they are not overly modified. Thus, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0041] As used in this specification and the appended claims, it should be noted that the singular forms "a," "an," and "the," as well as any singular use of any word, include plural referents unless clearly and unambiguously limited to one referent. As used herein, the term "comprising" and grammatical variations thereof are intended to be open-ended, such that the enumeration of items in a list does not exclude other similar items that may be substituted for or added to the listed items.

[0042] As used herein, a "biomolecule" includes, but is not limited to, synthetic or naturally occurring proteins or fragments thereof, 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. As used herein, the term "peptide" refers to a polymer in which the monomers are amino acids and are linked together through amide bonds, or referred to as a polypeptide. When the amino acids are α-amino acids, either the L-optical isomer or the D-optical isomer can be used. In addition, unnatural amino acids, such as β-alanine, phenylglycine and homoarginine, are also included. Commonly encountered amino acids that are not genetically encoded can also be used in the present invention. All amino acids used in the present invention can be either the D- or L-isomer. The L-isomer is commonly used. In addition, other peptidomimetics are also useful in the present invention. For a general review, 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 elicit a polyclonal antibody response, by chemical synthesis, and by recombinant host cells transformed with an expression vector encoding the 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 IgG4. 4に Mice generally have the same isotypes as humans, but the IgG isotypes are IgG1, IgG 2a , IgG2b , and IgG3 subtypes. Thus, as used herein, the term "antibody" will be understood to include within its scope (a) any of the various classes or subclasses of immunoglobulins (e.g., IgA, IgD, IgG, IgM, and IgE from any animal that produces antibodies), and (b) polyclonal and monoclonal antibodies, such as murine, chimeric, or humanized antibodies. Antibody molecules have regions of amino acid sequence that can act as antigenic determinants (e.g., Fc region, kappa light chain, lambda light chain, hinge region, etc.). Antibodies generated against selected regions are referred to as anti-[regions] (e.g., anti-Fc, anti-kappa light chain, anti-lambda light chain, etc.). Antibodies are typically generated against antigens by immunizing an organism with a macromolecule to initiate lymphocyte activation to express immunoglobulin proteins. As used herein, the term "antibody" also includes, but is not limited to, any polypeptide or protein having an antibody binding domain that is or is homologous thereto, including single chain Fv molecules (scFv), in which 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 can be derived from natural sources or they can be partially or wholly synthetically produced.

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

[0046] As used herein, the term "antibody fragment" refers to a fragment of an antibody that retains the primary selective binding properties of the whole antibody. Specific 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 the so-called "half molecule" fragments obtained by reductive cleavage of the 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 the light chain variable region, "Fv" fragments consisting of the variable regions of the heavy and light chains, and recombinant single chain polypeptide molecules in which the light chain variable region and the 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 the CH1 domain; (ii) dAb fragments consisting of the VH domain (Ward et al., Nature 341:544 (1989)), (iii) isolated CDR regions, and (iv) the single chain Fv molecules (scFv) described above. Furthermore, any fragment can be produced using recombinant techniques that retains the antigen recognition properties.

[0047] Exemplary VHH antibodies that can be used are single domain antibodies, which are antibody fragments consisting of a single monomeric variable antibody domain. 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 protein chains and two light chains.

[0048] As used herein, "antigen" refers to a molecule, including but not limited to a biological substance, that induces or is capable of inducing the formation of an antibody or to which an antibody selectively binds. Antigen is also referred to as "immunogen." An antibody selectively binds 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., reacts covalently under appropriate reaction conditions, and generally represents a point of attachment 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, 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, sulfenic acid isonitriles, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids, alkynes, and azides.

[0050] As used herein, a "spacer," "spacer molecule," or "spacer agent" refers to a compound (e.g., an organic compound) that can enhance the fluorescence emitted from a biomolecule when directly or indirectly conjugated to the biomolecule. This is believed to result from a reduction in the fluorescence quenching of the fluorescent label. Any number of compounds can act as a spacer, and exemplary compounds include NHS-acetate and various forms of polyethylene glycol (PEG). As used herein, the term "polyethylene glycol" or "PEG" refers to an oligomer or polymer of ethylene oxide. PEG polymer chain length can vary widely, but they tend to have high molecular weights, on the order of 10,000,000 g / mol. PEG is also available in a variety of forms. For example, branched PEGs typically have 3-10 PEG chains emanating from a central core group. Star PEGs have 3-100 PEG chains emanating from a central core group. Comb PEGs usually have multiple PEG chains grafted to the polymer backbone. Most PEGs include molecules with a distribution of molecular weights (i.e., they are polydisperse). The size distribution can be statistically characterized by its weight average molecular weight and its number average molecular weight, the ratio of which is called the polydispersity index. Exemplary PEG compounds that can be used in the practice of the present invention include MS(PEG)4, MS(PEG)8, and MS(PEG) 12 (Thermo Fisher Scientific, Waltham, MA, Cat. Nos. 22341, 22509B, and 22686, respectively), and (methyl-PEG 12 ) 3-PEG4-NHS ester (Thermo Fisher Scientific, Waltham, MA, catalog no. 22421).

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

[0052] As used herein, a "polymer" is a molecule that consists of repeating subunits (typically at least four repeating subunits). Polymers can be synthetic or naturally occurring. The repeating units of a polymer need not be identical. For example, proteins are polymers that consist of different amino acid subunits. Furthermore, polymers need not be completely linear molecules and thus may be branched, such as dextran.

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

[0054] As used herein, the term "multi-arm polymer" refers to a branched molecule having at least one fluorescent label attached thereto and having an unbranched structure (see FIG. 19). Examples of multi-arm polymers that can be used in the practice of the present invention are branched polysaccharides (e.g., dextran, glycogen), polyethylene glycol, branched long-chain carbon molecules (e.g., Ahx), and branched polypeptides.

[0055] As used herein, the term "conjugation molecule" or "conjugation arm" refers to a linker through which a dye is attached (e.g., covalently attached) to a molecule (e.g., a biomolecule). The conjugation molecule may attach to a single dye molecule or multiple dye molecules (the same dye or different dyes).

[0056] As used herein, the term "fluorescence" refers to the optical phenomenon in which a molecule absorbs a high-energy photon and re-emits it as a lower-energy (longer-wavelength) photon, with the difference in energy between the absorbed and emitted photons ending up as 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 inherently fluorescent. Fluorophores may contain substituents that modify the solubility, spectral properties, or physical properties of the fluorophore. Numerous fluorophores are known to those skilled in the art, including, but not limited to, coumarins, cyanines, benzofurans, quinolines, quinazolinones, indoles, furans, benzazoles, borapolyazaindacenes, and xanthenes, including fluoresceins, rhodamines, and rhodols, 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 chemistries such as N-hydroxysuccinimide (NHS), maleimides, and hydrazides as well as click chemistry (eg, SITECLICK™) are currently used for the conjugation of fluorescent labels to biomolecules.

[0058] As used herein, the term "conjugate" refers to a molecule that is attached to another molecule, either directly or indirectly, by either covalent or non-covalent bonds.

[0059] The term "dye conjugate" refers to a dye molecule covalently or non-covalently bound to another carrier molecule such as an antibody, and in many cases the dye is covalently attached. Dye conjugates can be linked by direct attachment via a single covalent bond, cross-linking or conjugation via a linker such as a series of stable covalent bonds incorporating 1-20 non-hydrogen atoms selected from the group consisting of C, N, O, S and P, covalently linking the fluorescent dye to another moiety or biological and non-biological components such as an antibody or chemically reactive group. The conjugation or linker can include a receptor binding motif such as biotin / avidin.

[0060] As used herein, the term "near infrared dye" or "near infrared reporter molecule" or "NIR dye" or "NIR reporter molecule" refers to a dye or reporter molecule having an excitation wavelength of about 580 nm to about 800 nm. Often, NIR dyes emit in the range of about 590 nm to about 860 nm. Often, NIR dyes are excited at about 680 to about 790 nm. Often, the 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 be selectively visualized without exciting endogenous substances present in the living body. Some NIR dyes have a large Stokes shift, so that the excitation and emission wavelengths are at least 20, 30, 40, 50, 60, 70, or 80 nm apart.

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

[0062] Solid support materials include, but are 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 noncovalent complexes, polystyrene-polyacrylamide covalent complexes, polystyrene-PEG [poly(ethylene glycol)] complexes, silicon, rubber, other materials used as supports for solid phase synthesis, affinity separation and purification, hybridization reactions, immunoassays and other such applications. The solid support may be particulate 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 materials.

[0063] By "kit" is meant a packaged set of related components, typically one or more compounds or compositions.

[0064] Detectable biomolecules In some embodiments, disclosed herein are biomolecules that are detectably labeled with multiple fluorescent labels that also include a spacer agent.

[0065] A spacer A spacer can be any molecule that, when conjugated to a biomolecule, can enhance the fluorescence emission resulting from excitation of multiple fluorescent labels independently conjugated to the biomolecule, believed to be due to reduced fluorescence quenching of the fluorescent labels.

[0066] In some embodiments, the spacer comprises 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 selected from the group consisting of alkanoyl, alkenoyl, and alkynoyl (-C(O)C n H m where n is 1-20 atoms, m>n, the carbon atoms can be bonded to each other by single, double, and / or triple bonds, and the alkyl, alkenyl, and / or alkynyl groups can be further substituted. In some embodiments, specific substitutions include -(OCH2CH2O) x -(CH2) y -OR, where x is 1 to 20, y is 1 to 6, and R is H or C. 1~6 In some embodiments, specific substitutions include ammonium (-NH3 + ), quaternary ammonium ((-NR3 + ) group, where R is C 1~6 alkyl), or a phosphonium group (-PQ3 + ) where Q is aryl, substituted aryl, or C 1~6 alkyl).

[0069] In some embodiments, the spacer is an alkyl, alkenyl, or alkynyl group (-C n H m where n is 1-20 atoms and m>n, the carbon atoms can be bonded to each other by single, double, and / or triple bonds, and the alkyl, alkenyl, and / or alkynyl groups can 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 -OR, where x is 1 to 20, y is 1 to 6, and R is H or C. 1~6 In some embodiments, other specific substitutions include ammonium (-NH3 + ), quaternary ammonium ((-NR3 + ) group, where R is C 1~6 alkyl), or a phosphonium group (-PQ3 + ) where Q is aryl, substituted aryl, or C 1~6 alkyl).

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

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

[0072] B. Fluorescent labeling The fluorescent dyes described herein function as reporter molecules that directly or indirectly provide detectable signals to samples as a result of conjugation to functional groups on proteins, including but not limited to amine or thiol groups.This provides the ability to detect all proteins in a sample, typically in combination with the detection of a subset of the total proteins of the sample.In such cases, the total protein label is distinguishable from the dye that labels a subset of the total proteins in the sample.

[0073] Typically, the detectable response is a change in fluorescence, for example 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 one or more aromatic or heteroaromatic rings that are optionally substituted one or more times with a variety of substituents, including, but not limited to, halogen, nitro, sulfo, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring systems, benzo, or other substituents typically present on chromophores or fluorophores known in the art.

[0075] A wide variety of fluorophores that may be suitable for whole 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 greater than 280 nm. Such chemical moieties include, but are not limited to, pyrene, sulfonated pyrene, sulfonated coumarin, sulfonated carbocyanine, sulfonated xanthene, anthracene, naphthalene, acridine, stilbene, indole, isoindole, indolizine, benzindole, oxazole or benzoxazole, thiazole or benzothiazole, 4-amino-7-nitrobenzo-2-oxa-1,3-diazole (NBD), carbocyanine, carbostyryl, porphyrin, salicylate, anthranilate, azulene, perylene, pyridine, quinoline, isoquinoline, chromene, borapolyazaindacene, xanthene, fluorescein, rosamine, rhodamine, rhodamine, benzo- or dibenzofluorescein, seminaphthofluorescein, naphthofluorescein, bimane, oxazine or benzoxazine, carbazine, phenalenone, coumarin, benzofuran, benzphenalenone) and derivatives thereof. As used herein, oxazines include resorufins, 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 a variety of substituents including halogen, nitro, sulfo, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring systems, benzo, or other substituents typically present on chromophores or fluorophores known in the art. In one embodiment, the fluorophore is a xanthene that comprises one or more julolidine rings.

[0077] In an exemplary embodiment, the dyes are independently substituted with substituents selected from the group consisting of hydrogen, halogen, amino, substituted amino, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, sulfo, reactive groups, solid supports, and carrier molecules. In another embodiment, the xanthene dyes of the present invention include compounds that are both substituted and unsubstituted on the carbon atoms of the central ring of the xanthene with substituents typically found in xanthene dyes, such as phenyl and substituted phenyl moieties. Often, the dyes are rhodamines, fluoresceins, borapolyazaindacenes, indoles, and derivatives thereof.

[0078] The choice of reactive group used to bind the whole protein label or expression tag label to the protein to be conjugated typically depends on the reactive or functional group on the substance to be conjugated and the type or length of the desired covalent bond.The types of functional groups typically present on organic or inorganic substrates (biomolecules or nonbiomolecules) include, but are not limited to, amines, amides, thiols, alcohols, phenols, aldehydes, ketones, phosphonates, imidazoles, hydrazines, hydroxylamines, disubstituted amines, halides, epoxides, silyl halides, carboxylates, sulfonates, purines, pyrimidines, carboxylic acids, olefinic bonds, or combinations of these groups.In proteins, various sites can occur, including, but 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, ... 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 These include, but are not limited to, RED™, TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED®, and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE).

[0080] In some embodiments, the fluorogenic reagent / dye that binds to a tag attached to a protein used in the protein labeling methods described herein can be a biarsenical fluorophore, including a biarsenical derivative of fluorescein, such as, by way of example only, FlAsH-EDT2 (4'-5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(2,2-ethanedithiol)2) (LUMIO™ Green, Life Technologies Corp., Carlsbad, Calif.), or a biarsenical derivative of resorufin, such as, by way of example only, ReAsh-EDT2 (LUMIO™ Red, Life Technologies Corp., Carlsbad, Calif.), or alternatively, an oxidized derivative such as ChoXAsH-EDT2 or HoXAsH-EDT2. Additionally, the biarsenical fluorophore may be a biarsenical derivative of other known fluorophores, including, by way of example only, 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 biarsenical fluorophore can be present at a concentration 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 more, and at a concentration 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 attached to the protein to which such a fluorescent dye is attached is a tetracysteine ​​peptide motif, cys-cys-Xn-cys-cys (SEQ ID NO: 1), where each X is any natural amino acid, unnatural amino acid, or 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. When such a motif, the 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 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 one another within the primary amino acid sequence of the protein, or may be directly multimerized in tandem as concatamers.

[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, unnatural amino acid, or 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 CCGGKGNGGCGC (SEQ ID NO: 4).

[0084] The tetracysteine ​​peptide tag(s) can be recombinantly fused to the protein desired to be labeled, either at the N-terminus, C-terminus, or in frame within the protein sequence, and expression vectors for making tetracysteine ​​fusion recombinant proteins can be readily constructed using techniques known to those skilled in the art. In certain embodiments, the tetracysteine ​​tagged protein is recombinantly expressed in a host cell, including, but not limited to, a bacterial host cell, a fungal host cell, an insect cell, a plant cell, or a mammalian cell. Such bacterial host cells include, by way of example only, species of the genera Escherichia (e.g., E. coli), Klebsiella, Streptomyces, Streptococcus, Shigella, Staphylococcus, Erwinia, Klebsiella, Bacillus (e.g., B. cereus, Bacillus subtilis ... The host cells include, but are not limited to, gram-negative and gram-positive bacteria of any genera, including, but not limited to, Bacillus subtilis, Bacillus megaterium, Serratia, Pseudomonas (e.g., P. aeruginosa, and P. syringae), and Salmonella (e.g., S. typhi, and S. typhimurium). Suitable bacterial strains and serotypes suitable for the present invention may include E. coli serotypes K, B, C, and W. An exemplary bacterial host is E. coli strain K-12. Fungal host cells include, by way of example only, Saccharomyces cerevisiae cells, while mammalian cells include, by way of example only, human cells. In such embodiments, the protein sample containing the protein of interest is a host cell lysate, which may be unpurified, partially purified, or substantially purified prior to labeling and analysis using the methods 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 in which translation (and optionally transcription) is performed, or a partially purified or purified fraction thereof. In embodiments in which the extract allows for coupled transcription and translation in a single cell-free extract, such as the E. coli-based EXPRESSWAY™ or EXPRESSWAY™ Plus system (Life Technologies Corp., Carlsbad, Calif.), the sample is the cell-free extract in which transcription and translation normally occur, or a fraction thereof.

[0086] Alternatively, GATEWAY® Technology (Life technologies Corp., Carlsbad, Calif.) is a universal cloning technology that can be used to express genes of interest in E. coli.

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

[0088] In addition to the tetracysteine ​​tag, other protein sequences can be usefully added recombinantly to the protein that is desired to be labeled.Among such additional protein sequences are linkers and / or short tags, usefully epitope tags such as FLAG tag or myc tag, or other sequences useful for purification, such as polyhistidine (e.g., 6xhis) tag.Alternatively, the tetracysteine ​​tag(s) can be chemically conjugated to the protein that is to be labeled using conjugation chemistry that is conventional 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 350-850 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™ fluorophore. In some embodiments, the DYLIGHT™ fluorophore 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™ fluorophore is conjugated to a PEG molecule (e.g., 2xPEG, 4xPEG, 8xPEG, or 12xPEG).

[0092] In some embodiments, the fluorescent label comprises ALEXA FLUOR®. In some embodiments, ALEXA FLUOR® is available as 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 FLUOR® 760. FLUOR® 790.

[0093] In some embodiments, the fluorescent label comprises a moiety selected from a xanthene, a coumarin, a cyanine, a pyrene, an oxazine, a borapolyazaindacene, a benzopyrylium, and a carbopyronine.

[0094] In some embodiments, the fluorescent label comprises fluorescein (e.g., Cy2 or FITC). In some embodiments, the fluorescent label comprises rhodamine (e.g., TRITC or Cy3). In some embodiments, the fluorescent label comprises 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 (eg, 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, B-phycoerythrin, phycoerythrocyanin, and b-phycoerythrin. The structures of phycobiliproteins have been studied and their fluorescence spectral properties are known. See A Glazer, "Photosynthetic Accessory Proteins with Bilin Prosthetic Groups", Biochemistry of Plants, Volume 8, M D Hatch and N K Boardman, EDS., Academic Press, pp. 51-96 (1981), and A Glazer, "Structure and Evolution of Photosynthetic Accessory Pigment Systems with Special Reference to Phycobiliproteins", The Evolution of Protein Structure and Function, B S Sigman and M A Brazier, EDS., Academic Press, pp. 221-244 (1980). In some embodiments, the fluorescent protein has an absorption maximum of 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 of at least about 500 nm, often at least about 550 nm.

[0097] In some embodiments, the fluorescent label is a dipyrrometheneboron difluoride dye, such as those disclosed in US 2014 / 0349,893, which is incorporated by reference in its entirety.

[0098] The amine-reactive fluorescent dyes used in the protein labeling methods described herein include, but are not limited to, aroyl-2-quinoline-carboxaldehyde type reagents. Such reagents are described in U.S. Patent No. 5,459,272 and U.S. Patent No. 5,631,374, each of which is incorporated herein by reference in its entirety. In some embodiments, the aroyl-2-quinoline-carboxaldehyde reagent used is 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde or 3-(2-furoyl)quinoline-2-carboxaldehyde. In certain embodiments, the amine-reactive fluorescent dye is 3-(2-furoyl)quinoline-2-carboxaldehyde, while in other embodiments, the amine-reactive fluorescent dye is 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde.

[0099] C. Biomolecules Biomolecules that can be used in the compositions and methods disclosed herein include any biomolecule that is useful in molecular biology 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 antibodies used in the practice of the invention can be polyclonal, monoclonal, or genetically engineered, and can be from any source (e.g., mammalian, such as shark, bird, or llama, human, mouse, rabbit, goat, rat, etc.). Additionally, humanized antibodies can be used.

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

[0102] In some embodiments, the biomolecule is a protein or polypeptide, hi 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., 15-50 nucleotides in length). In some embodiments, the nucleic acid molecule is more than 50 nucleotides in length, more than 100 nucleotides in length, more than 500 nucleotides in length, more than 1 kb in length, more than 2 kb in length, or more than 5 kb in length.

[0104] d. Conjugation of fluorescent dyes to biomolecules After selecting an appropriate dye with the desired spectral characteristics, typically with an excitation wavelength of at least 580 nm, the dye can be conjugated to the target carrier molecule using methods well known in the art (Haugland, MOLECULAR PROBES HANDBOOK, supra (2002)). In many cases, conjugation to form a covalent bond consists of simply mixing the reactive compounds of the present invention in a suitable solvent in which both the reactive compounds and the spacer molecule to be conjugated are soluble. In many cases, the reaction proceeds spontaneously at room temperature or below without the addition of reagents. For those reactive compounds that are photoactivated, the conjugation is facilitated by irradiation of the reaction mixture to activate the reactive compounds. Chemical modification of water-insoluble substances such that the desired compound-conjugates can be prepared is often carried out in aprotic solvents such as dimethylformamide, dimethylsulfoxide, acetone, ethyl acetate, toluene, or chloroform. Similar modification of water-soluble substances is easily achieved by using the present reactive compounds to make them more readily soluble in organic solvents.

[0105] Preparation of biomolecule (e.g., protein) conjugates typically involves first dissolving the biomolecule to be conjugated in an aqueous buffer at 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) are 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. An appropriate reactive compound is then dissolved in a non-hydroxylic solvent (usually DMSO or DMF) in an amount sufficient to give the appropriate degree of conjugation when added to a solution of the biomolecule to be conjugated. The appropriate amount of compound for any biomolecule (e.g., protein) or other moiety is conveniently predetermined by experimentation in which various amounts of compound are added to the biomolecule, the conjugate is chromatographically purified to separate unconjugated compound, and the compound-biomolecule conjugate is tested in the desired application.

[0106] Any number of buffers may be used for the conjugation reaction, and others as set forth herein. Using Examples 1 and 5 for illustrative purposes, phosphate buffered saline and borate buffers can and will be used for the conjugation reaction. It is also believed that a carbonate buffer at pH 9.5 can be used. As such, it is believed that higher pH conjugations require lower molar excesses of dye and spacer molecule. Thus, the present invention includes compositions and methods for carrying out conjugation reactions at a pH of 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 may be carried out in a variety of molar excesses of fluorescent dyes or mixtures of each fluorescent dye with NHS-acetate, NHS-MS(PEG)4, NHS-MS(PEG)8, or NHS-MS(PEG) 12The labeling reaction may be performed in borate buffer 50 mM, pH 8.5, with a spacer selected from. The labeling reaction may be incubated at room temperature (RT) for approximately 1 hour. The NHS-activated dye and NHS-activated spacer agent may be combined prior to addition to the antibody such that both reactions occur simultaneously, allowing for dye displacement and random spacing of the spacer.

[0107] Buffers that may be used in the practice of the present invention include, by way of example, 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 a concentration is obtained by dilution of a stock solution of the 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 concentration of the stock solution is 100 mM. In certain embodiments, the concentration of the stock solution is 50 mM. In certain embodiments, the concentration of the stock solution is 20 mM. In certain embodiments, the concentration of the stock solution is 10 mM. In certain embodiments, the concentration of the stock solution 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.

[0109] In some embodiments, the concentration of the amine-reactive fluorescent dye used in the methods of 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 100 μM, about 100 nM to about 5 mM, etc.). In certain embodiments, such concentrations are obtained by dilution of a stock solution of the fluorescent dye having a concentration in the range of 100 nM to 200 mM. In certain embodiments, the concentration of the stock solution is 100 mM. In certain embodiments, the concentration of the stock solution is 50 mM. In certain embodiments, the concentration of the stock solution is 20 mM. In certain embodiments, the concentration of the stock solution is 10 mM. In certain embodiments, the concentration of the stock solution 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 a protein or protein fragment (e.g., an antibody fragment) labeled using the methods 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 examples, more than one dye molecule can be attached to each location on a biomolecule. One way to attach more than one dye molecule to a single location on a biomolecule is by using conjugation molecules that bind more than one dye molecule. These conjugation molecules then bind to the biomolecule and carry multiple dye molecules with them. As an example, polymer dendrimers, such as those described in US Patent Application Publication No. 2012 / 0256102, can be used to attach multiple fluorescent dye molecules to a single polymer backbone or core (herein referred to as "dendrimer") for the attachment of these dye molecules to biomolecules. These dendrimers can have regular or irregular, branched polymer network structures that allow for the chemical attachment 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 that are used as spacers.Thus, in some examples, the spacer molecule and the conjugation molecule will have the same structure, except that the conjugation molecule has a dye molecule attached to it.Therefore, various forms of PEG molecules can be used as conjugation molecules.Thus, the spacer molecule can include dye molecules (e.g., fluorescent labels) (see Figure 18).

[0113] Thus, the present invention contemplates the use of conjugation molecules each having an average of about 2 to about 50 dye molecules attached (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 dye molecules attached to the conjugation molecules will be less than 10%, 15%, and / or 20%.

[0114] The degree of labeling can be measured for the labeled biomolecule. The degree of labeling can be calculated as follows: First, the molar concentration of the labeled biomolecule is calculated, for example, using the following formula: JPEG2025028883000010.jpg10128ε = Molar extinction coefficient of the 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, adjusting for the amount of absorbance at 280 nm caused by the dye (see Table 8) Dilution factor = the extent to which the protein:dye sample was diluted for absorbance measurement (if diluted at all)

[0115] The degree of labeling is then calculated using the following formula: JPEG2025028883000011.jpg10140ε' = Molar extinction coefficient of the fluorescent dye

[0116] Table 8. Properties of exemplary dyes JPEG2025028883000012.jpg88156

[0117] In some embodiments of the invention, enhanced fluorescence is observed for biomolecules containing a spacer that have a lower degree of labeling (DOL) than biomolecules without a spacer. As an example, assume there is an antibody labeled with a fluorescent dye with or without a spacer. On a comparable DOL basis, an antibody labeled with a dye that also has a spacer attached to it may show an enhancement of fluorescence between 1.5-3.5 fold, where 1 indicates the same level of fluorescence for both antibodies. Importantly, for biomolecules attached to both a dye and a spacer, the amount of fluorescent signal on a per dye molecule basis is increased.

[0118] Following the addition of reactive compound to the component solution, the mixture is incubated for a suitable period (typically about 1 hour at room temperature to several hours on ice), and excess compound is removed by gel filtration, dialysis, HPLC, ion exchange or adsorption to hydrophobic polymer, or other suitable means.Compound-conjugate is 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 temperature used in the methods described herein can be room temperature, ambient temperature, or a temperature above room temperature, such as, by way of example only, at least about 26°C, 27°C, 28°C, 29°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, or even higher, 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 can 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 at ambient 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 aspects, the second incubation temperature is ambient or room temperature.

[0120] 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 therein. The first incubation time and the second incubation time used in the methods described herein may be the same or different. In one embodiment, the first incubation time is 0-60 minutes, 5-10 minutes, and / or 5-10 minutes at room temperature. In some embodiments, the second incubation time is 0-20 minutes and / or about 10 minutes. In a particular example, the second incubation time is about 10 minutes at about 70°C. In another embodiment, the first incubation time is 1-3 hours at 25°C, the second incubation time is overnight at 25°C, and the third incubation time is 2-3 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 reactive site may be available, as is typical of polysaccharides, or multiple types of reactive sites may be available, as is typical of proteins (e.g., amines, thiols, alcohols, phenols). Selectivity of labeling is best obtained by selection of appropriate reactive dyes; for example, modification of thiols with thiol-selective reagents such as haloacetamides or maleimides, 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 careful control of reaction conditions.

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

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

[0124] In certain embodiments of the methods described herein, the proteins of the labeled sample can be usefully separated in parallel with a series of fluorescent molecular weight standards. Usefully, the standards are spectrally matched with at least one fluorophore used to label the proteins. Such spectral matching can be achieved, for example, by using tetracysteine ​​tagged protein standards that are labeled in parallel with the same biarsenical fluorophore used to label the protein sample, or by using standards that have fluorescent moieties that are spectrally matched with the biarsenical or other fluorophore used to label the sample proteins. Examples of standards useful in the practice of 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 can also be used to quantify the amount of fluorescently labeled protein present in a sample. In certain embodiments, the methods described herein further comprise quantifying the amount of fluorescence from the biarsenical fluorophore. In certain embodiments, the methods described herein further comprise quantifying the amount of fluorescence from the amine-reactive fluorescent dye. In certain embodiments, the methods described herein further comprise quantifying the amount of fluorescence from the fluorescent moiety of the amine-reactive fluorescent dye. Quantification can be performed without separating the proteins present in the protein sample, or after partial or complete separation of the proteins by electrophoresis, such as PAGE, 2D-PAGE, or IEF, or chromatography, or a combination thereof.

[0126] e. Conjugation of spacer molecules to biomolecules In some embodiments, the spacer molecule is conjugated to the biomolecule using NHS-ester chemistry as described in the Examples herein, although other chemistries, such as maleimides, pyridyl disulfides, and hydrazides, as well as SITECLICK™ technology including azide / alkynes, can also be used for this conjugation strategy.

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

[0128] In some embodiments, the concentration of a protein, protein fragment, or other biomolecule labeled using the methods 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 dye-to-protein ratio of the fluorescently labeled antibody is 1 to 50. In some embodiments, the dye-to-protein ratio of the fluorescently labeled antibody is 5 to 30. In some embodiments, the dye-to-protein ratio of the fluorescently labeled antibody is 1 to 20.

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

[0131] In some embodiments, the spacer agent is added in molar excess to the plurality of fluorescent labels in an amount of 0.1-25x, 1-15x, or 2.5-10x. In some embodiments, the spacer agent is in molar excess to the plurality of fluorescent labels in an amount of 2.5x. In some embodiments, the spacer agent is in molar excess to the plurality of fluorescent labels in an amount of 5x. In some embodiments, the spacer agent is in molar excess to the plurality of fluorescent labels in an amount of 7.5x. In some embodiments, the spacer agent is in molar excess to the plurality of fluorescent labels in an amount of 10x.

[0132] In some embodiments, the spacer agent is conjugated to the nucleic acid molecule. Protocols for conjugating moieties (e.g., fluorescent labels) to nucleic acids are 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 allow labeling of nucleic acid molecules. For example, 5-aminohexyl acrylamide-dUTP (aha-dUTP) and 5-aminohexyl acrylamide-dCTP (aha-dCTP) can be used to produce amine-modified DNA 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 technique consistently produces uniform and high degrees of DNA labeling that are difficult to obtain by other methods.

[0133] One method for achieving high DOL using spatially resolved fluorescent labels is shown in Figure 18, which illustrates the preparation of fluorescently labeled branched PEG molecules and the conjugation of the resulting PEG molecules (direct spacer) to an antibody, where the PEG molecules are covalently attached to the antibody via the CLICK-IT™ reaction (see Example 7).

[0134] The PEG molecules shown in FIG. 18 are each covalently attached to seven fluorescent labels. Additionally, fluorescent labels can be attached to individual PEG molecules such that the labels have a particular "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 about 3.5 angstroms, when n=1 in FIG. 18, the brush length of the fluorescent label would range from about 5 to about 10 angstroms. In many cases, n in FIG. 18 will not be 1. Additionally, PEG molecules typically vary in size and are described based on average molecular weight. Thus, using the PEG molecules shown in FIG. 18 for illustrative purposes, a population of PEG molecules with an average weight of 10,000 would have an n of about 30. Also, a population of PEG molecules with an average weight of 40,000 would have an n of about 120. Given the fact that both the arm of the branched PEG molecule and the other arm linking this molecule to the labeled molecule all have repeat 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.). Thus, the invention includes compositions and methods for producing and using molecules covalently attached to at least one fluorescent label, the fluorescent label having a brush length of about 22 angstroms to about 800 angstroms.

[0135] Alternatively, compositions of the invention may be described by the number of covalent bonds between the molecules and the fluorescent labels to which they are attached. Again using the branched PEG molecules shown in FIG. 18 for illustrative purposes, the number of intervening covalent bonds will range from about 24 to about 32, depending on where the fluorescent labels are attached to the branched PEG molecule. The invention includes compositions and methods for producing and using molecules covalently attached to at least one fluorescent label, where the fluorescent labels are attached to the molecule by about 16 to about 800 (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.) intervening covalent bonds.

[0136] The present invention also relates, in part, to spacing multiple fluorescent labels attached to the same location on each labeled molecule. Similar to that described above with respect to separation of fluorescent labels from labeled molecules, multiple fluorescent labels attached to the same location on each labeled molecule can be separated from one another by a distance of about 15 angstroms to about 800 angstroms (inclusive) and / or by about 16 to about 800 intervening covalent bonds (inclusive).

[0137] As described herein, an aspect of the invention relates 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 times. For example, the arms of the branched PEG molecules can be fully extended (brush) or fully coiled (mushroom), as well as virtually any conformation in between. Furthermore, each arm can be in a different extended or coiled state independent of the other arms at any given time. Thus, in Table 9, the term "brush" refers to a fully extended PEG arm, and the term "mushroom" refers to a fully coiled PEG arm. The mushroom state models a very large Flory radius (distance between adjacent PEG arms) of 80 Angstroms to obtain a minimum number.

[0138] The FF distance in Table 9 is the distance between two fluorophores on the ends of different arms, assuming all fluorophores are equidistant to each other. The maximum distance is twice the length of the arm. Furthermore, the nearest neighbor values ​​assume a tetrahedral arrangement for 4 arms and a sphere / cube for 8 arms. Of course, 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: Branched PEG arm lengths (estimated fluorophore-to-fluorophore (FF) distances) JPEG2025028883000013.jpg90155

[0140] Dextrans are another suitable material 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 inert due to their prominent poly-(α-D-1,6-glucose) bonds. These bonds make them resistant to cleavage by most endogenous cellular glycosidases. They also usually have low immunogenicity and are generally branched-chain molecules.

[0141] Dextrans are commercially available in nominal molecular weights (MW) ranging from 3000 daltons to 2,000,000 daltons. Dextrans suitable for use in the practice of the invention can be of any number of different molecular weights, including 3000, 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] Dextrans typically have a degree of substitution (e.g., DOL) of 0.2 to 2 dye molecules per dextran molecule for dextran in the 10,000 MW range. Additionally, dextrans typically contain 0.2 to 0.7 dyes per dextran in the 3000 MW range, 0.4 to 2 dyes per dextran in the 10,000 MW range, 1 to 4 dyes in the 40,000 MW range, and 2 to 6 dyes in the 70,000 MW range. Thus, dextrans, as well as other labeled polymers used in the practice of the present invention, may have a DOL in the range of about 0.03 to 0.3 per 1,000 MW (e.g., about 0.03 to about 0.25, about 0.08 to about 0.3, about 0.09 to about 0.3, about 0.1 to about 0.3, about 0.05 to about 0.2, about 0.07 to about 0.25, etc., per 1,000 MW) of fluorescent label.

[0143] Dextran, as well as other polymers, can be labeled in several ways. For example, fluorescently labeled dextran can be prepared by reaction of water-soluble aminodextran with a fluorescent label having a succinimidyl ester group. Fluorescently labeled dextran can also be prepared by reaction of native dextran with an isothiocyanate derivative of a fluorescent label such as FITC. Where appropriate, once the fluorescent label has been 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 that can render the dextran anionic or cationic.

[0144] Another type of polysaccharide that may be useful in the practice of the 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 six glucose units per turn. This type of molecule provides a structural regularity that allows the use of fluorescent labels arranged in a deliberately designed manner. Thus, the invention includes compositions with fairly static structural features that can be used to keep the distance between the fluorescent labels uniform, as well as methods of making and using such compositions. In many cases, the distance between two fluorescent labels bound to such molecules in a manner and under conditions that result in two fluorescent labels will 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 practice of the present invention. One example of such a molecule is the branched polylysine molecule shown in Figure 21. This molecule can be made according to the method described in US Patent Application Publication No. 2010 / 0278750. Several "R groups" are shown in Figure 21, which represent the positions where fluorescent labels can be attached. In such molecules, the R groups can be the same or different (e.g., R1 and R2). Furthermore, when several R groups are the same, these groups can act as fluorescently labeled attachment points where the R groups are not completely labeled.

[0146] As an example, FIG. 21 shows a polymer with 33 R groups, i.e., 32 R1 groups and 1 R2 group. 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 such that only a certain percentage of the R groups undergo fluorescent labeling. Furthermore, the labeling is semi-random since some R groups are more prone to undergo fluorescent labeling due to their position in the polymer. Thus, the present invention includes the design of fluorescently labeled polymers in which the positioning and DOL are tailored for the desired fluorescent effect. In some cases, the average number of binding sites available based on per polymer accepting labels is within 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 with R1 and R2 groups. If "directional" attachment of the polymer to a biomolecule (e.g., an antibody) is desired, the R2 group may be different from the R1 group. If the R groups are identical, the attachment will be "non-directional," in the sense that any R group at an appropriate position on the polymer can act as a conjugation point to the biomolecule. Of course, conditions are adjusted to achieve a high level of fluorescence while maintaining a high level of biological activity (e.g., antigen binding) to the biomolecule.

[0148] Maximizing biomolecular fluorescence can be partially independent of the number of fluorescent labels on the biomolecule of interest. For example, assume that there are seven fluorescent labels on a particular antibody when labeled under a first set of conditions, and ten fluorescent labels on the same antibody when labeled under a second set of conditions. Further assume that 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. Thus, the first set of conditions would be preferable to the second set of conditions, other factors being equal (e.g., functional activity of the biomolecule).

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

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

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

[0153] The core may already have functional groups for initiating polymerization or attachment onto the arms, or may require chemical modification to facilitate attachment of the arms, as shown in FIG.

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

[0155] The arms may consist of any number of linear polymers and will typically function as attachment points for the fluorescent labels and to space the labels from each other, as well as from other fluorescent labels attached to the same labeled molecule (e.g., biomolecule). 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 the present invention will be uncharged.

[0156] One region of an antibody that, when present, is particularly useful for attachment of a fluorescent label is the Fc (fragment crystallizable) region. The Fc region is at the distal end of the antibody relative to the antigen binding site. This region of the antibody interacts with cell surface receptors called Fc receptors and proteins of the complement system. Typically, attachment of a chemical at 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 attached to the antibody. In addition, large attached chemicals tend to interfere with each other in their ability to bind to the biomolecule due to steric hindrance. Thus, a series of factors may need to be balanced to produce a biomolecule (e.g., an antibody) that has both a high level of fluorescence and a high level of functional activity (e.g., antigen binding ability). Some of these factors are: (1) the size of the biomolecule, (2) the location of the attachment point on the biomolecule for fluorescent labeling, and (3) the size and three-dimensional structure of the fluorescently labeled molecule attached to the biomolecule. With respect to antibodies, the invention includes antibodies comprising one or more of the following features: - Fluorescently labeled polymers attached (e.g., covalently) at an average of 2-10 different positions on the antibody molecule. -An average of 3-80 fluorescent labels attached to each antibody molecule. - Antigen-binding affinity (K D ) is reduced by two orders of magnitude or less (e.g., about 0.5 to about 2.0, about 1.0 to about 2.0, about 0.5 to about 1.5, about 0.75 to about 1.5, etc.) compared to the antibody in its unlabeled form. - based on the fluorescent label bound to the antibody molecule, the average fluorescence emission is at least 60% (e.g., about 60% to about 98%, about 70% to about 98%, about 80% to about 98%, about 85% to about 98%, about 80% to about 93%, etc.) of the free fluorescent label.

[0157] f. Biomolecule / fluorescent label / spacer combination The present invention is based in part on the combination of three components: a biomolecule, a fluorescent label, and a spacer. For proteins (e.g., antibodies), the groups that can be used as binding sites for fluorescent labels and spacers are not always accessible for binding, especially when the protein is not denatured. Moreover, in many cases, it will be desirable to maintain the protein in its native form.

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

[0159] In some instances, the ratio may be based on the weight of each of the components. In other instances, the ratio may be based on a 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 multiple conjugation sites), the use of component weights may be more appropriate.

[0160] For antibodies, and other biomolecules, the ratio of biomolecule to fluorescent label to spacer used in the conjugation process can vary widely, but in most cases the amount of biomolecule will be less than the amount of both the fluorescent label and the spacer.

[0161] Additionally, the density and / or spacing of the conjugation sites on the biomolecule is one factor that often determines the optimal ratio of fluorescent label to spacer. This is because, assuming that the 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 biomolecule to fluorescent label to spacer ratio that can be used is shown as follows: B1:FL2~30 :S 2~20 , B is a biomolecule, FL is a fluorescent label, and S is a spacer. Specific ranges of ratios that may be used in the practice of the invention include ratios that fall within the range of 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 fluorescent label and spacer used for conjugation are such that the available binding sites on the biomolecule are effectively saturated (e.g., at least 95% of the available binding sites are bound to either the fluorescent label or the spacer). In such cases, the ratio of fluorescent label to spacer can be a determining factor in the level of fluorescence enhancement. In many cases, the ratio of fluorescent label to spacer will be 10:1 to 10:50 (e.g., about 10:1 to about 10:25, about 10:1 to about 10:10, about 10:1 to about 10:5, about 10:5 to about 10:50, about 10:5 to about 10:20, about 10:3 to about 10:30, about 10:5 to about 10:30, about 10:10 to about 10:25, etc.).

[0163] Spacer and dye can be conjugated to biomolecule simultaneously or sequentially, first conjugating either spacer or dye to biomolecule.In many cases, when spacer and dye are conjugated to biomolecule at the same place, they are conjugated to biomolecule simultaneously.However, sequential conjugation can be used when binding site is biomolecule and first conjugation reaction (e.g. by spacer) is carried out under non-saturating condition, thus making binding site available for second conjugation reaction (e.g. by dye).

[0164] G. Buffer In some embodiments, the compositions of the present invention comprise one or more spacers, one or more fluorescent labels in a buffer solution. 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 comprise any buffer suitable for molecular biology applications.

[0166] In some embodiments, the buffer is a suitable storage buffer (eg, a borate buffer, a phosphate buffer, or a carbonate buffer).

[0167] In some embodiments, the buffering agent is suitable for buffering a detectable biomolecule as disclosed herein during use in a detection assay.

[0168] method The invention has useful applications in basic research, high throughput screening, immunohistochemistry, fluorescence in situ hybridization (FISH), microarray technology, diagnostics, and medical therapy. The invention can be used in a variety of assay formats 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 biology application in which fluorescently labeled molecules are detected.For example, as disclosed herein, detectable biomolecules can be used in applications including Western blotting, ELISA, flow cytometry, flow cytometry, and FRET.As disclosed herein, detectable biomolecules can also be used in fluorescent immunohistochemistry (IHC), fluorescent 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, where the spacer and fluorescent labels are conjugated to a biomolecule but not to each other; b) detecting the fluorescence emitted by the plurality of fluorescent labels; and c) determining the presence of the desired target in the biological sample when the fluorescence emitted by the plurality of fluorescent labels is detected. In some embodiments, the biological sample comprises a cell lysate. In some embodiments, the biological sample comprises an intact cell. In some embodiments, the biological sample comprises a tissue sample. Furthermore, such tissue samples may be fixed. Furthermore, the compositions of the present invention can be used for applications such as immunohistochemistry. In some embodiments, the biological sample comprises an isolated protein. In some embodiments, the biological sample comprises a recombinant protein. In some embodiments, the biological sample is immobilized on a solid support. In some embodiments, the biological sample comprises an intact cell 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, lung, muscle, and skin.

[0171] In some embodiments, methods are disclosed herein for imaging a target antigen in vivo, the methods including: a) providing an antibody conjugated to a plurality of fluorescent labels and spacer agents that bind to the target antigen as disclosed herein; b) introducing the antibody into an organism to form a contact body; c) illuminating the contact body with an appropriate wavelength to form an illumination body; and d) observing the illumination body in which the target antigen is imaged. In some embodiments, these antibodies, or other target proteins or peptides, specific for targets or antigens in vivo, are conjugated to fluorescent dyes having excitation wavelengths compatible with in vivo imaging, typically about 580 nm to about 800 nm. The target-specific dye conjugates travel relatively freely in the circulation until their preferential sequestration occurs at the target pathological or non-pathological tissue site, such as a diseased or injured tissue site.

[0172] h.Kit The composition of the present invention can be incorporated into a kit that facilitates the implementation of various assays.The kit may be packaged with the composition in dry form or in solution.The kit 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.Additional optional agents include components for testing other cellular functions together with the compound.

[0173] In some embodiments, the kit comprises a biomolecule, a spacer agent, 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 a spacer agent and a fluorescent label. In some embodiments, the kit may comprise a fluorescent label and a polymer conjugated to a reactive group as a bioconjugation kit.

[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: fluorescent dyes, spacers that are either pre-labeled or labeled according to the instructions provided in the kit, and compounds that can be used to (1) conjugate the spacer to the biomolecule and / or (2) conjugate the fluorescent label to the biomolecule.

[0175] The present 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 expressing quantities, proportions, or ratios, and other numerical values ​​used in the specification and claims should be understood in all cases as modified by the term "about" to the extent that they are not overly modified. Thus, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. EXAMPLES

[0176] The following examples are provided to illustrate certain disclosed embodiments and should not be construed in any way as limiting the scope of the disclosure.

[0177] Example 1: Fluorescent Western Blotting a) NHS-activated fluorescent dyes and sulfo-NHS-acetate / NHS-acetate and NHS-MS(PEG)4, NHS-MS(PEG)8 and NHS-MS(PEG). 12 Antibody labeling with. NHS-activated fluorescent dyes such as DYLIGHT™ 650-4×PEG were reconstituted at 10 mg / ml in dimethylformamide (DMF). NHS-acetate was freshly prepared at 1 mg / ml in dimethylformamide (DMF). NHS-MS(PEG)4 (Cat. No. 22341 (Thermo Fisher Scientific), NHS-MS(PEG)8 (Cat. No. 22509 (Thermo Fisher Scientific)), NHS-MS(PEG) 12 (catalog no. 22686 (Thermo Fisher Scientific)) was reconstituted at 100 mg / ml in DMF. Just before use, the PEG reagent was further diluted to 1 mg / ml in DMF. One milligram of goat anti-mouse (GAM) and goat anti-rabbit (GAR) antibodies at 7–10 mg / ml in borate buffer 50 mM (catalog no. 28384 (Thermo Fisher Scientific), pH 8.5) was diluted with various molar excesses of fluorochromes or a mixture of each fluorochrome with NHS-acetate, NHS-MS(PEG)4, NHS-MS(PEG)8, or NHS-MS(PEG) 12The conjugates were labeled with a spacer selected from the group consisting of: 1) 100 mM MES buffer, pH 4.7, 2) 100 mM 100 mM 100 mM 100 mM MES, 1) 100 mM MES, 2) 100 mM MES, 3) 100 mM MES, 4) 100 mM MES, 5) 100 mM MES, 6) 100 mM MES, 7) 100 mM MES, 8) 100 mM MES, 9) 100 mM MES, 1) 100 mM MES, 1) 100 mM MES, 2) 100 mM MES, 3) 100 mM MES, 4) 100 mM MES, 5 ... The concentration of the conjugate was determined using an OD scan (252 nm-900 nm) and the ratio of dye moles / protein moles (D / P) was calculated. Finally, the conjugate was diluted to 1 mg / ml in STABILZYME® NOBLE Storage Buffer (Surmodics, Cat. No. SZ04) for long-term storage.

[0178] Serially diluted cell lysates (500ng–2ng) were mixed with SDS-PAGE sample buffer. Samples were heated at 95°C for 5 min and loaded onto Thermo Fisher Scientific Tris Glycine SDS-PAGE gels (Novex Gels, 4–20%, 10-well, Cat. No. WT4202BX10). Gels were electrophoresed according to the manufacturer's instructions and then transferred to nitrocellulose membranes using the semi-dry transfer method. Membranes were blocked with SEA BLOCK blocking buffer (Thermo Fisher Scientific, Cat. No. 0037527) for 30 min. Primary antibodies were prepared in Sea Block Blocking Buffer to a final concentration of 0.1–2.5 μg / ml. Blots were incubated with antibodies prepared in Sea Block Blocking Buffer for 1 h at room temperature (RT) with shaking. The antibody solution was decanted and the membrane was washed twice for 10 min in 10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.2 (TBST). Fluorescently labeled secondary antibody conjugates were diluted in SEA BLOCK Blocking Buffer to a final concentration of 20-1000 ng / mL. The washed membrane was incubated with the appropriate secondary antibody conjugate for 30-60 min with agitation. The buffer was decanted and the membrane was washed six times for 5 min with TBST. The membrane was imaged using a compatible fluorescent imager.

[0179] Results are shown in FIG. 8 for an experiment testing the effect of NHS acetate (2.5×, 5×, and 10×) and the addition of MS(PEG)4 (3.75×) spacers on GAM secondary antibodies conjugated to DYLIGHT™ 488 at 5×-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 1 / 5000 from 1 mg / ml. All DYLIGHT™ secondary antibodies were diluted 1 / 5000 from a 1 mg / ml stock. In Western blot applications, there is a significant increase in fluorescence intensity over the base conjugate (made without spacer) at 7.5×-20× molar excess of each dye for DYLIGHT™ 488-GAR conjugated with NHS acetate or the addition of MS(PEG)4.

[0180] Results are shown in FIG. 9 demonstrating the effect of adding an NHS acetate (5X) or MS(PEG)4 (5X) spacer to a GAM secondary antibody conjugated to DYLIGHT™ 650-4xPEG (with 7.5X 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 1 / 5000 of 1 mg / ml. All DYLIGHT™ secondary antibodies were diluted 1 / 5000 of 1 mg / ml stock. NHS acetate added at 5X molar excess to GAM-DYLIGHT™ 650-4XPEG-7.5X conjugate improved intensity by 1.5-fold. NHS acetate added at 3.75X molar excess to GAM-DYLIGHT™ 650-4XPEG-7.5X conjugate improved intensity by 1.4-fold.

[0181] Results demonstrating the effect of the addition of NHS acetate (2.5x, 5x) and MS(PEG)4 spacers to the GAR-DYLIGHT™ 800-4xPEG secondary antibody in a Western blot assay are shown in Figure 10 and Table 10. A431 cell lysates were serially diluted 1:1. Primary antibodies rabbit anti-Hsp90 and anti-cyclophilin B were diluted 1 / 5000. All DYLIGHT™ secondary antibodies were diluted 1 / 20,000 of the 1 mg / ml stock. In this Western blotting application, the addition of MS(PEG)4 (3.75x and 5x) and NHS acetate (2.5-5x) significantly enhanced the fluorescence intensity and sensitivity of the base DYLIGHT™ 800-4xPEG conjugate by 20-100% at different molar excesses of the dye.

[0182] Table 10. Effect of ±NHS acetate (2.5X, 5X) or MS(PEG)4 (5X) on GAR-DYLIGHT™ 800-4xPEG in a Western Blot Assay JPEG2025028883000014.jpg48153NA=No addition

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

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

[0185] Example 2: Dot blot assay Serial dilutions (1:1) of mouse or rabbit IgG were made from the selected stock concentrations. A 20 μL 12-channel multipipette was used to place the dilutions from highest to lowest into a 96-well plate. 1 or 2 μL of the 11 serial dilutions were carefully spotted onto a nitrocellulose membrane. The membrane was allowed to dry 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 in TBS or blocking buffer. 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 conjugates); and 1:20,000 (DYLIGHT™ 680-4×PEG and DYLIGHT™ 800-4×PEG conjugates). Membranes were incubated with the appropriate secondary antibody conjugate for 30-60 min with agitation. Membranes were washed 5 times for 5 min with TBST buffer. Membranes were imaged with an appropriate 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 effect of NHS acetate (2.5x, 5x and 10x) and MS(PEG)4 (3.75x) spacers on GAM-DYLIGHT™ 488 (5x to 20x molar excess of dye) in dot blot assays. DYLIGHT™ 488-GAM conjugates made with the addition of NHS acetate and MS(PEG)4 provided clear improvements in fluorescence intensity in the range of 1.2-1.8x over the base conjugate (made without spacer) at 7.5- to 20-fold molar excess of the various dyes in dot blot applications.

[0187] Table 11. Effect of NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x to 10x) addition on conjugation of GAM-DYLIGHT™ 488 at molar excesses of 5x to 20x in dot blots. JPEG2025028883000015.jpg57157NA=No addition

[0188] The results shown in Figure 4 and Table 12 demonstrate the effect of adding NHS acetate (2.5x and 5x) and MS(PEG)4 (3.75x) to GAM-DYLIGHT™ 488 (7.5x to 20x molar excess of dye) in a dot blot assay. Different sources of secondary antibodies were used in this experiment. Addition of NHS acetate and MS(PEG)4 to the conjugation mixture significantly improved signal intensity in the range of 1.2 to 2.6 fold compared to basic conjugation at 5x, 15x and 20x molar excess of dye.

[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 at molar excesses of 5x to 20x in a dot blot assay. JPEG2025028883000016.jpg30155

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

[0191] Table 13. Effect of NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x) addition on conjugation of GAM-DYLIGHT™ 550-2xPEG-GAR at 10x to 20x molar excess in a dot blot assay. JPEG2025028883000017.jpg48155

[0192] The results shown in Figure 6 and Table 14 [Surbhi and Marie: The data in Figure 6 and Table 14 are not completely consistent. I'm not sure which is correct. Please sort this out. This is not a big deal, but it needs to be done correctly.] demonstrate the effect of the addition of NHS acetate (2.5x, 5x, and 10x) and MS(PEG)4 (3.75x) to GAM-DYLIGHT™ 650-4xPEG-GAM (10x to 20x molar excess) in a dot blot assay. Mouse IgG was serially diluted 1:1 from 1000ng / dot. All DYLIGHT™ 650-4xPEG-GAR secondary antibodies were diluted 1 / 10000 of the 1mg / ml stock. Both NHS acetate and (MS)PEG4 provided notable improvements in sensitivity and signal / background over the initial base conjugates. NHS acetate added to GAM-DYLIGHT™ 650-4xPEG-15x at 2.5x molar excess improved intensity by 1.7x. The improvement provided by NHS acetate was 1.3x better than that achieved with the conjugate prepared with the highest molar excess of dye (20x). All MS(PEG)4 added to GAM-DYLIGHT™ 650-4xPEG-15x conjugates improved fluorescence intensity by 1.8-2.2x, performing better than the corresponding highest base conjugate GAM-DYLIGHT™ 650-4xPEG-20x.

[0193] Table 14. Effect of NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x) addition on 10x to 20x GAM-DYLIGHT™ 650-4xPEG-GAR conjugation in a dot blot assay. JPEG2025028883000018.jpg57155NA=No addition

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

[0195] Table 15. Effect of adding NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x, 5x, and 10x) to GAM-DYLIGHT™ 800-4xPEG in a dot blot assay. JPEG2025028883000019.jpg57152NA=No addition

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

[0197] The results shown in FIG. 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 of dye) in dot blot assays. Mouse IgG was serially diluted 1:2 from 1000 ng / dot. All DYLIGHT™ 680-4×PEG-GAR secondary antibodies were diluted 1 / 20,000 of the 1 mg / ml stock. These dot blot assays show that the addition of MS(PEG)4 (5×) and NHS acetate (2.5× and 5×) significantly enhanced the fluorescence intensity and sensitivity of the base DYLIGHT™ 680-4×PEG conjugate.

[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 multi-pipette, 100 μL of each dilution was placed into the corresponding wells of a 96-well plate, from highest to lowest, 1-11, with PBS added to the last column (#12; negative control). This was repeated for rows A through H. After overnight incubation, the plates were blocked and incubated with SUPERBLOCK™ Blocking Buffer (Thermo Fisher, Cat. No. 37515) as follows: 2 times with 200 μL for 5 min, followed by 1 time with 200 μL for 10 min. 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 PBST 20, then once with PBS. Secondary antibody conjugates were diluted in 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 in mouse IgG coated plates, GAR in rabbit IgG coated plates was added to the plate wells. Each dilution was added to a different row for each conjugate tested. All comparisons were made on the same plate. Plates were incubated for 60 minutes. Plates were washed 3 times with 200 μL with TBST or PBST buffer. 100 μL of PBS was added to each row of each well. Fluorescence intensity was measured using a VariosKan instrument or by imaging the fluorescent signal with an appropriate imaging device such as ChemiDoc MP (488, 550, 650, 680 nm) and LiCOR Odyssey CLx (650, 680, 800 nm).

[0200] Example 4: Immunofluorescence (IFC) (i.e., cell imaging) Method 1: Frozen U2OS cell plates stored at -80°C were thawed at 50°C for 30 min. Storage buffer (PBS) was removed and cells were permeabilized with 0.1% Triton-X100 in 1x PBS buffer for 15 min (100 μl / well). Plates were blocked with 2% BSA / PBS-0.1% Triton-X100 blocker for 30 min. Primary antibodies mouse anti-PDI or rabbit anti-HDAC2 (10 μg / ml) (Cat. no. PA1-861) diluted in 2% BSA / PBS-0.1% Triton-X100 were used. 、A 1×100μl / well PBST solution was added to the plate and incubated at room temperature for 1 hour. Negative controls contained 2% BSA / PBS-0.1% Triton-X100 blocker only. After incubation, the primary antibody solution was removed from the plate and the plate was washed 3 times with 100μl / well PBST and once with 100μl / well PBS. GAM or GAR secondary antibodies labeled with various molar excess dyes were then diluted to 4μg / ml in PBS and incubated at room temperature for 1 hour. The plate was washed 3 times with 100μl / well PBST and 1×100μl / well PBS and Hoechst33342 (catalog no. 62249, Thermo Scientific, Waltham, MA) (diluted to 0.1μg / ml in PBS) was added to each well (100μl / well). Plates were scanned with an ARRAYSCAN™ Plate Reader VTI3, 20× objective.

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

[0202] The results shown in Figure 13 and Tables 16 and 17 demonstrate the effect of NHS acetate (2.5x and 5x) and MS(PEG)4 (3.75x) spacer addition to GAM-DYLIGHT™ 488 (13A) and GAR-DYLIGHT™ 488 ((13B) at 7.5x-20x molar excess of dye) in cell imaging applications. DYLIGHT™ 488-GAM and DYLIGHT™ 488-GAR. A549 cells were stained with pH2Ax primary antibodies diluted 1 / 1000 of 1mg / ml stock. All DYLIGHT™ 488 secondary antibodies were diluted 1 / 250 of 1mg / ml stock. NHS acetate added to the conjugation mixture resulted in improvements in signal / background in the range of 1.4-1.5-fold (GAM) and 1.1-1.6-fold (GAR) compared to the base conjugate at 15x molar excess of dye. For the GAM conjugate, the most significant improvements were observed with NHS acetate at 5x and MS(PEG)4 at 3.75x for the GAR conjugate, with even more significant improvements observed with NHS acetate at 2.5x and MS(PEG)4 at 3.75x.

[0203] Table 16. Effect of NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x) addition on conjugation of GAM-DYLIGHT™ 488 at 5x to 20x molar excess in cell imaging applications - DYLIGHT™ 488-GAM JPEG2025028883000020.jpg27155NA=No addition

[0204] Table 17. Effect of NHS acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x) addition on conjugation of GAR-DYLIGHT™ 488 at 5x to 20x molar excess in cell imaging applications - DYLIGHT™ 488-GAR JPEG2025028883000021.jpg27155NA=No addition

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

[0206] Table 18. Effect of 2.5x to 10x NHS acetate and MS(PEG)4 (3.75x to 10x) on the fluorescence intensity of GAM-DYLIGHT™ 550-2xPEG in cell imaging applications. JPEG2025028883000022.jpg57155NA=No addition

[0207] The results of an experiment testing the effect of NHS acetate (2.5x, 5x, and 10x) and MS(PEG)4 (3.75x, 5x, 10x) spacers on GAM-DYLIGHT™ 650-4xPEG) in a cellular imaging application are shown in Figure 15 and Table 19. U2OS cells were stained with anti-PDI primary antibody diluted 1 / 100 of a 1 mg / ml stock. All DYLIGHT™ 650-4xPEG-GAM secondary antibodies were diluted 1 / 250 of a 1 mg / ml stock. In this cellular imaging application, the addition of NHS acetate-5x resulted in an approximately 70% improvement over the base conjugate (made without addition) for the DYLIGHT™ 650-4xPEG GAM conjugate at a 20x molar excess, and the addition of MS(PEG)4-3.75x at a 20-fold molar excess showed an approximately 90% improvement over the base conjugate.

[0208] Table 19. Effect of Addition of NHS Acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.75x, 5x, 10x) to GAM-DYLIGHT™ 650-4xPEG - Cell Imaging Applications JPEG2025028883000023.jpg57155NA=No addition

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

[0210] Table 20. Effect of Addition of NHS Acetate (2.5x, 5x, and 10x) or MS(PEG)4 (3.3.75x, 5x, 10x) to GAM-DYLIGHT™ 680-4xPEG - Cell Imaging Applications JPEG2025028883000024.jpg57155NA=No addition

[0211] result The use of spacer agents such as NHS-acetate, NHS-MS(PEG), and NHS-betaine increased the fluorescent signal sensitivity and intensity beyond the optimal D / P levels that typically result in quenching. This was achieved by using goat anti-mouse (GAM) and goat anti-rabbit (GAR) secondary antibodies with 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, NHS-DYLIGHT™ 800 4×PEG, in combination with spacer agents including, but not limited to, NHS-DYLIGHT™ 488, NHS-DYLIGHT™ 550 2×PEG, NHS-DYLIGHT™ 650 4×PEG, NHS-DYLIGHT™ 680 4×PEG, NHS-DYLIGHT™ 800 4×PEG. Our calculations of D / P values ​​after dye conjugation and purification demonstrated that the addition of the spacer agent did not result in 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 made with different dyes and spacer agents were tested in a variety of applications including IFC, Western blotting, dot blotting, or IgG-binding plate-based assays. In each case, for a particular spacer agent at a particular molar excess of spacer agent to dye, an increase in fluorescence intensity was observed when the spacer agent was used compared to a control lacking the spacer agent.

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

[0214] Table 21. Effect of 2.5x to 10x betaine on the fluorescence of TAMRA-GAM conjugates JPEG2025028883000025.jpg67160MR=molar ratio DOL=degree of labeling

[0215] Example 5: Reaction of Goat Anti-Mouse IgG (GAM) with and without N,N,N-Trimethylglycine-N-hydroxysuccinimide ester bromide (Betaine-SE) and 5-(and-6)-Carboxytetramethylrhodamine, succinimidyl ester (5(6)-TAMRA-SE) TAMRA-SE was weighed out and prepared as a 10 mg / mL stock solution in anhydrous DMSO, and Betaine-SE was weighed out and prepared as a 4 mg / mL stock in anhydrous DMSO. The DMSO solution was then transferred to reaction vials, and TAMRA-SE + / - Betaine-SE was measured into the vials based on a 5, 10, or 20 times dye to IgG molar ratio, and equivalents of 0 or 10 Betaine-SE to IgG molar ratio were also added to the vials.

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

[0217] Absorbance spectra were obtained on a Perkin-Elmer Lambda 35 UV / Vis spectrometer to determine the degree of substitution (DOS) or dye moles / GAM moles for each sample. Fluorescence emission spectra were obtained with a Perkin Elmer LS 55 fluorescence spectrometer using samples with 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 / area of ​​the sample standard spectrum. Total fluorescence was then calculated as the product of RQY*DOS.

[0218] Table 22. Total fluorescence output for different molar ratios of TAMRA / GAM JPEG2025028883000026.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 10 mg / mL stock solution in anhydrous DMSO, and propane-sultone was weighed and prepared as a 1 mg / mL stock in E-Pure H2O.

[0220] 0.357 mL (4.0 mg) of a 11.2 mg / mL solution of GAM in 10 mM potassium phosphate, 150 mM sodium chloride buffer (PBS) was measured into 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 of propane sultone for 2 min, then AF488 stock was added to the mixture at 8 or 15-fold molar excess over GAM and allowed to react for 1 h at room temperature. The dye-protein conjugate was separated from free dye and propane sultone by size-exclusion chromatography using 5-0.75 x 20 cm columns packed with BIORAD™ BIO-GEL® P-30 microparticles in PBS and eluted with the same. The first protein-containing band from each column was collected.

[0221] Absorbance spectra were obtained on a Perkin-Elmer Lambda 35 UV / Vis spectrometer to determine the degree of substitution (DOS) or dye moles / GAM moles for each sample. Fluorescence emission spectra were obtained using a Perkin Elmer LS 55 fluorescence spectrometer with samples having matched optical densities at 475 nm and excited at 475 nm. Emission data were collected from 480-800 nm. Relative quantum yields (RQY) were measured as the area of ​​the sample spectrum / area of ​​the sample standard spectrum. Total fluorescence was then calculated as the product of RQY*DOS.

[0222] Table 23. Total fluorescence output for different molar ratios of AF488 / GAM JPEG2025028883000027.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 No. A37573), abbreviated as "AF647-SE", was weighed and prepared as a 32 mM stock solution in anhydrous DMSO (Thermo Fisher Scientific, Catalog No. D12345). CLICK-IT™ SDP Ester sDIBO Alkyne (sDIBO) (Thermo Fisher Scientific, Catalog No. C20025) was prepared as a 9 mg / mL stock solution in anhydrous DMSO. 8-arm PEG amine (hexaglycerol), HCl salt, referred to as "20K8 PEG" (JenKem, Plano, TX 75024, Catalog No. 8ARM-NH2HCl), having the following structure: JPEG2025028883000028.jpg19128 was weighed out and prepared as a 40 mg / mL stock solution in anhydrous DMSO.

[0224] To a 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 reacted for 3 hours at 25° C. After 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 No. 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 for 2 hours at 25°C. The azide-SK3 antibody was purified using a 2 mL BioRad BIO-GEL® P-30M spin column. Azido-SK3 antibody was prepared with a 5- to 20-fold excess of azido-SE over 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 azido-SK3 were mixed in a plastic tube. 360 μL of PBS was added to bring the final concentration of the solution to 100 μM AF647-20K8 PEG-sDIBO (DIBO concentration) and 1 mg / mL azido-SK3 antibody. The click reaction was allowed to proceed for 2 h at 37 °C, followed by quenching with 5 mM NaN3 at room temperature for 1 h. The AF647-20K8 PEG-SK3 conjugate was diluted to 0.5 mg / mL in PBS. The conjugation reaction was carried out for 2 h to 20 h at a reaction temperature of 25 °C to 37 °C with a final DIBO concentration of 100 to 600 μM using 1 to 3 mg / mL azido-SK3 antibody. The specific conditions for each experiment performed are shown in Table 24.

[0227] 24.2 μL of a 2 mM solution of AF647-20K8 PEG-sDIBO (DIBO concentration) and 116 μL of 4.3 mg / mL azido-SK3 were mixed in a plastic tube. 360 μL of PBS was added to bring the final concentration of the solution to 100 μM AF647-20K8 PEG-sDIBO (DIBO concentration) and 1 mg / mL azido-SK3 antibody. The click reaction was allowed to proceed for 2 hours at 37° C., 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 centrifugal filter. The AF647-20K8 PEG-SK3 conjugate was diluted to 0.5 mg / mL in PBS. Using 1-3 mg / mL azido-SK3 antibody, the conjugation reaction was carried out for 2-20 hours at a final DIBO concentration of 100-600 μM and a reaction temperature of 25° C.-37° C. The specific conditions for each experiment performed are shown in Table 24.

[0228] Table 24: Conjugation conditions JPEG2025028883000029.jpg143170

[0229] Example 8: Analysis of quantum yield of branched PEG AF647 constructs To prepare samples for quantum yield measurements, solutions of AF647 branched 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 yields (Φ) were measured using a Hamamatsu Absolute PL Quantum Yield Spectrometer. Quantum yields for the branched PEG constructs were compared to those of the free dye to determine the degree of quenching in the final constructs. In addition, brightness was determined to determine the fluorescence enhancement achieved with the branched PEG spacer. The smallest construct, AF647-2K4 (2,000 molecular weight branched PEG with four arms), showed the greatest quenching (QY of 20% of the free dye, fluorescence ratio of 0.2) and the lowest overall improvement in total fluorescence. The greatest fluorescence enhancement was seen for high molecular weight constructs with either four or eight arms (AF647-10K4 and AF647-20K8), in which up to 89% of the fluorescence quantum yield was retained in the free dye (fluorescence ratio 0.9), and an improvement in brightness of up to 5.8-fold was seen.

[0230] Table 25. Effect of branched PEG spacer on percent quantum yield (QY) and brightness (B) of ALEXA FLUOR® 647 JPEG2025028883000030.jpg61153

[0231] Example 9: Flow cytometry evaluation of AF647-20K8 PEG-SK3 constructs Freshly drawn anticoagulated whole blood (human) was lysed with ACK lysis buffer for 20 min at room temperature. White blood cells were isolated by centrifugation (400 x g, 5 min) and washed twice in 1% bovine serum albumin / PBS (1% BSA / PBS). After isolation, the total number of white blood cells was determined using a COUNTESS® automated cell counter and then diluted to 10 million cells per mL. One million cells / well in a 96-well plate were stained with AF647-20K8 PEG-SK3 conjugate using a 7-point titration of antibody from 1 μg to 0.015 μg. Stained cells were washed twice with 1% BSA / PBS. Analysis of stained cells was performed using an ATTUNE™ NxT flow cytometer and compared to 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] FIG. 22 shows a histogram plot of CD4 positive lymphocyte cells 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 AF647-20K8 PEG-SK3 conjugate is shown as a dotted or solid line. Compared to the AF647 conjugate alone, the star PEG conjugate shows a greater than 0.5 log increase in brightness. FIG. 23 shows the signal to noise ratio (S / N) and percent positive (% positive) plotted as a function of conjugate concentration in a flow cytometry experiment. The star PEG constructs (here B1 and B2) have up to a 2.5-fold increase in S / N over the APC CD4 benchmark conjugate, and up to a 2-fold increase in S / N over the AF647 CD4 benchmark conjugate, while retaining the ability to accurately assess some CD4 positive cell counts in the sample.

[0233] Example 10: Conjugation of ALEXA FLUOR® 488 to an aminodextran scaffold Preparation of 70 kD aminodextran AF488 scaffold: 10 mg of aminodextran (70,000 MW, 20 amino groups; Thermo Fisher Scientific, Cat. No. D1862) was dissolved in 1.2 ml of dry DMSO containing 1.0 μl of DIEA. 0.9 mg of ALEXA FLUOR® 488 succinimidyl ester lithium salt (643MF; Thermo Fisher Scientific, Cat. 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 solid was shaken with 10 mL of fresh ethyl acetate and centrifuged. This wash was repeated three more times with 10 mL of fresh ethyl acetate and the resulting precipitate was dried in vacuum. The solid was redissolved in 0.5 ml water, and the solution was placed into a 10 cm Spectra / Por Dialysis membrane (Spectrum Labs, MWCO 12-14,000, flat, 10 mm wide) cut from both sides. The dialysis membrane was gently stirred in 1 L water for 1 week. The water was changed twice a day. The dialysis membrane was opened from one end, and the solution was lyophilized to obtain the aminodextran ALEXA FLUOR® scaffold. The measured DOL is 9.7, with a relative QY of 0.6 (based on the QY of ALEXA FLUOR® 488).

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

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

[0236] Table 26. Streptavidin labeled with 70 kD aminodextran AF488 scaffold (average of 9.7 dye molecules per scaffold) JPEG2025028883000031.jpg32170

[0237] Table 27. Streptavidin labeled with AF488 dye. JPEG2025028883000032.jpg29128

[0238] Results: As shown in Tables 26 and 27, the conjugates made from the scaffold are brighter compared to the conjugates made from the single AF488 dye. Similarly, in contrast to the nearly constant QY for labeling with the aminodextran scaffold, the QY of the AF488 fluorophore drops from 0.70 to 0.34 for the single dye conjugation.

[0239] The present invention is further presented in the following paragraphs.

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

[0241] 2. The composition described in item 1, wherein the first antibody exhibits a higher fluorescence emission level than a second antibody prepared with an equivalent amount of fluorescent label but without a spacer molecule.

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

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

[0244] 5. The composition according to any one of items 1 to 4, wherein the spacer molecule is conjugated to the antibody via a reactive group.

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

[0246] 7. The composition of claim 6, wherein the amine group is on a lysine residue.

[0247] 8. The composition described in paragraph 1, wherein the fluorescent label is conjugated to the antibody by a conjugation molecule.

[0248] 9. The composition according to any one of items 1 to 5, wherein the fluorescent label is positively charged.

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

[0250] 11. The fluorescent label is selected from the group consisting of 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 (trademark), TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED (registered trademark), or 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE), The composition according to items 1 to 9, which is a dye selected from the group consisting of 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 composition according to any one of items 1 to 12, wherein the spacer molecule is negatively charged or neutral.

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

[0254] 15. The composition according to any one of claims 1 to 12, wherein the spacer molecule comprises an acetyl group.

[0255] 16. The composition according to any one of claims 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 paragraphs 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 paragraphs 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. The spacer molecule is selected from the group consisting of alkanoyl, alkenoyl, and alkynoyl (-C(O)C n H mwherein 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.

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

[0261] 22. The alkyl, alkenyl, and / or alkynyl groups are ammonium (-NH3 + ), quaternary ammonium (-NR3 + ) groups, wherein R is C 1~6 Item 22. The composition according to item 21, wherein the alkyl is alkyl.

[0262] 23. The fluorescent dye is one or more of 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, tetramethyl rhodamine, FAM, TEXAS RED®, or 7-hydroxy-9H-(1,3-dichloro-9,9-Dimethylacridin-2-one) succinimidyl ester (DDAO-SE) and is 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 is Sulfo-NHS-acetate; (PEG)n, where 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 ) wherein n is 1 to 20 atoms, m>n, and the carbon atoms may be bonded to each other by single bonds, double bonds, and / or triple bonds; or -(OCH2CH2O) x -(CH2) y -OR (wherein x is 1 to 20, y is 1 to 6, and R is H or C 1~6 an alkyl, alkenyl, or alkynyl group further substituted with ammonium (-NH3 + ), quaternary ammonium ((-NR3 + ) groups, wherein R is C 1~6 an alkyl, alkenyl, or alkynyl group that is an alkyl The composition according to items 1 to 22, comprising one or more of the following:

[0263] 24. The alkyl, alkenyl, and / or alkynyl group is a phosphonium group (-PQ3 +), wherein Q is aryl, substituted aryl, or C 1~6 Item 24. The composition according to item 23, wherein the alkyl is alkyl.

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

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

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

[0267] 28. The composition according to any one of items 1 to 27, wherein the ratio of spacer molecule to antibody is 1 to 50.

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

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

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

[0271] 32. The composition according to paragraphs 1 to 31, wherein the spacer molecule is in molar excess over the plurality of fluorescent labels in an amount of 0.1 to 25 times, 1 to 15 times, or 2.5 to 10 times.

[0272] 33. The composition of claim 32, wherein the spacer molecule is in molar excess over the multiple fluorescent labels in an amount of 2.5 times.

[0273] 34. The composition of claim 33, wherein the spacer molecule is in molar excess over the multiple fluorescent labels in an amount of 5-fold.

[0274] 35. The composition of claim 33, wherein the spacer molecule is in molar excess over the multiple fluorescent labels in an amount of 7.5-fold.

[0275] 36. The composition of claim 33, wherein the spacer molecule is in molar excess over the multiple fluorescent labels in an amount of 10-fold.

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

[0277] 38. The composition according to paragraphs 1 to 37, 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%.

[0278] 39. A method for increasing the fluorescence of a fluorescently labeled biomolecule, comprising: (a) conjugating a spacer molecule to a biomolecule; (b) conjugating a fluorescent label to the biomolecule; Steps (a) and (b) may 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 according to paragraph 39, wherein the spacer molecule reduces quenching of the fluorescent label compared to quenching in the absence of the spacer molecule.

[0280] 41. The method of claim 39, wherein the spacer molecule is conjugated to the antibody via a reactive group.

[0281] 42. The method of claim 41, wherein the reactive group is an amine group.

[0282] 43. The method of claim 42, wherein the amine group is on a lysine residue.

[0283] 44. The method according to any one of claims 39 to 43, wherein the fluorescent label is positively charged.

[0284] 45. The method according to any one of claims 39 to 43, wherein the fluorescent dye is selected from ALEXA FLUOR (registered trademark) and DYLIGHT (trademark).

[0285] 46. ​​The fluorescent label is selected from the group consisting of 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 (trademark), TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED (registered trademark), and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE).

[0286] 47. The method according to paragraphs 39 to 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 any one of items 39 to 43, wherein the spacer molecule is negatively charged or neutral.

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

[0289] 50. The method according to any one of claims 39 to 48, wherein the spacer molecule comprises an acetyl group.

[0290] 51. The method of any one of claims 39 to 49, wherein the spacer molecule comprises an acetate molecule.

[0291] 52. The method of claim 51, wherein the acetate molecule is sulfo-NHS-acetate.

[0292] 53. The method of any one of paragraphs 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 of any one of paragraphs 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. The spacer molecule is selected from the group consisting of alkanoyl, alkenoyl, and alkynoyl (-C(O)C n H m49. The method according to any one of claims 39 to 48, wherein 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.

[0295] 56. The alkyl, alkenyl, and / or alkynyl groups are -(OCH2CH2O) x -(CH2) y -OR, where x is 1 to 20, y is 1 to 6, and R is H or C 1~6 49. The method according to any one of items 39 to 48, wherein the alkyl is alkyl.

[0296] 57. The alkyl, alkenyl, and / or alkynyl groups are ammonium (-NH3 + ), quaternary ammonium (-NR3 + ) groups, wherein R is C 1~6 Item 56. The method of item 55, wherein the alkyl group is alkyl.

[0297] 58. The alkyl, alkenyl, and / or alkynyl group is a phosphonium group (-PQ3 + ), wherein Q is aryl, substituted aryl, or C 1~6 Item 56. The method of item 55, wherein the alkyl group is alkyl.

[0298] 59. The method according to any one of claims 39 to 58, wherein the ratio of fluorescent label to antibody is 1 to 50.

[0299] 60. The method according to claim 59, wherein the ratio of fluorescent label to antibody is 5 to 30.

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

[0301] 62. The method according to any one of claims 39 to 61, wherein the ratio of spacer molecules to protein is 1 to 50.

[0302] 63. The method of claim 62, wherein the ratio of spacer molecules to protein is 5 to 30.

[0303] 64. The method of claim 62, wherein the ratio of spacer molecules to protein is 5 to 30.

[0304] 65. The method of claim 62, wherein the ratio of spacer molecules to protein is 1 to 20.

[0305] 66. The method according to any one of paragraphs 39 to 65, wherein the spacer molecule is in molar excess over the multiple fluorescent labels in an amount of 0.1 to 25 times, 1 to 15 times, or 2.5 to 10 times.

[0306] 67. The method of claim 66, wherein the spacer molecule is in molar excess over the multiple fluorescent labels at a quantity of 2.5.

[0307] 68. The method of claim 66, wherein the spacer molecule is in molar excess to the plurality of fluorescent labels in an amount of 5-fold.

[0308] 69. The method of claim 66, wherein the spacer molecule is in molar excess over the multiple fluorescent labels in an amount of 7.5-fold.

[0309] 70. The method of claim 66, wherein the spacer molecule is in molar excess to the plurality of fluorescent labels in an amount of 10-fold.

[0310] 71. The method according to any one of claims 39 to 70, wherein the percentage of binding sites on the antibody that are occupied by multiple fluorescent labels is 1% to 99%.

[0311] 72. The method according to any one of paragraphs 39 to 71, 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%.

[0312] 73. A method for identifying a spacer molecule capable of enhancing the fluorescence emission of a fluorescently labeled biomolecule, comprising: (a) conjugating a spacer molecule to the biomolecule independently of a plurality of fluorescent labels conjugated to the biomolecule; (b) testing whether the presence of a spacer molecule in addition to multiple fluorescent labels conjugated to a biomolecule increases the detectable fluorescence of the multiple fluorescent labels; and (c) identifying a spacer molecule as one that reduces quenching of a fluorescent label conjugated to a protein when the presence of the spacer molecule in addition to the multiple fluorescent labels conjugated to a biomolecule increases the detectable fluorescence of the multiple fluorescent labels.

[0313] 74. The method of claim 73, wherein the spacer molecule is conjugated to the biomolecule at the first lysine side chain present on the biomolecule.

[0314] 75. The method according to any one of claims 73 to 74, wherein the biological molecule is an antibody or an antibody fragment.

[0315] 76. The method according to any one of claims 73 to 75, wherein the multiple fluorescent labels are positively charged.

[0316] 77. The method according to any one of claims 73 to 76, wherein the spacer molecule is positively charged.

[0317] 78. The method according to any one of claims 73 to 74, wherein the multiple fluorescent labels are negatively charged.

[0318] 79. The method according to any one of items 73 to 76, wherein the spacer molecule is negatively charged.

[0319] 80. The method of any one of claims 73 to 79, wherein the multiple fluorescent labels are dyes selected from ALEXA FLUOR® and DYLIGHT® molecules.

[0320] 81. Multiple fluorescent labels may be used, including but not limited to 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 (trademark), TET, TAMRA, tetramethylrhodamine, FAM, TEXAS RED (registered trademark), and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE).

[0321] 82. The method of any one of claims 73 to 80, 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.

[0322] 83. The method of any one of paragraphs 73 to 82, wherein the spacer molecule is selected from acetate and PEG.

[0323] 84. The method according to any one of claims 73 to 83, wherein multiple fluorescent labels are conjugated to the antibody by conjugation molecules.

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

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

[0326] 87. The method according to any one of claims 73 to 86, wherein the dye-to-protein ratio for multiple fluorescent labels on an antibody is 1 to 50.

[0327] 88. The method according to item 87, wherein the dye-to-protein ratio of the multiple fluorescently labeled antibody is 5 to 30.

[0328] 89. The method according to item 87, wherein the dye-to-protein ratio of multiple fluorescent labels on an antibody is 1 to 20.

[0329] 90. The method according to any one of paragraphs 73 to 88, wherein the ratio of spacer molecules to protein is 1 to 50.

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

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

[0332] 93. The method of claim 90, wherein the ratio of spacer molecules to protein is 1 to 20.

[0333] 94. The method of any one of paragraphs 73 to 89, wherein the spacer molecule is in molar excess over the multiple fluorescent labels in an amount of 0.1 to 25 times, 1 to 15 times, or 2.5 to 10 times.

[0334] 95. The method of claim 94, wherein the spacer molecule is in molar excess to the plurality of fluorescent labels at a quantity of 2.5.

[0335] 96. The method of claim 94, wherein the spacer molecule is in molar excess to the plurality of fluorescent labels in an amount of 5-fold.

[0336] 97. The method of claim 94, wherein the spacer molecule is in molar excess over the plurality of fluorescent labels at a quantity of 7.5-fold.

[0337] 98. The method of claim 94, wherein the spacer molecule is in molar excess to the plurality of fluorescent labels in an amount of 10-fold.

[0338] 99. The method according to any one of claims 73 to 98, wherein the percentage of binding sites on the antibody that are occupied by multiple fluorescent labels is between 1% and 99%.

[0339] 100. The method of 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%.

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

[0341] 102. The method of claim 101, wherein the biological sample comprises a cell lysate.

[0342] 103. The method of claim 101, wherein the biological sample comprises intact cells.

[0343] 104. The method of claim 101, wherein the biological sample comprises an isolated protein.

[0344] 105. The method of claim 101, wherein the biological sample comprises recombinant protein.

[0345] 106. The method according to any one of claims 101 to 105, wherein the biological sample is immobilized on a solid support.

[0346] 107. The method of claim 101, wherein the biological sample comprises intact cells in a fluid.

[0347] 108. The method of claim 101, wherein the biological sample is a living animal.

[0348] 109. The method of claim 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 attached to the first nucleic acid molecule, and the fluorophores and spacer molecules are not covalently attached to each other.

[0350] 111. The composition of paragraph 110, wherein the first nucleic acid molecule exhibits a higher level of fluorescence emission than a second nucleic acid molecule prepared with an equivalent amount of fluorescent label but without a spacer molecule.

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

[0352] 113. A conjugated antibody comprising an antibody conjugated to multiple fluorescent labels, the antibody having the following characteristics: (a) a fluorescence ratio of 0.5 or greater based on a single fluorescent label; (b) conjugating at least four fluorescent labels to the antibody; and / or (c) a conjugated antibody, wherein the total fluorescence of the antibody is at least 20 percent greater than the fluorescence of the unconjugated fluorescent molecule.

[0353] 114. The conjugated antibody of paragraph 113, wherein the fluorescent label is conjugated to the antibody by one or more multi-arm polymers.

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

[0355] 116. The conjugated antibody according to paragraphs 113 and 114, wherein the fluorescent label is conjugated to the antibody by 2 to 10 multi-arm polymers.

[0356] 117. The conjugated antibody of paragraph 116, wherein two or more fluorescent labels are conjugated to the antibody.

[0357] 118. The arms of a multi-arm polymer are (a) polyethylene glycol, (b) polysaccharides, and (c) Polypeptide 118. The conjugated antibody according to any one of items 113 to 117, which is composed of a type of chemical substance selected from the group consisting of:

[0358] 119. A conjugated antibody according to any one of items 113 to 118, wherein the average brush distance between the fluorescent labels is 200 to 800 angstroms.

[0359] 120. The conjugated antibody of paragraphs 113 to 119, wherein the fluorescent label is separated from the antibody by at least 16 covalent bonds.

[0360] 121. The conjugated antibody of paragraph 120, wherein the fluorescent label is separated from the antibody by 16 to 800 covalent bonds.

[0361] 122. The fluorescent label is selected from the group consisting of 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-9,9-Dimethylacridin-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 a fluorescently labeled biomolecule, comprising: (a) conjugating a reactive group and two or more fluorescent labels to a spacer molecule, thereby forming a fluorescently labeled spacer molecule; (b) conjugating a fluorescently labeled spacer molecule to the biomolecule, thereby forming a fluorescently labeled biomolecule; A method wherein the individual fluorescent labels of the fluorescently labeled biomolecule have a fluorescence ratio of 0.5 or greater on a per fluorescent label basis.

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

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

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

[0366] 127. The method of claim 126, wherein the multi-arm polymer is a branched polyethylene glycol molecule.

[0367] 128. The method of claim 126, wherein the multi-arm polymer is conjugated to 4 to 10 fluorescent labels.

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

[0369] 130. A method for detecting a fluorescently labeled biomolecule, comprising: (a) exposing a fluorescently labeled biomolecule to light that excites a fluorescent label conjugated to the biomolecule; (b) detecting the emission produced by the fluorescent label conjugated to the biomolecule; A fluorescently labeled biomolecule is conjugated to four or more fluorescent labels, A method wherein the individual fluorescent labels of the fluorescently labeled biomolecule have a fluorescence ratio of 0.7 or greater on a per fluorescent label basis.

[0370] 131. The method of claim 130, wherein the fluorescently labeled biomolecule is an antibody.

Claims

1. A composition comprising a first antibody, A composition, wherein two or more fluorescent labels and two or more spacer molecules are covalently attached to said first antibody, and said fluorescent labels and spacer molecules are not covalently attached to each other.

2. The composition of claim 1 , wherein the first antibody exhibits a higher level of fluorescence emission than a second antibody prepared with an equivalent amount of fluorescent label but without the spacer molecule.

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

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

5. The composition of claim 1 , wherein the spacer molecule is conjugated to the antibody via a reactive group.

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

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

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

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

10. The composition of claim 1 , wherein the fluorescent label is an ALEXA FLUOR® dye or a DYLIGHT™ dye.

11. The fluorescent label is selected from the group consisting of 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 2. The composition of claim 1, wherein the dye is selected from the group consisting of OCTACH, OCTACH, OCTACH 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-dimethylacridin-2-one) succinimidyl ester (DDAO-SE).

12. 2. The composition of 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 of claim 1 , wherein the spacer molecule is negatively charged or neutral.

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

15. The composition of claim 1 , wherein the spacer molecule comprises an acetyl group.

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

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

18. 2. The composition of 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. 2. The composition of 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 selected from the group consisting of alkanoyl, alkenoyl, and alkynoyl (-C(O)C n H m 13. The composition of claim 1, comprising or consisting of a group selected from:

21. The alkyl, alkenyl, and / or alkynyl groups are -(OCH 2 CH 2 O) x - (CH 2 ) y -OR, where x is 1 to 20, y is 1 to 6, and R is H or C. 1~6 The composition of claim 1 wherein the alkyl group is an alkyl group.

22. The alkyl, alkenyl, and / or alkynyl groups are ammonium (-NH 3 + ), quaternary ammonium (-NR 3 + ) groups, wherein R is C 1~6 22. The composition of claim 21, wherein said alkyl is alkyl.

23. The fluorescent dye may be one or more of 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® 620-X, ALEXA FLUOR® 630-X, ALEXA FLUOR® 640-X, ALEXA FLUOR® 650-X, ALEXA FLUOR® 660-X, ALEXA FLUOR® 670-X, ALEXA FLUOR® 680-X, ALEXA FLUOR® 690-X, ALEXA FLUOR® 700-X, ALEXA FLUOR® 710-X, ALEXA FLUOR® 720-X, ALEXA FLUOR® 730-X, ALEXA FLUOR® 740-X, ALEXA FLUOR® 750-X, ALEXA FLUOR® 760-X, ALEXA FLUOR® 770-X, ALEXA FLUOR® 780-X, ALEXA FLUOR® 79 ...90-X, ALEXA FLUOR® 790-X, ALEXA FLUOR® 790-X, ALEXA FLUOR 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®, 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-9,9-Dimethylacridin-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 is Sulfo-NHS-acetate; (PEG)n, where 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 may be bonded to each other by single, double, and / or triple bonds; or - (OCH 2 CH 2 O) x - (CH 2 ) y -OR (wherein x is 1 to 20, y is 1 to 6, and R is H or C 1~6 an alkyl, alkenyl, or alkynyl group further substituted with ammonium (-NH 3 + ), quaternary ammonium ((-NR 3 + ) groups, wherein R is C 1~6 an alkyl, alkenyl, or alkynyl group that is an alkyl The composition of claim 1 comprising one or more of:

24. The alkyl, alkenyl, and / or alkynyl groups are phosphonium groups (-PQ 3 + wherein Q is aryl, substituted aryl, or C 1~6 24. The composition of claim 23, wherein the alkyl is alkyl.

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

26. 26. The composition of claim 25, wherein the ratio of fluorescent label to antibody is between 5 and 30.

27. 26. The composition of claim 25, wherein the ratio of fluorescent label to antibody is 1-20.

28. The composition of claim 1, wherein the ratio of spacer molecules to antibodies is 1-50.

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

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

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

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

33. 33. The composition of claim 32, wherein the spacer molecule is in molar excess to the multiple fluorescent labels in an amount of 2.5-fold.

34. 34. The composition of claim 33, wherein the spacer molecule is in molar excess to the multiple fluorescent labels in an amount of 5-fold.

35. 34. The composition of claim 33, wherein the spacer molecule is in molar excess to the multiple fluorescent labels in an amount of 7.5-fold.

36. 34. The composition of claim 33, wherein the spacer molecule is in molar excess to the multiple fluorescent labels in an amount of 10-fold.

37. The composition of claim 33, wherein the percentage of binding sites on the antibody that are occupied by the multiple fluorescent labels is between 1% and 99%.

38. 2. The composition of 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. 1. A method for increasing the fluorescence of a fluorescently labeled biomolecule, comprising: (a) conjugating a spacer molecule to a biomolecule; (b) conjugating a fluorescent label to the biomolecule, Steps (a) and (b) may be performed simultaneously or in any order; The method, wherein the spacer and the fluorescent label are not conjugated to each other.

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

41. 40. The method of claim 39, wherein the spacer molecule is conjugated to the antibody via a reactive group.

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

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

44. 40. The method of claim 39, wherein the fluorescent label is positively charged.

45. 40. The method of claim 39, wherein the fluorescent dye is selected from ALEXA FLUOR® and DYLIGHT™.

46. The fluorescent label is selected from the group consisting of 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 40. The method of claim 39, wherein the isopropyl alcohol is selected from the group consisting of OXYGREEN® 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-dimethylacridin-2-one) succinimidyl ester (DDAO-SE).

47. 40. The method of 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. 40. The method of claim 39, wherein the spacer molecule is negatively charged or neutral.

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

50. 40. The method of claim 39, wherein the spacer molecule comprises an acetyl group.

51. 40. The method of claim 39, wherein the spacer molecule comprises an acetate molecule.

52. 40. The method of claim 39, wherein the acetate molecule is sulfo-NHS-acetate.

53. 40. The method of 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. 40. The method of 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 selected from the group consisting of alkanoyl, alkenoyl, and alkynoyl (-C(O)C n H m wherein n is 1 to 20 atoms, m>n, and the carbon atoms may be bonded to each other 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 -OR, where x is 1 to 20, y is 1 to 6, and R is H or C. 1~6 56. The method of claim 55, wherein said alkyl is alkyl.

57. The alkyl, alkenyl, and / or alkynyl groups are ammonium ((-NH 3 + ), quaternary ammonium (-NR 3 + ) groups, wherein R is C 1~6 56. The method of claim 55, wherein said alkyl is alkyl.

58. The alkyl, alkenyl, and / or alkynyl groups are phosphonium groups (-PQ 3 + wherein Q is aryl, substituted aryl, or C 1~6 56. The method of claim 55, wherein said alkyl is alkyl.

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

60. 60. The method of claim 59, wherein the ratio of fluorescent label to antibody is between 5 and 30.

61. 60. The method of claim 59, wherein the ratio of fluorescent label to antibody is 1 to 20.

62. 40. The method of claim 39, wherein the ratio of spacer molecules to protein is 1-50.

63. 63. The method of claim 62, wherein the ratio of spacer molecules to protein is between 5 and 30.

64. 63. The method of claim 62, wherein the ratio of spacer molecules to protein is between 5 and 30.

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

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

67. 67. The method of claim 66, wherein the spacer molecule is in molar excess to the multiple fluorescent labels in an amount of 2.5 fold.

68. 67. The method of claim 66, wherein the spacer molecule is in molar excess to the multiple fluorescent labels in an amount of 5-fold.

69. 67. The method of claim 66, wherein the spacer molecule is in molar excess to the multiple fluorescent labels in an amount of 7.5 fold.

70. 67. The method of claim 66, wherein the spacer molecule is in molar excess to the multiple fluorescent labels in an amount of 10-fold.

71. 40. The method of claim 39, wherein the percentage of binding sites on the antibody that are occupied by the multiple fluorescent labels is between 1% and 99%.

72. 40. The method of 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. 1. A method for identifying a spacer molecule capable of enhancing the fluorescence emission of a fluorescently labeled biomolecule, comprising the steps of: (a) conjugating a spacer molecule to a biomolecule independently of a plurality of fluorescent labels conjugated to said biomolecule; (b) testing 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) identifying the spacer molecule as one that reduces quenching of the fluorescent label conjugated to the protein when the presence of the spacer molecule in addition to the multiple fluorescent labels conjugated to the biomolecule increases the detectable fluorescence of the multiple fluorescent labels.

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

75. 74. The method of claim 73, wherein the biological molecule is an antibody or an antibody fragment.

76. 74. The method of claim 73, wherein said plurality of fluorescent labels are positively charged.

77. 74. The method of claim 73, wherein the spacer molecule is positively charged.

78. 74. The method of claim 73, wherein said plurality of fluorescent labels are negatively charged.

79. 74. The method of claim 73, wherein the spacer molecule is negatively charged.

80. 74. The method of claim 73, wherein said multiple fluorescent labels are dyes selected from ALEXA FLUOR® and DYLIGHT™ molecules.

81. The plurality of fluorescent labels may be selected from the group consisting of 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 74. The method of claim 73, wherein the isopropyl ether is selected from the group consisting of RED 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-dimethylacridin-2-one) succinimidyl ester (DDAO-SE).

82. 74. The method of 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. 74. The method of claim 73, wherein the spacer molecule is selected from acetate and PEG.

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

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

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

87. 74. The method of claim 73, wherein the dye to protein ratio of the multiple fluorescent labels on the antibody is between 1 and 50.

88. 88. The method of claim 87, wherein the dye to protein ratio of the multiple fluorescent labels to the antibody is between 5 and 30.

89. 88. The method of claim 87, wherein the ratio of dye to protein for multiple fluorescent labels on the antibody is 1-20.

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

91. 91. The method of claim 90, wherein the ratio of spacer molecules to protein is between 5 and 30.

92. 91. The method of claim 90, wherein the ratio of spacer molecules to protein is between 5 and 30.

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

94. 74. The method of claim 73, wherein the spacer molecule is in molar excess over the plurality of fluorescent labels in an amount of 0.1 to 25 fold, 1 to 15 fold, or 2.5 to 10 fold.

95. 95. The method of claim 94, wherein said spacer molecule is in molar excess to said plurality of fluorescent labels in an amount of 2.5 fold.

96. 95. The method of claim 94, wherein said spacer molecule is in molar excess to said plurality of fluorescent labels in an amount of 5-fold.

97. 95. The method of claim 94, wherein said spacer molecule is in molar excess to said plurality of fluorescent labels in an amount of 7.5 fold.

98. 95. The method of claim 94, wherein said spacer molecule is in molar excess to said plurality of fluorescent labels in an amount of 10-fold.

99. 74. The method of claim 73, wherein the percentage of binding sites on the antibody that are occupied by the multiple fluorescent labels is between 1% and 99%.

100. 100. The method of 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. 1. A method for determining the presence of a desired target in a biological sample, comprising: (a) contacting the biological sample with a composition, an antibody, wherein two or more fluorescent labels and two or more spacer molecules are covalently attached to the antibody, and the fluorophores and spacer molecules are not covalently attached to each other; (b) detecting fluorescence emitted by the plurality of fluorescent labels; and (c) determining the presence of the desired target in the biological sample when fluorescence emitted by the plurality of fluorescent labels is detected.

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

103. 102. The method of claim 101, wherein the biological sample comprises intact cells.

104. 102. The method of claim 101, wherein the biological sample comprises an isolated protein.

105. 102. The method of claim 101, wherein the biological sample comprises recombinant protein.

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

107. 102. The method of claim 101, wherein the biological sample comprises intact cells in a fluid.

108. 102. The method of claim 101, wherein the biological sample is from a living animal.

109. 109. The method of 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 attached to the first nucleic acid molecule; A composition wherein said fluorophore and spacer molecule are not covalently bonded to one another.

111. 111. The composition of claim 110, wherein the first nucleic acid molecule exhibits a higher level of fluorescence emission than a second nucleic acid molecule prepared with an equivalent amount of fluorescent label but not containing the spacer molecule.

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

113. A conjugated antibody comprising an antibody conjugated to multiple fluorescent labels, the antibody having the following characteristics: (a) a fluorescence ratio of 0.5 or greater on a per fluorescent label basis; (b) conjugating at least four fluorescent labels to said antibody; and / or (c) a conjugated antibody, wherein the total fluorescence of the antibody is at least 20 percent greater than the fluorescence of the unconjugated fluorescent molecule.

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

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

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

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

118. the arms of the multi-arm polymer (a) polyethylene glycol, (b) a polysaccharide, and (c) Polypeptide The conjugated antibody of claim 113, which is composed of a type of chemical selected from the group consisting of:

119. The conjugated antibody of claim 113, wherein the average brush distance between said fluorescent labels is between 200 and 800 angstroms.

120. The conjugated antibody of claim 113, wherein the fluorescent label is separated from the antibody by at least 16 covalent bonds.

121. The conjugated antibody of claim 120, wherein the fluorescent label is separated from the antibody by a covalent bond of 16 to 800.

122. The fluorescent label is selected from the group consisting of 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-9,121. The conjugated antibody of claim 120, wherein the dye is selected from the group consisting of 9-dimethylacridin-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.

123. 1. A method for preparing a fluorescently labeled biomolecule, comprising: (a) conjugating a reactive group and two or more fluorescent labels to a spacer molecule, thereby forming a fluorescently labeled spacer molecule; (b) conjugating the fluorescently labeled spacer molecule to the biomolecule, thereby forming the fluorescently labeled biomolecule; A method wherein each individual fluorescent label of said fluorescently labeled biomolecule has a fluorescence ratio of 0.5 or greater on a per fluorescent label basis.

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

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

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

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

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

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

130. 1. A method for detecting a fluorescently labeled biomolecule, comprising: (a) exposing a fluorescently labeled biomolecule to light that excites a fluorescent label conjugated to the biomolecule; (b) detecting the emission produced by the fluorescent label conjugated to the biomolecule; the fluorescently labeled biomolecule is conjugated to four or more fluorescent labels; A method wherein each individual fluorescent label of said fluorescently labeled biomolecule has a fluorescence ratio of 0.7 or greater on a per fluorescent label basis.

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

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