Compositions and methods for enhanced fluorescence

Abstract

This disclosure relates to the field of fluorescent dyes, and in particular, compositions and methods for increasing fluorescent signals and the reduction of fluorescent quenching.

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   wherein the fluorescent label and spacer molecules are not covalently attached to each other.   
     
     
         2 . The composition of  claim 1 , wherein the first antibody exhibits higher fluorescent emission levels than a second antibody prepared with an equivalent amount of fluorescent label but without the spacer molecule. 
     
     
         3 . The composition of  claim 1 , wherein the first antibody exhibits higher fluorescent emission levels than a second antibody, wherein the first antibody and the second antibody each have the same number of covalently bound fluorescent labels, and wherein the second antibody does not have a covalently bound spacers molecule. 
     
     
         4 . The composition of  claim 1 , wherein the spacer molecule reduces quenching of the fluorescent labels as compared to the quenching in the absence of the spacer molecule. 
     
     
         5 . The composition of  claim 1 , wherein the spacer molecule is conjugated to the antibody to 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 .- 13 . (canceled) 
     
     
         14 . The composition of  claim 1 , wherein the spacer molecule is selected from acetate and polyethylene glycol (PEG). 
     
     
         15 .- 23 . (canceled) 
     
     
         25 . The composition of  claim 1 , wherein the ratio of fluorescent label to antibody is from 1 to 50. 
     
     
         26 . The composition of  claim 25 , wherein the ratio of fluorescent label to antibody is from 5 to 30. 
     
     
         27 .- 38 . (canceled) 
     
     
         39 . A method for increasing fluorescence of a fluorescently labeled biomolecule, the method comprising:
 (a) conjugating a spacer molecule to a biomolecule; and   (b) conjugating a fluorescent label to the biomolecule;   wherein steps (a) and (b) can be done simultaneously or in any order, and   wherein the spacer and fluorescent label are not conjugated to each other.   
     
     
         40 . The method of  claim 39 , wherein the spacer molecule reduces quenching of the fluorescent labels as compared to the quenching in the absence of the spacer molecule. 
     
     
         41 . The method of  claim 39 , wherein the spacer molecule is conjugated to the antibody to a reactive group. 
     
     
         42 . The method of  claim 41 , wherein the reactive group is an amine group. 
     
     
         43 . The method of  claim 42 , wherein the amine group is on a lysine residue. 
     
     
         44 .- 122 . (canceled) 
     
     
         123 . A method for preparing a fluorescently labeled biomolecule, the method comprising:
 (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 the biomolecule, thereby forming the fluorescently labeled biomolecule,   and   wherein the individual fluorescent labels of the fluorescently labeled biomolecule have a Fluorescent Ratio on a per fluorescent label basis that is equal to or greater than 0.5.   
     
     
         124 . The method of  claim 123 , wherein an average of from one to ten fluorescently labeled spacer molecules are conjugated to each biomolecule. 
     
     
         125 . The method of  claim 123 , wherein an average of from three to ten fluorescently labeled spacer molecules are conjugated to each biomolecule. 
     
     
         126 . The method of  claim 123 , wherein the fluorescently labeled spacer molecule is a multi-armed polymer. 
     
     
         127 . The method of  claim 126 , wherein the multi-armed polymer is a branched polyethylene glycol molecule. 
     
     
         128 - 131 . (canceled)