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.

We claim: 
     
         1 . A conjugated antibody, comprising an antibody that is conjugated to a plurality of fluorescent labels, wherein the conjugated antibody comprises one or more of the following characteristics:
 (i) a Fluorescent Ratio on a per fluorescent label basis that is equal to or greater than 0.5;   (ii) at least four fluorescent labels are conjugated to the antibody; and/or   (iii) a total fluorescence of the antibody is at least 20 percent greater than fluorescence of an unconjugated fluorescent label.   
     
     
         2 . The conjugated antibody of  claim 1 , wherein individual fluorescent labels of the plurality of fluorescent labels are conjugated to the antibody by one or more multi-armed polymers. 
     
     
         3 . The conjugated antibody of  claim 1 , wherein individual fluorescent labels of the plurality of fluorescent labels are conjugated to the antibody by two to ten multi-armed polymers. 
     
     
         4 . The conjugated antibody of  claim 3 , wherein the arms of the multi-armed polymer are composed of a chemical of a type selected from a polyethylene glycol, a polysaccharide, or a polypeptide. 
     
     
         5 . The conjugated antibody of  claim 1 , wherein an average brush distance between the fluorescent labels ranges from 200 to 800 Angstroms. 
     
     
         6 . The conjugated antibody of  claim 1 , wherein individual fluorescent labels of the plurality of fluorescent labels are separated from the antibody by at least 16 covalent bonds. 
     
     
         7 . The conjugated antibody of  claim 1 , wherein individual fluorescent labels of the plurality of fluorescent labels are separated from the antibody by 16 to 800 covalent bonds. 
     
     
         8 . The conjugated antibody of  claim 1 , wherein the fluorescent labels are positively charged. 
     
     
         9 . The conjugated antibody of  claim 1 , wherein the fluorescent labels are dyes independently selected from a cyanine dye; a benzo-rhodamine dye; a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dye; a fluorescein dye; a benzopyrillium dye, including derivatives thereof; a coumarin dye, a benzofuran dye; a quinoline dye; a quinazolinone dye; an indole dye; a furan dye; a benzazole dye; a borapolyazaindacene dye; or a xanthene dye. 
     
     
         10 . The conjugated antibody of  claim 1 , wherein the fluorescent labels comprise a moiety selected from xanthene, coumarin, cyanine, pyrene, oxazine, borapolyazaindacene, benzopyrylium, or carbopyronine. 
     
     
         11 . The conjugated antibody of  claim 1 , wherein the fluorescent labels comprise a moiety selected from dinitrophenyl, Fluorescein, hexachlorofluorescein, 4′,5′-dichloro-2′,7′-dimethoxy-5(6)-carboxyfluorescein, 6-carboxyl-X-rhodamine, tetrachlorofluorescein, tetramethyl rhodamine, or 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE). 
     
     
         12 . The conjugated antibody of  claim 1 , wherein a number of the fluorescent labels conjugated to the first antibody is from 2 to 20. 
     
     
         13 . The conjugated antibody of  claim 1 , wherein a number of the fluorescent labels conjugated to the first antibody is from 4 to 10. 
     
     
         14 . A conjugated antibody, comprising a plurality of fluorescent labels and a spacer molecule covalently attached to a first antibody, wherein the conjugated antibody exhibits increased fluorescence relative to a second conjugated antibody that is the same as and has a same degree of fluorescent labeling as the first conjugated antibody but that is lacking a spacer molecule covalently attached to the second conjugated antibody. 
     
     
         15 . The conjugated antibody of  claim 14 , wherein the increased fluorescence exhibited by the conjugated antibody ranges from 1.5 to 3.5 times more fluorescence than the second conjugated antibody. 
     
     
         16 . The conjugated antibody of  claim 14 , wherein the presence of the spacer molecule increases detectable fluorescence of the fluorescent labels by 20% to 500%. 
     
     
         17 . The conjugated antibody of  claim 14 , wherein the spacer molecule comprises polyethylene glycol (PEG). 
     
     
         18 . The conjugated antibody of  claim 14 , wherein the fluorescent labels are positively charged.