Reagents for improved cell-based assays

Abstract

The ability to efficiently determine the state of enzyme expression in cells has long been desired as material to the diagnosis of disease. This invention relates to cytoenzymology, and more particularly to improved reagents for use in cell-based assays, especially those using fluorogenic substrates.

1 . A reagent-containing composition for assaying the activity of an enzyme in an intact metabolically active whole cell in medium, said reagent-containing composition comprising a substrate of said enzyme or an analyte compound and an agent that enhances uptake of said substrate; 
 wherein said agent is present at a concentration sufficient to enhance the uptake of said substrate or analyte compound in a metabolically active cell, and is selected from the group consisting of glycerol, dimethyl sulfoxide (DMSO), trehalose, glutamate, betaine, ethylene glycol, threitol, ribose, and trimethylamine N-oxide, with the proviso that when said agent is dimethyl sulfoxide (DMSO), said agent will be present at a concentration of between about 20% and about 60% (v/v); and    wherein said reagent-containing composition, when added to said medium with minimal mixing, forms a layer over said intact metabolically active whole cell.    
     
     
         2 . The reagent of  claim 1 , wherein said enzyme is selected from the group consisting of a 5′ nucleotidase, acetylcholinesterase, an acid phosphatase, an acidic esterase, an acidic esterase I, an acidic esterase II, an acidic non-specific esterase, an adenosine deaminase, an adenosine monophosphate deaminase, an alkaline phosphatase, an aminopeptidase A, an aminopeptidase B, an aminopeptidase M, an Aminopeptidase N, an angiotensin converting enzyme, a caspase, a cathepsin B, a cathepsin B1, a cathepsin C, a cathepsin D, a cathepsin H, a cathepsin L, a cholinesterase, a cholinesterase, a chymotrypsin, a collagenase, a cytosine deaminase, a DPP I, a DPP II, a DPP IV, an elastase, an endopeptidase I, an endopeptidase II, an ester proteinase, a galactopyranosidase, a glucoronidase, a glycopyranossidase, a guanine deaminase, an HIV Protease, a lipase, a membrane associated endopeptidase I, a membrane associated endopeptidase II, a neutral endopeptidase, a neutral esterase, a neutral esterase I, a neutral esterase II, a neutral non-specific esterase, a nucleosidase, a pancreatin, a phospholipase A, a phospholipase C, a phospholipase D, a plasmin, a serine phosphatase, a tartrate resistant phosphatase, a tartrate resistant phosphatase, a threonine phosphatase, a thymidine deaminase, a tripeptidyl peptidase, a trypsin, a tyrosine phosphatase, a urokinase, and a γ-GT.  
     
     
         3 . The reagent of  claim 2 , wherein said enzyme is a caspase.  
     
     
         4 . The reagent of  claim 3 , wherein said caspase is caspase 1, caspase 3, caspase 6, caspase 8 or caspase 9.  
     
     
         5 . The reagent of  claim 1 , wherein said substrate or analyte compound comprises comprising an indicator group and one or more leaving groups, each of said leaving groups being selected for cleavage by said enzyme, said indicator group being in a first state when bonded to a leaving group, and being in a second state when said leaving group is cleaved from said indicator group by said enzyme; and 
 wherein said step (b) comprises sensing whether said second state of said indicator group is produced; wherein the production of said second state of said indicator group is indicative of the presence or activity of said enzyme in said metabolically active whole cell.    
     
     
         6  The reagent of  claim 5 , wherein said indicator group is a fluorescent, colorimetric, bioluminescent or chemiluminescent indicator group.  
     
     
         7 . The reagent of  claim 6 , wherein said indicator group is a fluorescent or chemiluminescent indicator group.  
     
     
         8 . The reagent of  claim 1 , wherein said uptake-enhancing agent is glycerol.  
     
     
         9 . The reagent of  claim 8 , wherein said glycerol concentration is between about 5% and about 60% (v/v).  
     
     
         10 . The reagent of  claim 9 , wherein said glycerol concentration is between about 20% and about 60% (v/v).  
     
     
         11 . The reagent of  claim 10 , wherein said glycerol concentration is between about 25% and about 40% (v/v).  
     
     
         12 . The reagent of  claim 1 , wherein said uptake-enhancing agent is dimethyl sulfoxide (DMSO).  
     
     
         13 . The reagent of  claim 1 , wherein said uptake-enhancing agent is glutamate.  
     
     
         14 . The reagent of  claim 13 , wherein said glutamate concentration is between about 0.25 M and about 2.0 M.  
     
     
         15 . The reagent of  claim 14 , wherein said glutamate concentration is between about 1 M and about 2 M.  
     
     
         16 . The reagent of  claim 1 , wherein said uptake-enhancing agent is betaine.  
     
     
         17 . The reagent of  claim 16 , wherein said betaine concentration is about 0.3 M or greater.  
     
     
         18 . The reagent of  claim 1 , wherein said uptake-enhancing agent is trehalose.  
     
     
         19 . The reagent of  claim 18 , wherein said trehalose concentration is between about 0.1 M and about 1.5 M.  
     
     
         20 . The reagent of  claim 1 , wherein said uptake-enhancing agent is ethylene glycol.  
     
     
         21 . The reagent of  claim 20 , wherein said ethylene glycol concentration is between about 2 M and about 7 M.  
     
     
         22 . The reagent of  claim 1 , wherein said uptake-enhancing agent is threitol.  
     
     
         23 . The reagent of  claim 22 , wherein said threitol concentration is between about 1 M and about 5 M.  
     
     
         24 . The reagent of  claim 1 , wherein said uptake-enhancing agent is ribose.  
     
     
         25 . The reagent of  claim 24 , wherein said ribose concentration is between about 0.4 M and about 4 M.  
     
     
         26 . The reagent of  claim 1 , wherein said uptake-enhancing agent is triethylamine N-oxide.  
     
     
         27 . The reagent of  claim 26 , wherein said triethylamine N-oxide concentration is between about 0.4 M and about 4 M.  
     
     
         28 . The reagent of  claim 6 , wherein said enzyme is selected from the group consisting of a 5′ nucleotidase, acetylcholinesterase, an acid phosphatase, an acidic esterase, an acidic esterase I, an acidic esterase II, an acidic non-specific esterase, an adenosine deaminase, an adenosine monophosphate deaminase, an alkaline phosphatase, an aminopeptidase A, an aminopeptidase B, an aminopeptidase M, an Aminopeptidase N, an angiotensin converting enzyme, a caspase, a cathepsin B, a cathepsin B1, a cathepsin C, a cathepsin D, a cathepsin H, a cathepsin L, a cholinesterase, a cholinesterase, a chymotrypsin, a collagenase, a cytosine deaminase, a DPP I, a DPP II, a DPP IV, an elastase, an endopeptidase I, an endopeptidase II, an ester proteinase, a galactopyranosidase, a glucoronidase, a glycopyranossidase, a guanine deaminase, an HIV Protease, a lipase, a membrane associated endopeptidase I, a membrane associated endopeptidase II, a neutral endopeptidase, a neutral esterase, a neutral esterase I, a neutral esterase II, a neutral non-specific esterase, a nucleosidase, a pancreatin, a phospholipase A, a phospholipase C, a phospholipase D, a plasmin, a serine phosphatase, a tartrate resistant phosphatase, a tartrate resistant phosphatase, a threonine phosphatase, a thymidine deaminase, a tripeptidyl peptidase, a trypsin, a tyrosine phosphatase, a urokinase, and a γ-GT.  
     
     
         29 . The reagent of  claim 7 , wherein said substrate or analyte compound comprises more than one leaving group, and wherein each of said substrate's leaving groups is cleaved consecutively by said enzyme to ultimately yield a free dye.  
     
     
         30 . The reagent of  claim 7 , wherein said indicator group is selected from the group consisting of rhodamine 110, rhodol, fluorescein, coumarin, and derivatives thereof.  
     
     
         31 . The reagent of  claim 30 , wherein said derivatives of rhodamine 110, rhodol, fluorescein and coumarin are selected from the group consisting of 4′(5′)thiofluorescein, 4′(5′)-aminofluorescein, 4′(5′)-carboxyfluorescein, 4′(5′)-chlorofluorescein, 4′(5′)-methylfluorescein, 4′(5′)-sulfofluorescein, 4′(5′)-aminorhodol, 4′(5′)-carboxyrhodol, 4′(5′)-chlororhodol, 4′(5′)-methylrhodol, 4′(5′)-sulforhodol; 4′(5′)-aminorhodamine 110, 4′(5′)-sulforhodamine 110, 4′(5′)thiorhodamine 110, 7-aminocoumarin, and sulfonated coumarin.  
     
     
         32 . The reagent of  claim 6 , wherein said assay detects the presence or absence of an abnormality in the activity of said enzyme by comparing the production of said second state of said indicator group by said test cell to the production of said second state of said indicator group by a reference normal cell.  
     
     
         33 . The reagent of  claim 32 , wherein said abnormality is a morphological or disease state.  
     
     
         34 . The reagent of  claim 33 , wherein said morphological state is an apoptotic state.  
     
     
         35 . The reagent of  claim 33 , wherein said disease state is a tumorigenic state.  
     
     
         36 . The reagent of  claim 6 , wherein said substrate or analyte compound contains a blocking group.  
     
     
         37 . The reagent of  claim 36 , wherein said blocking group is a Cbz blocking group.