Direct cholesterol esterase assay

Abstract

A direct assay for cholesterol esterase is provided wherein the assay reagent comprises a tetrazolium salt, a cholesterol ester an exogenous electron carrier to create an assay sample. In one embodiment the reagent is mixed with a test sample and the presence of cholesterol esterase is detected by an optical response. In a second embodiment, the reagent is mixed with a test sample and the optical response is quantitated by comparison with standards to determine the cholesterol esterase activity in the test sample. Kits are also provided which comprise the reagent components.

We claim:  
     
         1 . A non-radioactive method of detecting an enzyme-substrate reaction comprising coupling said reaction with a tetrazolium salt and the transfer of free electrons from the reaction to the tetrazolium salt to produce a single colored or fluorescent formazan wherein the improvement comprises assaying a plurality of compounds for enzyme activity.  
     
     
         2 . The method of    claim 1    wherein said formazan produces a visual or instrumental monitoring at a single wavelength with a single extinction coefficient.  
     
     
         3 . The method of    claim 1    wherein said tetrazolium salt has the structure of:  
                 
 
       where R 1  is aryl or heteroaryl, and R 2  is aryl or heteroaryl, X can be any common counter ion.  
     
     
         4 . The method according to    claim 1    wherein the tetrazolium salt is dimethylthiazol tetrazolium (MTT).  
     
     
         5 . The method of    claim 1    wherein said test sample is further reacted with an exogenous electron carrier.  
     
     
         6 . The method of    claim 6    wherein said exogenous electron carrier is a phenazine salt of the structure:  
                 
 
       where R is an alkyl, Z is an alkyl, alkoxyl, halo, or nitro, and X is any common counter ion.  
     
     
         7 . The compound of    claim 6    wherein the phenazine is Phenazine Methosulfate.  
     
     
         8 . The method of    claim 1    where the enzyme is Esterase (EC3.1.1.1).  
     
     
         9 . The method of    claim 1    wherein the enzyme is Beta-glucuronidase (EC 3.2.1.31).  
     
     
         10 . The method of    claim 1    wherein the enzyme is Beta-glucosidase (EC 3.2.1.21).  
     
     
         11 . The method of    claim 1    where the enzyme is Cholesterol esterase (EC 3.1.1.13).  
     
     
         12 . The method of    claim 1    where the enzyme is Cholesterol oxidase (EC 1.1.3.6).  
     
     
         13 . The method of    claim 1    where the enzyme is Glucose oxidase (EC 1.1.3.4).  
     
     
         14 . The method of    claim 1    where the enzyme is Chloramphenicol acetyltransferase (EC 2.3.1.28).  
     
     
         15 . The method of    claim 1    where the enzyme is Beta-N-acetylglucosaminidase (EC 3.2.1.30).  
     
     
         16 . The method of    claim 1    where the enzyme is Neuraminidase (EC 3.2.1.18).  
     
     
         17 . The method of    claim 1    wherein said reaction concerns two or more enzymes which can be detected with a single substrate, wherein that substrate contains one or more sites for enzymatic action.  
     
     
         18 . The method of    claim 9    wherein said test sample is further reacted with an exogenous electron carrier.  
     
     
         19 . The method of    claim 1    wherein said test sample contains two or more enzymes and two or more substrates.  
     
     
         20 . The method of    claim 11    wherein said test sample is further reacted with an exogenous electron carrier.  
     
     
         21 . The method of    claim 1    wherein said test sample contains two or more enzymes, two or more substrates, and one or more inhibitors of said enzymes.  
     
     
         22 . The method of    claim 13    wherein said test sample is further reacted with an exogenous electron carrier.  
     
     
         23 . A kit of materials for performing the method of detection of enzyme reaction, consisting of a vial or packet of tetrazolium salt in an amount sufficient, when reacted with said test sample, to produce a colored or fluorescent formazan or a color or fluorescent change indicative of the transfer of electrons in the sample.  
     
     
         24 . A kit of materials for performing the method of detection of enzyme reaction, consisting of a vial or packet of tetrazolium salt and a vial or packet of exogenous electron carrier, in an amounts sufficient, when reacted with said test sample, to produce a colored or fluorescent formazan or a color or fluorescent change indicative of the transfer of electrons in the sample.