Direct assay of cholesterol in skin removed by tape stripping

Abstract

Skin cholesterol is measured by applying an adhesive tape onto a selected area of the skin to adhere the tape to the selected skin area and stripping the tape off the selected skin area to obtain a sample representative of the outer stratum corneum layer of the skin, the sample adhering to the tape so as to have exposed skin constituents. The sample is assayed using a detector reagent that specifically binds to cholesterol and in addition has an indicator component that allows quantitation of the amount of cholesterol present in the exposed skin constituents.

1 . A method of measuring skin cholesterol, which comprises the steps of: 
 a) providing a tape comprising a backing member coated on at least one side thereof with a medical adhesive;    b) applying the tape onto a selected area of skin to adhere the tape to the selected skin area;    c) stripping the tape off the selected skin area to obtain a sample representative of an outer stratum corneum layer of the skin, the sample adhering to the tape so as to have exposed skin constituents;    d) providing a source of an affinity-enzymatic compound of formula A-C-B, wherein A is a detecting agent having affinity for cholesterol, B is an enzymatic visualizing agent and C is a binding agent linking said detecting agent and said visualizing agent to one another;    e) applying a predetermined amount of said affinity-enzymatic compound onto a predetermined surface area of said sample and allowing said compound to remain in contact therewith for a period of time sufficient to cause binding of said detecting agent to cholesterol present in said exposed skin constituents; and    f) applying a predetermined amount of a color developing agent onto the predetermined surface area of said sample, whereby said color developing agent reacts with said enzymatic visualizing agent to form a colored product having a color indicative of cholesterol level.    
     
     
         2 . A method as claimed in  claim 1 , wherein said detecting agent is selected from the group consisting of steroid glycosides, triterpene glycosides, hydrophobic proteins, polyene antibiotics and anti-cholesterol antibodies.  
     
     
         3 . A method as claimed in  claim 2 , wherein said detecting agent is a steroid glycoside.  
     
     
         4 . A method as claimed in  claim 3 , wherein said steroid glycoside is digitonin.  
     
     
         5 . A method as claimed in  claim 1 , wherein said enzymatic visualizing agent is an enzyme selected from the group consisting of peroxidase, alkaline phosphatase, urease, galactosidase, glucose oxidase and acetylcholinesterase.  
     
     
         6 . A method as claimed in  claim 5 , wherein said enzyme is peroxidase.  
     
     
         7 . A method as claimed in  claim 6 , wherein said peroxidase is horseradish peroxidase.  
     
     
         8 . A method as claimed in  claim 6 , wherein after step (e) said peroxidase is activated with hydrogen peroxide to form an activated peroxidase, and wherein the color developing agent used in step (f) reacts with said activated peroxidase to form said colored product.  
     
     
         9 . A method as claimed in  claim 8 , wherein in step (f) a predetermined amount of an aqueous solution containing hydrogen peroxide and said color developing agent is applied onto said predetermined surface area of said sample.  
     
     
         10 . A method as claimed in  claim 9 , wherein said color developing agent is selected from the group consisting of  2 , 2 ′-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) and 3,3′,5,5′-tetramethyl benzidine.  
     
     
         11 . A method as claimed in  claim 10 , wherein said color developing agent is 3,3′5,5′-tetramethyl benzidine.  
     
     
         12 . A method as claimed in  claim 1 , wherein said binding agent is a copolymer of maleic anhydride and N-vinylpyrrolidone.  
     
     
         13 . A method as claimed in  claim 1 , wherein said backing member is formed of polyester.  
     
     
         14 . A method as claimed in  claim 1 , wherein said medical adhesive is a pressure-sensitive adhesive.  
     
     
         15 . A method as claimed in  claim 1 , wherein said medical adhesive is an acrylic based adhesive.  
     
     
         16 . A method as claimed in  claim 1 , wherein said medical adhesive is a synthetic rubber elastomer adhesive.  
     
     
         17 . A method as claimed in  claim 1 , wherein said medical adhesive is a silicone based adhesive.  
     
     
         18 . A method as claimed in  claim 1 , wherein said medical adhesive comprises an elastomer formed of block polymers of styrene-isoprene-styrene or styrene-butadiene-styrene.  
     
     
         19 . A kit for use in carrying out a method as defined in  claim 1 , comprising: 
 said tape;    said source of affinity-enzymatic compound of formula A-C-B, wherein A, B and C are as defined in  claim 1;  and    a source of said color developing agent.    
     
     
         20 - 35 . (canceled)  
     
     
         36 . A method of measuring skin cholesterol, which comprises the steps of: 
 a) providing a tape comprising a backing member coated on at least one side thereof with a medical adhesive;    b) applying the tape onto a selected area of skin to adhere the tape to the selected skin area;    c) stripping the tape off the selected skin area to obtain a sample representative of an outer stratum corneum layer of the skin, the sample adhering to the tape so as to have exposed skin constituents;    d) providing a source of an affinity signal-generating compound of formula A-C-B′, wherein A is a detecting agent having affinity for cholesterol, B′ is a signal-generating indicator agent and C is a binding agent linking said detecting agent and said indicator agent to one another;    e) applying a predetermined amount of said affinity signal-generating compound onto a predetermined surface area of said sample and allowing said compound to remain in contact therewith for a period of time sufficient to cause binding of said detecting agent to cholesterol present in said exposed skin constituents; and    f) measuring the signal generated by said indicator agent to provide a value indicative of cholesterol level.    
     
     
         37 - 58 . (canceled)  
     
     
         59 . A kit for use in carrying out a method as defined in  claim 36 , comprising: 
 said tape; and    said source of affinity signal-generating compound of formula A-C-B′, wherein A, B′ and C are as defined in  claim 36 .    
     
     
         60 - 75 . (canceled)  
     
     
         76 . A method of measuring skin cholesterol, which comprises the steps of: 
 a) providing a tape comprising a backing member coated on at least one side thereof with a medical adhesive;    b) applying the tape onto a selected area of skin to adhere the tape to the selected skin area;    c) stripping the tape off the selected skin area to obtain a sample representative of an outer stratum corneum layer of the skin, the sample adhering to the tape so as to have exposed skin constituents;    d) providing a source of cholesterol oxidase as a detecting agent having affinity for cholesterol;    e) applying a predetermined amount of cholesterol oxidase onto a predetermined surface area of said sample and allowing the cholesterol oxidase to remain in contact therewith for a period of time sufficient to cause oxidation of cholesterol and formation of hydrogen peroxide; and    f) measuring the amount of hydrogen peroxide formed in step (e), the amount of hydrogen peroxide measured being indicative of cholesterol level.    
     
     
         77 - 109 . (canceled)  
     
     
         110 . A tape stripping device for use in obtaining skin samples, the device comprising: 
 a) a sampling member having a surface,    b) a tape provided on at least a portion of the surface of the sampling member, the tape having a medical adhesive presented away from the surface; and    c) a closure member adapted to engage the sampling member and sealing the tape within the device.    
     
     
         111 . A device as claimed in  claim 110 , wherein said tape is sealed within the device when the closure member engages the sampling member.  
     
     
         112 . A device as claimed in  claim 111 , wherein at least the closure member or the sampling member is provided with a peripheral rim, and the other of the closure member or the sampling member is provided with a peripheral groove adapted to receive the rim so that the tape is sealed within the device.  
     
     
         113 . A device as claimed in  claim 110 , wherein the closure member is connected to the sampling member by a hinge.  
     
     
         114 . A device as claimed in  claim 110 , wherein at least a portion of the sampling member is adapted to be cut from the closeable device to form a dipstick, said dipstick having a first end thereof devoid of tape, and a second end thereof with tape.  
     
     
         115 . A device as claimed in  claim 110 , wherein at least a portion of the sampling member is adapted to be cut from the closeable device to form a disk, said disk having the tape provided on one face thereof.