WO2005106018A1 - Dosage direct du cholesterol cutane dans des echantillons de peau preleves par decollement de bande - Google Patents

Dosage direct du cholesterol cutane dans des echantillons de peau preleves par decollement de bande Download PDF

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Publication number
WO2005106018A1
WO2005106018A1 PCT/CA2005/000642 CA2005000642W WO2005106018A1 WO 2005106018 A1 WO2005106018 A1 WO 2005106018A1 CA 2005000642 W CA2005000642 W CA 2005000642W WO 2005106018 A1 WO2005106018 A1 WO 2005106018A1
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WO
WIPO (PCT)
Prior art keywords
kit
tape
agent
skin
adhesive
Prior art date
Application number
PCT/CA2005/000642
Other languages
English (en)
Inventor
Peter Horsewood
Robert Zawydiwski
Original Assignee
Premd Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Premd Inc. filed Critical Premd Inc.
Priority to BRPI0510352-5A priority Critical patent/BRPI0510352A/pt
Priority to CA002563973A priority patent/CA2563973A1/fr
Priority to CN2005800138932A priority patent/CN1961078B/zh
Priority to JP2007509839A priority patent/JP2007534326A/ja
Priority to AU2005238099A priority patent/AU2005238099B2/en
Priority to EP05738502A priority patent/EP1745146A4/fr
Priority to MXPA06012326A priority patent/MXPA06012326A/es
Publication of WO2005106018A1 publication Critical patent/WO2005106018A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/02—Instruments for taking cell samples or for biopsy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B10/0045—Devices for taking samples of body liquids
    • A61B10/0064—Devices for taking samples of body liquids for taking sweat or sebum samples
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B17/32—Surgical cutting instruments
    • A61B17/3205—Excision instruments
    • A61B17/32053—Punch like cutting instruments, e.g. using a cylindrical or oval knife
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • A61B2010/0003—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements including means for analysis by an unskilled person
    • A61B2010/0006—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements including means for analysis by an unskilled person involving a colour change

Definitions

  • TITLE DIRECT ASSAY OF SKIN CHOLESTEROL IN SKIN SAMPLES REMOVED BY TAPE STRIPPING
  • the present invention relates to a method of measuring skin cholesterol. More particularly, the invention pertains to a method for the direct assay of cholesterol in skin samples removed by tape stripping, with a view to identifying individuals at risk of having atherosclerosis as well as those at risk of developing atherosclerosis and similar diseases associated with and attributable to high cholesterol levels.
  • Cost effective prevention of atherosclerosis requires the identification of individuals at risk, thereby allowing their medical treatment and change of life style.
  • a desired goal is identifying those individuals belonging to the high-risk group but there are difficulties in selecting optimum methods for discriminating individuals at risk.
  • a widely used method for identifying individuals at risk of having atherosclerosis is based on the measurement of total cholesterol levels in venous blood plasma (Consensus Conference on Lowering Blood Cholesterol to Prevent Heart Disease, JAMA, 1985, 253, pg. 2080). Patients are considered to be at high-risk if their cholesterol level is over 240 mg/dL and there have been recent moves to lower this threshold level to lower values. [0005] However, total cholesterol levels alone do not accurately predict a patient's risk level.
  • HDL high- density lipoprotein
  • U. S. Patent No. 4,458,686 describes a method of quantifying various compounds in the blood directly under the skin or on its surface. The method is based on measuring oxygen concentration changes electrochemically, for instance, via polarography. In the case of non-volatile substances that do not diffuse through the skin, it is necessary to implant enzymes under the skin to effect oxygen changes at the skin surface. This patent also discloses the potential of using such methods to quantify the amount of cholesterol using cholesterol oxidase. The complex instrumentation and procedures needed require the services of highly skilled personnel for making measurements, thus limiting the usefulness of the method for screening large numbers of people.
  • U. S. Patent No. 5,489,510 describes a non-invasive method for the visual identification of cholesterol on skin using a reagent having a specific cholesterol binding component in combination with a reagent having an indicator component to provide a visual color change corresponding to the presence of the component bound to cholesterol of the skin.
  • the method overcomes many of the objections of earlier procedures and meets many of the desired goals required for a simple mass screening to identify individuals at risk of having atherosclerosis. The procedure is done directly on the palmar skin and, while it is quick and simple, it requires all individuals to be tested to be present at a doctor's office or clinic where the test is conducted. This of course limits effective large scale screening.
  • High performance liquid chromatography (HPLC) and gas liquid chromatography in conjunction with mass spectrometry were used to separate and analyze the lipids.
  • the analytical methods are complex, but more importantly, the use of corrosive and irritant organic solvent systems to extract human skin for routine determinations is not practical.
  • the lipid profile of the stratum corneum layer of skin has been determined using a tape stripping method as described by A. Weerheim and M. Ponec (Arch. Dermatol. Res., 191-199, 293, 2001).
  • lipids, including cholesterol were solvent extracted from stratum corneum after tape stripping of skin.
  • the resultant lipid extract was separated by high performance thin-layer chromatography. This method is very laborious.
  • 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 the detecting agent and the visualizing agent to one another; e) applying a predetermined amount of the affinity- enzymatic compound onto a predetermined surface area of the sample and allowing the compound to remain in contact therewith for a period of time sufficient to cause binding of the detecting agent to cholesterol present in the
  • the detecting agent in the aforesaid method is selected from the group consisting of steroid glycosides, triterpene glycosides, hydrophobic proteins, polyene antibiotics and anti-cholesterol antibodies.
  • the detecting agent is a steroid glycoside, and the steroid glycoside is digitonin.
  • the enzymatic visualizing agent is an enzyme selected from the group consisting of peroxidase, alkaline phosphatase, urease, galactosidase, glucose oxidase and acetylcholinesterase.
  • the enzyme is peroxidase, and the peroxidase is horseradish peroxidase.
  • step (e) the peroxidase is activated with hydrogen peroxide to form an activated peroxidase, and wherein the color developing agent used in step (f) reacts with the activated peroxidase to form the colored product.
  • step (f) a predetermined amount of an aqueous solution containing hydrogen peroxide and the color developing agent is applied onto the predetermined surface area of the sample.
  • the 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.
  • the color developing agent is 3,3'5,5'-tetramethyl benzidine.
  • the binding agent is a copolymer of maleic anhydride and N- vinylpyrrolidone.
  • the backing member of the tape is formed of polyester.
  • the medical adhesive is a pressure-sensitive adhesive.
  • the medical adhesive is an acrylic based adhesive.
  • the medical adhesive is a synthetic rubber elastomer adhesive.
  • the medical adhesive is a silicone based adhesive.
  • the medical adhesive comprises an elastomer formed of block polymers of styrene- isoprene-styrene or styrene-butadiene-styrene.
  • 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 1 is a signal-generating indicator agent and C is binding agent linking the detecting agent and the indicator agent to one another; e) applying a predetermined amount of the affinity signal- generating compound onto a predetermined surface area of the sample and allowing the compound to remain in contact therewith for a period of time sufficient to cause binding of the detecting agent to cholesterol present in the exposed skin constituents
  • the detecting agent in the aforesaid method is selected from the group consisting of steroid glycosides, triterpene glycosides, hydrophobic proteins, polyene antibiotics and anti-cholesterol antibodies.
  • the detecting agent is a steroid glycoside, and the steroid glycoside is digitonin.
  • the indicator agent in the aforesaid method is selected from the group consisting of dyes, fluorophores, radioisotopes, metal sol compounds and chemiluminescent compounds.
  • the indicator agent is a dye.
  • the indicator agent is a fluorophore.
  • the indicator agent is a radioisotope.
  • the indicator agent is a metal- sol compound.
  • the indicator agent is a chemiluminescent compound.
  • step (f) is carried out by spectrophotometry. In another aspect of the invention, step (f) is carried out by colorimetry. In yet a further aspect of the invention, step (f) is carried out by fluorometry. A further aspect of the invention has step (f) is carried out by means of a radioactivity sensor. In a further aspect of this invention, step (f) is carried out by luminometry.
  • the binding agent is a copolymer of maleic anhydride and N-vinylpyrrolidone.
  • the backing member of the tape is formed of polyester.
  • the medical adhesive is a pressure-sensitive adhesive.
  • the medical adhesive is an acrylic based adhesive.
  • the medical adhesive is a synthetic rubber elastomer adhesive.
  • the medical adhesive is a silicone based adhesive.
  • the medical adhesive comprises an elastomer formed of block polymers of styrene- isoprene-styrene or styrene-butadiene-styrene.
  • 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 the 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.
  • step (f) is carried out by means of an electrochemical sensor. In another aspect of the method, step (f) is carried out amperometrically using an electrode. In a further aspect of the method, step (f) is carried out by spectrophotometry after addition of peroxidase and a colorimetric indicator.
  • the peroxidase is horseradish peroxidase.
  • the colorimetric indicator is 2,2'- azino-di-(3-ethylbenzthiazoline-6-sulfonic acid). In yet a further aspect of the invention, the colorimetric indicator is 3, 3', 5, 5'- tetramethyl benzidine. In a further aspect of the invention, the colorimetric indicator is a multicomponent oxidative coupling reagent of Trinder or Ngo-Lenhoff type.
  • the backing member of the tape is formed of polyester.
  • the medical adhesive is a pressure-sensitive adhesive.
  • the medical adhesive is an acrylic based adhesive.
  • the medical adhesive is a synthetic rubber elastomer adhesive.
  • the medical adhesive is a silicone based adhesive.
  • the medical adhesive comprises an elastomer formed of block polymers of styrene- isoprene-styrene or styrene-butadiene-styrene.
  • the present invention also provides, in a fourth aspect thereof, a kit for use in carrying out a method according to the first aspect.
  • the kit comprises: the aforesaid tape; the aforesaid source of affinity-enzymatic compound of formula A-C-B, wherein A, B and C are as defined above; and - a source of the aforesaid color developing agent.
  • the detecting agent in the aforesaid kit is selected from the group consisting of steroid glycosides, triterpene glycosides, hydrophobic proteins, polyene antibiotics and anti-cholesterol antibodies.
  • the detecting agent is a steroid glycoside
  • the steroid glycoside is digitonin.
  • the enzymatic visualizing agent is an enzyme selected from the group consisting of peroxidase, alkaline phosphatase, urease, galactosidase, glucose oxidase and acetylcholinesterase.
  • the enzyme is peroxidase, and the peroxidase is horseradish peroxidase.
  • the aforesaid kit further includes an aqueous solution containing hydrogen peroxide, the color developing agent being present in said solution.
  • the 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.
  • the color developing agent is 3,3'5,5'-tetramethyl benzidine.
  • the binding agent is a copolymer of maleic anhydride and N-vinylpyrrolidone.
  • the backing member of the tape is formed of polyester.
  • the medical adhesive is a pressure-sensitive adhesive.
  • the medical adhesive is an acrylic based adhesive.
  • the medical adhesive is a synthetic rubber elastomer adhesive.
  • the medical adhesive is a silicone based adhesive.
  • the medical adhesive comprises an elastomer formed of block polymers of styrene- isoprene-styrene or styrene-butadiene-styrene.
  • the invention further provides, in a fifth aspect thereof, a kit for use in carrying out a method according to the second aspect.
  • the kit comprises: - the aforesaid tape; and the aforesaid source of affinity signal-generating compound of formula A-C-B', wherein A, B' and C are as defined above.
  • the detecting agent in the aforesaid kit is selected from the group consisting of steroid glycosides, triterpene glycosides, hydrophobic proteins, polyene antibiotics and anti-cholesterol antibodies.
  • the detecting agent is a steroid glycoside, and the steroid glycoside is digitonin.
  • the indicator agent in the aforesaid kit is selected from the group consisting of dyes, fluorophores, radioisotopes, metal sol compounds and chemiluminescent compounds.
  • the indicator agent is a dye.
  • the indicator agent is a fluorophore.
  • the indicator agent is a radioisotope.
  • the indicator agent is a metal- sol compound.
  • the indicator agent is a chemiluminescent compound.
  • the binding agent is a copolymer of maleic anhydride and N-vinylpyrrolidone.
  • the backing member of the tape is formed of polyester.
  • the medical adhesive is a pressure-sensitive adhesive.
  • the medical adhesive is an acrylic based adhesive.
  • the medical adhesive is a synthetic rubber elastomer adhesive.
  • the medical adhesive is a silicone based adhesive.
  • the medical adhesive comprises an elastomer formed of block polymers of styrene- isoprene-styrene or styrene-butadiene-styrene.
  • the invention additionally provides, in a sixth aspect thereof, a kit for use in carrying out a method according to the third aspect.
  • the kit comprises: - the aforesaid tape; and the aforesaid source of cholesterol oxidase.
  • the peroxidase is horseradish peroxidase.
  • colorimetric indicator is of 2,2'-azino-di-(3- ethylbenzthiazoline-6-sulfonic acid).
  • the colorimetric indicator is 3,3'5,5'-tetramethyl benzidine.
  • the backing member of the tape is formed of polyester.
  • the medical adhesive is a pressure-sensitive adhesive.
  • the medical adhesive is an acrylic based adhesive.
  • the medical adhesive is a synthetic rubber elastomer adhesive.
  • the medical adhesive is a silicone based adhesive.
  • the medical adhesive comprises an elastomer formed of block polymers of styrene- isoprene-styrene or styrene-butadiene-styrene.
  • the tape is carried by a closeable device, the closeable device having a sampling member that carries the tape, and a closure member adapted to engage the sampling member and retain the tape within the device. It is preferable that the tape is sealed within the device when the closure member engages the sampling member.
  • 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.
  • the closure member can be connected to the sampling member by a hinge.
  • At least a portion of the sampling member is adapted to be cut from the closeable device to form a dipstick, the dipstick having a first end thereof devoid of tape, and a second end thereof with the tape.
  • kits can further comprise a cutting tool adapted to cut the disk from the device.
  • the closeable device can be provided with a marker on an outside surface thereof.
  • the cutting tool can be provided with a plunger to eject the disk from the end of the cutter after the disk is cut.
  • the invention also provides for 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 retain the tape within the device.
  • the tape is sealed within the device when the closure member engages the sampling member.
  • 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.
  • the closure member can be connected to the sampling member by a hinge.
  • At least a portion of the sampling member is adapted to be cut from the closeable device to form a dipstick, the dipstick having a first end thereof devoid of tape, and a second end thereof with the tape.
  • the sampling member is adapted to be cut from the closeable device to form a disk, the disk having the tape provided on one face thereof.
  • Applicant has found quite surprisingly that the measurement of skin cholesterol can be carried out directly on the skin sample adhering to the aforementioned tape.
  • the procurement of skin samples removed by tape stripping from donor individuals allows assays to be conducted at distant and centralized sites and also allows assays from many individuals to be run concurrently.
  • the method according to the invention is suitable for large scale screening of individuals for assessing their risk of cardiovascular disease.
  • Figure 1 is a top view of a sampling device as used in Example
  • Figure 2 is a fragmentary view of the sampling device illustrated in Figure 1 , showing details of the sampling member thereof;
  • Figure 3 is a perspective view of a dipstick cut from the sampling device of this invention.
  • Figure 4 is a perspective view of a disk cut from the sampling device of this invention in an alternative embodiment
  • Figure 5 is a cross-sectional view of a disk from Fig. 4 in the well of a microwell plate;
  • Figure 6 is a perspective view of the sampling device with cutting tool to produce a disk of Figure 4;
  • Figure 7 is a cross-sectional view of the sampling device with cutting tool to produce a disk of Figure 4; and [0063] Figure 8 is a cross-sectional view showing the cutting tool placing the disk in the well of a microwell plate.
  • a tape comprising a backing member formed of polyester.
  • the tape is coated on at least one side thereof with a medical adhesive.
  • medical adhesive refers to an adhesive which is hypoallergic and safe for application to the skin.
  • Such an adhesive is preferably a pressure-sensitive adhesive, for example, an adhesive comprising an elastomer formed of block polymers of styrene- isoprene-styrene or styrene-butadiene-styrene.
  • any adhesive suitable for use with this invention is a medical adhesive as defined above to ensure there will be generally no problems with allergic reactions when the adhesive was applied to the skin for sampling.
  • the inventors tested several types of adhesives for use in taking a skin sample; the majority of these were pressure sensitive acrylic based adhesives, but several synthetic rubber type elastomer adhesives and silicone based adhesives were also tested.
  • the inventors have found that synthetic rubber adhesives based on block copolymers of styrene and butadiene or styrene and isoprene perform well for this invention.
  • An example of a synthetic rubber adhesive is a synthetic KratonTM type adhesive (latex free) based on a block copolymer of styrene and butadiene. Such an adhesive provided better stability for skin samples to facilitate transportation of the samples for subsequent analysis.
  • a further preferred adhesive tape for use in the method of the invention is a double-coated pressure-sensitive medical grade tape.
  • a medical grade tape examples are those sold by 3M under Product #9877, or by Adhesive Research, Inc. under Product #8570.
  • a list of some of the other tapes that have been tested by the inventors is shown in the accompanying table. The one requirement that is constant is the use of a medical grade tape that is hypoallergenic. •
  • Double-coated pressure-sensitive tapes are generally available with an easily removable protective liner.
  • the liner protects the tape from adhering until it is removed and keeps the adhesive from becoming contaminated. Liners may be placed on either side of the double-coated tape or the tape may have a single liner and be wound onto itself, thereby protecting both surfaces.
  • Liners with differential release properties may be used so that a first side of adhesive may be exposed while protecting the second adhesive surface.
  • a double-coated tape with differential liners is particularly advantageous for skin sampling. Removal of the first liner allows the tape to be stuck onto the backing support of a sampling device and leaves the skin- sampling side covered with the second liner. This second liner protects the skin sampling adhesive area from sticking and from contamination until it is to be used. When required for skin sampling, the second liner is removed.
  • the tape can be applied onto any part of skin, but the most suitable part is the surface of a palm because the palm does not have sebaceous glands whose secretions contain cholesterol which may affect results. Additionally, the skin on the palm is readily accessible for sampling.
  • the total amount of cholesterol present in the skin sample on the adhesive tape is related to the size of the skin sample obtained. Moreover, a consistent skin sample size is required in order to compare relative levels of skin cholesterol between different individuals.
  • Obtaining consistently sized skin samples from various individuals (or repeated samples from the same individual) is accomplished by the following steps. First, as previously described, the skin sample is taken by applying the adhesive tape repeatedly to the skin such that it becomes saturated with skin and is no longer sticky. The tape becomes saturated with skin after about three to seven applications and ten applications are routinely done to ensure saturation.
  • a fixed sized area for example, as will be hereinafter become apparent from Examples 2 and 3
  • a fixed sized area for example, as will be hereinafter become apparent from Examples 2 and 3
  • immersed in standardized volumes of detector and indicator reagents as will also be described hereinafter.
  • the sampling device After skin sampling, the sampling device is closed and shipped to a central laboratory for assay of cholesterol.
  • the detecting agent A can be for example a steroid glycoside, a triterpene glycoside, a hydrophobic protein, a polyene antibiotic or an anti-cholesterol antibody.
  • Use is preferably made of a steroid glycoside, such as digitonin.
  • the binding agent C is preferably a copolymer of maleic anhydride and N-vinylpyrrolidone.
  • the enzymatic visualizing agent B is preferably an enzyme selected from the group consisting of peroxidase, alkaline phosphatase, urease, galactosidase, glucose oxidase and acetylcholinesterase.
  • Peroxidase such as horseradish peroxidase is preferred.
  • the peroxidase is activated with hydrogen peroxide to form an activated peroxidase, and the color developing agent used in step (f) reacts with the activated peroxidase to form the aforesaid colored product.
  • a predetermined amount of an aqueous solution containing hydrogen peroxide and the color developing agent is applied in step (f) onto the predetermined surface area of the sample.
  • suitable color developing agents which can be used in step (f) include 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) and 3, 3', 5,5'- tetramethyl benzidine. 3,3'5,5'-Tetramethyl benzidine is preferred.
  • the indicator agent B' can be for example a dye, a fluorophore, a radioisotope, a metal sol compound or a chemiluminescent compound.
  • step (f) can be carried out by spectrophotometry, such as colorimetry.
  • step (f) can be carried out by fluorometry.
  • step (f) can be carried out by means of a radioactivity sensor.
  • step (f) can be carried out by colorimetry.
  • step (f) can be carried out by luminometry.
  • step (f) is preferably carried out by means of an electrochemical sensor, for instance, amperometrically using an electrode.
  • Step (f) can also be carried out by spectrophotometry after addition of peroxidase and a colorimetric indicator.
  • the peroxidase used is preferably horseradish peroxidase.
  • suitable colorimetric indicators which can be used include 2,2'-azino-di-(3-ethylbenzthiazoline-6- sulfonic acid) and 3,3', 5, 5'- tetramethyl benzidine.
  • a colorimetric indicator consisting of a multicomponent oxidative coupling reagent of Trinder or Ngo- Lenhoff type can also be used.
  • the aforementioned kit for carrying out the method according to the third aspect of the invention further comprises a source of peroxidase and a source of the colorimetric indicator.
  • EXAMPLE 1 A double-coated pressure-sensitive medical grade tape having a protective release liner on an upper sampling side and sold by Adhesive Research, Inc. was used. A piece of tape 1 inch by 1 inch was cut. The piece of tape was stuck, using the exposed, lower adhesive surface to one end of a 1 inch by 3 inch thin plastic (white polystyrene) member, leaving a 1 inch by 2 inch piece of uncovered plastic as a handle for applying the tape to the skin and for labeling the sample.
  • a 1 inch by 3 inch thin plastic white polystyrene
  • the protective liner was removed and the exposed adhesive area applied to a clean dry section of skin. Pressure was applied to the back of the plastic member over the adhesive area to effect good contact of the adhesive with the skin. The plastic member with the attached tape and stratum corneum sample was then peeled from the skin.
  • the sample was cut into four equal pieces each measuring ⁇ inch by ⁇ inch.
  • One piece was placed in a well of a 12 well tissue culture plate, or similar container, with the skin sampling side facing up.
  • An aliquot of reagent of the type A-C-B was then applied onto a predetermined surface area of the skin sample.
  • the A-C-B reagent used was a conjugate of digitonin (A) linked to horseradish peroxidase (B) through a maleic anhydride-N- vinylpyrrolidone copolymer (C).
  • the reagent was left in contact with the skin sample for fifteen minutes at room temperature, after which it is removed by aspiration.
  • the sample was washed with three separate aliquots of a wash solution to remove non-specifically bound reagent.
  • the piece was then placed in a new, clean well of a 12 well tissue culture plate, or similar container, with the skin sampling side facing up.
  • An aliquot of substrate solution was applied to the sample and left in contact with the skin sample for about fifteen minutes at room temperature.
  • the substrate, solution used was Enhanced K-Blue reagent available from Neogen Corp.(Lexington, KY.USA) and containing hydrogen peroxide and tetramethyl benzidine as color developing agent.
  • An aliquot of the developed substrate solution was removed from the well and added to an aliquot of 1 N sulfuric acid in a well of a 96 well microwell plate.
  • the optical density of the resulting solution which is a measure of the amount of cholesterol in the skin sample, was read at about 450 nm on a plate reading spectrophotometer.
  • the sampling device which is generally designated by reference numeral 10, is formed of plastic (polypropylene) and comprises a sampling member 12 connected to a closure member 14 by an integral hinge 16.
  • the closure member 14 has a peripheral rim 18 and four pins 20, adapted to lock into, respectively, a peripheral groove 22 and four holes 24 formed in the sampling member 12. Folding the hinge 16 causes engagement of the rim 18 with the groove 22 and of the pins 20 with the holes 24, thereby ensuring that the two halves of the device 10 remain closed and sealed to prevent dust and contamination of the interior surfaces.
  • the outer surface (not shown in Figs. 1 and 2) of the closure member 14 has a flat area for receiving a label and barcode strip, for sample identification.
  • the sampling member 12 and closure member 14 are respectively provided with finger-tabs 26 and 28 for opening the device 10.
  • the release liner 32 is wider than the adhesive tape 30, thereby defining a strip 32' along one edge with no attached tape. This strip 32' of liner overhangs the edge of the device to form a tab for easy removal of the liner. Immediately before use, the liner 32 is removed using the overhanging tab 32' and this exposes the adhesive of the tape 30 for skin sampling.
  • the palmar skin area for sampling was cleaned and dried.
  • the tape 30 with the exposed adhesive was applied onto the palm.
  • the tape 30 was pressed against the skin by applying pressure to the back of the sampling member 12 above the adhesive area, thereby causing adherence of the stratum corneum layer.
  • the device 10 was peeled away, reapplied to a new area of the palm and again pressed to the skin.
  • the device is peeled away and applied to the palmar skin in this way for a total of 10 applications.
  • At least two small dipsticks 40 (see Fig. 3) about four mm in width were cut from the device 10 after application to the skin as follows. Referring to Fig. 2, an end portion of the sampling member 12 was removed by cutting along the portion of groove 22, which is adjacent to the tab 26. Three cuts were then made along guide lines 36 (shown in Fig. 2) molded into the sampling member 12, to delineate the four mm sticks, cutting from the edge to just past the centre line. The two 4 mm wide sticks were released from the sampling member 12 by making a third cut across the center of the member 12, using guide line 38 molded into the member 12. Sticks 40 had a first end portion 42 devoid of tape and a second end portion 44 with tape having the skin sample adhered thereto.
  • the sticks were each placed into approximately 100 ⁇ L solution of an A-C-B reagent in wells of a 96 well microwell plate (not illustrated).
  • the reagent was a conjugate of digitonin (A) linked to horseradish peroxidase (B) through a maleic anhydride-N-vinylpyrrolidone copolymer (C) and was used at a concentration of approximately 1 ⁇ g/mL.
  • the sticks were left in the solution for about fifteen minutes at room temperature, after which they were removed and placed into new wells of a microwell plate containing approximately 200 ⁇ L of wash solution.
  • the microwell plate was agitated to effect washing and after about one minute the sticks were removed to new wells containing approximately 200 ⁇ L of fresh wash solution and again agitated for about one minute. Washing with agitation was done a third time, after which the sticks were removed and placed in approximately 100 ⁇ L of a substrate solution (Enhanced K-Blue reagent). The sticks were then incubated with the substrate solution, in the dark, for about fifteen minutes at room temperature. The microwell plate can be shaken during this step.
  • a substrate solution Enhanced K-Blue reagent
  • the sticks can then be removed. Approximately one hundred (100) ⁇ L of 1 N sulfuric acid is then added to the wells with the substrate solution to stop further reaction, and the optical density of the resulting solution was read at about 450 nm on a plate reading spectrophotometer, to provide a measure of the amount of cholesterol in the skin sample.
  • EXAMPLE 3 To allow many samples from Example 2 to be processed together requires that the dipsticks 40 be held in a configuration that matches that of a standard 96 well (8 x 12) microplate. Instruments are available that can dispense reagents into these plates and also to wash the wells, a requirement that is necessary to prevent reagent carry-over between assay steps.
  • Spectrophotometers that can read the coloured solutions directly in the wells at the final step of the assay are also readily available. However, for such an application, the protruding part of the dipsticks from the wells, and the fixtures that hold them, prevent easy access to the wells for dispensing and washing steps. This results in a dipstick assay that requires customized equipment and/or more manual steps than conventional assays run in microwells.
  • the disks are sized to fit into the wells of a microplate, yet remain free and not become wedged or trapped within the well.
  • disks that are smaller than 6.0 mm diameter will fit into the wells of all commonly manufactured microplates. It can be appreciated, however, that disks that are too small will have insufficient amount of skin that will compromise assay sensitivity and reproducibility. It has been found that disks 5 to 6 mm in diameter are best suited for assay in 96 well microplates. However, it can be appreciated that the invention is not limited to these dimensions, and that other disk sizes are contemplated for different wells and microplates, as would be apparent to those skilled in the art.
  • the disks when the disks are placed in the well, they should not float with the skin-side up since this will contact the dispensing and aspiration tubes that are inserted in the wash steps. Therefore, if the sampling device is constructed of materials that are less dense than water the disks should be added to the well with the skin side down. If the sampling device is constructed of materials that are more dense than water, then the disk is best added with the skin side up and the height of the dispensing and aspiration tubes adjusted so that they do not touch the skin surface.
  • the disks must be cut from the device such that any anvil-type part used to eject the disk from a punch or cutting tool must not contact the skin.
  • any anvil-type part used to eject the disk from a punch or cutting tool must not contact the skin.
  • the anvil should contact the back of the device (non-skin side) when cutting and ejecting a disk.
  • a cutting tool 60 removes a disk from the device 10 of Fig. 1 when the device 10 is in a folded over (closed) position, as illustrated.
  • the closed device is placed on a firm surface (not illustrated) with the outer surface 62 of the sampling member 12 of the device facing up.
  • the cutting tool 60 is inserted in a circular depression 64 that can be provided on the outer surface 62 of the sampling member 12 of device 10 and the cutting tool 60 is then pressed down to cut through the plastic and the tape 30 / skin 70 sample.
  • the cutting tool 60 is not pressed down so far, however, so as to cut through the plastic of the closure member 14 of the device 10.
  • the end 66 of the cutting tool 60 with the disk 50 is placed into a designated well 54 of the microwell plate 56 (see Fig. 8) and plunger 68 of the cutting tool 60 is depressed to eject the disk with the skin sample on the adhesive tape into the well 54.
  • the microwell plate is placed on automated plate reader/washer and approximately 100 ⁇ L of detector reagent is added to all the wells and the disks are incubated for approximately fifteen minutes at generally room temperature (20-24°C).
  • the reagent can be a solution of an A-C-B reagent as defined in Example 2.
  • the detector reagent is then aspirated and approximately 250 ⁇ L of wash buffer is added to the wells.
  • the plate can be shaken for approximately thirty seconds after the addition of the wash buffer, removing excess detector reagent, and then left for approximately a further ninety seconds.
  • the wash step can be repeated two or more times as necessary. It is found that three wash steps are satisfactory.
  • K-Blue substrate is added to the wells and allowed to incubate with the washed disk for approximately fifteen minutes at generally ambient room temperature (as previously described).
  • the microwell plate can be shaken during this step, and, as in Example 2, the incubation can be in the dark.
  • the reaction is then stopped by the addition of approximately 100 ⁇ L of 1 N sulphuric acid to the wells, and the plate is shaken to mix the solutions. Approximately 100 ⁇ L of the stopped substrate is then removed and transferred to the wells of a new plate and read at about 450 nm on a plate reading spectrophotometer and analyzed as previously described to determine the relative level of skin cholesterol for each donor.

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Abstract

Cette invention concerne un procédé permettant de mesurer le cholestérol cutané et consistant à appliquer une bande adhésive sur une zone sélectionnée de l'épiderme de façon que la bande adhère à la zone sélectionnée de l'épiderme, puis à décoller la bande de la zone sélectionnée de l'épiderme afin qu'on obtienne un échantillon représentatif de la couche cornée externe de l'épiderme, l'échantillon adhérant à la bande de façon que les constituants de l'épiderme soient exposés. L'échantillon est analysé à l'aide d'un réactif détecteur qui se lie spécifiquement au cholestérol et qui comporte en outre un composant indicateur qui permet d'effectuer une analyse quantitative du cholestérol présent dans les constituants de l'épiderme exposés.
PCT/CA2005/000642 2004-04-28 2005-04-28 Dosage direct du cholesterol cutane dans des echantillons de peau preleves par decollement de bande WO2005106018A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
BRPI0510352-5A BRPI0510352A (pt) 2004-04-28 2005-04-28 análise direta de colesterol na pele em amostras de pele retiradas ao desmantar a fita
CA002563973A CA2563973A1 (fr) 2004-04-28 2005-04-28 Dosage direct du cholesterol cutane dans des echantillons de peau preleves par decollement de bande
CN2005800138932A CN1961078B (zh) 2004-04-28 2005-04-28 直接分析通过胶带剥离获得的皮肤样品中的皮肤胆固醇
JP2007509839A JP2007534326A (ja) 2004-04-28 2005-04-28 テープ剥離により取除いた皮膚試料における皮膚コレステロールの直接分析
AU2005238099A AU2005238099B2 (en) 2004-04-28 2005-04-28 Direct assay of skin cholesterol in skin samples removed by tape stripping
EP05738502A EP1745146A4 (fr) 2004-04-28 2005-04-28 Dosage direct du cholesterol cutane dans des echantillons de peau preleves par decollement de bande
MXPA06012326A MXPA06012326A (es) 2004-04-28 2005-04-28 Ensayo directo del colesterol en la piel en muestras de la piel removidas por separacion con una cinta.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CA002465427A CA2465427A1 (fr) 2004-04-28 2004-04-28 Dosage direct du cholesterol dans des echantillons de peau preleves au moyen d'un ruban adhesif
CA2465427 2004-04-28

Publications (1)

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WO2005106018A1 true WO2005106018A1 (fr) 2005-11-10

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PCT/CA2005/000642 WO2005106018A1 (fr) 2004-04-28 2005-04-28 Dosage direct du cholesterol cutane dans des echantillons de peau preleves par decollement de bande

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Country Link
EP (1) EP1745146A4 (fr)
JP (1) JP2007534326A (fr)
CN (2) CN1961078B (fr)
AU (1) AU2005238099B2 (fr)
BR (1) BRPI0510352A (fr)
CA (1) CA2465427A1 (fr)
MX (1) MXPA06012326A (fr)
RU (1) RU2006137332A (fr)
WO (1) WO2005106018A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2893419A1 (fr) * 2005-11-14 2007-05-18 Chanel Parfums Beaute Soc Par Procede de determination qualitatif et/ou quantitatif d'au moins une molecule presente sur une surface solide.
WO2008086596A1 (fr) * 2007-01-16 2008-07-24 Premd, Inc. Appareil d'échantillonnage et test de peau non invasifs
KR101092000B1 (ko) * 2009-05-26 2011-12-08 고려대학교 산학협력단 나노 물질의 피부 침투 평가 방법
JP2012193992A (ja) * 2011-03-15 2012-10-11 Canon Inc 対象物質捕集装置および捕集方法

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006122430A1 (fr) * 2005-05-20 2006-11-23 Premd, Inc. Dosage direct de proteine cutanee dans des echantillons de peau preleves par pelliculage a bande
CN102749462B (zh) * 2012-07-20 2014-08-06 常熟柏宇医疗电子有限公司 基于生物芯片的无创胆固醇智能监测系统
CN104181312B (zh) * 2014-08-08 2016-06-01 安徽易康达光电科技有限公司 一种用于动脉粥样硬化风险评估的皮肤游离胆固醇无创检测装置
CN107421905A (zh) * 2017-09-15 2017-12-01 中国科学院合肥物质科学研究院 一种用于皮肤角质层成分测量的样品测量平台和无创测量装置及方法
JP2019158497A (ja) * 2018-03-12 2019-09-19 株式会社島津製作所 生体試料採取方法及び生体試料採取用器具
JP6963285B2 (ja) * 2018-08-24 2021-11-05 国立大学法人 東京大学 対象の皮膚情報を検査するための検査キット

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US5489510A (en) * 1988-01-19 1996-02-06 2860601 Canada Inc. Method for visual indication of cholesterol on skin surface agents used therefor and methods for producing such agents
US20030045810A1 (en) * 1999-11-10 2003-03-06 Piotr Borkowski Highly sensitive, practical, widely available diagnostic kit for fungal skin infections

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SE8401798L (sv) * 1984-04-02 1985-10-03 Erik Dahlgren Indikeringsbricka for nervgas och andra kolinesterashemmare
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JPH06238008A (ja) * 1993-02-15 1994-08-30 Yuutoku Yakuhin Kogyo Kk パッチテスト用具
JP3635742B2 (ja) * 1995-09-22 2005-04-06 花王株式会社 角質細胞採取用シート
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US5489510A (en) * 1988-01-19 1996-02-06 2860601 Canada Inc. Method for visual indication of cholesterol on skin surface agents used therefor and methods for producing such agents
US20030045810A1 (en) * 1999-11-10 2003-03-06 Piotr Borkowski Highly sensitive, practical, widely available diagnostic kit for fungal skin infections

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WEERHEIM A. ET AL: "Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography.", ARCH DERMATOL RES., vol. 293, no. 4, 2001, pages 191 - 199, XP001181113 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2893419A1 (fr) * 2005-11-14 2007-05-18 Chanel Parfums Beaute Soc Par Procede de determination qualitatif et/ou quantitatif d'au moins une molecule presente sur une surface solide.
WO2007057556A1 (fr) * 2005-11-14 2007-05-24 Chanel Parfums Beaute Procede de determination qualitatif et/ou quantitatif d'au moins une molecule presente sur une surface solide
WO2008086596A1 (fr) * 2007-01-16 2008-07-24 Premd, Inc. Appareil d'échantillonnage et test de peau non invasifs
KR101092000B1 (ko) * 2009-05-26 2011-12-08 고려대학교 산학협력단 나노 물질의 피부 침투 평가 방법
JP2012193992A (ja) * 2011-03-15 2012-10-11 Canon Inc 対象物質捕集装置および捕集方法

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CN1961078B (zh) 2012-05-02
CA2465427A1 (fr) 2005-10-28
BRPI0510352A (pt) 2007-10-30
JP2007534326A (ja) 2007-11-29
MXPA06012326A (es) 2007-06-05
EP1745146A4 (fr) 2008-07-09
CN102776267A (zh) 2012-11-14
AU2005238099B2 (en) 2011-02-03
CN1961078A (zh) 2007-05-09
RU2006137332A (ru) 2008-06-10
AU2005238099A1 (en) 2005-11-10
EP1745146A1 (fr) 2007-01-24

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