JP2009210411A - Skin cathepsin analysis method, skin optical-stress determining method, and kit for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a kit for the same, can simply and speedily quantitatively-analyze skin cathepsin in a sample collected from skin and simply and speedily determining a level of skin optical-stress from a sample collected from skin. <P>SOLUTION: The method analyzes skin cathepsin in a sample collected from skin by immunochromatography using localized plasmon resonance; the method measures skin cathepsin and total protein in a sample collected from skin and determines a skin optical-stress from the ratio of the skin cathepsin to the total protein; and the method also measures a skin cathepsin inhibiting substance and determines a skin optical-stress from the ratio of the skin cathepsin to the total protein, the ratio of the skin cathepsin inhibiting substance to the total protein, and the ratio of the skin cathepsin to the skin cathepsin inhibiting substance; and a kit is constructed to carry out these methods. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for analyzing skin cathepsin and a kit therefor, and a method for judging light stress in skin and a kit therefor.

  It is known that the stratum corneum of the skin is thickened by light stress such as ultraviolet exposure, and the fall off of the stratum corneum is promoted. Conventionally, it has been possible to judge such skin light stress from changes in skin moisture and melanin content in the skin, but this is only a qualitative evaluation and can quantitatively evaluate skin light stress. It wasn't.

  In recent years, skin cathepsin has attracted attention as an enzyme responsible for the production of keratin in skin metabolism due to light stress. Cathepsins are proteases secreted from lysosomes of organelles, and are classified into approximately 18 types according to their structures and functions (see Table 1 below).

  Cathepsin L is a cysteine protease having a molecular weight of 24 kDa and an optimum pH of 5.0 to 6.0, and strongly hydrolyzes proteins such as collagen, azocasein and hemoglobin (for example, Tsuru Dainori et al., “Proteolytic Enzyme I”, (See Publishing Center, pages 51-55). It is also known to increase in skin that has been inflamed due to trauma, etc., and in cancer cells, and it also acts as a regulator of the cell cycle (for example, Thomas Reinheckell et al., “The lysosomal cysteine protease cathepsin L “Regulates keratinocyte proliferation by control of growth factor recycling”, Journal of cell science, vol. 118 (15), pp 3387-3395 (non-patent document 2)).

  It is thought that when skin is exposed to ultraviolet light, the amount of cathepsin secreted increases in stimulated skin cells, which triggers signal transduction in a chain and ultimately leads to apoptosis ( For example, Cecilia A. Bivik et al., “UVA / B-Induced Apoptosis in Human Melanocytes Involves Translocation of Cathepsins and Bcl-2 Family Members”, Journal of Investigative Dermatology, Vol. 126, pp. 1119–1127 (2006) (non- See Patent Document 3). When the skin is exposed to ultraviolet light, the amount of melanin secretion from melanocytes increases to prevent cell damage, but there is a difference in cathepsin L activity between races (black and white) with different melanin levels. (For example, refer to Nannan Chen et al., “Cathepsin L2 Levels Inversely Correlate with Skin Color, Journal of Investigative Dermatology, Vol. 126, pp2345-2347 (2006) (non-patent document 4).) An attempt has been made to develop a method that can easily and quickly determine the light stress of the skin by analyzing the skin cathepsin in a sample collected from the skin.

  In general, an enzyme-linked immunosorbent assay (ELISA) method is often used as a technique for analyzing a trace amount of protein contained in a biological sample. The enzyme immunoassay is roughly divided into a competitive method and a sandwich method, both of which are widely known, mainly focusing on improving sensitivity, and various methods centering on improving sensitivity of the label itself. Improvements have been made (see, for example, Japanese Patent Application Laid-Open No. 2000-180448 (Patent Document 1) and Japanese Patent Application Laid-Open No. 2005-337960 (Patent Document 2)).

Regarding the analysis of skin cathepsins described above, the development and sales of analysis kits have not been realized at present, and the fact is that the presence or absence in biological samples is qualitatively determined by gene expression analysis of skin cathepsins. is there. However, such a method not only requires many hours for pretreatment of the sample, but quantitative evaluation of skin cathepsin is difficult.
JP 2000-180448 A JP 2005-337960 A Tsuru Dainori et al., “Proteolytic Enzyme I”, Academic Publishing Center, 51-55 Thomas Reinheckell et al., `` The lysosomal cysteine protease cathepsin L regulates keratinocyte proliferation by control of growth factor recycling, '' Journal of cell science, vol. 118 (15), pp 3387-3395. Cecilia A. Bivik et al., `` UVA / B-Induced Apoptosis in Human Melanocytes Involves Translocation of Cathepsins and Bcl-2 Family Members '', Journal of Investigative Dermatology, Vol. 126, 1191-1127 (2006) Nannan Chen et al., “Cathepsin L2 Levels Inversely Correlate with Skin Color, Journal of Investigative Dermatology, Vol.126, pp2345-2347 (2006) Hashimoto et al., “Absorptiometric and visual quantification of urinary protein using tetrabromophenol”, Analytical Chemistry, Vol. 54, No. 9, 2005, pages 783-788 Welss T et al., `` Hurpin is a selective inhibitor of lysosomal cathepsin L and protects keratinocytes from ultraviolet-induced apoptosis '', Biochemistry 42 (24), 2003, pp. 7381-7389

  The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a method and a kit for the same that can easily and quickly quantitatively analyze skin cathepsin in a sample collected from the skin. It is to be.

  Another object of the present invention is to provide a method and a kit therefor that can easily and quickly determine the level of light stress in the skin from a sample collected from the skin.

  The present invention is characterized in that it is a method for analyzing skin cathepsin contained in a sample collected from skin by immunochromatography using localized plasmon resonance.

  In the skin cathepsin analysis method of the present invention, the skin cathepsin is preferably skin cathepsin L.

  The present invention is a method for measuring skin cathepsin and total protein contained in a sample collected from the skin, and determining the skin light stress from the ratio of the skin cathepsin to the total protein (hereinafter referred to as “first determination method”). Is also provided.

  In the first determination method of the present invention, skin cathepsin is preferably measured by immunochromatography using localized plasmon resonance.

  In the first determination method of the present invention, the skin cathepsin is preferably skin cathepsin L.

  The present invention also measures skin cathepsin, skin cathepsin inhibitor and total protein contained in a sample collected from the skin, ratio of skin cathepsin to total protein, ratio of skin cathepsin inhibitor to total protein, and skin cathepsin inhibitor There is also provided a method for determining the light stress of the skin from the ratio of skin cathepsin to (hereinafter referred to as “second determination method”).

  In the second determination method of the present invention, it is preferable to measure skin cathepsin and a skin cathepsin inhibitor by immunochromatography using localized plasmon resonance.

  In the second determination method of the present invention, the skin cathepsin is preferably skin cathepsin L.

  The present invention relates to a conjugate pad preliminarily impregnated with a conjugate of a skin cathepsin antibody and a gold colloid on a substrate configured to allow liquid to move by capillary action, and the antibody used for the conjugate. There is also provided a skin cathepsin analysis kit (hereinafter referred to as “first kit”) in which a detection unit to which an antibody for skin cathepsin recognizing different sites is previously immobilized is formed.

  The present invention also provides a conjugate pad in which a conjugate of a skin cathepsin antibody and a gold colloid is pre-impregnated on a substrate configured to allow liquid to move by capillary action, and the antibody used for the conjugate. Is a skin light stress determination kit (hereinafter referred to as “first”), which is formed with a first detection unit in which antibodies for skin cathepsin recognizing different sites are immobilized in advance and a second detection unit including an acid-base indicator. Also referred to as “2 kits”).

  The present invention further includes a conjugate pad preliminarily impregnated with a conjugate of an antibody of skin cathepsin and a gold colloid on a substrate configured to allow liquid to move by capillary action, and the antibody used for the conjugate. A first detection unit in which antibodies for skin cathepsin recognizing different sites are immobilized in advance, a second detection unit including an acid-base indicator, and a third detection unit in which antibodies for a skin cathepsin inhibitor are immobilized in advance A kit for determining the light stress of the skin (hereinafter referred to as “third kit”) is also provided.

  According to the method for analyzing skin cathepsin of the present invention and the kit for the same (first kit), the skin in a sample collected from the skin by a highly specific immunoassay by immunochromatography using localized plasmon resonance Cathepsin can be molecularly recognized and quantitatively analyzed easily and quickly. Such methods and kits are useful for simple and quick determination of the level of skin light stress.

  Moreover, according to the method for determining the light stress of the skin of the present invention (including both the first determination method and the second determination method), skin cathepsin and total protein in the sample collected from the skin, or the skin By measuring cathepsin, skin cathepsin inhibitor and total protein, and calculating the ratio of each, it is possible to quickly and significantly determine the level of light stress in the skin. Further, according to the kit for performing these methods (including both the second kit and the third kit), skin cathepsin and total protein, or skin cathepsin, skin cathepsin inhibitor and total protein are 1 Composing with dry chemistry on two test papers has the advantage that an inexpensive disposable test strip can be realized.

<Skin cathepsin analysis method and kit therefor>
The skin cathepsin analysis method of the present invention is characterized in that skin cathepsin contained in a sample collected from the skin is analyzed by immunochromatography using localized plasmon resonance. According to such a method for analyzing skin cathepsin of the present invention, skin cathepsin in a sample collected from the skin can be quantitatively analyzed easily and rapidly.

  The skin cathepsin to be analyzed in the analysis method of the present invention is an enzyme that exists in human skin and is responsible for the production of keratin in skin metabolism due to light stress. As shown in Table 1 above, skin cathepsin L Skin cathepsin A, skin cathepsin B, skin cathepsin C, skin cathepsin D, skin cathepsin E, skin cathepsin F, skin cathepsin G, skin cathepsin K, skin cathepsin L1, skin cathepsin L2, skin cathepsin O, skin Types such as cathepsin R, cutaneous cathepsin S, cutaneous cathepsin T, cutaneous cathepsin W, cutaneous cathepsin X are known. In the analysis method of the present invention, any of these types of skin cathepsins may be the object of analysis, but since the highest concentration can be collected from the skin, skin cathepsin L is the object of analysis. It is preferable that

  As a sample containing skin cathepsin to be analyzed in the analysis method of the present invention, a sample collected from human skin, in particular, from the skin of human skin is used. The part of the human body from which the sample is collected is not particularly limited as long as the skin is exposed, such as the arm, face, hand, and neck, but it is the longest exposure time and is subject to light stress. Arms or faces are preferred. Further, the sample collecting method is not particularly limited, but for example, a pad having an adhesive layer (D-Squame D100 (made by Cuderm) (diameter: 22 mm × thickness: about 0.1 mm) as a specific example) is described above. After sticking on the skin of such a human body part, it can extract | collect by collect | recovering the deposits to an adhesion layer by peeling this. When analyzing the collected sample by immunochromatography, a liquid sample is prepared by dissolving the sample in an extract. As this extract, for example, an extract containing 0.1% Triton X-100 in an acetate buffer and having the composition shown in Table 2 below (per 100 mL) is preferably used. In the acetate buffer of the example shown in Table 2, the concentration is reduced to a low concentration (for example, 4 mM), thereby making it less susceptible to subsequent concentration treatment. Moreover, the acetate buffer of the example shown in Table 2 makes pH 5 which is the optimum pH of skin cathepsin L.

In the analysis method of the present invention, the skin cathepsin contained in the sample is molecularly recognized by a highly specific immunoassay, and the detection sensitivity of the skin cathepsin contained in the sample is increased (sensitized) by localized plasmon resonance. For example, rare metal particles such as gold colloid and silver colloid are used. Of these, gold colloid is preferred for reasons of chemical stability. A commercially available product can be suitably used as the gold colloid, and specific examples thereof include a gold colloid stock solution for immunochromatography (WRGH1, manufactured by Wine Red Chemical Co., Ltd.) having an OD ₅₂₀ = 12.

  In immunochromatography using localized plasmon resonance using colloidal gold, specifically, a conjugate of a skin cathepsin antibody and a colloidal gold is prepared in advance, and this is first performed on the skin cathepsin and antigen in the sample (liquid sample). Binding by an antibody reaction, and then this skin cathepsin-conjugate conjugate is further trapped by binding to an antibody of skin cathepsin that recognizes a site different from the antibody of the conjugate by further antigen-antibody reaction. This can be done by quantitative analysis of the body. Here, in the skin cathepsin analysis method of the present invention, commercially available products are preferably used for the antibody used for the conjugate and the antibody for the skin cathepsin that recognizes a site different from the antibody of the conjugate. it can.

Moreover, the immunochromatographic test paper used for the immunochromatography by the localized plasmon resonance using the gold colloid in this invention can be specifically produced suitably by the following procedures. First, 500 μL of an immunochromatographic gold colloid stock solution (WRGH1, manufactured by Wine Red Chemical Co., Ltd.) with an OD ₅₂₀ = 12 was added to 500 μL of Tris-HCl buffer (pH 9.2, 20 mM) and diluted 2 times to obtain a colloidal gold solution ( OD ₅₂₀ = 6) is prepared. In 1 mL of this colloidal gold solution, in a siliconized tube (1.5 mL), an antibody solution of skin cathepsin L (anti-cathepsin L antibody) (0.5 mg / mL, Anti Catthepsin L, model number: MAB952, manufactured by R & D SYSTEMS Inc.) ) Add 100 μL with stirring and let stand at 4 ° C. for 20 minutes. Thereafter, 123 μL of 20 mM Tris-HCl buffer (pH 9.8) containing 2.5% sodium caseinate is added with stirring, and the mixture is allowed to stand again at 4 ° C. for 20 minutes. This is centrifuged at 14,000 × g for 15 minutes at 10 ° C. The supernatant after centrifugation is removed, and 1 mL of 20 mM Tris-HCl buffer (pH 9.2) containing 1% of BSA (Bovine serum albumin) (hereinafter referred to as BSA solution) is added and suspended. This is again centrifuged at 14,000 × g for 15 minutes at 10 ° C., the supernatant after centrifugation is removed, and 267 μL of a BSA solution containing 5% of D (+)-trehalose dihydrate is added. This is a gold colloid labeled antibody solution. A glass fiber pad (5 × 5 × 0.41 mm ³ , GFCP103000, manufactured by Millipore) is immersed in this gold colloid-labeled antibody solution and dried at room temperature for 30 minutes to prepare a conjugate pad.

Next, a strip-shaped nitrocellulose membrane (50 × 5 × 0.24 mm ³ , HF12004XSS, manufactured by Millipore) was produced, and anti-cathepsin L antibody (0.5 mg / mL, Anti Catthepsin) was placed 20 mm from the end. L, model number: MAB952, R & D SYSTEMS Inc.) is applied in a line shape. This is dried at room temperature for 30 minutes, and then a glass petri dish is filled with 10 mM phosphate buffer (pH 7) containing 1% sodium caseinate, and a nitrocellulose membrane is placed and shaken at 50 rpm for 30 minutes. Thereafter, the nitrocellulose membrane is immersed in a glass petri dish filled with 10 mM phosphate buffer (pH 7) and shaken for 30 minutes for washing. After the washing is completed, moisture on the surface of the nitrocellulose membrane is sucked off with Kimwipe and dried at room temperature for 30 minutes. Onto a nitrocellulose membrane, conjugate pad, a sample pad ^{(5 × 10 × 0.83mm 3,} CFSP203000, Millipore), an absorbent pad ^{(5 × 5 × 0.83mm 3,} CFSP203000, Millipore) superimposed, An immunochromatographic test paper can be suitably prepared by fixing with an adhesive tape.

  Here, FIG. 1 is a diagram schematically showing a skin cathepsin analysis kit (first kit) 1 as a preferred example of the present invention, and FIG. 1 (a) is a state before the liquid sample 8 is injected. FIG. 1B schematically shows the state after the liquid sample 8 is injected. The present invention is a kit (first kit) particularly useful for the above-described method for analyzing skin cathepsin of the present invention, wherein a skin cathepsin antibody is formed on a substrate configured to allow liquid to move by capillary action. For analysis of skin cathepsin, in which a conjugate pad pre-impregnated with a conjugate of gold and colloid of gold and a detection part to which an antibody of skin cathepsin recognizing a site different from the antibody used in the conjugate is preliminarily formed are formed Kits are also provided. FIG. 1 shows a sample pad 3 for dropping a liquid sample 8 on a substrate 2 configured to allow liquid to move by capillary action, a conjugate 11 of a skin cathepsin antibody 13 and a gold colloid 12. Of the present invention in which the conjugate pad 4 previously impregnated with and the detection part 5 to which the antibody 14 of skin cathepsin recognizing a site different from the antibody 13 used in the conjugate 11 is immobilized are formed. 1 kit is shown. With such a first kit of the present invention, molecular recognition of skin cathepsin in a sample collected from the skin is performed with a highly specific immunoassay by immunochromatography using localized plasmon resonance using colloidal gold. An inexpensive disposable test strip that can perform quantitative analysis simply and quickly is provided.

  The substrate 2 used in the first kit 1 of the present invention is configured so that the liquid can move by capillary action. As a material for forming such a substrate 2, for example, a nitrocellulose film or a cellulose film cut into a strip shape as described above is preferably used, but is not limited thereto. Further, the substrate 2 may be realized by forming a nitrocellulose film on a base substrate formed of polyethylene, polyester, fluororesin, polycarbonate, glass or the like.

  The sample pad 3 is configured such that when the liquid sample 8 is injected, the skin cathepsin to be analyzed contained in the liquid sample 8 passes through the sample pad 3 and reaches the substrate 2. From the viewpoint of enabling a more accurate competitive reaction, the sample pad 3 is a contaminant (for example, mucin, pecryl, lysozyme, etc.) having a relatively large molecular weight contained in the liquid sample 8 collected from the skin. ) Is preferably made of a material that can be removed by filtration or adsorption. Examples of the material for forming the sample pad 3 that can filter or adsorb impurities having a relatively high molecular weight include glass fiber and cellulose, but are not limited thereto. The example shown in FIG. 1 shows an example in which the sample pad 3 is provided separately from the conjugate pad 4. However, the liquid sample is directly injected into the conjugate pad 4, and the conjugate pad 4 serves as the sample pad. Of course, it may be configured to serve as both.

  The conjugate pad 4 is previously impregnated with the conjugate 11 of the gold colloid 12 and the skin cathepsin antibody 13 described above. 2A shows the antigen-antibody reaction in the conjugate pad 4 of the first kit 1 of the present invention, and FIG. 2B shows the antigen-antibody in the detection unit 5 of the first kit 1 of the present invention. It is a figure which shows reaction typically. As shown in FIG. 1B, when the liquid sample 8 was injected into the sample pad 3 described above, the liquid sample 8 first moved to the conjugate pad 4 and was previously impregnated in the conjugate pad 4. A conjugate 11 of colloidal gold 12 and skin cathepsin antibody 13 dissolves in the liquid sample 8. As a result, as shown in FIG. 2A, in the conjugate pad 4, the skin cathepsin 9 and the antibody 13 of the conjugate 11 undergo an antigen-antibody reaction, and a conjugate of the skin cathepsin 9 and the conjugate 11 is formed. The As a material for forming such a conjugate pad 4, the materials described above as suitable materials for forming the sample pad 3 can be suitably used. In addition, as a method of pre-impregnating the conjugate 11 to the conjugate pad 4, a conventionally known appropriate method can be applied and is not particularly limited. For example, the method described above is preferable.

  As described above, the skin cathepsin antibody 14 that recognizes a site different from the skin cathepsin antibody 13 used in the conjugate 11 is fixed to the detection unit 5 in the first kit 1 of the present invention. The antibody 14 is fixed to the detection unit 5 by using a conventionally known appropriate method such as fixation by chemical bond such as covalent bond, ionic bond, coordination bond, van der Waals force, or film formation such as coating. can do. As described above, the conjugate of the skin cathepsin 9 and the conjugate 11 formed by the antigen-antibody reaction in the conjugate pad 4 moves to the detection unit 5 substantially along the horizontal direction by capillary action, and in the detection unit 5, As shown in FIG. 2 (b), an antigen-antibody reaction with the antibody 14 occurs and is trapped by the detection unit 5.

  FIG. 3 is a diagram schematically showing the measurement of the amount of skin cathepsin in the detection unit 5 of the kit 1 of the present invention of the example shown in FIG. 1, FIG. 3 (a) is a perspective view, and FIG. It is sectional drawing. As described above, the conjugate of skin cathepsin 9 and conjugate 11 moves to the detection unit 5 generally along the horizontal direction by capillary action, and is trapped by the antibody 14 that has been fixed to the detection unit 5 in advance. The conjugate of the trapped skin cathepsin 9, conjugate 11 and antibody 14 includes a light emitting element 21 and a light receiving element 22 as shown in FIG. 3, and further controls the temperature for chemical reaction and stabilization of color density. Skin cathepsins are quantitatively analyzed using an optical measuring instrument with a built-in mechanism 23. In other words, light is emitted from the light emitting element 21 to the combined body trapped in the detection unit 5 and the reflected light is received by the light receiving element 22 to measure the concentration of colloidal gold in the combined body. Skin cathepsin can be analyzed quantitatively.

  In the first kit 1 of the present invention, the absorbent pad 10 is usually provided at the end of the substrate 2 opposite to the sample pad 3 with respect to the detection unit 5. The absorption pad 10 is for moving the liquid sample substantially along the horizontal direction by capillary action and absorbs the liquid sample after passing through the detection unit 5, and is described above as a suitable material for forming the sample pad 3 described above. The absorbent pad 10 formed of the above material can be preferably used.

<Determination Method of Skin Light Stress (First Determination Method) and Kit for It>
The present invention also provides a method (first determination method) for measuring skin cathepsin and total protein contained in a sample collected from the skin, and determining skin light stress from the ratio of skin cathepsin to total protein. . According to the first determination method of the present invention, it is possible to quickly and significantly determine the level of skin photostress by measuring skin cathepsin and total protein and calculating the ratio thereof. Here, “total protein” refers to the total amount of protein in a sample collected from the skin. When the ratio of skin cathepsin to this total protein is low, it can be determined that acute stress is low, and vice versa. In addition, when the ratio of skin cathepsin to the total protein is high, it can be determined that the acute stress is high (for the correlation between the ratio of the skin cathepsin to the total protein and the acute stress, see, for example, Non-Patent Document 3). reference).

  In the first determination method of the present invention, the sample can be collected by the same method as described above in the method for analyzing skin cathepsin of the present invention, and the kind of skin cathepsin to be measured is also the same. Skin cathepsin L is preferred for reasons. In the first determination method of the present invention, the method for measuring skin cathepsin in a sample collected from the skin is not particularly limited. Immunochromatography using localized plasmon resonance, microelectrophoresis, surface An appropriate method such as plasmon resonance can be used, but from the viewpoint of simplicity and speed, immunochromatography by localized plasmon resonance using a gold colloid is used in the same manner as the method for analyzing skin cathepsin of the present invention described above. Is preferably used.

In the first determination method of the present invention, the method for measuring the total protein contained in the sample collected from the skin is not particularly limited. For example, Hashimoto et al., “Absorptiometry of urine protein using tetrabromophenol” Quantitative and visual quantification ", Analytical Chemistry, Vol. 54, No. 9, 2005, pages 783-788 (Non-patent Document 5) can be suitably employed. For example, tetrabromophenol blue (C ₁₉ H ₁₀ Br ₄ O ₅ S, TBPB, molecular weight: 669.96), which is one of acid-base indicators, is used as H ₂ TBPB, HTBPB ⁻ , or TBPB ^{2− in} an aqueous solution. Exists. In the presence of the nonionic surfactant Triton X-100, it dissolves in the micelle in the state of the following formula (A). Next, when protein is added to the acid-base indicator, a reaction of the following formula (B) occurs. As a result of this reaction, the acid-base indicator changes from yellow to dark green according to the protein concentration. By observing this color change, the total protein contained in the sample collected from the skin is quantitatively analyzed. It becomes possible to analyze.

HTBPNB ^- + _x TritonX-100
_{^{→ (HTBPB - · x TritonX-}} 100) m (A)
_{^{y (HTBPB - · x TritonX-}} 100) m + Protein
→ _{^{[y (HTBPB - · x TritonX-}} 100 · Protein) m (B)
(The above formula (A), in (B), x is HTBPB ^- 1mol moles of TritonX-100 to capture, y is HTBPB that reacts with Protein 1 mol ^- moles of, m represents a micelle.)
Here, FIG. 4 is a perspective view schematically showing a skin light stress determination kit (second kit) 51 of a preferred example of the present invention, FIG. 4 (a) is an external view, and FIG. FIG. 4B is a view showing the upper body with the housing 52 removed from the state of FIG. The present invention is a kit that can suitably perform the above-described first determination method of the present invention. As shown in FIG. 4, a liquid is placed on a substrate 55 configured so that the liquid can move by capillary action. A sample pad 53 for dropping the sample 54, a conjugate pad 56 pre-impregnated with a conjugate of a skin cathepsin antibody and a gold colloid, and a skin cathepsin recognizing a site different from the antibody used for the conjugate Also provided is a kit 51 in which a first detection unit 57 to which an antibody is immobilized in advance and a second detection unit 58 containing an acid-base indicator are formed. With such a second kit of the present invention, molecular analysis of skin cathepsin in a sample collected from the skin is performed with a highly specific immunoassay by immunochromatography using localized plasmon resonance for quantitative analysis. An inexpensive disposable test strip is provided that can simultaneously measure the total protein in the sample and simultaneously and quickly determine the light stress of the skin.

  In the second kit 51 of the present invention, the substrate 55, the sample pad 53, the conjugate pad 56, and the first detection unit 57 are the same as the substrate 2, the sample pad 3, and the conjugate described above in the first kit 1 of the present invention. The pad 4 and the detection unit 5 are as described above, and the same applies to the gold colloid used for the conjugate pad 56, the skin cathepsin antibody, and the skin cathepsin antibody used for the detection unit 57. In the second kit 51 of the present invention, as shown in FIG. 4, a sample pad 53 for injecting a liquid sample 54 is provided near the center of the substrate 55, and the conjugate described above is provided in about half of the substrate 55. A pad 56 and a first detection unit 57 are formed and configured to quantitatively analyze the skin cathepsin in the liquid sample, and a second half containing an acid-base indicator is included in the remaining half of the substrate 55. The detection unit 58 is formed so that the total protein in the liquid sample can be measured simultaneously.

  Here, for example, a total protein test paper using tetrabromophenol blue, which is one of the acid-base indicators described above, is installed in the second detection unit 58. This total protein test paper is, for example, (1) a TBPB solution in which tetrabromophenol blue is dissolved in ethanol to 74 μM, a 1% surfactant solution in which Triton X-100 is dissolved in water, 0.5 M acetic acid -After preparing the sodium acetate buffer solution, (2) TBPB solution: surfactant solution: buffer solution = 5: 1: 5 was mixed to adjust the indicator, and (3) the indicator was impregnated into the filter paper. It can be suitably produced by drying at a temperature of 15 ° C. and a humidity of 15% for 20 minutes.

<Determination method of skin light stress (second determination method) and kit therefor>
The present invention further measures skin cathepsin, skin cathepsin inhibitor and total protein contained in a sample collected from skin, and ratio of skin cathepsin to total protein, ratio of skin cathepsin inhibitor to total protein and skin cathepsin inhibitor There is also provided a method (second determination method) for determining the skin light stress from the ratio of the skin cathepsin to the skin. According to the second determination method of the present invention, skin cathepsin, skin cathepsin inhibitor and total protein are measured, the ratio of skin cathepsin to total protein, the ratio of skin cathepsin inhibitor to total protein and the skin to skin cathepsin inhibitor By calculating the ratio of cathepsin, not only acute stress but also chronic stress can be determined quickly and significantly from the correlation shown in Table 3 below. Regarding the correlation shown in Table 3 below, for example, Welss T et al., “Hurpin is a selective inhibitor of lysosomal cathepsin L and protects keratinocytes from ultraviolet-induced apoptosis”, Biochemistry 42 (24), 2003, 7381-7389. Page (Non-Patent Document 6) can be referred to.

  Also in the second determination method of the present invention, the sample can be collected by the same method as described above in the method for analyzing skin cathepsin of the present invention, and the type of skin cathepsin to be measured is also the same reason. To skin cathepsin L. Also in the second determination method of the present invention, the method for measuring skin cathepsin in a sample collected from the skin is not particularly limited, and immunochromatography using localized plasmon resonance, microelectrophoresis, Although an appropriate method such as surface plasmon resonance can be used, from the viewpoint of simplicity and speed, immunity by localized plasmon resonance using gold colloid is used in the same manner as the method for analyzing skin cathepsin of the present invention described above. It is preferable to use chromatography. Further, even in the second determination method of the present invention, the method for measuring the total protein in the sample collected from the skin is not particularly limited, but as in the above-described first determination method of the present invention, an acid-base indicator A method of using is preferable.

  According to the second determination method of the present invention, the skin cathepsin inhibitor contained in the sample collected from the skin is also measured, and the ratio of the skin cathepsin inhibitor to the total protein and the ratio of the skin cathepsin to the skin cathepsin inhibitor are also determined. Use as judgment material. Here, in Non-Patent Document 6 described above, skin cathepsin L (CatL) is an enzyme that promotes apoptosis of skin, while skin keratinization resulting from apoptosis caused by UV stimulation is described by Hurpin ( It has been pointed out that it is prevented by an inhibitor (inhibitCatL) called headpin, PI13 or serpin). That is, it is suggested that the secretion balance of CatL and inhibitCatL changes depending on the degree of damage of skin cells by ultraviolet rays, and as a result, the apoptosis of skin cells may be regulated. Therefore, it may be possible to more accurately determine whether the light stress caused on the skin of the subject is acute or chronic by determining the skin cathepsin / cathepsin inhibitor ratio.

  In the second determination method of the present invention, the skin cathepsin inhibitor to be measured is not particularly limited, and examples thereof include Hurpin.

  Skin cathepsin inhibitors contained in samples collected from the skin can be measured using appropriate methods such as immunochromatography by local plasmon resonance using gold colloid, microelectrophoresis, and surface plasmon resonance. The measurement method is not particularly limited, but from the viewpoint of simplicity and rapidity, it is preferable to use immunochromatography as in the case of measuring skin cathepsin contained in a sample collected from the skin. In this case, a commercially available product can be preferably used as the antibody for the skin cathepsin inhibitor (specific examples are described above).

  The present invention further provides a kit that can suitably perform the above-described second determination method of the present invention, and is a sample for dropping a liquid sample onto a substrate configured such that the liquid can move by capillary action. First detection wherein a pad, a conjugate pad pre-impregnated with a conjugate of a skin cathepsin antibody and a gold colloid, and a skin cathepsin antibody recognizing a site different from the antibody used for the conjugate are fixed There is also provided a kit (third kit) in which a part, a second detection part containing an acid-base indicator, and a third detection part to which an antibody of a skin cathepsin inhibitor is immobilized in advance are formed. Even with such a third kit of the present invention, the skin cathepsin in the sample collected from the skin is molecularly recognized and quantitatively analyzed by immunochromatography using localized plasmon resonance and by a highly specific immunoassay. In addition, a low-cost disposable test strip is provided that can simultaneously measure the total protein and skin cathepsin inhibitor in the sample and determine light stress of the skin easily and quickly.

  In the third kit of the present invention, the substrate, the sample pad, the conjugate pad, the first detection unit, and the second detection unit are the substrate, the sample pad, and the conjugate pad described above in the second kit 51 of the present invention. The first detection unit and the second detection unit are as described above, and the gold colloid used for the conjugate pad, the skin cathepsin antibody, the skin cathepsin antibody used for the first detection unit, and the second detection unit. The same applies to the total protein test paper used in the above. In the third kit of the present invention, for example, a sample pad for injecting a liquid sample is provided in the vicinity of the center of the substrate, and the substrate is divided into approximately three regions centering on the sample pad, about 3 minutes. In one part, the above-described conjugate pad and the first detection part are formed and configured to quantitatively analyze the skin cathepsin in the liquid sample, and in about one third part, The second detection unit provided with the total protein test paper is formed so that the total protein in the liquid sample can be quantitatively analyzed, and the remaining one-third portion includes The third detection unit is formed and configured to quantitatively analyze the skin cathepsin inhibitor in the liquid sample.

  Here, for example, an antibody of a skin cathepsin inhibitor (preferably, Hurpin described above) is immobilized in advance on the third detection unit. The antibody is attached to the third detection unit using a conventionally known appropriate method such as immobilization by chemical bond such as covalent bond, ionic bond, coordinate bond, van der Waals force, and film formation such as coating. Can be fixed.

  The embodiments disclosed herein are illustrative in all respects and should not be considered as restrictive. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

  Skin cathepsin is an enzyme responsible for the production of keratin in skin metabolism due to light stress. If simple and rapid analysis of skin cathepsin contained in human keratin is realized, it is useful for quantitative evaluation of skin light stress. is there. In addition, this technology is considered to contribute to the rapid analysis of not only skin cathepsins but also other skin enzymes, and has a wide range of applications in the cosmetics and medical fields.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows typically the analysis kit (1st kit) 1 of the skin cathepsin of a preferable example of this invention, Fig.1 (a) is a state before inject | pouring the liquid sample 8, FIG.1 (b) is FIG. The state after injecting the liquid sample 8 is typically shown. 2A schematically shows the antigen-antibody reaction in the conjugate pad 4 of the first kit 1 of the present invention, and FIG. 2B schematically shows the antigen-antibody reaction in the detection unit 5 of the first kit 1 of the present invention. FIG. It is a figure which shows typically the measurement of the amount of skin cathepsins in the detection part 5 of the kit 1 of this invention of the example shown in FIG. 1, FIG. 3 (a) is a perspective view, FIG.3 (b) is sectional drawing. . FIG. 5 is a perspective view schematically showing a skin light stress determination kit (second kit) 51 of a preferred example of the present invention, FIG. 5 (a) is an external view, and FIG. 5 (b) is FIG. It is a figure which shows the upper body which removed the housing 52 from the state of ().

Explanation of symbols

  1 Kit, 2 Substrate, 3 Sample Pad, 4 Conjugate Pad, 5 Detector, 8 Liquid Sample, 9 Skin Cathepsin, 10 Absorption Pad, 11 Conjugate, 12 Gold Colloid, 13 Skin Cathepsin Antibody, 14 Skin Cathepsin Antibody , 21 Light emitting element, 22 Light receiving element, 51 Kit, 52 Housing, 53 Sample pad, 54 Liquid sample, 55 Substrate, 56 Conjugate pad, 57 First detection unit, 58 Second detection unit.

Claims (11)

  A method for analyzing skin cathepsin contained in a sample collected from skin by immunochromatography using localized plasmon resonance.   2. The method of claim 1, wherein the skin cathepsin is skin cathepsin L.   A method of measuring skin cathepsin and total protein contained in a sample collected from the skin, and determining the light stress of the skin from the ratio of the skin cathepsin to the total protein.   The method according to claim 3, wherein the skin cathepsin is measured by immunochromatography using localized plasmon resonance.   The method according to claim 3 or 4, wherein the skin cathepsin is skin cathepsin L.   Measure skin cathepsin, skin cathepsin inhibitor and total protein in samples taken from skin, ratio of skin cathepsin to total protein, ratio of skin cathepsin inhibitor to total protein and ratio of skin cathepsin to skin cathepsin inhibitor To determine skin light stress.   The method according to claim 6, wherein the skin cathepsin and the skin cathepsin inhibitor are measured by immunochromatography using localized plasmon resonance.   The method according to claim 6 or 7, wherein the skin cathepsin is skin cathepsin L.   A conjugate pad pre-impregnated with a conjugate of a skin cathepsin antibody and a gold colloid on a substrate that is configured to allow liquid movement by capillary action, and recognizes a different site from the antibody used for the conjugate A kit for analyzing skin cathepsin, wherein a detection part to which an antibody for skin cathepsin to be immobilized is previously formed is formed.   A conjugate pad pre-impregnated with a conjugate of a skin cathepsin antibody and a gold colloid on a substrate that is configured to allow liquid movement by capillary action, and recognizes a different site from the antibody used for the conjugate A skin photo-stress determination kit in which a first detection unit to which an antibody for skin cathepsin is immobilized in advance and a second detection unit including an acid-base indicator are formed.   A conjugate pad pre-impregnated with a conjugate of a skin cathepsin antibody and a gold colloid on a substrate that is configured to allow liquid movement by capillary action, and recognizes a different site from the antibody used for the conjugate The first detection unit in which the antibody of the skin cathepsin to be immobilized is formed in advance, the second detection unit including the acid-base indicator, and the third detection unit in which the antibody of the skin cathepsin inhibitor is immobilized in advance are formed. , Kit for light stress determination of skin.

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