WO2020045364A1 - Method and system for analyzing skin sample for determining psoriasis - Google Patents

Method and system for analyzing skin sample for determining psoriasis Download PDF

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Publication number
WO2020045364A1
WO2020045364A1 PCT/JP2019/033364 JP2019033364W WO2020045364A1 WO 2020045364 A1 WO2020045364 A1 WO 2020045364A1 JP 2019033364 W JP2019033364 W JP 2019033364W WO 2020045364 A1 WO2020045364 A1 WO 2020045364A1
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amount
serine
psoriasis
amino acid
unit
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PCT/JP2019/033364
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French (fr)
Japanese (ja)
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洋介 東條
智恵子 水本
秀　道広
崇暢 菅
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株式会社 資生堂
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Priority to JP2020539451A priority Critical patent/JP7366428B2/en
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    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids

Definitions

  • the present invention relates to a skin sample analysis method for determining psoriasis from a skin sample using the amount of D-serine as an index, a psoriasis test method, and a sample analysis system that outputs pathological information about psoriasis.
  • the determination of psoriasis can be made using not only the amount of D-serine but also parameters calculated from the amount of D-serine.
  • Psoriasis is an inflammatory skin disease with cutaneous keratinization associated with keratinocyte overgrowth, characterized primarily by erythema covered by scales. Although the cause of psoriasis has not yet been identified, genetic factors and multiple factors may contribute. For the treatment of psoriasis, topical therapy, phototherapy, internal therapy, surgery and the like are used. In particular, although immunosuppressants such as cyclosporine and infliximab and adalimumab, which are TNF- ⁇ antibodies, have high therapeutic effects, high drug prices place a heavy burden on patients, and apply these treatment methods after accurately diagnosing psoriasis. Is needed.
  • Diagnosis of psoriasis is made based on the appearance and distribution of lesions.Diseases characterized by scale-covered erythema include psoriasis, rose pityriasis, and pirates Different skin disorders are also known, such as rash, lichen pityriasis, lichen planus, and lichen sclerosus.
  • Diagnosis of psoriasis includes atopic dermatitis, seborrheic dermatitis, dermatophytosis, cutaneous lupus erythematosus, eczema, lichen planus, rose pityriasis, squamous cell carcinoma in the epidermis, chronic lichen simplest
  • Differential diagnosis from diseases such as second-stage syphilis is required, and the judgment of an experienced physician is required.
  • inflammatory markers such as TNF- ⁇ , IFN- ⁇ , IL-6, IL-8, IL-10 and IL-12 have been used as psoriasis markers (Patent Document 1).
  • inflammatory markers such as TNF- ⁇ , IFN- ⁇ , IL-6, IL-8, IL-10 and IL-12 have been used as psoriasis markers (Patent Document 1).
  • D-amino acids which were thought to be absent in mammalian organisms, exist in various tissues along with the development of detection technology (Patent Document 2). It was expected to carry. D-amino acids are broadly classified into bound forms, which are present as amino acid residues in proteins, and free forms, which dissolve in tissue fluids and cells. Some free D-amino acids in body fluids are independent of L-amino acids. Thus, it was found that the content fluctuated, and that the content fluctuated depending on the type of disease (Patent Document 3).
  • Non-Patent Document 1 Biotechnology (2014), Vol. 92, No. 12, pp. 653-656; Non-Patent Document 2: Fragrance ⁇ Journal ⁇ (2016), vol. 4, pages 14-18).
  • the present inventors have analyzed chiral amino acids in skin samples of patients with various skin diseases, and found that the amount of D-serine is reduced specifically for psoriasis, leading to the present invention.
  • the present invention relates to a method for analyzing a skin sample for determining psoriasis using the amount of D-serine as an index.
  • a decrease in the amount of D-serine can be associated with psoriasis.
  • the determination of psoriasis can be made using not only the amount of D-serine but also parameters calculated from the amount of D-serine.
  • Still another embodiment relates to a sample analysis system capable of performing the analysis method of the present invention.
  • a sample analysis system includes a storage unit, an input unit, a data processing unit, and an output unit.
  • the analysis result of the skin sample is input from the input unit, and the pathological information of psoriasis is output to the output unit. can do.
  • the present invention relates to a program that can be installed in the sample analysis system of the present invention and a storage medium that stores the program. Yet another aspect relates to a method of operating the sample analysis system of the present invention.
  • the present invention also relates to a kit for determining psoriasis, comprising an anti-D-serine antibody.
  • ⁇ Psoriasis markers different from conventionally known inflammatory markers can be provided.
  • FIG. 1 is a graph showing the ratio of D-serine in a skin sample obtained from a rash and a rash in psoriatic patients, atopic patients, and xeroderma patients.
  • FIG. 2 is an example of a configuration diagram of the analysis system of the present invention.
  • FIG. 3 is a flowchart illustrating an example of an operation for determining psoriasis.
  • the present invention relates to a method for analyzing a skin sample for determining psoriasis using the amount of D-serine in the skin sample as an index.
  • “Using the amount of D-serine as an index” refers to any embodiment in which the amount of D-serine is used as an index for determination. Therefore, it is not intended to use only the amount of D-serine, but includes using a parameter calculated from the amount of D-serine.
  • the analysis method of the present invention relates to an analysis method including a step of measuring the amount of D-serine in a skin sample and a step of relating a decrease in the amount of D-serine to psoriasis. Since psoriasis can be determined by the analysis method of the present invention, in another aspect, the present invention can also be referred to as a diagnostic method.
  • the psoriasis that can be determined in the present invention can be any disease classified into psoriasis such as psoriasis vulgaris, psoriatic arthritis, pustular psoriasis, and guttate psoriasis.
  • psoriasis vulgaris psoriasis vulgaris
  • psoriatic arthritis psoriatic arthritis
  • pustular psoriasis psoriasis
  • guttate psoriasis guttate psoriasis.
  • the analysis method of the present invention makes it possible to distinguish psoriasis from atopic dermatitis, which is an inflammatory skin disease, and xeroderma.
  • Psoriasis can also be specifically distinguished and determined from diseases.
  • the subject may be any subject, for example, a patient suffering from a skin disease of unknown etiology, the analysis method of the present invention may be performed, or a patient diagnosed clinically with psoriasis may be confirmed.
  • the analysis method of the present invention may be performed for diagnosis.
  • the target chiral amino acid may be measured alone, or may be measured together with another chiral amino acid.
  • chiral amino acids can be diagnostic markers for other diseases. Therefore, from the viewpoint of analyzing a plurality of diseases at once, it is preferable to collectively measure D-form and L-form for amino acids serving as diagnostic markers for other diseases, for example, 20 kinds of protein constituent amino acids.
  • D-serine, D-alanine, D-glutamic acid, and D-aspartic acid are present as D-amino acids in skin, and these D-amino acids and corresponding L-amino acids Is more preferably measured collectively.
  • a step of obtaining a skin sample and a step of processing the obtained skin sample may be performed.
  • the skin sample include a skin section and a stratum corneum sample obtained by a tape strip.
  • the skin sample may be obtained from the rash or the rash, but is preferably obtained from the rash to increase the accuracy of the determination of psoriasis. Since the rash of psoriasis is covered with scales, a horny layer sample may be obtained after performing a tape strip several times for the purpose of removing scales.
  • the stratum corneum sample obtained by the tape strip can be treated with a solvent such as methanol to extract amino acids contained in the sample.
  • the amount of each chiral amino acid can be measured by subjecting the extracted chiral amino acid to HPLC using a chiral column or immunological technique using an antibody capable of detecting the chiral amino acid.
  • HPLC is used in measuring the amount of D-serine
  • multi-step HPLC can be performed using another column before the chiral column.
  • the multi-step HPLC is performed by, after separation on a column, holding the separated fractions in a multi-loop unit and sequentially introducing the fractions into the next column.
  • the amount of D-serine in a sample may be measured using any method known to those skilled in the art.
  • D- and L-amino acids are derivatized stereoisomerically in advance with o-phthalaldehyde (OPA), N-tert-butyloxycarbonyl-L-cysteine (Boc-L-Cys) or another modifying reagent, and then A method of separating a mixture of 100 mM acetate buffer (pH 6.0) and acetonitrile by gradient elution using an analytical column such as ODS-80TsQA for simultaneous measurement of D-form and L-form of amino acids. Can be used.
  • OPA o-phthalaldehyde
  • Boc-L-Cys N-tert-butyloxycarbonyl-L-cysteine
  • D- and L-amino acids are previously derivatized with a fluorescent reagent such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and then ODS-80TsQA, Mightysil @ RP
  • a fluorescent reagent such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F)
  • NBD-F 4-fluoro-7-nitro-2,1,3-benzoxadiazole
  • ODS-80TsQA ODS-80TsQA
  • Mightysil @ RP A method of separating each amino acid non-stereospecifically using an analytical column such as -18GP and the like, followed by optical resolution using a Pirkle type chiral stationary phase column (for example, Sumichiral @ OA-2500S or R), It can be used for the trace measurement of constituent amino acids (Kenji Hamase and Kiyoshi Zaitsu, Analytical Chemistry, 53, 677-690 (2004)).
  • the optical resolution column system in this specification refers to a separation analysis system using at least an optical resolution column, and may include a separation analysis using an analysis column other than the optical resolution column. More specifically, passing a sample containing a component having an optical isomer, together with a first liquid as a mobile phase, through a first column packing material as a stationary phase, and separating the components of the sample, Holding each of the components of the sample individually in a multi-loop unit, each of the components of the sample individually held in the multi-loop unit, together with a second liquid as a mobile phase, as a stationary phase Supplying the second column packing material having an optically active center through a channel to split the optical isomer contained in each of the components of the sample, and the optical isomer contained in each of the components of the sample.
  • a method for analyzing an optical isomer which comprises a step of detecting an isomer, the D- / L-amino acid concentration in a sample can be measured ( Patent No. 4,291,628).
  • D-amino acid can be alternatively quantified by an immunological technique using a monoclonal antibody that discriminates optical isomers of D-serine (JP-A-2009-184981).
  • detection can be performed by an ELISA method using a monoclonal antibody that binds to D-serine and a secondary antibody that binds to the antibody and has a label.
  • the step of associating with psoriasis may be performed using a parameter calculated from the amount of D-serine.
  • a parameter calculated from the amount of D-serine for example, a parameter obtained by normalizing the amount of D-serine using the amount of D-serine and an internal standard contained in a skin sample can be used.
  • the ratio or ratio between D-serine and the internal standard may be calculated, or an arbitrary constant may be added, subtracted, multiplied, or divided.
  • the amount of a skin sample, particularly a stratum corneum sample obtained by a tape strip, is not constant depending on the condition of the skin.
  • a stratum corneum sample obtained by a tape strip is not constant depending on the condition of the skin.
  • since the rash is covered with scale it is difficult to obtain a certain amount by using any technique. Therefore, by normalizing using the internal standard in the skin sample, a stable result can be obtained regardless of the technique of obtaining the skin sample.
  • Examples of the internal standard that can be used include a protein amount, a gene amount, a total amino acid amount, a total D-amino acid amount, a total L-amino acid amount, an arbitrary L-amino acid amount, a stratum corneum weight, and the like. Any substance can be used as long as the substance is contained at a certain ratio. In the present invention, it is particularly preferable to use the total amount of L-amino acids or the amount of individual L-amino acids as an internal standard. More preferably, L-serine can be used as an internal standard.
  • the presence or absence of psoriasis can be determined by comparing with a cutoff value of a parameter calculated from the amount of D-serine derived as described above. . Therefore, the judgment may be made by a medical assistant or the like who is not a doctor, or may be made by an analysis institution or the like. Therefore, the analysis method of the present invention can be said to be a preliminary method or an auxiliary method of diagnosis.
  • the analysis method of the present invention may include a step of calculating as a parameter for determining psoriasis instead of the step of associating a decrease in the amount of D-serine with psoriasis. By comparing a parameter calculated by this analysis method with a predetermined cutoff value, psoriasis can be determined.
  • the cutoff value for the determination of psoriasis can be arbitrarily determined by analyzing the cohort and performing statistical processing.
  • a method of the statistical processing a method well-known to those skilled in the art may be used.
  • ROC analysis, t-test, or the like may be used, and the average value, median value, X percentile value of a healthy group or a psoriasis patient group may be used.
  • any numerical value can be selected for X, and 3, 5, 10, 15, 20, 30, 40, 60, 70, 80, 85, 90, 95, and 97 can be appropriately used.
  • the cutoff value may be one, or the disease state may be classified according to the severity of the disease. Further, the cutoff value can be determined by comparison with a patient group having a skin disease other than psoriasis.
  • a treatment suitable for psoriasis is selected and performed.
  • At least one psoriasis treatment step selected from the group consisting of, but not limited to, topical therapy, phototherapy, oral therapy, injection therapy, surgery, and lifestyle improvement is further performed.
  • topical therapy include steroidal external preparations, immunosuppressant external preparations, and vitamin D3 and its derivatives.
  • Phototherapy includes ultraviolet irradiation, for example, irradiation with medium wavelength ultraviolet (UVB) and long wavelength ultraviolet (UVA).
  • UVB medium wavelength ultraviolet
  • UVA long wavelength ultraviolet
  • vitamin A derivatives such as retinoids, steroids, immunosuppressants such as cyclosporine, and PDE4 inhibitors are administered.
  • an antibody drug such as an antibody against an inflammatory cytokine is administered.
  • the treatment steps for psoriasis are not intended to be limited to those described above, and new therapies and agents may be used in accordance with medical advances.
  • anti-TNF- ⁇ monoclonal antibodies adalimumab and infliximab, anti-human IL-12 / 23p40 monoclonal antibody ustekinumab, anti-IL-23p19 monoclonal antibody guselkumab, anti-IL-17A monoclonal antibodies secukinumab and ixekizumab, anti-IL Brodalumab, a -17A receptor A monoclonal antibody, is effective.
  • the method of the present invention may include a therapeutic step including administering these antibody drugs after the determination or diagnosis.
  • FIG. 2 is a configuration diagram of the sample analysis system of the present invention.
  • a sample analysis system 10 includes a storage unit 11, an input unit 12, a data processing unit 14, and an output unit 15, and can analyze a target skin sample and output disease state information. More specifically, in the sample analysis system 10 of the present invention, The storage unit 11 stores a cutoff value of the amount of D-serine and pathological information of psoriasis for determining psoriasis input from the input unit 12, A measurement value of the amount of D-serine in the target skin sample is input from the input unit 12 and stored in the storage unit.
  • the data processing unit 14 determines the disease state information of the target psoriasis by comparing the measured value of the amount of D-serine with the cutoff value stored in the storage unit 11,
  • the output unit 15 can output the disease state information about the target psoriasis.
  • a further aspect of the present invention may relate to a program for operating such a sample analysis system, and also relates to a medium recording such a program.
  • FIG. 3 is a flowchart showing an example of an operation for determining psoriasis according to the program of the present invention.
  • Such programs include: An instruction to store the association between the cutoff value of the amount of D-serine and the pathological information of psoriasis in the storage unit 11; A command to read a measured value of the amount of D-serine in the target skin sample from the input unit 12 and store the measured value in the storage unit 11; A command to activate the data processing unit 14 to compare the measured value of the amount of D-serine stored in the storage unit with the cut-off value to determine pathological information of control psoriasis and store it in the storage unit 11; And a command to operate the output unit 15 to output the stored pathological information about psoriasis.
  • Such a program may be stored in a storage medium, or may be provided via an electric communication line such as the Internet or a LAN.
  • the storage unit 11 includes a memory device such as a RAM, a ROM, and a flash memory, a fixed disk device such as a hard disk drive, or a portable storage device such as a flexible disk and an optical disk.
  • the storage unit 11 includes data measured by the analysis and measurement unit, data and instructions input from the input unit 12, calculation results performed by the data processing unit 14, and other computer programs used for various processes of the information processing apparatus. Stores a database and the like.
  • the computer program may be installed via a computer-readable recording medium such as a CD-ROM, a DVD-ROM, or the Internet. The computer program is installed in the storage unit using a known setup program or the like.
  • the input unit 12 is an interface or the like, and includes an operation unit such as a keyboard and a mouse.
  • an operation unit such as a keyboard and a mouse.
  • the analysis / measurement unit 13 When the analysis / measurement unit 13 is provided outside, the measured data or the like can be input directly or via a network or a storage medium via an interface of the input unit.
  • the input unit can input data measured by the analysis and measurement unit 13, instructions for calculation processing performed by the data processing unit 14, and the like.
  • the analysis system of the present invention may include the analysis and measurement unit 13 via the input unit, or may include the analysis and measurement unit 13 without the input unit.
  • the analysis system of the present invention includes the analysis / measurement unit 13
  • a measurement value of the amount of D-serine in the target skin sample is input from the input unit 12 and stored in the storage unit.
  • 13 includes a step of measuring the amount of the chiral amino acid in the skin sample and storing it in the storage unit.
  • the program includes a command to operate the analysis and measurement unit 13 to determine the measured value of the amount of D-serine and store the measured value in the storage unit.
  • the analysis and measurement unit 13 has a configuration that enables separation and measurement of chiral amino acids.
  • Amino acids may be analyzed one by one, but some or all types of amino acids can be analyzed together.
  • the analysis and measurement unit 13 is not intended to be limited to the following, but may be, for example, a high-performance liquid chromatography system (HPLC) including a sample introduction unit, an optical separation column, and a detection unit.
  • HPLC is preferably a two-dimensional HPLC including a column for separating amino acids separately from the optical resolution column.
  • the analysis and measurement unit 13 may be included in the sample analysis system 10. Further, it may be configured separately from the sample analysis system 10, and the measured data or the like may be input via the input unit 12 using a network or a storage medium.
  • the data processing unit 14 is configured to determine psoriasis by comparing the measured amount of the chiral amino acid with the cutoff value stored in the storage unit 11.
  • the data processing unit 14 performs various arithmetic processes on the data measured by the analysis and measurement unit 13 and stored in the storage unit 11 according to the program stored in the storage unit 11.
  • the arithmetic processing is performed by a CPU included in the data processing unit 14.
  • the CPU includes a functional module that controls the analysis and measurement unit 13, the input unit 12, the storage unit 11, and the output unit 15, and can perform various controls. Each of these units may be configured by an independent integrated circuit, microprocessor, firmware, or the like.
  • the output unit 15 is configured to output a disease state index value and / or disease state information that is a result of performing the arithmetic processing in the data processing unit 14.
  • the calculation processing result in the data processing unit 14 may be directly output to the output unit 15 or may be stored in the storage unit 11 once and then output to the output unit 15 as needed.
  • the output unit 15 may be a display device such as a liquid crystal display that directly displays the result of the arithmetic processing, an output unit such as a printer, or an interface unit for outputting to an external storage device or outputting via a network. There may be.
  • a further aspect of the invention may relate to a kit for determining psoriasis.
  • a kit for determining psoriasis contains an antibody for detecting D-serine, and can detect D-serine in a sample.
  • Antibodies for detecting D-serine include anti-D-serine antibodies.
  • the antibody described in JP-A-2009-184981 can be used.
  • the composition further comprises a control antibody that allows detection of a substance present in the skin sample in an amount. Thereby, even if D-serine has not been detected, it can be determined whether or not the sample preparation has failed.
  • the kit for determining psoriasis may further include, in addition to the antibody, a tape for a tape strip and a solvent for amino acid extraction.
  • a kit may further include a substrate further provided with a detection portion, wherein the detection portion has immobilized thereon an antibody that captures D-serine.
  • the stratum corneum sample obtained by the tape strip is treated with a solvent for amino acid extraction, the solution is developed on a base material, and D-serine is supplemented to the antibody in the detection section. Thereafter, by developing a label capable of detecting the captured D-serine, the captured D-serine can be detected.
  • An example of such a label is an antibody labeled with colloidal gold or the like.
  • a horny layer sample was obtained by a tape strip method from the eruption part and the eruption part of 21 patients with psoriasis vulgaris, 31 patients with atopic dermatitis, and 8 patients with xeroderma. .
  • a horny layer sample was obtained from the skin of 42 healthy subjects as a control.
  • the severity of psoriasis patients was 8 in mild, 7 in moderate, 3 in severe, and 3 in unknown.
  • the horny layer sample was a corneal tape having a sampling area of 5.6 cm 2, and the tape was stripped three times at the same location, and the third sample was used.
  • the stratum corneum sample attached to the stratum corneum tape was treated with 1500 ⁇ l of 95% methanol, subjected to ultrasonic treatment, and centrifuged at 15000 rpm for 10 minutes. 250 ⁇ l of the supernatant was taken and dried under reduced pressure. A 200 mM aqueous sodium borate solution (pH 8.0: 30 ⁇ l) was added to the dried product to dissolve it. To the obtained solution, a 40 mM NBD-F acetonitrile solution (5 ⁇ l) was added, reacted at 60 ° C. for 2 minutes, and 2% (v / v) aqueous trifluoroacetic acid solution (90 ⁇ l) was added thereto to obtain a two-dimensional solution. It was subjected to HPLC analysis.
  • ML-750 Gradient elution using an aqueous mobile phase containing
  • the fraction of the target amino acid is automatically collected by using a multi-loop valve, and the optical resolution column (OA2500S-15250, 1.5 mmi.e. ⁇ 250 mm) (Distributor: Daicel Industries).
  • the mobile phase was a mixed solution of MeOH-MeCN containing citric acid or formic acid, and the fluorescence of NBD-amino acids was excited at 470 nm and detected at 530 nm. All quantitative data was obtained by fluorescence detection. HPLC-MS / MS was used to confirm the presence of D-amino acids in the actual biological matrix.
  • the concentration of D-serine and L-serine in the sample was measured, and the following formula: , The content of D-serine was measured.
  • the measurement results for each disease are shown in FIG.
  • the discrimination between a healthy subject and psoriasis can also be performed using another index including the amount of D-Ser in the calculation formula.
  • a parameter obtained by multiplying the amount of D-Ser by another amount of amino acid here, for example, D-Ser / D-Ala
  • a parameter (D-Ser / cm 2 ) obtained by normalizing the amount of D-Ser in terms of the amount per horny layer area is also useful for diagnosis and discrimination since a significant difference was observed between a healthy subject and a psoriatic eruption area. You can see that there is.

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Abstract

The present invention provides a method for analyzing a skin sample for determining psoriasis in a subject, wherein the amount of D-serine is used as an index. The present invention also provides a system for analyzing a skin sample for determining psoriasis in a subject, the system comprising a storage unit, an input unit, a data processing unit, and an output unit. Further, the present invention provides a kit for determining psoriasis, the kit containing an anti-D-serine antibody.

Description

乾癬を判定するための皮膚試料の分析方法及びシステムMethod and system for analyzing skin samples to determine psoriasis
　本発明は、皮膚試料からＤ－セリン量を指標として乾癬を判定するための皮膚試料の分析方法、乾癬の検査方法、並びに乾癬ついての病態情報を出力する試料分析システムに関する。乾癬の判定は、Ｄ－セリン量のみならず、Ｄ－セリン量から算出されるパラメータを用いて行うことができる。 The present invention relates to a skin sample analysis method for determining psoriasis from a skin sample using the amount of D-serine as an index, a psoriasis test method, and a sample analysis system that outputs pathological information about psoriasis. The determination of psoriasis can be made using not only the amount of D-serine but also parameters calculated from the amount of D-serine.
　乾癬は、ケラチノサイトの異常増殖に伴う皮膚角化を伴う炎症性皮膚疾患であり、主に鱗屑で覆われた紅疹を特徴とする。乾癬の原因はいまだ特定されていないものの、遺伝的要因と、複数の要因が寄与すると考えられる。乾癬の治療には、外用療法、光線療法、内服療法、外科療法などが用いられている。特にシクロスポリンなどの免疫抑制剤、ＴＮＦ－α抗体であるインフリキシマブやアダリムマブなどは治療効果も高いものの、高い薬価のため患者の負担が大きく、乾癬を正確に診断した上でこれらの治療方法を適用することが必要とされている。 Psoriasis is an inflammatory skin disease with cutaneous keratinization associated with keratinocyte overgrowth, characterized primarily by erythema covered by scales. Although the cause of psoriasis has not yet been identified, genetic factors and multiple factors may contribute. For the treatment of psoriasis, topical therapy, phototherapy, internal therapy, surgery and the like are used. In particular, although immunosuppressants such as cyclosporine and infliximab and adalimumab, which are TNF-α antibodies, have high therapeutic effects, high drug prices place a heavy burden on patients, and apply these treatment methods after accurately diagnosing psoriasis. Is needed.
　乾癬の診断は、病変の外観や分布に基づいて行われるが、鱗屑で覆われた紅疹を特徴とする疾患としては、乾癬の他に、類乾癬、ばら色粃糠疹、毛孔性紅色粃糠疹、苔癬状粃糠疹、扁平苔癬、および硬化性苔癬といった異なる皮膚疾患も知られている。また、乾癬の診断には、アトピー性皮膚炎、脂漏性皮膚炎、皮膚糸状菌症、皮膚エリテマトーデス、湿疹、扁平苔癬、ばら色粃糠疹、表皮内有棘細胞癌、慢性単純性苔癬、第２期梅毒といった疾患との鑑別診断が必要となり、経験を有する医師の判断が必要となる。これまで、ＴＮＦ－α、ＩＦＮ－γ、ＩＬ－６、ＩＬ－８、ＩＬ－１０及びＩＬ－１２といった炎症性マーカーを乾癬マーカーとすることは行われてきている（特許文献１）が、他の炎症性疾患と区別しうる乾癬を特異的に診断できる実用化されたマーカーは存在していなかった。 Diagnosis of psoriasis is made based on the appearance and distribution of lesions.Diseases characterized by scale-covered erythema include psoriasis, rose pityriasis, and pirates Different skin disorders are also known, such as rash, lichen pityriasis, lichen planus, and lichen sclerosus. Diagnosis of psoriasis includes atopic dermatitis, seborrheic dermatitis, dermatophytosis, cutaneous lupus erythematosus, eczema, lichen planus, rose pityriasis, squamous cell carcinoma in the epidermis, chronic lichen simplest Differential diagnosis from diseases such as second-stage syphilis is required, and the judgment of an experienced physician is required. Until now, inflammatory markers such as TNF-α, IFN-γ, IL-6, IL-8, IL-10 and IL-12 have been used as psoriasis markers (Patent Document 1). There has been no practical marker capable of specifically diagnosing psoriasis that can be distinguished from inflammatory diseases.
　従来、哺乳類の生体には存在しないと考えられていたＤ-アミノ酸が、検出技術の発達に伴い（特許文献２）、様々な組織において存在することが明らかにされてきており、何らかの生理機能を担うことが予測されていた。Ｄ－アミノ酸はタンパク質中にアミノ酸残基として存在する結合型と、組織液や細胞内に溶解する遊離型に大別され、体液中の幾つかの遊離Ｄ－アミノ酸は、Ｌ－アミノ酸とは独立して含量が変動することが分かっており、疾病の種類に応じても変動することが示された（特許文献３）。また、皮膚においては、Ｄ－セリン、Ｄ－アラニン、Ｄ－グルタミン酸、Ｄ－アスパラギン酸が遊離型として存在することが示されていた（非特許文献１：生物工学（２０１４）、第９２巻、第１２号、第６５３～６５６頁；非特許文献２：Fragrance Journal (2016), vol. 4, pages 14-18)。 Conventionally, it has been revealed that D-amino acids, which were thought to be absent in mammalian organisms, exist in various tissues along with the development of detection technology (Patent Document 2). It was expected to carry. D-amino acids are broadly classified into bound forms, which are present as amino acid residues in proteins, and free forms, which dissolve in tissue fluids and cells. Some free D-amino acids in body fluids are independent of L-amino acids. Thus, it was found that the content fluctuated, and that the content fluctuated depending on the type of disease (Patent Document 3). In the skin, D-serine, D-alanine, D-glutamic acid, and D-aspartic acid have been shown to exist as free forms (Non-Patent Document 1: Biotechnology (2014), Vol. 92, No. 12, pp. 653-656; Non-Patent Document 2: Fragrance {Journal} (2016), vol. 4, pages 14-18).
国際公開第２００９／０９３２８８号International Publication No. 2009/093288 特許第４２９１６２８号公報Japanese Patent No. 4291628 国際公開第２０１３／１４０７８５号International Publication No. 2013/140785
　従来の熟練の専門医による外観判定や炎症性マーカーなどに基づく乾癬診断に代わり、より乾癬に特徴的な診断マーカーの開発、並びにそれにより乾癬を正確に判定、検査又は診断する技術の開発が望まれている。 Instead of the appearance judgment by a conventional expert and the diagnosis of psoriasis based on inflammatory markers, etc., the development of a diagnostic marker that is more characteristic of psoriasis, and the development of a technique for accurately determining, examining or diagnosing psoriasis are desired. ing.
　本発明者らは、様々な皮膚疾患の患者の皮膚試料において、キラルアミノ酸の解析をしていたところ、乾癬特異的に、Ｄ－セリン量が減少することを見出し、本発明に至った。 The present inventors have analyzed chiral amino acids in skin samples of patients with various skin diseases, and found that the amount of D-serine is reduced specifically for psoriasis, leading to the present invention.
　したがって、本発明は、Ｄ－セリン量を指標として乾癬を判定するための皮膚試料の分析方法に関する。本発明によれば、Ｄ－セリン量の低下と、乾癬とを関連付けることができる。乾癬の判定は、Ｄ－セリン量のみならず、Ｄ－セリン量から算出されるパラメータを用いて行うことができる。 Therefore, the present invention relates to a method for analyzing a skin sample for determining psoriasis using the amount of D-serine as an index. According to the present invention, a decrease in the amount of D-serine can be associated with psoriasis. The determination of psoriasis can be made using not only the amount of D-serine but also parameters calculated from the amount of D-serine.
　さらに別の態様では、本発明の分析方法を実施することができる試料分析システムに関する。このような試料分析システムは、記憶部と、入力部と、データ処理部と、出力部とを含んでおり、入力部から皮膚試料の分析結果が入力され、乾癬の病態情報を出力部に出力することができる。 Still another embodiment relates to a sample analysis system capable of performing the analysis method of the present invention. Such a sample analysis system includes a storage unit, an input unit, a data processing unit, and an output unit. The analysis result of the skin sample is input from the input unit, and the pathological information of psoriasis is output to the output unit. can do.
　さらに別の態様では、本発明の試料分析システムにインストールされうるプログラム及び当該プログラムを格納する記憶媒体にも関する。さらに、別の態様では、本発明の試料分析システムを作動させる方法にも関する。 In yet another aspect, the present invention relates to a program that can be installed in the sample analysis system of the present invention and a storage medium that stores the program. Yet another aspect relates to a method of operating the sample analysis system of the present invention.
　さらに別の態様では、本発明は、抗Ｄ－セリン抗体を含む、乾癬を判定するためのキットにも関する。 で は In yet another aspect, the present invention also relates to a kit for determining psoriasis, comprising an anti-D-serine antibody.
　従来知られていた炎症性マーカーとは異なる乾癬マーカーを提供することができる。 乾 Psoriasis markers different from conventionally known inflammatory markers can be provided.
図１は、乾癬患者、アトピー性患者、乾皮症患者の皮疹部と、無疹部とから取得された皮膚試料中におけるＤ－セリンの割合を示すグラフである。FIG. 1 is a graph showing the ratio of D-serine in a skin sample obtained from a rash and a rash in psoriatic patients, atopic patients, and xeroderma patients. 図２は、本発明の分析システムの構成図の一例である。FIG. 2 is an example of a configuration diagram of the analysis system of the present invention. 図３は、乾癬を決定するための動作の例を示すフローチャートである。FIG. 3 is a flowchart illustrating an example of an operation for determining psoriasis.
　本発明は、皮膚試料中のＤ－セリン量を指標とする乾癬を判定するための皮膚試料の分析方法に関する。「Ｄ－セリン量を指標とする」とは、Ｄ－セリン量を判定のための指標として使用する任意の態様を指す。したがって、Ｄ－セリン量のみを用いることを意図するものではなく、Ｄ－セリン量から算出されるパラメータを用いることを包含する。本発明の分析方法は、具体的には、皮膚試料においてＤ－セリンの量を測定する工程、及びＤ－セリン量の低下と乾癬とを関連付ける工程を含む分析方法に関する。本発明の分析方法によって、乾癬を判定できることから、別の態様では、本発明は診断方法ということもできる。 The present invention relates to a method for analyzing a skin sample for determining psoriasis using the amount of D-serine in the skin sample as an index. “Using the amount of D-serine as an index” refers to any embodiment in which the amount of D-serine is used as an index for determination. Therefore, it is not intended to use only the amount of D-serine, but includes using a parameter calculated from the amount of D-serine. Specifically, the analysis method of the present invention relates to an analysis method including a step of measuring the amount of D-serine in a skin sample and a step of relating a decrease in the amount of D-serine to psoriasis. Since psoriasis can be determined by the analysis method of the present invention, in another aspect, the present invention can also be referred to as a diagnostic method.
　本発明で、判定できる乾癬は、尋常性乾癬、関節症性乾癬、膿疱性乾癬、滴状乾癬など乾癬に分類するすべての疾患を判定することができる。本実施例では、本発明の分析方法により、乾癬と、炎症性の皮膚疾患であるアトピー性皮膚炎や、乾皮症との区別が可能であることが示されており、他の炎症性皮膚疾患からも、乾癬を特異的に区別し、判定しうる。 乾 The psoriasis that can be determined in the present invention can be any disease classified into psoriasis such as psoriasis vulgaris, psoriatic arthritis, pustular psoriasis, and guttate psoriasis. In this example, it has been shown that the analysis method of the present invention makes it possible to distinguish psoriasis from atopic dermatitis, which is an inflammatory skin disease, and xeroderma. Psoriasis can also be specifically distinguished and determined from diseases.
　本発明において、対象とは、任意の対象であってよく、例えば病因不明の皮膚疾患を患う患者について、本発明の分析方法が行われてもよいし、乾癬と臨床診断された患者において、確定診断のために、本発明の分析方法が行われてもよい。 In the present invention, the subject may be any subject, for example, a patient suffering from a skin disease of unknown etiology, the analysis method of the present invention may be performed, or a patient diagnosed clinically with psoriasis may be confirmed. The analysis method of the present invention may be performed for diagnosis.
　Ｄ－セリンの量を測定する工程は、目的のキラルアミノ酸のみを測定してもよいし、他のキラルアミノ酸とまとめて測定することもできる。また、キラルアミノ酸は、他の疾患の診断マーカーになりうる。したがって、複数の疾患を一度に解析する観点から、他の疾患の診断マーカーとなるアミノ酸、例えば２０種のタンパク質構成アミノ酸について、Ｄ体及びＬ体をまとめて測定することが好ましい。特に皮膚には、Ｄ－アミノ酸としてＤ－セリン、Ｄ－アラニン、Ｄ－グルタミン酸、Ｄ－アスパラギン酸が存在することが示されていることから、これらのＤ－アミノ酸と、対応するＬ－アミノ酸とをまとめて測定することがより好ましい。 In the step of measuring the amount of D-serine, the target chiral amino acid may be measured alone, or may be measured together with another chiral amino acid. In addition, chiral amino acids can be diagnostic markers for other diseases. Therefore, from the viewpoint of analyzing a plurality of diseases at once, it is preferable to collectively measure D-form and L-form for amino acids serving as diagnostic markers for other diseases, for example, 20 kinds of protein constituent amino acids. In particular, it has been shown that D-serine, D-alanine, D-glutamic acid, and D-aspartic acid are present as D-amino acids in skin, and these D-amino acids and corresponding L-amino acids Is more preferably measured collectively.
　Ｄ－セリンの量を測定する工程の前に、皮膚試料を取得する工程、取得された皮膚試料を処理する工程が行われてもよい。皮膚試料は、皮膚切片や、テープストリップにより取得された角層試料が挙げられる。皮膚試料は、皮疹部から取得されてもよいし、無疹部からとられてもよいが、乾癬の判定の正確度を高めるために、皮疹部から取得されることが好ましい。乾癬の皮疹部は、鱗屑で覆われていることから、鱗屑を除去する目的で複数回テープストリップを行ったうえで、角層試料を取得してもよい。テープストリップにより取得された角層試料は、メタノールなどの溶媒で処理することで、試料に含まれるアミノ酸を抽出することができる。抽出されたキラルアミノ酸を、キラルカラムを用いたＨＰＬＣやキラルアミノ酸を検出可能な抗体を用いた免疫学的手法に供して、各キラルアミノ酸量を測定することができる。Ｄ－セリン量の測定においてＨＰＬＣを用いる場合、キラルカラムの前に別のカラムを用いて、多段階ＨＰＬＣを行うことができる。多段階ＨＰＬＣは、一例として、カラムでの分離後に、分離された画分をマルチループユニット中に保持し、順次、次のカラムに導入することでおこなわれる。 Before the step of measuring the amount of D-serine, a step of obtaining a skin sample and a step of processing the obtained skin sample may be performed. Examples of the skin sample include a skin section and a stratum corneum sample obtained by a tape strip. The skin sample may be obtained from the rash or the rash, but is preferably obtained from the rash to increase the accuracy of the determination of psoriasis. Since the rash of psoriasis is covered with scales, a horny layer sample may be obtained after performing a tape strip several times for the purpose of removing scales. The stratum corneum sample obtained by the tape strip can be treated with a solvent such as methanol to extract amino acids contained in the sample. The amount of each chiral amino acid can be measured by subjecting the extracted chiral amino acid to HPLC using a chiral column or immunological technique using an antibody capable of detecting the chiral amino acid. When HPLC is used in measuring the amount of D-serine, multi-step HPLC can be performed using another column before the chiral column. As an example, the multi-step HPLC is performed by, after separation on a column, holding the separated fractions in a multi-loop unit and sequentially introducing the fractions into the next column.
　試料中のＤ－セリン量の測定は、当業者に周知ないかなる方法を用いて実施してもよい。例えば、予めｏ－フタルアルデヒド（ＯＰＡ）、Ｎ－ｔｅｒｔ－ブチルオキシカルボニル－Ｌ－システイン（Ｂｏｃ－Ｌ－Ｃｙｓ）その他の修飾試薬で立体異性特異的にＤ－及びＬ－アミノ酸を誘導体化し、その後、ＯＤＳ－８０ＴｓＱＡのような分析カラムを用いて１００ｍＭの酢酸塩緩衝液（ｐＨ６．０）とアセトニトリルの混液をグラジエント溶離して分離する方法により、アミノ酸のＤ－体及びＬ－体の同時測定に用いることができる。また、予め４－フルオロ－７－ニトロ－２，１，３－ベンゾキサジアゾール（ＮＢＤ－Ｆ）のような蛍光試薬でＤ－及びＬ－アミノ酸を誘導体化し、その後、ＯＤＳ－８０ＴｓＱＡ、Ｍｉｇｈｔｙｓｉｌ　ＲＰ－１８ＧＰ等のような分析カラムを用いて立体異性非特異的に各アミノ酸を分離した後、Ｐｉｒｋｌｅ型キラル固定相カラム（例えばＳｕｍｉｃｈｉｒａｌ　ＯＡ－２５００Ｓ又はＲ）を用いて光学分割する方法が、タンパク質を構成するアミノ酸の微量測定に用いることができる（浜瀬健司及び財津潔、分析化学、５３巻、６７７－６９０（２００４））。本明細書における光学分割カラム系は、少なくとも光学分割カラムを用いる分離分析系をいい、光学分割カラム以外の分析カラムによる分離分析を含む場合がある。より具体的に、光学異性体を有する成分を含む試料を、移動相としての第一の液体と共に、固定相としての第一のカラム充填剤に通じて、前記試料の前記成分を分離するステップ、前記試料の前記成分の各々をマルチループユニットにおいて個別に保持するステップ、前記マルチループユニットにおいて個別に保持された前記試料の前記成分の各々を、移動相としての第二の液体と共に、固定相としての光学活性中心を有する第二のカラム充填剤に流路を通じて供給し、前記試料の成分の各々に含まれる前記光学異性体を分割するステップ、及び前記試料の成分の各々に含まれる前記光学異性体を検出するステップを含むことを特徴とする光学異性体の分析方法を用いることにより、試料中のＤ-／Ｌ-アミノ酸濃度を測定することができる（特許第４２９１６２８号）。 (4) The amount of D-serine in a sample may be measured using any method known to those skilled in the art. For example, D- and L-amino acids are derivatized stereoisomerically in advance with o-phthalaldehyde (OPA), N-tert-butyloxycarbonyl-L-cysteine (Boc-L-Cys) or another modifying reagent, and then A method of separating a mixture of 100 mM acetate buffer (pH 6.0) and acetonitrile by gradient elution using an analytical column such as ODS-80TsQA for simultaneous measurement of D-form and L-form of amino acids. Can be used. Further, D- and L-amino acids are previously derivatized with a fluorescent reagent such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and then ODS-80TsQA, Mightysil @ RP A method of separating each amino acid non-stereospecifically using an analytical column such as -18GP and the like, followed by optical resolution using a Pirkle type chiral stationary phase column (for example, Sumichiral @ OA-2500S or R), It can be used for the trace measurement of constituent amino acids (Kenji Hamase and Kiyoshi Zaitsu, Analytical Chemistry, 53, 677-690 (2004)). The optical resolution column system in this specification refers to a separation analysis system using at least an optical resolution column, and may include a separation analysis using an analysis column other than the optical resolution column. More specifically, passing a sample containing a component having an optical isomer, together with a first liquid as a mobile phase, through a first column packing material as a stationary phase, and separating the components of the sample, Holding each of the components of the sample individually in a multi-loop unit, each of the components of the sample individually held in the multi-loop unit, together with a second liquid as a mobile phase, as a stationary phase Supplying the second column packing material having an optically active center through a channel to split the optical isomer contained in each of the components of the sample, and the optical isomer contained in each of the components of the sample. By using a method for analyzing an optical isomer, which comprises a step of detecting an isomer, the D- / L-amino acid concentration in a sample can be measured ( Patent No. 4,291,628).
　Ｄ－セリン量の測定については、代替的に、Ｄ－セリンの光学異性体を識別するモノクローナル抗体を用いる免疫学的手法によってＤ－アミノ酸を定量することができる（特開2009-184981）。具体的に、一例として、Ｄ－セリンに結合するモノクローナル抗体と、当該抗体に結合し、標識を有する２次抗体とを用いてＥＬＩＳＡの手法により検出することができる。 For the measurement of 測定 D-serine amount, D-amino acid can be alternatively quantified by an immunological technique using a monoclonal antibody that discriminates optical isomers of D-serine (JP-A-2009-184981). Specifically, as an example, detection can be performed by an ELISA method using a monoclonal antibody that binds to D-serine and a secondary antibody that binds to the antibody and has a label.
　Ｄ－セリン量の低下と乾癬とを関連付ける工程は、Ｄ－セリン量から算出されるパラメータを用いて、乾癬との関連付けを行ってもよい。Ｄ－セリン量から算出されるパラメータとして、例えばＤ－セリン量と、皮膚試料中に含まれる内部標準を用いて、Ｄ－セリン量を正規化して得たパラメータを用いることができる。このようなパラメータとして、Ｄ－セリンと内部標準の比や割合を算出してもよいし、さらに任意の定数を加減乗除してもよい。皮膚試料、特にテープストリップにより得られた角層試料は、皮膚の状態に応じ、取得できる量が一定にはならない。特に皮疹部では、鱗屑で覆われていることから、どのような手技を用いても、一定量を取得することは困難である。したがって、皮膚試料中の内部標準を用いて正規化することで、皮膚試料の取得の手技にかかわらず安定した結果を得ることができる。 In the step of associating a decrease in the amount of D-serine with psoriasis, the step of associating with psoriasis may be performed using a parameter calculated from the amount of D-serine. As a parameter calculated from the amount of D-serine, for example, a parameter obtained by normalizing the amount of D-serine using the amount of D-serine and an internal standard contained in a skin sample can be used. As such a parameter, the ratio or ratio between D-serine and the internal standard may be calculated, or an arbitrary constant may be added, subtracted, multiplied, or divided. The amount of a skin sample, particularly a stratum corneum sample obtained by a tape strip, is not constant depending on the condition of the skin. In particular, since the rash is covered with scale, it is difficult to obtain a certain amount by using any technique. Therefore, by normalizing using the internal standard in the skin sample, a stable result can be obtained regardless of the technique of obtaining the skin sample.
　使用しうる内部標準としては、タンパク質量、遺伝子量、総アミノ酸量、総Ｄ－アミノ酸量、総Ｌ－アミノ酸量、任意のＬ－アミノ酸の量、角層重量といった、皮膚試料中に、通常、一定の割合で含まれる物質であれば任意のものを使用することができる。本発明では特に、総Ｌ－アミノ酸量や、個別のＬ－アミノ酸の量を内部標準として用いることが好ましい。さらに好ましくはＬ－セリンを内部標準として用いることができる。一例として、Ｌ－セリン量を内部標準として用いた場合、Ｄ－セリン量／Ｌ－セリン量や、Ｄ－セリン％（＝１００×Ｄ－セリン量／Ｄ－セリン量＋Ｌ－セリン量）を用いることができる。 Examples of the internal standard that can be used include a protein amount, a gene amount, a total amino acid amount, a total D-amino acid amount, a total L-amino acid amount, an arbitrary L-amino acid amount, a stratum corneum weight, and the like. Any substance can be used as long as the substance is contained at a certain ratio. In the present invention, it is particularly preferable to use the total amount of L-amino acids or the amount of individual L-amino acids as an internal standard. More preferably, L-serine can be used as an internal standard. As an example, when the amount of L-serine is used as an internal standard, D-serine amount / L-serine amount or D-serine% (= 100 × D-serine amount / D-serine amount + L-serine amount) is used. be able to.
　Ｄ－セリン量の低下と乾癬とを関連付ける工程において、上述のようにして導いたＤ－セリン量から算出されるパラメータのカットオフ値と比較することで乾癬の罹患の有無を判定することができる。したがって、判定は、医師ではない医療補助者などにより行われてもよいし、分析機関などが行うこともできる。したがって、本発明の分析方法は、診断の予備的方法又は補助方法ということもできる。 In the step of associating a decrease in the amount of D-serine with psoriasis, the presence or absence of psoriasis can be determined by comparing with a cutoff value of a parameter calculated from the amount of D-serine derived as described above. . Therefore, the judgment may be made by a medical assistant or the like who is not a doctor, or may be made by an analysis institution or the like. Therefore, the analysis method of the present invention can be said to be a preliminary method or an auxiliary method of diagnosis.
　本発明の別の態様では、本発明の分析方法は、Ｄ－セリン量の低下と乾癬とを関連付ける工程に代わり、乾癬を判定するためのパラメータとして算出する工程を含んでもよい。この分析方法で算出されたパラメータを、予め決定されたカットオフ値と比較することで、乾癬を判定することができる。 In another aspect of the present invention, the analysis method of the present invention may include a step of calculating as a parameter for determining psoriasis instead of the step of associating a decrease in the amount of D-serine with psoriasis. By comparing a parameter calculated by this analysis method with a predetermined cutoff value, psoriasis can be determined.
　乾癬の判定のためのカットオフ値は、コホートを解析し、統計処理することで任意に決定することができる。統計処理の手法は、当業者に周知のものを使用すればよく、例えばＲＯＣ解析、ｔ検定などが用いられてもよいし、健常者群又は乾癬患者群の平均値、中央値、Ｘパーセンタイル値を使用することもできる。ここでＸは任意の数値を選択することができ、３、５、１０、１５、２０、３０、４０、６０、７０、８０、８５、９０、９５、９７を適宜使用することができる。カットオフ値は１つであってもよいし、疾患の重篤度に応じて病態を分類することもできる。また、乾癬以外の皮膚疾患を有する患者群との比較によりカットオフ値を決定することができる。 カ ッ ト The cutoff value for the determination of psoriasis can be arbitrarily determined by analyzing the cohort and performing statistical processing. As a method of the statistical processing, a method well-known to those skilled in the art may be used. For example, ROC analysis, t-test, or the like may be used, and the average value, median value, X percentile value of a healthy group or a psoriasis patient group may be used. Can also be used. Here, any numerical value can be selected for X, and 3, 5, 10, 15, 20, 30, 40, 60, 70, 80, 85, 90, 95, and 97 can be appropriately used. The cutoff value may be one, or the disease state may be classified according to the severity of the disease. Further, the cutoff value can be determined by comparison with a patient group having a skin disease other than psoriasis.
　本発明の分析方法で乾癬を判定した場合に、乾癬に適した治療が選択して行われる。以下のものに限定されるものではないが、外用療法、光線療法、内服療法、注射療法、外科療法、生活習慣の改善からなる群から選ばれる少なくとも１の乾癬の治療工程がさらに行われる。外用療法としては、ステロイド系の外用剤、免疫抑制剤の外用剤、ビタミンＤ３およびその誘導体外用剤が挙げられる。光線療法としては、紫外線照射、例えば中波長紫外線（ＵＶＢ）と長波長紫外線（ＵＶＡ）の照射が挙げられる。内服療法としては、レチノイドなどのビタミンＡ誘導体や、ステロイド、シクロスポリンなどの免疫抑制剤、ＰＤＥ４阻害剤が投与される。注射療法としては、炎症性サイトカインに対する抗体などの抗体医薬が投与される。乾癬の治療工程は、上述のものに限定されることを意図しておらず、医療の進歩に応じて新たな療法及び薬剤が用いられてもよい。 治療 When psoriasis is determined by the analysis method of the present invention, a treatment suitable for psoriasis is selected and performed. At least one psoriasis treatment step selected from the group consisting of, but not limited to, topical therapy, phototherapy, oral therapy, injection therapy, surgery, and lifestyle improvement is further performed. Examples of the topical therapy include steroidal external preparations, immunosuppressant external preparations, and vitamin D3 and its derivatives. Phototherapy includes ultraviolet irradiation, for example, irradiation with medium wavelength ultraviolet (UVB) and long wavelength ultraviolet (UVA). For internal therapy, vitamin A derivatives such as retinoids, steroids, immunosuppressants such as cyclosporine, and PDE4 inhibitors are administered. As an injection therapy, an antibody drug such as an antibody against an inflammatory cytokine is administered. The treatment steps for psoriasis are not intended to be limited to those described above, and new therapies and agents may be used in accordance with medical advances.
　近年、乾癬は、インターロイキン１７を産生するＴｈ１７細胞性慢性疾患と考えられるようになっており、その治療には、Ｔｈ１７細胞の活性を抑制する薬剤が用いられている。具体的には、抗ＴＮＦ－αモノクローナル抗体のアダリムマブとインフリキシマブ、抗ヒトＩＬ－１２／２３ｐ４０モノクローナル抗体のウステキヌマブ、抗ＩＬ－２３ｐ１９モノクローナル抗体のグセルクマブ、抗ＩＬ－１７Ａモノクローナル抗体のセクキヌマブとイキセキズマブ、抗ＩＬ－１７Ａ受容体Ａモノクローナル抗体のブロダルマブなどが効果をあげている。一方これらの抗体医薬は、非常に高額であり、その効果が乾癬と誤診されうる皮膚疾患に対しては低いか全くないことが多いため、乾癬を正確に判定又は診断後に行うことが肝要となる。したがって、本発明の方法により、判定又は診断後に、これらの抗体医薬を投与することを含む治療工程を含んでもよい。 In recent years, psoriasis has been considered to be a Th17 cell chronic disease that produces interleukin 17, and a drug that suppresses the activity of Th17 cells has been used for its treatment. Specifically, anti-TNF-α monoclonal antibodies adalimumab and infliximab, anti-human IL-12 / 23p40 monoclonal antibody ustekinumab, anti-IL-23p19 monoclonal antibody guselkumab, anti-IL-17A monoclonal antibodies secukinumab and ixekizumab, anti-IL Brodalumab, a -17A receptor A monoclonal antibody, is effective. On the other hand, these antibody drugs are very expensive, and their effects are often low or not at all for skin diseases that can be misdiagnosed as psoriasis, so it is important to accurately determine or carry out psoriasis after diagnosis. . Therefore, the method of the present invention may include a therapeutic step including administering these antibody drugs after the determination or diagnosis.
　本発明の試料分析システムは、本発明の方法を実施するように構成される。図２は、本発明の試料分析システムの構成図である。このような試料分析システム１０は、記憶部１１と、入力部１２、データ処理部１４と、出力部１５とを含んでおり、対象の皮膚試料を分析し、病態情報を出力することができる。より具体的に、本発明の試料分析システム１０において、
　記憶部１１は、入力部１２から入力された乾癬を判別するための、Ｄ－セリン量のカットオフ値と乾癬の病態情報を記憶し、
　入力部１２から、対象の皮膚試料中のＤ－セリンの量の測定値が入力され、記憶部に記憶し、
　データ処理部１４は、Ｄ－セリン量の測定値を、記憶部１１に記憶されたカットオフ値と比較することにより、対象の乾癬の病態情報を決定し、
　出力部１５が対象の乾癬についての病態情報を出力することができる。 The sample analysis system of the present invention is configured to perform the method of the present invention. FIG. 2 is a configuration diagram of the sample analysis system of the present invention. Such a sample analysis system 10 includes a storage unit 11, an input unit 12, a data processing unit 14, and an output unit 15, and can analyze a target skin sample and output disease state information. More specifically, in the sample analysis system 10 of the present invention,
The storage unit 11 stores a cutoff value of the amount of D-serine and pathological information of psoriasis for determining psoriasis input from the input unit 12,
A measurement value of the amount of D-serine in the target skin sample is input from the input unit 12 and stored in the storage unit.
The data processing unit 14 determines the disease state information of the target psoriasis by comparing the measured value of the amount of D-serine with the cutoff value stored in the storage unit 11,
The output unit 15 can output the disease state information about the target psoriasis.
　本発明のさらなる態様では、かかる試料分析システムを作動させるプログラムに関していてもよく、かかるプログラムを記録した媒体にも関する。図３は、本発明のプログラムによる乾癬を決定するための動作の例を示すフローチャートである。かかるプログラムは、以下の：
　Ｄ－セリン量のカットオフ値と、乾癬の病態情報との関連を記憶部１１に記憶させる指令；
　入力部１２から、対象の皮膚試料中のＤ－セリンの量の測定値を読みだして記憶部１１に記憶させる指令；
　データ処理部１４を作動させて、記憶部に記憶されたＤ－セリン量の測定値を、カットオフ値と比較させて、対照の乾癬の病態情報を決定させて記憶部１１に記憶させる指令；及び
　出力部１５を作動させて、記憶された乾癬についての病態情報を出力させる指令
　を含む。このようなプログラムは、記憶媒体に格納されてもよいし、インターネット又はＬＡＮ等の電気通信回線を介して提供されてもよい。 A further aspect of the present invention may relate to a program for operating such a sample analysis system, and also relates to a medium recording such a program. FIG. 3 is a flowchart showing an example of an operation for determining psoriasis according to the program of the present invention. Such programs include:
An instruction to store the association between the cutoff value of the amount of D-serine and the pathological information of psoriasis in the storage unit 11;
A command to read a measured value of the amount of D-serine in the target skin sample from the input unit 12 and store the measured value in the storage unit 11;
A command to activate the data processing unit 14 to compare the measured value of the amount of D-serine stored in the storage unit with the cut-off value to determine pathological information of control psoriasis and store it in the storage unit 11; And a command to operate the output unit 15 to output the stored pathological information about psoriasis. Such a program may be stored in a storage medium, or may be provided via an electric communication line such as the Internet or a LAN.
　記憶部１１は、ＲＡＭ、ＲＯＭ、フラッシュメモリ等のメモリ装置、ハードディスクドライブ等の固定ディスク装置、又はフレキシブルディスク、光ディスク等の可搬用の記憶装置などを有する。記憶部１１は、分析測定部で測定したデータ、入力部１２から入力されたデータ及び指示、データ処理部１４で行った演算処理結果等の他、情報処理装置の各種処理に用いられるコンピュータプログラム、データベースなどを記憶する。コンピュータプログラムは、例えばＣＤ－ＲＯＭ、ＤＶＤ－ＲＯＭ等のコンピュータ読み取り可能な記録媒体や、インターネットを介してインストールされてもよい。コンピュータプログラムは、公知のセットアッププログラム等を用いて記憶部にインストールされる。 The storage unit 11 includes a memory device such as a RAM, a ROM, and a flash memory, a fixed disk device such as a hard disk drive, or a portable storage device such as a flexible disk and an optical disk. The storage unit 11 includes data measured by the analysis and measurement unit, data and instructions input from the input unit 12, calculation results performed by the data processing unit 14, and other computer programs used for various processes of the information processing apparatus. Stores a database and the like. The computer program may be installed via a computer-readable recording medium such as a CD-ROM, a DVD-ROM, or the Internet. The computer program is installed in the storage unit using a known setup program or the like.
　入力部１２は、インターフェイス等であり、キーボード、マウス等の操作部を含む。また、分析測定部１３が外部にある場合は、入力部のインターフェイスを介し、測定したデータ等を直接、又はネットワークや記憶媒体を介して入力することができる。これにより、入力部は、分析測定部１３で測定したデータ、データ処理部１４で行う演算処理の指示等を入力することができる。 The input unit 12 is an interface or the like, and includes an operation unit such as a keyboard and a mouse. When the analysis / measurement unit 13 is provided outside, the measured data or the like can be input directly or via a network or a storage medium via an interface of the input unit. Thus, the input unit can input data measured by the analysis and measurement unit 13, instructions for calculation processing performed by the data processing unit 14, and the like.
　本発明の分析システムは、入力部を介して分析測定部１３を備えてもよいし、入力部を介さず分析測定部１３を備えていてもよい。本発明の分析システムが分析測定部１３を備える場合、入力部１２から、対象の皮膚試料中のＤ－セリンの量の測定値が入力され、記憶部に記憶する工程に代えて、分析測定部１３が、皮膚試料中のキラルアミノ酸の量を測定し、記憶部に記憶する工程を含む。その場合の、プログラムは、分析測定部１３を作動させて、Ｄ－セリン量の測定値を決定し、記憶部に記憶させる指令を含む。 The analysis system of the present invention may include the analysis and measurement unit 13 via the input unit, or may include the analysis and measurement unit 13 without the input unit. When the analysis system of the present invention includes the analysis / measurement unit 13, a measurement value of the amount of D-serine in the target skin sample is input from the input unit 12 and stored in the storage unit. 13 includes a step of measuring the amount of the chiral amino acid in the skin sample and storing it in the storage unit. In this case, the program includes a command to operate the analysis and measurement unit 13 to determine the measured value of the amount of D-serine and store the measured value in the storage unit.
　分析測定部１３は、キラルアミノ酸の分離及び測定を可能にする構成を有する。アミノ酸は、１つずつ分析されてもよいが、一部又は全ての種類のアミノ酸についてまとめて分析することができる。分析測定部１３は、以下のものに限定されることを意図するものではないが、例えば試料導入部、光学分割カラム、検出部を備えた高速液体クロマトグラフィーシステム（ＨＰＬＣ）であってもよい。かかるＨＰＬＣは、光学分割カラムとは別にアミノ酸を分離するカラムを含む２次元ＨＰＬＣであることが好ましい。分析測定部１３は、試料分析システム１０に含まれていてもよい。また、試料分析システム１０とは別に構成されていてもよく、測定したデータ等をネットワークや記憶媒体を用いて入力部１２を介して入力されてもよい。 The analysis and measurement unit 13 has a configuration that enables separation and measurement of chiral amino acids. Amino acids may be analyzed one by one, but some or all types of amino acids can be analyzed together. The analysis and measurement unit 13 is not intended to be limited to the following, but may be, for example, a high-performance liquid chromatography system (HPLC) including a sample introduction unit, an optical separation column, and a detection unit. Such HPLC is preferably a two-dimensional HPLC including a column for separating amino acids separately from the optical resolution column. The analysis and measurement unit 13 may be included in the sample analysis system 10. Further, it may be configured separately from the sample analysis system 10, and the measured data or the like may be input via the input unit 12 using a network or a storage medium.
　データ処理部１４は、測定されたキラルアミノ酸の量を、記憶部１１に記憶されたカットオフ値と比較することにより、乾癬を判定するように構成される。データ処理部１４は、記憶部１１に記憶しているプログラムに従って、分析測定部１３で測定され記憶部１１に記憶されたデータに対して、各種の演算処理を実行する。演算処理は、データ処理部１４に含まれるＣＰＵによりおこなわれる。このＣＰＵは、分析測定部１３、入力部１２、記憶部１１、及び出力部１５を制御する機能モジュールを含み、各種の制御を行うことができる。これらの各部は、それぞれ独立した集積回路、マイクロプロセッサ、ファームウェアなどで構成されてもよい。 The data processing unit 14 is configured to determine psoriasis by comparing the measured amount of the chiral amino acid with the cutoff value stored in the storage unit 11. The data processing unit 14 performs various arithmetic processes on the data measured by the analysis and measurement unit 13 and stored in the storage unit 11 according to the program stored in the storage unit 11. The arithmetic processing is performed by a CPU included in the data processing unit 14. The CPU includes a functional module that controls the analysis and measurement unit 13, the input unit 12, the storage unit 11, and the output unit 15, and can perform various controls. Each of these units may be configured by an independent integrated circuit, microprocessor, firmware, or the like.
　出力部１５は、データ処理部１４で演算処理を行った結果である病態指標値及び／又は病態情報を出力するように構成さる。データ処理部１４における演算処理結果は、直接出力部１５に出力されてもよいし、いったん記憶部１１に記憶された上で、必要に応じて出力部１５に出力されてもよい。出力部１５は、演算処理の結果を直接表示する液晶ディスプレイ等の表示装置、プリンタ等の出力手段であってもよいし、外部記憶装置への出力又はネットワークを介して出力するためのインターフェイス部であってもよい。 The output unit 15 is configured to output a disease state index value and / or disease state information that is a result of performing the arithmetic processing in the data processing unit 14. The calculation processing result in the data processing unit 14 may be directly output to the output unit 15 or may be stored in the storage unit 11 once and then output to the output unit 15 as needed. The output unit 15 may be a display device such as a liquid crystal display that directly displays the result of the arithmetic processing, an output unit such as a printer, or an interface unit for outputting to an external storage device or outputting via a network. There may be.
　本発明のさらなる態様では、乾癬を判定するためのキットに関していてもよい。かかるキットは、Ｄ－セリン検出用の抗体を含み、試料中のＤ－セリンの検出ができる。Ｄ－セリン検出用の抗体として、抗Ｄ－セリン抗体が挙げられ、一例として特開2009-184981号公報に記載の抗体を使用しうる。乾癬患者から得られた試料では、Ｄ－セリン量が極めて少ないか、又は存在しないため、Ｄ－セリンの存在を検出することで、乾癬の判定が可能である。より好ましい態様では、皮膚試料中に一定量存在する物質の検出を可能にする対照抗体をさらに含む。これにより、Ｄ－セリンが検出できていない場合であっても、試料調製の失敗か否かを判定することができる。 さ ら な る A further aspect of the invention may relate to a kit for determining psoriasis. Such a kit contains an antibody for detecting D-serine, and can detect D-serine in a sample. Antibodies for detecting D-serine include anti-D-serine antibodies. As an example, the antibody described in JP-A-2009-184981 can be used. In a sample obtained from a psoriasis patient, the amount of D-serine is extremely small or absent, and thus, the presence of D-serine can be detected to determine psoriasis. In a more preferred embodiment, the composition further comprises a control antibody that allows detection of a substance present in the skin sample in an amount. Thereby, even if D-serine has not been detected, it can be determined whether or not the sample preparation has failed.
　乾癬を判定するためのキットは、抗体に加えて、テープストリップ用のテープ、及びアミノ酸抽出用の溶媒をさらに含んでいてもよい。このようなキットは、さらに検出部を備える基材であって、検出部においてＤ－セリンを補足する抗体を固定化した基材さらに含んでいてもよい。この場合、テープストリップにより取得された角層試料は、アミノ酸抽出用溶媒で処理され、かかる溶液を基材上で展開され、検出部の抗体にＤ－セリンが補足される。そののちに、補足されたＤ－セリンを検出可能な標識を展開させることで、補足されたＤ－セリンを検出することができる。このような標識として、金コロイドなどで標識された抗体が挙げられる。 (4) The kit for determining psoriasis may further include, in addition to the antibody, a tape for a tape strip and a solvent for amino acid extraction. Such a kit may further include a substrate further provided with a detection portion, wherein the detection portion has immobilized thereon an antibody that captures D-serine. In this case, the stratum corneum sample obtained by the tape strip is treated with a solvent for amino acid extraction, the solution is developed on a base material, and D-serine is supplemented to the antibody in the detection section. Thereafter, by developing a label capable of detecting the captured D-serine, the captured D-serine can be detected. An example of such a label is an antibody labeled with colloidal gold or the like.
　本明細書において言及される全ての文献はその全体が引用により本明細書に取り込まれる。 All documents referred to herein are hereby incorporated by reference in their entirety.
　以下に説明する本発明の実施例は例示のみを目的とし、本発明の技術的範囲を限定するものではない。本発明の技術的範囲は特許請求の範囲の記載によってのみ限定される。本発明の趣旨を逸脱しないことを条件として、本発明の変更、例えば、本発明の構成要件の追加、削除及び置換を行うことができる。 実 施 The embodiments of the present invention described below are for the purpose of illustration only, and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Modifications of the present invention, for example, additions, deletions, and substitutions of constituent elements of the present invention, can be made on condition that they do not depart from the spirit of the present invention.
集団及びサンプルの分析
　２１名の尋常性乾癬患者、３１名のアトピー性皮膚炎患者、８名の乾皮症患者の皮疹部と、無疹部とから、テープストリップ法により角層試料を取得した。同様に、対照として４２名の健常者の皮膚から角層試料を取得した。乾癬患者の重症度は、軽症８名、中等症７名、重症３名、不明３名であった。角層試料は、５．６ｃｍ2の採取面積の角層テープを用い、同じ個所で３回テープストリップを行い、３枚目の試料を用いた。角層テープに付着した角層試料を、９５％メタノール１５００μｌで処理し、超音波処理を行い、１５０００ｒｐｍで１０分遠心した。上清を２５０μｌ分取し、減圧下で乾固させた。乾固物に対し、２００ｍＭホウ酸ナトリウム水溶液（ｐＨ８．０：３０μｌ）を添加し溶解させた。得られた溶液に対し、４０ｍＭのＮＢＤ－Ｆアセトニトリル溶液（５μｌ）を添加して、６０℃で２分間反応させ、２％（v/v)トリフルオロ酢酸水溶液９０μｌ）を添加して、２次元ＨＰＬＣ分析に供した。 Analysis of Population and Samples A horny layer sample was obtained by a tape strip method from the eruption part and the eruption part of 21 patients with psoriasis vulgaris, 31 patients with atopic dermatitis, and 8 patients with xeroderma. . Similarly, a horny layer sample was obtained from the skin of 42 healthy subjects as a control. The severity of psoriasis patients was 8 in mild, 7 in moderate, 3 in severe, and 3 in unknown. The horny layer sample was a corneal tape having a sampling area of 5.6 cm 2, and the tape was stripped three times at the same location, and the third sample was used. The stratum corneum sample attached to the stratum corneum tape was treated with 1500 μl of 95% methanol, subjected to ultrasonic treatment, and centrifuged at 15000 rpm for 10 minutes. 250 μl of the supernatant was taken and dried under reduced pressure. A 200 mM aqueous sodium borate solution (pH 8.0: 30 μl) was added to the dried product to dissolve it. To the obtained solution, a 40 mM NBD-F acetonitrile solution (5 μl) was added, reacted at 60 ° C. for 2 minutes, and 2% (v / v) aqueous trifluoroacetic acid solution (90 μl) was added thereto to obtain a two-dimensional solution. It was subjected to HPLC analysis.
２Ｄ-ＨＰＬＣによるアミノ酸エナンチオマーの測定
　アミノ酸のエナンチオマーを、J Chromatogr A 1217, 1056-1062 (2010)やJournal of chromatography. B, Analytical technologies in the biomedical and life sciences 877, 2506-2512 (2009)に記載される通りに、マイクロ２Ｄ-ＨＰＬＣプラットフォームを用いて定量した。簡潔に記載すると、アミノ酸のＮＢＤ-誘導体を、逆相カラム（モノリシックＯＤＳカラム、０．５３ｍｍi.d.×１００ｍｍ（ＭＬ－７５０）販売元：非売品・資生堂試作品）を用い、ＭｅＣＮ、ＴＨＦ及びＴＦＡを含む水性移動相を用いて勾配溶出した。Ｄ体及びＬ体を分離して測定するために、標的アミノ酸の画分をマルチループバルブを用いて自動的に回収し、そして光学分割カラム（ＯＡ２５００Ｓ－１５２５０，　１．５ｍｍi.e. ×２５０ｍｍ）販売元：ダイセル工業）に供した。移動相は、クエン酸又はギ酸を含むＭｅＯＨ-ＭｅＣＮの混合溶液であり、そしてＮＢＤ-アミノ酸の蛍光を、４７０ｎｍで励起し、５３０ｎｍで検出した。全ての定量データを蛍光検出により取得した。ＨＰＬＣ－ＭＳ／ＭＳを用いて、実際の生物的マトリクス中のＤ-アミノ酸の存在を確認した。 Measurement of amino acid enantiomers by 2D-HPLC The enantiomers of amino acids are described in J Chromatogr A 1217, 1056-1062 (2010) and Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 877, 2506-2512 (2009). Quantification was performed using a micro 2D-HPLC platform as described. Briefly, NBD-derivatives of amino acids were converted to MeCN, THF and TFA using a reversed-phase column (monolithic ODS column, 0.53 mm i.d. × 100 mm (ML-750); Gradient elution using an aqueous mobile phase containing In order to separate and measure the D-form and the L-form, the fraction of the target amino acid is automatically collected by using a multi-loop valve, and the optical resolution column (OA2500S-15250, 1.5 mmi.e. × 250 mm) (Distributor: Daicel Industries). The mobile phase was a mixed solution of MeOH-MeCN containing citric acid or formic acid, and the fluorescence of NBD-amino acids was excited at 470 nm and detected at 530 nm. All quantitative data was obtained by fluorescence detection. HPLC-MS / MS was used to confirm the presence of D-amino acids in the actual biological matrix.
　試料中におけるＤ－セリン及びＬ－セリンの濃度を測定し、下記の式：
Figure JPOXMLDOC01-appb-M000001
 に基づいて、Ｄ－セリンの含有率を測定した。各疾患における測定結果を、図１に示した。 The concentration of D-serine and L-serine in the sample was measured, and the following formula:
Figure JPOXMLDOC01-appb-M000001
 , The content of D-serine was measured. The measurement results for each disease are shown in FIG.
統計処理
　健常者と、各疾患の皮疹部と無疹部とに分けて、各群における％Ｄ-Ｓｅｒについて、Student’s t-testによる群間比較（等分散仮定）を行い、有意差を検証した（表１）。これらの結果は％Ｄ（Ｓｅｒ）が健常者と乾癬の区別に利用できることを示すだけでなく、乾癬特徴的なパラメータ変動であるためにそのほかの皮膚疾患との鑑別にも有用であることを端的に示している。
Figure JPOXMLDOC01-appb-T000002
 Statistical processing Normal subjects were divided into eruptions and eruptions of each disease, and% D-Ser in each group was compared between groups by Student's t-test (equal variance assumption), and significant differences were verified. (Table 1). These results not only indicate that% D (Ser) can be used to distinguish healthy subjects from psoriasis, but also indicate that it is useful for discriminating psoriatic from other skin diseases due to its characteristic parameter variation. Is shown in
Figure JPOXMLDOC01-appb-T000002
　健常者と乾癬の鑑別はＤ－Ｓｅｒ量を計算式に含む別の指標でも行うことができる。たとえばＤ－Ｓｅｒ量を別のアミノ酸量で乗除したパラメータ（ここでは例としてＤ－Ｓｅｒ／Ｄ－Ａｌａ）は、健常者と乾癬皮疹部の間に有意な差を認めた。また、Ｄ－Ｓｅｒ量を角層面積当たりの量でノーマライズしたパラメータ（Ｄ－Ｓｅｒ／ｃｍ２）も、健常者と乾癬皮疹部の間に有意な差を認めたことから診断、鑑別に有用であることがわかる。
Figure JPOXMLDOC01-appb-T000003
 The discrimination between a healthy subject and psoriasis can also be performed using another index including the amount of D-Ser in the calculation formula. For example, a parameter obtained by multiplying the amount of D-Ser by another amount of amino acid (here, for example, D-Ser / D-Ala) showed a significant difference between a healthy subject and a psoriatic rash. Further, a parameter (D-Ser / cm 2 ) obtained by normalizing the amount of D-Ser in terms of the amount per horny layer area is also useful for diagnosis and discrimination since a significant difference was observed between a healthy subject and a psoriatic eruption area. You can see that there is.
Figure JPOXMLDOC01-appb-T000003

Claims (12)

  1. 　対象において、乾癬を判定するための皮膚試料の分析方法であって、Ｄ－セリンの量を指標とする、前記分析方法。 (4) A method for analyzing a skin sample for determining psoriasis in a subject, wherein the amount of D-serine is used as an index.
  2. 　以下の：
    　皮膚試料においてＤ－セリンの量を測定する工程；
    　Ｄ－セリン量の低下と、乾癬とを関連付ける工程
    　を含む、請求項１に記載の分析方法。     below:
    Measuring the amount of D-serine in the skin sample;
    The analysis method according to claim 1, further comprising a step of associating a decrease in the amount of D-serine with psoriasis.
  3. 　皮膚試料の内部標準の量を定量し、内部標準の量を用いて、Ｄ－セリン量を正規化する工程をさらに含む、請求項１又は２に記載の分析方法。 3. The analysis method according to claim 1, further comprising a step of quantifying the amount of the internal standard of the skin sample and normalizing the amount of D-serine using the amount of the internal standard.
  4. 　前記内部標準が、タンパク質量、遺伝子量、総アミノ酸量、総Ｄ－アミノ酸量、総Ｌ－アミノ酸量、任意のＬ－アミノ酸の量、角層重量、からなる群から選ばれる少なくとも１である、請求項３に記載の分析方法。 The internal standard is at least one selected from the group consisting of protein amount, gene amount, total amino acid amount, total D-amino acid amount, total L-amino acid amount, arbitrary L-amino acid amount, and stratum corneum weight; The analysis method according to claim 3.
  5. 　前記内部標準が、Ｌ－セリン量、又はＤ，Ｌ－セリン量である、請求項４に記載の分析方法。 (5) The method according to (4), wherein the internal standard is an amount of L-serine or an amount of D, L-serine.
  6. 　Ｄ－セリン量の低下が、予め決定されたカットオフ値と比較することで、決定される、請求項２～５のいずれか一項に記載の分析方法。 The analysis method according to any one of claims 2 to 5, wherein the decrease in the amount of D-serine is determined by comparing with a predetermined cutoff value.
  7. 　記憶部、入力部、データ処理部、及び出力部を含む、対象において、乾癬を判定するための皮膚試料分析システムであって、
    　入力部から、乾癬を判定するための皮膚試料中のＤ－セリンの量のカットオフ値と乾癬の病態情報が入力され、記憶部に記憶し、
    　入力部から、対象の皮膚試料中のＤ－セリンの量の測定値が入力され、記憶部に記憶し、
    　データ処理部が、記憶されたＤ－セリンの量の測定値を、記憶されたカットオフ値と比較して、対象の乾癬の病態情報を決定し；
    　乾癬の病態情報を出力部に出力する
    　を含む、前記皮膚試料分析システム。     A skin sample analysis system for determining psoriasis in a subject, comprising a storage unit, an input unit, a data processing unit, and an output unit,
    A cutoff value of the amount of D-serine in the skin sample for determining psoriasis and pathological information of psoriasis are input from the input unit, and stored in the storage unit.
    From the input unit, a measured value of the amount of D-serine in the target skin sample is input and stored in the storage unit,
    A data processor comparing the stored measure of D-serine to a stored cut-off value to determine psoriasis pathology information for the subject;
    Outputting the psoriasis disease state information to an output unit.
  8. 　分析測定部をさらに含み、該分析測定部が、対象の皮膚試料からＤ－セリンを定量して測定値を決定し、入力部に代わり又は入力部を介して測定値を入力する、請求項７に記載の分析システム。 8. The method according to claim 7, further comprising an analysis and measurement unit, wherein the analysis and measurement unit determines the measurement value by quantifying D-serine from the target skin sample, and inputs the measurement value instead of or via the input unit. Analysis system according to 1.
  9. 　入力部から内部標準の量が入力され、データ処理部において内部標準の量に基づきＤ－セリン量を正規化する工程をさらに含む、請求項７又は８に記載の分析システム。 9. The analysis system according to claim 7, further comprising a step of inputting the amount of the internal standard from the input unit and normalizing the amount of D-serine based on the amount of the internal standard in the data processing unit.
  10. 　前記内部標準が、タンパク質量、遺伝子量、総アミノ酸量、総Ｄ－アミノ酸量、総Ｌ－アミノ酸量、任意のＬ－アミノ酸の量、角層重量、からなる群から選ばれる少なくとも１である、請求項９に記載の分析システム。 The internal standard is at least one selected from the group consisting of protein amount, gene amount, total amino acid amount, total D-amino acid amount, total L-amino acid amount, arbitrary L-amino acid amount, and stratum corneum weight; The analysis system according to claim 9.
  11. 　前記内部標準が、Ｌ－セリン量、又はＤ，Ｌ－セリン量である、請求項１０に記載の分析システム。 11. The analysis system according to claim 10, wherein the internal standard is an amount of L-serine or an amount of D, L-serine.
  12. 　抗Ｄ－セリン抗体を含む、乾癬を判定するためのキット。 キ ッ ト A kit for determining psoriasis, comprising an anti-D-serine antibody.
PCT/JP2019/033364 2018-08-27 2019-08-26 Method and system for analyzing skin sample for determining psoriasis WO2020045364A1 (en)

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