Method and kit for diagnosing vitiligo

Abstract

The invention relates to a method and a kit for diagnosing vitiligo. Specifically, the invention provides an application of a reagent for detecting arginine in a blood-derived sample, or a reagent for detecting arginine in a blood-derived sample and an arginine standard substance in preparation of a kit, and the kit is used for diagnosing vitiligo, identifying the stage of a vitiligo patient, or identifying the severity of the vitiligo patient. Wherein the vitiligo patient stage comprises a progression stage and a stable stage, and the severity comprises the vitiligo area of the patient. The method is simple and rapid, the result is accurate, the vitiligo patient can be detected in real time, and the disease activity and severity of the vitiligo patient can be judged in an assisted mode.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to methods and reagent kits for diagnosing vitiligo. Background Technology

[0002] Vitiligo is an acquired depigmentation disorder with a global prevalence of 0.5% to 2.0%, which can significantly impact patients' mental health and quality of life. Based on clinical presentation, vitiligo is generally classified into segmental vitiligo (SV) and non-segmental vitiligo (NSV) (the most common form). Segmental vitiligo is characterized by a distinctive unilateral and segmental distribution, rapid progression, and early involvement of the follicular melanocyte reservoir; while non-segmental vitiligo has an unpredictable clinical course, presenting as a stable disease interspersed with slow or rapid flare-ups.

[0003] Early identification of vitiligo classification and disease activity is crucial for developing appropriate intervention strategies. However, early diagnosis of focal vitiligo lesions is challenging. Furthermore, there are no sensitive and specific biomarkers to assess disease progression, severity, and prognosis in patients with vitiligo.

[0004] Mounting evidence suggests that arginine is an essential amino acid for regulating energy metabolism and immune responses in mammals. Circulating arginine is a key indicator of systemic immune metabolism, and its measurement is highly diagnostic for immune-related diseases such as vitiligo. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the first aspect of the present invention provides a reagent for detecting arginine in blood-derived samples and its use in the diagnosis of vitiligo.

[0006] A first aspect of this invention provides reagents and optional arginine standards for detecting arginine in blood-derived samples in the preparation of kits for diagnosing vitiligo, identifying vitiligo patient stages (disease activity), or identifying vitiligo patient severity; wherein the vitiligo patient type includes segmental and non-segmental vitiligo patients, the disease activity includes active vitiligo and stable vitiligo, and the severity includes the size of the vitiligo patch area. In one or more embodiments, the sample is whole blood, serum, and / or plasma.

[0007] In one or more embodiments, the kit is used to diagnose vitiligo and assist in determining the disease activity and severity of vitiligo; wherein the disease activity includes progressive and stable phases, and the severity includes the size of the vitiligo patches in the patient.

[0008] In one or more embodiments, the vitiligo patient phase includes the progressive phase and the stable phase of non-segmental vitiligo.

[0009] In one or more embodiments, the reagents for detecting arginine include those required for detecting arginine using one or more methods selected from: spectrophotometry, capillary electrophoresis, small molecule chemical probe methods, mass spectrometry, and optical probe methods.

[0010] In one or more embodiments, the reagent for detecting arginine includes reagents for transforming, enriching, separating, or recognizing arginine. Preferably, the reagent for detecting arginine includes one or more selected from: arginine-specific antibodies, arginine deiminases, phthalaldehyde, sulfites, borates, small chemical probes ThiCS, formic acid, acetonitrile, isopropanol, arginine-binding proteins, or functional variants thereof.

[0011] In one or more embodiments, the reagent for detecting arginine includes one or more selected from the following: arginine-specific antibodies.

[0012] In one or more embodiments, the reagent for detecting arginine includes one or more selected from the following: arginine deiminase, phthalaldehyde, and sulfite.

[0013] In one or more embodiments, the reagent for detecting arginine includes a borate.

[0014] In one or more embodiments, the reagent for detecting arginine includes one or more selected from the following: small chemical molecule probe ThiCS, acetonitrile.

[0015] In one or more embodiments, the reagent for detecting arginine includes one or more selected from the following: formic acid, acetonitrile, and isopropanol.

[0016] In one or more embodiments, functional variants of the arginine-binding protein include arginine optical probes.

[0017] In one or more embodiments, the arginine optical probe is selected from: QBP / Citrine / ECFP (Bogner et al., 2007), FLIP-cpArgT (Okada et al., 2009), cpFLIPR (Whitfield et al., 2015), and FLIPR-AhrC (Vanoaica et al., 2016).

[0018] In one or more embodiments, the arginine optical probe comprises a fused arginine-binding protein and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the arginine-binding protein, at its N-terminus or C-terminus.

[0019] In one or more embodiments, the arginine optical probe is as described in any of the embodiments CN113336854A and CN118324869A.

[0020] In one or more embodiments, arginine levels are significantly elevated in samples from vitiligo patients compared to control levels. The control levels are derived from arginine levels in corresponding samples from non-vitiligo patients or healthy subjects. In one or more embodiments, the control levels are values ​​ranging from approximately 113 ± 21 μM to 123 ± 19 μM.

[0021] In one or more embodiments, the arginine level in samples from patients with progressive vitiligo is significantly increased compared to a control level. The control level is derived from the arginine level in corresponding samples from non-vitiligo patients or healthy subjects, preferably from approximately 121 ± 19 μM to 123 ± 20 μM. The control level may also be derived from the arginine level in corresponding samples from patients with stable vitiligo, preferably from approximately 127 ± 23 μM to 130 ± 22 μM.

[0022] In one or more embodiments, arginine levels are significantly increased in patient samples representing at least 1% (1% to 3% or at least 3%) of the vitiligo area, compared to a control level. The control level is derived from arginine levels in corresponding samples from non-vitiligo patients or healthy subjects. Preferably, the control level is approximately 121 ± 18 μM to 124 ± 19 μM.

[0023] A second aspect of the present invention also provides a kit for detecting arginine, the kit comprising reagents for detecting arginine and instructions, the instructions describing a method for diagnosing vitiligo, identifying the stage of vitiligo, or identifying the severity of vitiligo by detecting arginine.

[0024] In one or more embodiments, the reagent for detecting arginine is as described in the first aspect herein.

[0025] In one or more embodiments, the kit further includes reagents for processing a test sample, wherein the detection is for detecting arginine in a blood-derived sample, and the reagents for processing the test sample include, but are not limited to, 3NPH_HCl, EDC, and buffer solutions. The sample includes whole blood, serum, and / or plasma.

[0026] In one or more embodiments, the buffer is a phosphate-based buffer or Tris, such as HEPES, PBS, etc.

[0027] In one or more embodiments, the kit further comprises an arginine standard.

[0028] In one or more embodiments, the reagent for detecting arginine includes reagents for transforming, enriching, separating, or recognizing arginine. Preferably, the reagent comprises one or more selected from: arginine deiminase, phthalaldehyde, sulfite, borate, small chemical probe ThiCS, formic acid, acetonitrile, isopropanol, arginine-binding protein, or functional variants thereof.

[0029] In one or more embodiments, the kit further includes a reagent for detecting vitiligo.

[0030] The present invention also provides a method for diagnosing vitiligo, identifying the stage (disease activity) of a vitiligo patient, or identifying the severity of a vitiligo patient, comprising: (1) detecting arginine in a blood-derived sample, and (2) comparing it with a control level, wherein the arginine level in the serum of a vitiligo patient is higher than the control level, the arginine level in a patient with progressive vitiligo is higher than the control level, and the arginine level in a patient with at least 1% vitiligo lesions is higher than the control level. The disease activity includes progressive and stable phases, and the severity includes the size of the vitiligo lesion area.

[0031] In one or more embodiments, the step of detecting arginine is performed by detecting arginine using one or more methods selected from: spectrophotometry, capillary electrophoresis, small molecule chemical probe method, mass spectrometry, and optical probe method.

[0032] In one or more embodiments, the optical probe method includes: mixing a sample with an arginine optical probe, detecting fluorescence intensity, and determining the arginine content based on the fluorescence intensity.

[0033] In one or more embodiments, the arginine level in blood samples from vitiligo patients is significantly increased compared to a control level. The control level is derived from the arginine level in corresponding samples from non-vitiligo patients or healthy subjects. Preferably, the control level is a value between approximately 113 ± 21 μM and 123 ± 19 μM.

[0034] In one or more embodiments, the arginine level in samples from patients with progressive vitiligo is significantly increased compared to a control level. The control level is derived from the arginine level in corresponding samples from non-vitiligo patients or healthy subjects. Preferably, the control level is approximately 121 ± 19 μM to 123 ± 20 μM. The control level may also be derived from the arginine level in corresponding samples from patients with stable vitiligo, preferably approximately 127 ± 23 μM to 130 ± 22 μM.

[0035] In one or more embodiments, arginine levels are significantly increased in patient samples with at least 1% (1% to 3% or at least 3%) vitiligo area compared to a control level. The control level is derived from arginine levels in corresponding samples from patients with less than 1% vitiligo area, non-vitiligo patients, or healthy subjects. Preferably, the control level is approximately 121 ± 18 μM to 124 ± 19 μM.

[0036] In one or more embodiments, the arginine optical probe comprises a fused arginine-binding protein and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the arginine-binding protein, at its N-terminus, or at its C-terminus. Preferably, the arginine optical probe is as described in any of the embodiments of CN113336854A and CN118324869A. Attached Figure Description

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0038] Figure 1 The clinical characteristics of healthy individuals, non-segmental vitiligo (NSV), and segmental vitiligo (SV) in this article are described.

[0039] Figure 2 This is a flowchart of a probe-based method for point-of-care testing of clinical samples.

[0040] Figure 3 This is a comparison chart of serum arginine levels in vitiligo samples of different types, different activity levels within the same type, and different areas within the same type and condition, compared with corresponding healthy samples, based on the serum test results of the STAR-H probe.

[0041] Figure 4 This is the serum validation result for the probe STAR-H.

[0042] Figure 5 This study aims to analyze the consistency of detection results for all serum samples using both probe detection and high-performance liquid chromatography (HPLC) methods. Detailed Implementation

[0043] When a value or range is given, the term “about” as used herein means that the value or range is within 20%, 10%, and 5% of the given value or range.

[0044] The terms “comprising,” “including,” and their equivalents as used herein include the meanings of “containing” and “composed of,” for example, a composition “comprising” X may consist of only X or may contain other substances, such as X+Y.

[0045] Early identification of vitiligo classification and disease activity is crucial for developing appropriate intervention strategies. However, early diagnosis of focal vitiligo lesions is challenging. Furthermore, there are no sensitive and specific biomarkers to assess disease progression, severity, and prognosis in vitiligo patients. This invention analyzes the differences in serum arginine levels among different vitiligo patients, including those with different types (NSV and segmental vitiligo), different disease activities (progressive and stable phases), and different depigmentation areas. The invention finds that serum arginine levels in vitiligo patients are correlated with disease activity and severity; therefore, arginine can serve as a potential screening biomarker for this disease, enabling a re-identification of the clinical manifestations of vitiligo subtypes and facilitating rapid screening for vitiligo.

[0046] Therefore, the present invention provides a method for diagnosing vitiligo, identifying the stage of vitiligo, or identifying the severity of vitiligo, comprising: (1) detecting arginine in a sample, and (2) comparing it with a control level, wherein the arginine level in the vitiligo patient sample is higher than the control level. In one or more embodiments, the sample is whole blood, serum, and / or plasma.

[0047] In this article, the criteria for judging the type, activity, and severity of vitiligo are based on the "Consensus on the Diagnosis and Treatment of Vitiligo (2021 Edition)". Vitiligo patients can be classified into segmental vitiligo and non-segmental (common) vitiligo. Segmental vitiligo typically refers to unilateral asymmetrical vitiligo distributed along a specific dermal nerve segment (completely or partially matching a skin segment). A few cases may involve bilateral multi-segmental distribution. Non-segmental vitiligo includes disseminated, generalized, facial and neck, acral, and mucosal types. The progression stage (disease activity) of vitiligo can be divided into the progressive stage and the stable stage. During the progressive stage, the number of white patches increases, and existing white patches may gradually migrate and expand into normal skin, with blurred borders. The progressive stage is prone to isomorphic reactions, which worsen the condition. In the stable stage, the white patches stop developing, the borders are clearer, and the pigmentation at the edges is relatively deeper. The isomorphic reaction is the most significant clinical manifestation distinguishing the progressive and stable stages. Clinically, the progression and stable phases can be determined by referring to the Vitiligo Activity Scale (VIDA), clinical characteristics, isomorphic response, and Wood's lamp examination results.

[0048] In this article, "area of ​​vitiligo" or "vitiligo lesions" refers to the area of ​​vitiligo affected on the body surface of vitiligo patients. The inventors found that patients with vitiligo area >1% had significantly different serum arginine levels compared to healthy individuals, and the difference in serum arginine levels between patients with vitiligo area >3% and healthy individuals was greater than the difference between patients with vitiligo area 1-3% and healthy individuals.

[0049] In this article, the control level is a reference arginine level derived from a blood sample (e.g., whole blood, serum, and / or plasma) that can be used as a diagnostic criterion. Such a level can be obtained by comparing a sample from a vitiligo subject with a sample from a healthy or non-vitiligo subject. Alternatively, the control level can also be the level of a healthy or non-vitiligo subject. The control level can be derived from a single subject or a group of at least two subjects. Those skilled in the art can select the reference level based on the desired sensitivity and specificity.

[0050] In one or more embodiments, vitiligo patients exhibit significantly elevated arginine levels compared to control levels. The control levels are derived from arginine levels in corresponding samples from healthy subjects. In an exemplary embodiment, the control level is a value ranging from approximately 113 ± 21 μM to 123 ± 19 μM.

[0051] In one or more embodiments, arginine levels are significantly increased in patients with progressive vitiligo (NSV) compared to a control level. The control level is derived from arginine levels in corresponding samples from non-vitiligo patients or healthy subjects, preferably from approximately 121 ± 19 μM to 123 ± 20 μM. The control level may also be derived from arginine levels in corresponding samples from patients with stable vitiligo, preferably from approximately 127 ± 23 μM to 130 ± 22 μM.

[0052] In one or more embodiments, arginine levels are significantly increased in patient samples representing at least 1% (1% to 3% or at least 3%) of the vitiligo area, compared to a control level. The control level is derived from arginine levels in corresponding samples from non-vitiligo patients or healthy subjects. Preferably, the control level is approximately 121 ± 18 μM to 124 ± 19 μM.

[0053] In an exemplary embodiment, arginine is detected using chromatography and probe methods. However, those skilled in the art will understand that other methods for detecting arginine in the art can also be used in this invention, such as spectrophotometry, capillary electrophoresis, small molecule chemical probe methods, mass spectrometry, and optical probe methods. Other methods for detecting arginine are within the knowledge of those skilled in the art. The steps and reagents for detecting arginine using these methods are known in the art. An exemplary description of the above methods can be found in: The most commonly used methods for detecting arginine are spectrophotometry (Casadebaig et al., 1979; Goldschmidt and Lockhart, 1971; Khramov et al., 1980; Micklus and Stein, 1973; Sakaguchi, 1925; Sastry and Tummuru, 1994; Wang et al., 2008; Yamasaki et al., 1981), capillary electrophoresis, and other methods are also available. Electrophoresis (Narezhnaya, Askalepova, Nikashina, Krukier and Pogorelova, 2010), liquid chromatography such as HPLC (Chu, Huang, Pao, & Li, 2003), liquid chromatography-mass spectrometry such as HPLC-MS (Aldamiz-Echevarria and Andrade, 2012; El-Khory et al., 2011), and small molecule chemical probe methods (Mohammadi et al., 2019) are all methods used in live cell studies. However, these methods have significant limitations in live cell research, requiring time-consuming sample processing steps such as cell disruption, separation, extraction, and purification. They cannot provide in situ, real-time, dynamic, high-throughput, and high spatiotemporal resolution detection in live cells and subcellular organelles. There is still a need in this field for methods that can detect arginine in situ, quantitatively, and at high throughput both intracellularly and extracellularly.

[0054] Arginine detection

[0055] Spectrophotometry:

[0056] Arginine deiminase (ADI) breaks down arginine into citrulline and ammonium ions. In the presence of sulfite, the ammonium ions selectively react with o-phthalaldehyde (OPA) in an alkaline medium. The resulting product can be detected by spectrophotometry and fluorescence methods. By detecting the reaction product and absorbance or fluorescence intensity, the arginine content in the sample can be calculated. The spectrophotometric method requires, but is not limited to, arginine deiminase (ADI), o-phthalaldehyde, sulfite, and arginine standard solution.

[0057] Capillary electrophoresis:

[0058] A quartz capillary (coated with a polyamide membrane, 60cm × 75μm, effective length 50cm) was used as the separation channel. Borate buffer solution with a pH of 9.33 was used as the running buffer. The sample was injected into the capillary via pneumatic injection, and a stable separation voltage was applied to both ends. Electroosmotic flow was generated within the capillary, causing the liquid to move towards the negative electrode. Simultaneously, due to the different mass-to-charge ratios of the components in the sample entering the capillary, different substances migrated at different rates. Direct spectrophotometry at 200nm was used to detect the signal intensity at different time points to obtain electrophoretic patterns. By analyzing the electrophoretic patterns, the arginine migration time was determined, thereby quantitatively analyzing the arginine content in the sample. Required reagents include, but are not limited to: arginine standard solution and borate buffer solution.

[0059] Detection methods based on small chemical molecule probes:

[0060] ThiCS, a small chemical probe for arginine based on the optical properties of a thiazole group (see Journal of Photochemistry and Photobiology A: Chemistry, Volume 384, 2019, 112035, ISSN 1010-6030, https: / / doi.org / 10.1016 / j.jphotochem.2019.112035), can selectively detect arginine in an acetonitrile-aqueous medium. Only after arginine reacts with ThiCS does the UV-Vis absorption spectrum exhibit a significant red shift, resulting in a visible color change. Furthermore, the interaction between ThiCS and arginine leads to a red shift in the fluorescence emission wavelength, accompanied by fluorescence enhancement at 545 nm and fluorescence quenching at 486 nm. Required reagents include, but are not limited to: the small chemical probe ThiCS, arginine standard solution, and an acetonitrile-aqueous solution (8:2, v / v).

[0061] Chromatography:

[0062] Arginine can be detected by various chromatographic methods, such as HPLC, LC-MS, and UHPLC-MS. For example, the UHPLC-MS procedure for arginine detection includes: mixing the sample or standard solution sequentially with 3NPH_HCl solution and EDC solution; freezing the mixture and then centrifuging to collect the supernatant for quantitative analysis; injecting the supernatant into the UHPLC system for separation with water and elution using a gradient of formic acid and acetonitrile / isopropanol; the ion pairing for arginine quantification is 310.2 / 293.

[0063] Optical probe method:

[0064] This invention relates to a method for the point-of-care detection of arginine in clinical samples based on a genetically encoded optical probe. The method includes: contacting an optionally diluted blood-derived sample with an arginine optical probe, and quantifying arginine by detecting changes in the fluorescence of the arginine optical probe.

[0065] In this article, arginine optical probes refer to peptide probes that quantitatively detect arginine levels by utilizing changes in optical properties. Such probes typically contain a protein that recognizes arginine (i.e., an arginine-binding protein) and a protein that undergoes a change in optical properties in response to the binding of this protein to arginine (i.e., an optically active peptide, such as a fluorescent protein). Generally, arginine optical probes contain one or more arginine-binding proteins and one or more optically active peptides, wherein the one or more optically active peptides are located within the sequence, at the N-terminus, or at the C-terminus of one or more arginine-binding proteins. Various optical probes for detecting arginine are known in the art, see, for example: CN113336854A, QBP / Citrine / ECFP (J Fluoresc. 2007, 17, 350), FLIP-cpArgT (Protein Sci. 2009, 18, 2518), cpFLIPR (Protein Sci. 2015, 24, 1412), and FLIPR-AhrC (Pflugers Arch. 2016, 468, 563). Those skilled in the art will understand that, in addition to the optical probes used in the embodiments, other arginine optical probes can also be used in this invention for arginine level detection.

[0066] The arginine optical probe used in this invention can be any of the optical probes described in embodiments of CN113336854A and CN118324869A, which are incorporated herein by reference in their entirety. The arginine optical probe comprises an arginine-binding protein or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or functional variant thereof is located within the sequence, at the N-terminus, or at the C-terminus of the arginine-binding protein or its functional variant thereof. Preferably, the probe of this invention comprises a probe from CN113336854A and CN118324869A with a response fold greater than 1.2 or less than 0.8.

[0067] In one or more embodiments, the arginine-binding protein has the sequence shown in SEQ ID NO:1 or a functional fragment thereof, or a variant thereof having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity and retaining arginine-binding function. In a preferred embodiment, the arginine-binding protein has the sequence shown in SEQ ID NO:1, or a sequence thereof having at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity and retaining the sequence sensitive to arginine.

[0068] In one embodiment, the optically active polypeptide is a fluorescent protein or a functional fragment or variant thereof that retains its fluorescence response capability. In one embodiment, the fluorescent protein is selected from yellow fluorescent protein, green fluorescent protein, blue fluorescent protein, and apple red fluorescent protein (such as cpYFP, ​​cpGFP, cpBFP, and cpmApple shown in SEQ ID NO:2-5 of CN113336854A, and SEQ ID NO:52-55 of this application).

[0069] In one embodiment, the arginine optical probe further comprises one or more linkers flanking the optically active polypeptide. The linkers of this invention can be any amino acid sequence of any length. In one embodiment, the optically active polypeptide flanking linkers comprise no more than 5 amino acids, such as linkers of 0, 1, 2, 3, or 4 amino acids. In one embodiment, the linkers flanking the optically active polypeptide comprise amino acid Y. In one embodiment, linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the arginine optical probe is as follows: arginine-binding protein first portion B1 - Y - optically active polypeptide A - arginine-binding protein second portion B2. In one embodiment, the arginine optical probe does not comprise a linker.

[0070] In one embodiment, the optically active peptide is located between residues 103-111 and / or 197-209 of the arginine-sensitive peptide, with the numbering corresponding to the full length of the arginine-binding protein. In another embodiment, the optically active peptide replaces one or more amino acids between residues 102-112 and / or 196-210 of the arginine-sensitive peptide, with the numbering corresponding to the full length of the arginine-binding protein.

[0071] In one embodiment, the optically active polypeptide is located at one or more sites of the arginine-binding protein selected from the following: 103 / 104, 103 / 105, 103 / 106, 103 / 107, 103 / 108, 103 / 109, 103 / 110, 103 / 111, 104 / 105, 104 / 106, 104 / 107, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 105 / 111, 106 / 107, 106 / 108, 106 / 109, 106 / 110, 106 / 111, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 109, 108 / 110, 108 / 111, 109 / 110, 109 / 111, 110 / 111, 197 / 198, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 197 / 205, 197 / 206, 197 / 207, 197 / 208, 197 / 209, 198 / 199, 198 / 200, 198 / 201, 198 / 202, 198 / 203, 198 / 204, 198 / 205, 198 / 2 06, 198 / 207, 198 / 208, 198 / 209, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 199 / 207, 199 / 208, 199 / 209, 200 / 201, 200 / 202, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 200 / 207, 200 / 208, 200 / 209, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 201 / 207, 201 / 208, 201 / 20 9, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 202 / 207, 202 / 208, 202 / 209, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 203 / 208, 203 / 209, 204 / 205, 204 / 206, 204 / 207, 204 / 208, 204 / 209, 205 / 206, 205 / 207, 205 / 208, 205 / 209, 206 / 207, 206 / 208, 206 / 209, 207 / 208, 207 / 209 or 208 / 209, the numbers correspond to the full length of the arginine-binding protein.

[0072] Preferably, the optically active polypeptide is located at one or more sites of the arginine-binding protein selected from the following: 103 / 104, 103 / 106, 103 / 107, 103 / 108, 103 / 109, 103 / 110, 104 / 105, 104 / 106, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 1 05 / 111, 106 / 107, 106 / 108, 106 / 109, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 109, 108 / 111, 109 / 110, 109 / 111, 110 / 111, 197 / 198, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 197 / 205, 197 / 206, 197 / 208, 198 / 201, 198 / 202, 198 / 203, 198 / 204, 198 / 205, 198 / 206, 198 / 208, 198 / 209, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 199 / 208, 200 / 203, 200 / 204, 200 / 205, 200 / 2 06, 200 / 207, 200 / 208, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 201 / 208, 201 / 209, 202 / 203, 202 / 205, 202 / 206, 203 / 204, 203 / 206, 204 / 205, 204 / 206, 204 / 208, 205 / 206, 205 / 207, 205 / 208, or 206 / 207. These probes responded to arginine more strongly than the control.

[0073] Preferably, the optically active polypeptide is located at one or more sites selected from the following arginine-sensitive polypeptides: 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 110, 106 / 108, 106 / 109, 107 / 109, 107 / 111, 110 / 111, 197 / 199, 197 / 203, 197 / 204, 198 / 202, 198 The probes were 203, 198 / 204, 199 / 200, 199 / 201, 199 / 202, 199 / 204, 199 / 205, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 202 / 205, 203 / 206, 204 / 205, or 204 / 206. These probes responded to arginine more than 1.5 times that of the control.

[0074] Preferably, the optically active peptide is located at one or more sites selected from the following arginine-sensitive peptides: 104 / 110, 104 / 111, 105 / 106, 105 / 110, 106 / 109, 107 / 109, 107 / 111, 197 / 203, 197 / 204, 198 / 202, 199 / 200, 199 / 202, 200 / 203, 200 / 205, 201 / 202, 201 / 205, 202 / 205, 203 / 206, 204 / 205, or 204 / 206. These probes respond to arginine more than twice as strongly as the control.

[0075] In an exemplary embodiment, the optical probe of the present invention may be a probe where cpYFP is located at positions 104 / 110, 104 / 111, 105 / 106, 105 / 110, 106 / 109, 107 / 109, 107 / 111, 197 / 203, 197 / 204, 198 / 202, 199 / 200, 199 / 202, 200 / 203, 200 / 205, 201 / 202, 201 / 205, 202 / 205, 203 / 206, 204 / 205, or 204 / 206 of the arginine-binding protein. In one embodiment, the optical probe of the present invention has or is composed of the sequence shown in SEQ ID NO: 6-41 of CN113336854A (SEQ ID NO: 4-39 of this application).

[0076] In exemplary embodiments, the optical probe of the present invention may be a probe in which cpYFP is located at position 200 / 203 of an arginine-binding protein and has one or more mutations selected from T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, and D177A. In some embodiments, the optical probe of the present invention has or is composed of the sequence described in SEQ ID NO: 4-15 of CN118324869A (SEQ ID NO: 40-51 of this application).

[0077] In one or more embodiments, the B1-A-B2 type arginine optical probe of the present invention can be located at 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 110, 106 / 108, 106 / 109, 107 / 109, 107 / 111, 110 / 111, 197 / 199, 197 / 203, 197 / 204, 198 when cpYFP is located at arginine-binding protein or its functional fragment. Probes at / 202, 198 / 203, 198 / 204, 199 / 200, 199 / 201, 199 / 202, 199 / 204, 199 / 205, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 202 / 205, 203 / 206, 204 / 205, or 204 / 206. In an exemplary embodiment, the B1-A-B2 type arginine optical probe may be a probe where cpYFP is located at positions 104 / 110, 104 / 111, 105 / 106, 105 / 110, 106 / 109, 107 / 109, 107 / 111, 197 / 203, 197 / 204, 198 / 202, 199 / 200, 199 / 202, 200 / 203, 200 / 205, 201 / 202, 201 / 205, 202 / 205, 203 / 206, 204 / 205, or 204 / 206 of the arginine-binding protein or a functional fragment thereof. In one or more embodiments, the arginine-binding protein has a sequence as shown in SEQ ID NO:1.

[0078] The arginine-binding protein in the arginine optical probe may have one or more mutations. These amino acid mutations include amino acid modification, substitution, deletion, or truncation. An arginine optical probe containing a mutated arginine-binding protein may be used to detect arginine if its response to arginine is higher or lower than that of its unmutated counterpart. Preferably, an arginine optical probe whose response to arginine (see CN113336854A) exceeds 1.2 times (e.g., 1.4, 1.5, 2, 2.2, 3, 4.3, 8.6, 11, 15, etc.) or is lower than 0.8 times (e.g., 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, etc.) to arginine can be used to detect arginine. In one embodiment, the mutation is located at one, two, or three of the following positions in the arginine-binding protein: position 30(S), position 32(T), position 37(E), position 71(F), position 89(G), position 90(M), position 91(D), position 96(R), position 135(E), position 138(T), position 139(T), position 140(H), position 177(D), position 96(R), and position 177(D). For example, in one or more embodiments, the mutation is selected from one, two or three of S30N, T32A, E37A, F71A, G89A, M90A, D91A, R96M, R96K, R96A, E135A, T138A, T139A, H140A, D177A, and D177N.

[0079] In an exemplary embodiment, the arginine optical probe may be a probe with cpYFP inserted at sites 104 / 110 and / or 107 / 111 of the arginine-binding protein functional fragment and having one or more mutations selected from the following: S30N, D177N, R96M, R96K.

[0080] In an exemplary embodiment, the arginine optical probe may be a probe with cpYFP inserted at position 200 / 203 of the arginine-binding protein functional fragment and having one or more mutations selected from the following: T32A, E37A, F71A, G89A, M90A, D91A, R96M, R96K, R96A, E135A, T138A, H140A, D177N.

[0081] In some embodiments, the optical probe comprises an arginine-sensitive peptide and an optically active peptide, wherein the optically active peptide is located within the sequence of the arginine-sensitive peptide, as shown in SEQ ID NO:1, and the optically active peptide is selected from any of the following: cpYFP, ​​cpGFP, cpBFP, or cpmApple.

[0082] The optically active polypeptide is cpYFP, ​​located at one or more of the following sites on the arginine-sensitive polypeptide: 103 / 104, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 111, 109 / 110, 110 / 111, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 197 / 205, 198 / 201, 198 / 2 02, 198 / 203, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 204, 201 / 20 5, 201 / 206, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 204 / 205, 204 / 206, 204 / 207, 205 / 206, 206 / 207, 207 / 208 or 208 / 209;

[0083] The optically active polypeptide is cpGFP, located at one or more of the following sites on the arginine-sensitive polypeptide: 103 / 104, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 108, 107 / 109, 107 / 110, 108 / 111, 110 / 111, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 1 98 / 201, 198 / 202, 198 / 203, 199 / 200, 199 / 201, 199 / 202, 199 / 204, 199 / 205, 199 / 206, 200 / 202, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 203 / 205, 203 / 206, 203 / 207, 204 / 205, 204 / 206 or 205 / 206;

[0084] The optically active polypeptide is cpBFP, located at one or more of the following sites on the arginine-sensitive polypeptide: 103 / 104, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 109, 107 / 110, 107 / 111, 108 / 111, 110 / 111, 197 / 199, 197 / 201, 197 / 203, 197 / 204, 198 / 201, 198 / 202, 1 98 / 203, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 203 / 205, 203 / 206, 203 / 207, 204 / 205, 204 / 206, 205 / 206, 206 / 207 or 207 / 208; or

[0085] The optically active polypeptide is cpBFP, located at one or more of the following sites on the arginine-sensitive polypeptide: 103 / 104, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 111, 109 / 110, 110 / 111, 197 / 199, 197 / 201, 197 / 203, 197 / 204, 197 / 205, 198 / 2 01, 198 / 202, 198 / 203, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 202 / 203, 202 / 205, 202 / 206, 203 / 204, 203 / 206, 203 / 207, 204 / 205, 204 / 206, 205 / 206, 206 / 207 or 207 / 208.

[0086] In one or more embodiments, the optically active polypeptide is located at position 107 / 111 of the arginine-sensitive polypeptide shown in SEQ ID NO:1, and the arginine-sensitive polypeptide further comprises a mutation selected from the following: (1) S30N, (2) D177N, and (3) R96M or R96K.

[0087] In one or more embodiments, the optical probe is a probe in which cpYFP is inserted at position 200 / 203 of the arginine-sensitive polypeptide shown in SEQ ID NO:1 and has any of the following mutations: T32A, E37A, F71A, G89A, M90A, D91A, R96A, E135A, T138A, T139A, H140A, or D177A.

[0088] In specific embodiments, the arginine optical probe comprises an arginine optical probe having the sequence shown in SEQ ID NO:6-41 of CN113336854A or any one of the sequences shown in SEQ ID NO:4-15 of CN118324869A, the full text of which is incorporated herein by reference. In exemplary embodiments, the arginine optical probe comprises the amino acid sequence SEQ ID NO:2 (also referred to herein as STAR-H, STM4351-105 / 110-cpYFP) or SEQ ID NO:3 (also referred to herein as STAR, STM4351-200 / 203-E37A-cpYFP) or a variant thereof or composed thereof. In one embodiment, the arginine optical probe comprises a sequence having at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the amino acid sequence SEQ ID NO:2 or 3.

[0089] The arginine optical probe can be fused with other functional peptides, for example, the functional peptides located at the N-terminus and / or C-terminus of the arginine optical probe. In some embodiments, the functional peptides include tags for purification or for immunoblotting. A linker may be present between the optical probe and the other functional peptides.

[0090] As used herein, the terms “functional variant,” “derivative,” and “analyte” refer to a protein that substantially retains the same biological function or activity as the original polypeptide or protein (e.g., arginine-binding protein or fluorescent protein). Functional variants, derivatives, or analogs of the polypeptides or proteins (e.g., arginine-binding proteins or fluorescent proteins) of the present invention may be (i) proteins in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by fusing a mature protein with another compound (e.g., a compound that extends the protein's half-life, such as polyethylene glycol); or (iv) proteins formed by fusing an additional amino acid sequence to this protein sequence (e.g., a secreted sequence or a sequence used to purify this protein or a proteogenic sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these functional variants, derivatives, and analogs are within the scope well known to those skilled in the art.

[0091] The difference between the analogue and the original polypeptide or protein can be a difference in amino acid sequence, a difference in modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.

[0092] The "response fold" mentioned in this article refers to the standardized fluorescence ratio. The greater the deviation of the probe's response fold from 1 (whether it increases or decreases), the greater the change in the probe's response relative to the control, or the greater its responsiveness. For example, the "change in the ratio of fluorescence intensity at 528nm emission site excited by 420nm to fluorescence intensity at 528nm emission site excited by 485nm" is mentioned multiple times in the examples of CN113336854A and CN118324869A (taking cpYFP as an example). This detection method is well-known in the art and is briefly described below:

[0093] Determination of probe fold change (using cpYFP as an example): Fluorescence signal values ​​were corrected by subtracting the detection signal values ​​from cells not expressing the probe protein. pH-sensitive interference was eliminated by dividing the probe detection signals in parallel experimental groups by the control detection signals to obtain corrected data.

[0094] F = F sample -F BLK

[0095]

[0096] F represents fluorescence intensity.sample F represents the total fluorescence intensity of the sample expressing the fluorescent probe. BLK F represents the background fluorescence intensity of samples without expressed fluorescent probes. cpYFP This indicates the fluorescence intensity of the sample used as a pH control. F 485 F represents the fluorescence intensity of a fluorescent protein sample excited at 485 nm and emitted at 528 nm. 420 Ratio represents the fluorescence intensity of a fluorescent protein sample excited at 420 nm and emitted at 528 nm. sensor Ratio represents the ratio of fluorescence intensity of the probe. cpYFP This represents the ratio of the fluorescence intensity of the corresponding probe to the pH control fluorescent protein.

[0097] In this article, the probe change factor or response factor refers to the Normalized Ratio. 485 / 420 (i.e., the normalized fluorescence ratio), when the probe's Normalized Ratio 485 / 420 The greater the deviation from 1 (whether it increases or decreases), the greater the change factor or response factor of the probe.

[0098] In some implementations, the detection of arginine includes the step of establishing an arginine standard curve. The determination of an arginine standard curve is a standard practice in the art; it is a curve obtained by detecting different known concentrations of arginine standards and mapping the concentrations to the detection results or calculations derived from those results.

[0099] For arginine analysis based on optical probes, after the optical probe is brought into contact with arginine in the sample, the appropriate excitation and emission wavelengths are selected according to the different fluorescent proteins to detect the fluorescence intensity, and quantitative analysis is performed according to the arginine standard curve.

[0100] In an exemplary implementation, for fluorescent proteins with a single excitation wavelength (e.g., cpBFP and cpmApple), the data processing procedure includes:

[0101] F = F Sample -F BLK

[0102] F represents the actual fluorescence intensity of a single channel. Sample F represents the sample fluorescence intensity representing the expression probe. BLKThe fluorescence intensity represents the fluorescence intensity of the sample without expressed probes. A standard curve is established using the fluorescence intensity of standards, and then quantitative analysis of arginine in the sample is performed based on the standard curve. In an exemplary embodiment, for cpBFP, the excitation wavelength is 360 nm BP 10 nm and the emission wavelength is 450 nm BP 10 nm; for cpmApple, the excitation wavelength is 540 nm BP 25 nm and the emission wavelength is 590 nm BP 20 nm.

[0103] For fluorescent proteins with multiple excitation wavelengths (e.g., cpYFP and cpGFP), the data processing procedure includes:

[0104] F = F Sample -F BLK

[0105] R = F 激发波长1 / F 激发波长2

[0106] F represents the actual fluorescence intensity of a single channel. Sample F represents the sample fluorescence intensity representing the expression probe. BLK F represents the fluorescence intensity of samples without expressed probes. 激发波长1 F represents the fluorescence intensity emitted by the probe at the emission wavelength (528 nm for cpYFP or cpGFP) after excitation at the first excitation wavelength (485 nm for cpYFP, ​​20 nm for BP). 激发波长2 This represents the fluorescence intensity emitted by the probe at the emission wavelength (528 nm for cpYFP or cpGFP) after excitation at the second excitation wavelength (420 nm for cpYFP, ​​20 nm for BP). The first and second excitation wavelengths can be determined based on the spectral properties of the fluorescent protein carried by the probe.

[0107] R (Ratio) represents the fluorescence ratio of the probe. The bandwidth (BP) of the filter represents the total range on both sides of the median, for example, 485BP 20nm is 475-495nm.

[0108] [Arg] = K d (R-Rmin) / (Rmax-R)

[0109] [Arg] represents arginine levels; K d R represents the dissociation constant of the probe. min and R max The values ​​represent the fluorescence ratios of the probe proteins with and without saturated concentrations of arginine, respectively; R represents the fluorescence ratio of the sample.

[0110] Typically, before arginine detection, samples can be pretreated to remove substances that may affect the detection. Such pretreatment methods can be adjusted depending on the specific detection method. Those skilled in the art are familiar with the procedures and reagents required for such pretreatment. For example, in a UHPLC-MS analysis method as described by Xie et al., 2021, sample pretreatment includes: mixing the sample (e.g., serum and / or plasma) sequentially with 3 NPH-HCl solution and EDC solution, then freezing at -20°C and collecting the supernatant for analysis. As another example, when using an optical probe to detect arginine in a sample, the sample (e.g., serum and / or plasma) is first diluted with a buffer solution (e.g., HEPES), then mixed with the optical probe, and the fluorescence intensity is measured.

[0111] Furthermore, the present invention also provides a kit for detecting arginine, comprising reagents for detecting arginine and instructions for use, the instructions describing methods for diagnosing vitiligo, identifying the stage of vitiligo, or identifying the severity of vitiligo by detecting arginine. In one or more embodiments, the reagents for detecting arginine include reagents required for detecting arginine using one or more methods selected from: spectrophotometry, capillary electrophoresis, ThiCS small molecule probe method, mass spectrometry, and optical probe method.

[0112] As used herein, the terms "detection substance," "detection reagent," and "reagent for detecting arginine" are used interchangeably, all referring to substances that are specific to arginine and can be used to directly or indirectly detect the presence and / or content of arginine. For ease of detection, the detection reagents of this invention may also be equipped with detectable markers, including but not limited to: radioactive isotopes, fluorophores, chemiluminescent components, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (e.g., biotin or haptens), etc.

[0113] In some embodiments, the reagents for detecting arginine include reagents for transforming, enriching, separating, or recognizing arginine. As described above, those skilled in the art will understand that methods for detecting arginine, including chromatography and probe methods, can be used in this invention, such as spectrophotometry, capillary electrophoresis, small molecule chemical probe methods, mass spectrometry, and optical probe methods. The steps and reagents for detecting arginine using these methods are known in the art. Therefore, by way of example, the reagents for detecting arginine herein are those used in the methods described below: spectrophotometry (Casadebaig et al., 1979; Goldschmidt and Lockhart, 1971; Khramov et al., 1980; Micklus and Stein, 1973; Sakaguchi, 1925; Sastry and Tummuru, 1994; Wang et al., 2008; Yamasaki et al., 1981), capillary electrophoresis (Olso... n et al., 1999; Narezhnaya, Askalepova, Nikashina, Krukier and Pogorelova, 2010), liquid chromatography such as HPLC (Chu, Huang, Pao, & Li, 2003), liquid chromatography-mass spectrometry such as HPLC-MS (Aldamiz-Echevarria and Andrade, 2012; El-Khory et al., 2011), and chemical small molecule probe methods (Mohammadi et al., 2019). Reagents used in the above methods for detecting arginine include, but are not limited to: arginine-specific antibodies, arginine deiminase, phthalaldehyde, sulfite, borate, chemical small molecule probes ThiCS, arginine-binding proteins, or functional variants thereof. In an exemplary embodiment, the kit further comprises the actual treatment of the test sample, wherein the detection is the detection of arginine in a blood-derived sample, and the reagent for treating the test sample may also be selected from one or more of the following: 3NPH-HCl, EDC, formic acid, acetonitrile, and isopropanol.

[0114] After using the actual reagents for processing the sample, the arginine in the sample may be more suitable for subsequent detection. Such pretreatment methods can be adjusted according to the specific detection method. Those skilled in the art are familiar with the procedures and reagents required for such pretreatment. For example, in a method using UHPLC-MS analysis as described by Xie et al., 2021, sample pretreatment includes: mixing the sample (e.g., serum and / or plasma) sequentially with 3NPH_HCl solution and EDC solution, then freezing at -20°C and taking the supernatant for analysis. As another example, when using an optical probe to detect arginine in a sample, the sample (e.g., serum and / or plasma) is first diluted with a buffer solution (e.g., HEPES), then mixed with the optical probe, and the fluorescence intensity is measured.

[0115] The kit may also include a buffer solution. The buffer solution provides a stable buffering environment for the reactions involved in arginine detection. Those skilled in the art can select a suitable buffer solution based on experience, such as phosphate-based buffers (HEPES, PBS) or Tris.

[0116] The kit may also include reagents required for other auxiliary tests that may be involved in the diagnosis of vitiligo, such as a Wood's lamp. These are all within the knowledge of those skilled in the art.

[0117] The present invention also provides the use of the above-described reagent for detecting arginine in the preparation of a kit for diagnosing vitiligo or for classifying and diagnosing the progression of vitiligo in patients.

[0118] In this document, concentrations, contents, percentages, and other values ​​are expressed in range form. It should also be understood that this range form is used for convenience and brevity only, and should be flexibly interpreted to include the values ​​explicitly mentioned at the upper and lower limits of the range, as well as all individual values ​​or subranges included within that range.

[0119] The present invention has the following beneficial effects:

[0120] Probe-based assays of body fluid samples are very fast and convenient because they eliminate the need for time-consuming sample preparation (i.e., pretreatment or purification). Typically, the entire process from pipetting to measurement for a single sample takes about one minute, and for 96 samples in an automated microplate assay, it usually takes about three minutes. These advantages make it a promising technology for metabolic diagnostics and screening.

[0121] The present invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and reagents used in the embodiments are conventional methods and reagents in the art.

[0122] Detection method:

[0123] 1. Protein induction, expression, and purification

[0124] The probe plasmid was transformed into BL21(DE3) or JM109(DE3) expression strains, and then single clones were picked and cultured in test tubes for primary culture. The next day, the primary cultured bacteria were inoculated into Erlenmeyer flasks at a dilution of 1:100 and allowed to grow until OD (Organic Dysplasia) was achieved. 600 When the concentration is 0.4-0.6, add IPTG to a final concentration of 1 mM and incubate at 18°C ​​to induce the expression of the target protein.

[0125] (1) Collect bacterial cells: After protein expression for 24-48 hours, collect the expressed bacterial cells by centrifuging at 4000 rpm for 10 minutes and resuspending them in buffer A.

[0126] (2) Ultrasonic crushing: The ultrasonic crusher program is set to ultrasonic 1s stop 3s, one working cycle time is 300s, using Φ15 probe, power is 55%.

[0127] (3) Centrifugation: When the bacterial cells are sonicated to clarity, centrifuge at 9600 rpm and 4℃ for 30 min, retain the supernatant and discard the precipitate.

[0128] (4) Column pretreatment: The self-packed nickel ion affinity chromatography column was first rinsed with 5 column volumes (CV) of deionized water, and then the affinity chromatography column was equilibrated with 5 column volumes (CV) of buffer A.

[0129] (5) Protein loading: Add the supernatant after crushing and centrifuging to the pretreated nickel column.

[0130] (6) Washing: Add 50mM imidazole washing buffer to remove impurities.

[0131] (7) Elution: Add elution buffer containing 300mM imidazole to elute the target protein from the nickel column.

[0132] (8) Nickel column treatment: After use, the nickel column is treated with 5 column volumes (CV) of buffer B (500 mM imidazole buffer), then 5 column volumes (CV) of deionized water is added, and finally the column packing is immersed in 20% ethanol solution.

[0133] Buffer A: 20mM phosphate, 0.5M NaCl, 10mM imidazole.

[0134] Buffer B: 20mM phosphate, 0.5M NaCl, 500mM imidazole.

[0135] After purification using a nickel column, the protein is dissolved in the elution solution. However, when performing property analysis or storing the protein, the solution needs to be changed. Therefore, the purified protein needs to be desalted. First, the desalting column needs to be pretreated by rinsing with 5 column volumes (CV) of deionized water, followed by treatment with 5 column volumes (CV) of desalting buffer. Second, the protein to be desalted is added to the desalting column, and the protein is collected. Finally, the desalting column is treated with 10 column volumes (CV) of deionized water.

[0136] 2. Clinical Sample Collection

[0137] Serum samples from the vitiligo and healthy groups were provided by the Ninth People's Hospital affiliated with Shanghai Jiao Tong University and the Suzhou Ruijin Vitiligo Research Institute. The sample collection and subsequent experiments were approved by the institution's review committee, and informed consent was obtained from all participants.

[0138] 3. Sample testing methods

[0139] For arginine analysis based on fluorescent protein probes, plasma or serum samples were diluted 100-fold in HEPES buffer. Measurements were performed using 96-well black plates with different concentration gradients of arginine standards. 50 μL of diluted sample and 50 μL of 0.4 μM probe protein solution were added. Alternatively, 1 μL of serum sample was directly mixed with 100 μL of probe protein (0.2 μM) using an Echo 650 ultrasonic pipetting system combined with a BioTek MultifloFX automated dispensing system. Fluorescence intensity was immediately measured using a Synergy neo2 multi-plate reader with a 485BP 20nm or 420BP 10nm excitation and 532BP 40nm emission bandwidth filter. Quantitative analysis was performed according to the arginine standard curve.

[0140] Fluorescence detection data processing (taking STAR-H probe as an example)

[0141] F = F Sample -F BLK

[0142] R(Ratio) = F 485 / F 420

[0143] F represents the actual fluorescence intensity of a single channel. Sample F represents the sample fluorescence intensity representing the expression probe. BLK F represents the fluorescence intensity of the sample without the probe. 420 The fluorescence intensity F represents the fluorescence intensity emitted at 532 nm when the fluorescent protein sample is excited at 420 nm. 485 Ratio represents the fluorescence intensity emitted at 532 nm when a fluorescent protein sample is excited at 485 nm. STAR-H This represents the ratio of fluorescence intensity of the probe.

[0144] The bandwidth (BP) of a filter represents the total range on both sides of the median value; 485BP 20nm is equivalent to 475-495nm.

[0145] [Arg] = K d (RR min ) / (R max -R)

[0146] [Arg] represents arginine levels; K dR represents the dissociation constant of the STAR-H probe. min and R max The values ​​represent the fluorescence ratios of the probe proteins with and without saturated concentrations of arginine, respectively; R represents the fluorescence ratio of the probe proteins after the addition of serum.

[0147] 4. UHPLC-MS analysis of arginine in blood-derived samples

[0148] Arginine in blood-derived samples was determined by UHPLC (Agilent, 1290) and triple quadrupole mass spectrometry (Agilent, 6460C). The detection method included (see Xie et al., 2021): 5 μL of sample or standard solution was mixed sequentially with 25 μL of 160 mM 3NPH HCl solution and 25 μL of 120 mM EDC solution; the mixture was frozen at -20 °C for 20 minutes, and then centrifuged to obtain the supernatant for quantitative analysis.

[0149] Five μL of sample was injected into an Agilent 1290U HPLC system for separation with water and a gradient elution of 0.1% formic acid and acetonitrile / isopropanol (7:3, v / v). Mass analysis was performed using ESI negative mode with multiple reaction monitoring. The ion pairing for arginine quantification was 310.2 / 293.

[0150] Example 1

[0151] Serum samples from vitiligo patients provided by the Ninth People's Hospital affiliated with Shanghai Jiao Tong University were analyzed, including 124 cases of non-segmental vitiligo (NSV) and 15 cases of segmental vitiligo (SV). To exclude metabolic differences caused by age, sex, and other metabolic indicators, each vitiligo patient was matched with a corresponding healthy individual as a control group based on their sex and age. Their clinical characteristics and baseline parameters are shown in Table 1 below. Figure 1 As shown.

[0152] Table 1

[0153]

[0154] Data are mean ± SD, or n (%).

[0155] First, a probe-based point-of-care testing method for clinical samples was established. When using the STAR-H probe to detect arginine in serum, only 1 μL of serum and 0.2 μM of STAR-H protein are required for a 96-well plate at room temperature, and the measurement time is less than 1 minute. The detection procedure is as follows: Figure 2 As shown.

[0156] Example 2

[0157] In serum tests of vitiligo patients and corresponding healthy controls, using STAR-H assays, it was found that serum arginine levels in the NSV group (136±31 μM) and SV group (131±21 μM) were higher than those in the healthy control group (121±19 μM, p<0.001; 115±18 μM, p<0.05). To compare the differences in serum arginine levels in vitiligo patients under different disease states, this example divided the NSV group into progressive and stable phases. Compared with the control group (121±19 μM, p<0.001; 120±16 μM, p>0.05), patients with progressive vitiligo had higher serum arginine levels (137±31 μM), while the serum arginine level in patients with stable vitiligo (127±23 μM) showed no significant difference from that in healthy controls, indicating that serum arginine levels are related to the disease activity of vitiligo.

[0158] Furthermore, serum arginine levels were also correlated with the affected area of ​​vitiligo lesions. In this study, patients with progressive non-segmental vitiligo were divided into three groups based on the area of ​​the lesions: <1%, 1-3%, and >3%. The results showed that patients with larger lesions (>3%) had the highest serum arginine levels (144±33 μM); followed by those with medium-sized lesions (1-3%), with serum arginine levels of 135±27 μM. These two groups showed significant differences in serum arginine levels compared to the healthy group (121±24 μM, p<0.01; 121±18 μM, p<0.001), with the larger lesion area showing a greater difference. However, patients with lesions <1% showed no significant difference in serum arginine levels (133±39 μM) compared to the healthy group (122±16 μM, p>0.05). Figure 3 As shown in (BD).

[0159] Table 2 Arginine Levels

[0160]

[0161]

[0162] To evaluate the diagnostic performance of serum arginine, receiver operating characteristic (ROC) analysis was further performed in this embodiment. Serum arginine was able to moderately distinguish the NSV or SV group from the healthy control group, with areas under the curve (AUC) of 0.635 (p<0.001) and 0.769 (p<0.01), respectively; however, serum arginine did not show significant evidence of distinguishing the NSV group from the SV group. Figure 3 Interestingly, serum arginine levels helped differentiate between the progressive and stable phases of vitiligo (0.814, p<0.01). Figure 3Furthermore, compared with corresponding healthy controls, the AUC values ​​of patients with non-segmental vitiligo covering <1%, 1-3%, and >3% of the area were 0.540, 0.648, and 0.706, respectively (p>0.05, p<0.001, p<0.001). Figure 3 These results further demonstrate that elevated serum arginine levels are associated with the progression and severity of vitiligo.

[0163] Example 3

[0164] To verify the above results, this embodiment collaborated with Suzhou Ruijin Vitiligo Research Institute to collect a new batch of serum samples from vitiligo patients and healthy controls as a validation set for independent testing. This embodiment includes 123 samples of non-segmental vitiligo (NSV) and 13 samples of segmental vitiligo (SV). Similarly, to exclude metabolic differences caused by factors such as age and gender, this embodiment matched each vitiligo patient with a corresponding healthy control based on their gender and age. Their clinical characteristics and baseline parameters are shown in the table below.

[0165] Table 3

[0166]

[0167] Data a re mean±SD, or n(%).

[0168] Example 4

[0169] Through analysis of the validation set samples, the inventors found that serum arginine levels in the NSV group (139±30 μM) and SV group (131±13 μM) were higher than those in the healthy control group (123±19 μM, p<0.001; 113±21 μM, p<0.05). Serum arginine levels in patients with progressive vitiligo (140±31 μM) were higher than those in healthy controls (123±20 μM, p<0.001), while there was no significant difference in serum arginine levels between patients with stable vitiligo (130±22 μM) and healthy controls (122±18 μM, p>0.05). These results are consistent with the training set results, indicating that serum arginine levels in vitiligo patients are higher than those in healthy controls, and that serum arginine levels are related to the disease activity of vitiligo.

[0170] Table 4 Arginine Levels

[0171]

[0172] Similarly, this embodiment analyzed the validation set sample test results based on the affected area of ​​vitiligo lesions. It was found that patients with larger lesion areas had higher serum arginine levels (>3%, 148±31μM; 1-3%, 139±30μM; <1%, 134±32μM), and the difference from healthy controls (122±17μM, p<0.001; 124±19μM, p<0.01; 122±23μM, p>0.05) was greater. Figure 4 As shown in (BD).

[0173] Furthermore, this embodiment performed receiver operating characteristic (ROC) analysis on the validation set detection results. Similar to the training set detection results, serum arginine could distinguish the NSV or SV group from the healthy control group, with areas under the curve (AUC) of 0.662 (p<0.001) and 0.790 (p<0.01), respectively. Serum arginine did not show significant evidence of distinguishing the NSV group from the SV group. Figure 4 Unlike the training set, in this embodiment, the area under the curve (AUC) between the progressive vitiligo group and the stable vitiligo group was 0.715, but there was no statistically significant difference. Figure 4 (F). Furthermore, compared with the corresponding healthy controls, the AUC values ​​of patients with non-segmental vitiligo covering <1%, 1-3%, and >3% of the affected area increased sequentially, to 0.605, 0.654, and 0.781, respectively (p>0.05, p<0.01, p<0.001). Figure 4 G).

[0174] Example 5

[0175] To verify the detection method of the embodiments, samples were analyzed in parallel using an ultra-high performance liquid chromatography (UPLC) system and a triple quadrupole mass spectrometer (UPLC-MS). The results showed a strong correlation between the two independent methods (r = 0.836, p < 0.001), as... Figure 5 As shown.

[0176] These results indicate that serum arginine levels in vitiligo patients are correlated with disease activity and severity, and that serum arginine levels have the potential to synergistically detect vitiligo disease activity or severity.

[0177] Part of the sequence in this article

[0178] SEQ ID NO:1

[0179] Arginine-binding protein

[0180] ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDPDYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ

[0181] SEQ ID NO:2 (SEQ ID NO:11 of CN113336854A)

[0182] STAR-H is STM4351-105 / 110-cpYFP

[0183] ASVSARTLHFGTSATYAPYEFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDPDYYGKGLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ

[0184] SEQ ID NO:3 (SEQ ID NO:5 of CN118324869A)

[0185] STAR is STM4351-200 / 203-E37A-cpYFP

[0186] ASVSARTLHFGTSATYAPYAFVDADNKIVGFDIDVANAVCKEMQAECSFTNQSFDSLIPSLRFKKFDAVIAGMDMTPKREQQVSFSQPYYEGLSAVVVTRKGAYHTFADLKGKKVGLENGTTHQRYLQDKQQAITPVAYDSYLNAFTDLKNNRLEGVFGDVAAIGKWLKNNPDYAIMDERASDYNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNYYGK GLGIAVRKDNDALLQEINAALDKVKASPEYAQMQEKWFTQ

[0187] SEQ ID NO: 52 (cpYFP shown in SEQ ID NO: 2 of CN113336854A)

[0188] YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0189] SEQ ID NO: 53 (cpGFP shown in SEQ ID NO: 3 of CN113336854A)

[0190] NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0191] SEQ ID NO: 54 (cpBFP shown as SEQ ID NO: 4 in CN113336854A)

[0192] NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0193] SEQ ID NO: 55 (cpmApple shown as SEQ ID NO: 5 in CN113336854A)

[0194] VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA

Claims

1. The use of a reagent for detecting arginine in blood-derived samples, or a reagent and arginine standard for detecting arginine in blood-derived samples, in the preparation of a kit for diagnosing vitiligo, identifying the stage of vitiligo, or identifying the severity of vitiligo; wherein, The stages of vitiligo patients include the progressive stage and the stable stage, and the severity includes the size of the white patches on the patient's skin. Preferably, the sample is whole blood, serum, and / or plasma.

2. The use as described in claim 1, characterized in that, The reagents used for detecting arginine include those required for detecting arginine using one or more methods selected from: spectrophotometry, capillary electrophoresis, small molecule chemical probe methods, mass spectrometry, and optical probe methods.

3. The use as described in claim 1 or 2, characterized in that, The reagents used for detecting arginine include reagents for transforming, enriching, separating, or recognizing arginine. Preferably, the reagent for detecting arginine includes one or more selected from the following: arginine-specific antibodies, arginine deiminase, phthalaldehyde, sulfite, borate, small chemical probe ThiCS, formic acid, acetonitrile, isopropanol, arginine-binding protein, or functional variants thereof.

4. The use as described in claim 3, characterized in that, Functional variants of the arginine-binding protein include arginine optical probes. Preferably, The arginine optical probe comprises an arginine-binding protein and an optically active polypeptide. More preferably, the optical probe has one or more features selected from the following: The arginine-binding protein has the sequence shown in SEQ ID NO:1 or a functional fragment thereof, or a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with it; The optically active polypeptide is located at one or more sites of the arginine-binding protein selected from the following: 103 / 104, 103 / 105, 103 / 106, 103 / 107, 103 / 108, 103 / 109, 103 / 110, 103 / 111, 104 / 105, 104 / 106, 104 / 107, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 105 / 111, 106 / 107, 106 / 108, 106 / 109, 106 / 110, 106 / 111, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 109, 108 / 110, 108 / 111, 109 / 110, 109 / 111, 110 / 111, 197 / 198, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 197 / 205, 197 / 206, 197 / 207, 197 / 208, 197 / 209, 198 / 199, 198 / 200, 198 / 201, 198 / 202, 198 / 203, 198 / 204, 198 / 205 , 198 / 206, 198 / 207, 198 / 208, 198 / 209, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 199 / 207, 199 / 208, 199 / 209, 200 / 201, 200 / 202, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 200 / 207, 200 / 208, 200 / 209, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 201 / 207, 201 / 208, 201 / 209, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 202 / 207, 202 / 208, 202 / 209, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 203 / 208, 203 / 209, 204 / 205, 204 / 206, 204 / 207, 204 / 208, 204 / 209, 205 / 206, 205 / 207, 205 / 208, 205 / 209, 206 / 207, 206 / 208, 206 / 209, 207 / 208, 207 / 209 or 208 / 209 The arginine-sensitive polypeptide contains mutations at one, two, three, or more of the following positions: S30, T32, E37, F71, G89, M90, D91, R96, R96, E135, T138, T139, H140, D177, wherein the amino acid mutations include modifications, substitutions, or deletions of amino acids. Preferably, the mutation is selected from one, two, three or more of S30N, T32A, E37A, F71A, G89A, M90A, D91A, R96M, R96K, R96A, E135A, T138A, T139A, H140A, D177A, and D177N.

5. A kit for detecting arginine, the kit comprising reagents for detecting arginine and instructions, the instructions describing a method for diagnosing vitiligo, identifying the stage of vitiligo, or identifying the severity of vitiligo by detecting arginine. Preferably, the reagents for detecting arginine include reagents required for detecting arginine using one or more methods selected from: spectrophotometry, capillary electrophoresis, small molecule chemical probe method, mass spectrometry, and optical probe method; More preferably, the reagent for detecting arginine includes reagents for converting, enriching, separating, or recognizing arginine.

6. The reagent kit as described in claim 5, characterized in that, The reagents for detecting arginine include one or more selected from the following: arginine-specific antibodies, arginine deiminase, phthalaldehyde, sulfite, borate, small chemical probe ThiCS, formic acid, acetonitrile, isopropanol, arginine-binding protein, or functional variants thereof. The kit also includes reagents for processing the test samples. The test detects arginine in a blood-derived sample. The reagents used to process the test samples include: 3NPH_HCl, EDC, and buffer solution; preferably, the buffer solution is a phosphate-based buffer solution or Tris.

7. The kit according to claim 5, characterized in that, The kit contains an arginine optical probe and a buffer solution. The arginine optical probe comprises an arginine-binding protein and an optically active peptide. Preferably, the optically active polypeptide is located within the sequence of the arginine-binding protein, at its N-terminus or C-terminus. Preferably, the buffer solution is a phosphate-based buffer or Tris, such as HEPES, PBS, etc.

8. The kit according to claim 7, characterized in that, The optical probe has one or more features selected from the following: The arginine-binding protein has the sequence shown in SEQ ID NO:1 or a functional fragment thereof. The optically active polypeptide is located at one or more sites of the arginine-binding protein selected from the following: 103 / 104, 103 / 106, 103 / 107, 103 / 108, 103 / 109, 103 / 110, 104 / 105, 104 / 106, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 1 05 / 111, 106 / 107, 106 / 108, 106 / 109, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 109, 108 / 111, 109 / 110, 109 / 111, 110 / 111, 197 / 198, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 197 / 205, 197 / 206, 197 / 208, 198 / 201, 198 / 202, 198 / 203, 198 / 204, 198 / 205, 198 / 206, 198 / 208, 198 / 209, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 199 / 208, 200 / 203, 200 / 204, 200 / 205, 200 / 2 06, 200 / 207, 200 / 208, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 201 / 208, 201 / 209, 202 / 203, 202 / 205, 202 / 206, 203 / 204, 203 / 206, 204 / 205, 204 / 206, 204 / 208, 205 / 206, 205 / 207, 205 / 208 or 206 / 207, The arginine-sensitive polypeptide contains mutations at one, two, three, or more of the following positions: S30, T32, E37, F71, G89, M90, D91, R96, R96, E135, T138, T139, H140, D177, wherein the amino acid mutations include modifications, substitutions, or deletions of amino acids. Preferably, the mutation is selected from one, two, three or more of S30N, T32A, E37A, F71A, G89A, M90A, D91A, R96M, R96K, R96A, E135A, T138A, T139A, H140A, D177A, and D177N.

9. The kit according to any one of claims 5-9, characterized in that, The kit also includes a reagent for detecting vitiligo.

10. A method for diagnosing vitiligo, identifying the stage of a vitiligo patient, or identifying the severity of a vitiligo patient, wherein the stage of the vitiligo patient includes a progressive phase and a stable phase, and the severity includes the size of the area of ​​white patches on the patient's skin, the method comprising: (1) Detection of arginine in blood-derived samples, (2) Comparison with control levels, where, Patients with vitiligo have higher arginine levels than the control group; preferably, the control group is the arginine level of healthy individuals. Patients with progressive vitiligo have higher arginine levels than control patients. Preferably, the control level is the arginine level of healthy individuals or the arginine level of patients with stable vitiligo. Patients with at least 1% vitiligo area had higher arginine levels than the control level. Preferably, the control level was the arginine level of a healthy subject or the arginine level of a patient with less than 1% vitiligo area.

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