Marker group and kit for predicting sensitivity of bicitinib combined with narrow-spectrum medium-wave ultraviolet rays in treatment of leucoderma and application of marker group and kit

Abstract

The invention discloses an application of a substance for detecting a marker group. The application comprises one or more of the following applications: A1) an application in preparation of a product for predicting sensitivity of bicitinib combined with narrow-spectrum medium-wave ultraviolet rays in treatment of leucoderma, and A2) an application in preparation of a product for predicting sensitivity of bicitinib combined with narrow-spectrum medium-wave ultraviolet rays in treatment of leucoderma; a2) is applied to preparation of a product for screening sensitivity of bicitinib combined with narrow-spectrum medium-wave ultraviolet rays in treatment of leucoderma; a3) in preparation of a product for evaluating sensitivity of bicitinib combined with narrow-spectrum medium-wave ultraviolet rays in treatment of leucoderma; the marker group is composed of CCL5, FGL1, EZR and GAPDH (glyceraldehyde-phosphate dehydrogenase). The invention screens and verifies the plasma and urine biomarker group capable of predicting the sensitivity of treating leucoderma by combining baricitinib with narrow-spectrum medium-wave ultraviolet rays of a subject through ELISA (Enzyme-Linked Immunosorbent Assay), and the plasma and urine biomarker group provided by the invention has higher prediction accuracy, sensitivity and specificity; the individualized treatment of vitiligo can be improved, and good clinical application and popularization prospects are achieved.

Description

Technical Field

[0001] The present invention relates to a group of sensitivity markers for predicting the treatment of vitiligo with baricitinib combined with narrow-band ultraviolet B, and also relates to a corresponding kit and application, belonging to the field of biological testing technology. Background Art

[0002] Vitiligo is a common acquired pigment loss disease with an incidence rate of about 0.5% - 2%. It is easy to diagnose but difficult to treat, seriously affecting the quality of life of patients, and there is still a lack of specific treatment regimens. Currently, it is generally believed that the IFN-γ / JAK / STAT pathway plays an important role in the pathogenesis of vitiligo. In recent years, a variety of topical and oral JAK inhibitors have been proven to control the disease progression and promote repigmentation. Combined with phototherapy, such as narrow-band ultraviolet B (NB-UVB), it can enhance the efficacy. The inventor previously confirmed through prospective and controlled clinical experiments that baricitinib combined with NB-UVB is an effective method for treating progressive non-segmental vitiligo and has good safety; at the same time, it was also found that different patients have different responses to this combined therapy, with obvious individual differences, namely the fast-response group and the slow-response group. Therefore, by screening the differential factors in the body fluids of patients in the fast-response group and the slow-response group before treatment and finding biomarkers that can predict the curative effect, it has important clinical value for the individualized treatment of vitiligo.

[0003] As the executor of biological structure and function, the expression difference of proteins can more reflect the changes in the role of downstream biological processes. Mass spectrometry (MS) is the most commonly used technology in proteomics. Data-independent acquisition (DIA) tandem mass spectrometry can basically achieve continuous and unbiased acquisition of information on all secondary mass spectra and is currently the most widely used in clinical research. For clinical research, body fluids such as blood and urine are convenient to obtain, minimally invasive, and the research results are easy to be clinically translated and applied. Currently, there is still a lack of research on vitiligo-related proteomics, and there is no relevant research on using blood and urine proteomic biomarkers to predict the sensitivity of JAK inhibitor combined with phototherapy. Summary of the Invention

[0004] The primary technical problem to be solved by the present invention is to provide a new use of a new marker group, which can be used for the new use in preparing substances for predicting, screening, and evaluating the sensitivity of treating vitiligo with baricitinib combined with narrow-band ultraviolet B.

[0005] Another technical problem to be solved by the present invention is to provide a kit for detecting the new marker group. This kit can be used for predicting, screening, and evaluating the sensitivity of treating vitiligo with baricitinib combined with narrow-band ultraviolet B.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] Use of a substance for a marker group, including one or more of the following uses:

[0008] A1) Use in the preparation of a product for predicting the sensitivity of baricitinib combined with narrowband ultraviolet B for the treatment of vitiligo;

[0009] A2) Use in the preparation of a product for screening the sensitivity of baricitinib combined with narrowband ultraviolet B for the treatment of vitiligo;

[0010] A3) Use in the preparation of a product for evaluating the sensitivity of baricitinib combined with narrowband ultraviolet B for the treatment of vitiligo;

[0011] The marker group is chemokine ligand 5 (C-C motif chemokine ligand 5, CCL5), fibrinogen-like protein 1 (Fibrinogen-like protein 1, FGL1), ezrin (Ezrin, EZR), and glyceraldehyde-3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase, GAPDH).

[0012] Preferably, the substance is a reagent for detecting the expression level of the marker group, or a reagent that specifically binds to and recognizes the marker group, or a reagent for detecting the content of the marker group.

[0013] Preferably, the substance is a mass spectrometry identification reagent, an antibody, or an antigen-binding fragment thereof.

[0014] Preferably, the substance for detecting the marker group is one of the following a), b), or c):

[0015] a) A reagent for detecting the expression level of the marker group;

[0016] b) A reagent set containing the reagent of a);

[0017] c) A kit containing the reagent of a) or the reagent set of b).

[0018] Preferably, the detection sample for the markers CCL5 and FGL1 is plasma, and the detection sample for the markers EZR and GAPDH is urine.

[0019] A kit for detecting a marker group, including one or more of the following uses:

[0020] A1) Use in the preparation of a product for predicting the sensitivity of baricitinib combined with narrowband ultraviolet B for the treatment of vitiligo;

[0021] A2) Use in the preparation of products for screening the sensitivity of baricitinib combined with narrow-band ultraviolet B for the treatment of vitiligo;

[0022] A3) Use in the preparation of products for evaluating the sensitivity of baricitinib combined with narrow-band ultraviolet B for the treatment of vitiligo;

[0023] The marker group is CCL5, FGL1, EZR and GAPDH, and the kit includes reagents for detecting the expression levels of the marker group.

[0024] Preferably, the detection samples for the markers CCL5 and FGL1 are plasma, and the detection samples for EZR and GAPDH are urine.

[0025] Using the kit provided by the present invention, the expression levels of the markers CCL5 and FGL1 proteins in the peripheral blood of the subject and the expression levels of the EZR and GAPDH proteins in the urine of the subject can be detected. Then, based on the information of up-regulation or down-regulation of these protein expressions, the sensitivity of the subject to the treatment of baricitinib combined with narrow-band ultraviolet B for vitiligo can be discriminated, so as to realize the prediction, screening and treatment effect evaluation of the sensitivity of baricitinib combined with narrow-band ultraviolet B for the treatment of vitiligo.

[0026] The kit provided by the present invention may include appropriate packaging and instructions for use in the methods disclosed in the present invention. The kit provided by the present invention includes identification reagents for the expression levels of the above-mentioned marker proteins, and the identification reagents can be mass spectrometry identification reagents, antibodies or their antigen-binding fragments. The kit can also be a chip fixed with the above-mentioned protein identification reagents.

[0027] Compared with the prior art, the present invention takes patients with progressive non-segmental vitiligo as the research object. By analyzing the results of plasma and urine proteomics before treatment in the fast-response group and slow-response group of patients, plasma and urine biomarkers that can predict the sensitivity of the subject to the treatment of baricitinib combined with narrow-band ultraviolet B are screened and verified by ELISA. Moreover, the combination of plasma and urine biomarkers proposed by the present invention has higher prediction accuracy, sensitivity and specificity, which helps to improve the individualized treatment of vitiligo and has good clinical application and popularization prospects. Brief Description of the Drawings

[0028] Figure 1A It is a volcano plot of differential proteins in the fast-response group and slow-response group of plasma proteomics before treatment;

[0029] Figure 1B It is a volcano plot of differential proteins in the fast-response group and slow-response group of urine group before treatment;

[0030] Figure 2AAnalysis of the correlation between CCL5 biomarker and clinical indicators for efficacy prediction;

[0031] Figure 2B Analysis of the correlation between FGL1 biomarker and clinical indicators for efficacy prediction;

[0032] Figure 2C Analysis of the correlation between EZR biomarker and clinical indicators for efficacy prediction;

[0033] Figure 2D Analysis of the correlation between GAPDH biomarker and clinical indicators for efficacy prediction;

[0034] Figure 2E AUC curve of CCL5 biomarker for efficacy prediction;

[0035] Figure 2F AUC curve of FGL1 biomarker for efficacy prediction;

[0036] Figure 2G AUC curve of EZR biomarker for efficacy prediction;

[0037] Figure 2H AUC curve of GAPDH biomarker for efficacy prediction;

[0038] Figures 3A - 3B Determination of plasma CCL5 and FGL1, urine EZR and GAPDH levels in the fast response group and slow response group before treatment by ELISA method;

[0039] Figures 3C - 3D Determination of urine EZR and GAPDH levels in the fast response group and slow response group before treatment by ELISA method;

[0040] Figure 4 ROC curve of biomarker combination for plasma and urine efficacy prediction before treatment. Detailed implementation manner

[0041] The present invention will be further described below in conjunction with specific embodiments. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described herein are only for demonstration purposes.

[0042] Example 1: Screening of Biomarkers for Predicting the Sensitivity of Baricitinib Combined with Narrow - band Ultraviolet B (NB - UVB) in the Treatment of Vitiligo Biomarker Screening

[0043] 1. Research objects

[0044] 16 patients receiving baricitinib combined with NB-UVB treatment.

[0045] 1.1 Specimen collection

[0046] Collect 4 ml of peripheral blood samples (EDTA anticoagulant) from patients before treatment and at follow-up nodes, gently invert the samples up and down to mix evenly, centrifuge at 1600 g for 10 min at 4°C, and collect the upper plasma. At the same time, collect 12 ml of clean fasting midstream urine samples from patients. After numbering and aliquoting the plasma and urine samples, store them in a -80°C refrigerator for plasma and urine proteomics research.

[0047] 1.2 Efficacy evaluation grouping criteria

[0048] According to whether the patients in the combined treatment group reach T-VSAI 50 after 3 months of baricitinib combined with NB-UVB treatment, the patients are divided into a fast-response group and a slow-response group.

[0049] 2. Main experimental reagents and equipment

[0050] 2.1 Main experimental instruments are shown in Table 1.

[0051] Table 1

[0052] Instrumentation Supplier Q - Exactive HF Mass Spectrometer Thermo Fisher Scientific, USA EASY - nLC 1200 Liquid Chromatograph Thermo Fisher Scientific, USA Ultracentrifuge Thermo Fisher Scientific, USA Vacuum Centrifugal Concentrator Savant, USA Vortex Shaker SCILOGEX, USA −80°C Ultra - Low Temperature Refrigerator Thermo Fisher Scientific, USA Adjustable Range Pipette Eppendorf, Germany Electronic Analytical Balance Sartorius Instrument Systems Co., Ltd., Beijing Magnetic Rack BioMS Technology Co., Ltd., Beijing Constant Temperature Water Bath Oriental Electric Instrument Factory, Yuyao Ice Maker Sanyo, Japan

[0053] 2.2 Main reagents and consumables are shown in Table 2.

[0054] Table 2

[0055] Main Reagents and Consumables Supplier High - Depth Blood Proteome Sample Preparation Reagent BioMS Technology Co., Ltd., Beijing Chromatographic - Grade Acetonitrile (ACN) Thermo Fisher, USA EasyPeptide Micro Pretreatment Preparation Kit eBioeasy Biotechnology Co., Ltd., Shanghai C18 Solid - Phase Extraction Column Thermo Fisher, USA nanoViper C18 Chromatographic Column Thermo Fisher, USA Dithiothreitol [[ID=5 ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0056] 3. Experimental methods

[0057] 3.1 Sample preparation <000>

[0058] 3.1.1 Depletion of high-abundance proteins from plasma samples <00> <00>

[0059] This part is strictly operated according to the instructions of the high-depth blood proteomics sample preparation kit. <00> <00>

[0060] (1) Protein preparation <00> <00>

[0061] ① Take out the plasma specimen from the -80°C refrigerator and place it at room temperature; <00> <00>

[0062] ②Vortex the nano - magnetic beads to mix well, transfer 50 μL to a new EP tube, place it on a magnetic stand until it becomes clear, remove the preservation solution, add 200 μL of Resuspension Buffer to resuspend the nano - magnetic beads, vortex to mix evenly, and perform water - bath sonication for 10 min. Place the EP tube on the magnetic stand, let it stand for 3 - 5 min, discard the supernatant, and repeat once;

[0063] ③Add 100 μL of Resuspension Buffer to the magnetic beads to resuspend them, perform water - bath sonication for 10 min, then add 100 μL of plasma, vortex to mix well, incubate at 37 °C for 1 h at 1000 rpm;

[0064] ④Centrifuge briefly (at a speed not exceeding 1000 rpm), place the tube on the magnetic stand for 3 - 5 min, and remove the supernatant. Add 500 μL of Washing Buffer, vortex to mix well, shake at 1000 rpm for 5 min, use the magnetic stand to assist in discarding the supernatant, and repeat the washing twice;

[0065] (2) Protease digestion:

[0066] ①Mix the Lysis Buffer evenly and add 50 μL of Lysis Buffer to the magnetic bead precipitate;

[0067] ②Incubate in a metal bath at 95 °C at 1000 rpm for 10 min;

[0068] ③Let the sample cool naturally to room temperature and centrifuge briefly (at a speed not exceeding 1000 rpm). Add 2.5 μL of Digestion Buffer, incubate in a metal bath at 37 °C at 1000 rpm for 2 h for enzymatic digestion;

[0069] ④Centrifuge the sample briefly, add 5 μL of Stop Buffer, and mix well. Terminate the enzymatic digestion reaction, which will produce a large amount of precipitate. After centrifuging at 16000 g for 5 min, aspirate the supernatant for subsequent desalting.

[0070] 3.1.2 Urine protein extraction and pretreatment

[0071] Some of the reagents in this process are from the Easy - Peptide Micro pretreatment preparation kit:

[0072] Table 3

[0073] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0074] ①Take out the urine specimen from the - 80 °C refrigerator, warm it in a 37 °C water bath, vortex evenly, then centrifuge at 5000 g for 10 min. Take 500 μL of urine supernatant, add 3 volumes of pre - cooled acetone, vortex to mix well, and place it in a - 20 °C refrigerator to precipitate overnight;

[0075] ② Centrifuge at 14000g for 15min, discard the acetone, and place the precipitate in a ventilated place to evaporate until the acetone smell disappears. Add 30μL of 20mM Tris buffer (AMRESCO, C4H 11 NO3, purity ≥99%), dissolve and add 1.2 μL of reagent B, mix well;

[0076] ③ Heat in a 95℃ metal bath for 5 minutes. After returning to room temperature, add 2μL of reagent C and 5μL of reagent D respectively, mix well, and incubate at 37℃ for overnight enzymatic hydrolysis.

[0077] ④ After the enzymatic hydrolysis is completed, add 3 μL of reagent E, mix well, stop the enzymatic hydrolysis, centrifuge at 14000g for 5 minutes, and then aspirate the supernatant for subsequent desalting.

[0078] 3.1.3C 18 Solid Phase Extraction

[0079] ①C 18 Activation of solid phase extraction column: Pipette 100 μl 100% ACN to activate the extraction column;

[0080] ②C 18 Equilibrate the solid phase extraction column: pipette 100 μl of 50% ACN 0.1% FA to clean the residual ACN in the extraction column;

[0081] ③C 18 Equilibrate the solid phase extraction column: pipette 100 μl of 2% ACN 0.1% FA to equilibrate the extraction column;

[0082] ④ Loading: Pipette the enzyme-cleaved peptide solution into the extraction column and enrich it. Repeat 3 times.

[0083] ⑤ Washing and desalting: aspirate 100 μl of 2% ACN 0.1% FA to wash the salt in the sample, repeat twice; ⑥ Elution: aspirate 50 μl of 50% ACN 0.1% FA, repeat twice, and collect the eluate with an EP tube; ⑦ Place the collected eluate in a rotary vacuum dryer, vacuum dry without heating, and store in a -80°C refrigerator for later use.

[0084] 3.2 LC-MS / MS analysis

[0085] 3.2.1 Liquid chromatography analysis

[0086] ① Redissolve the peptide components in 10 μL 0.1% FA solution;

[0087] ② After U3000 liquid phase, nano Viper C 18Separation was performed on a chromatographic column (75μm * 250mm, 2μm). Phase A was an aqueous solution of 0.1% FA, and phase B was an aqueous solution of 80% acetonitrile and 0.1% FA. The effective elution gradient was 1% - 35%, the total elution time was 90 min, and the flow rate was 1.2 μL / min. The liquid phase conditions were as follows:

[0088] Table 4 Low pH Reversed-Phase Chromatographic Gradient

[0089] ​ A％ B％ ​ 0 100 0 1.2 5:00 99 1 1.2 6:00 92 8 1.2 30:00 85 15 1.2 52:00 70 30 1.2 56:00 65 35 1.2 58:00 10 90 1.2 60:00 100 0 1.2

[0090] 3.2.2 Mass Spectrometry Analysis

[0091] The separated peptide fragments were injected into an NSI ion source (voltage 2.1 kv) for ionization and then identified using a Q-Exactive HF mass spectrometer.

[0092] In the DDA mode, a spectral library was established. The MS parameters were set as follows: full scan with a resolution of 60,000 in the range of 350 - 1500 m / z; cycle time set to 3 s; automatic gain control (AGC) of 1e6; injection time < 50 ms; HCD collision energy of 32%. Precursors were screened for charge states with a dynamic exclusion duration of 30 s and charge states of +2 to +6. Separation of precursors was performed by a quadrupole with a 1.6 m / z separation window, and the strongest ions in each measurement scan were used for fragmentation. The generated fragments were analyzed in the Q-Exactive HF analyzer. According to the m / z distribution of precursor ions in the mixed sample, the number of precursor ions in each isolation window was balanced.

[0093] For DIA analysis, to maintain the stability of retention time, the LC settings were the same as those in the DDA experiment. In addition, an iRT kit was added to all samples to correct the retention time between samples. The MS parameters were set as follows: full scan with a resolution of 120,000 in the range of 350 - 1500 m / z; cycle time set to 3 s; AGC of 3e6; injection time < 100 ms; HCD collision energy of 32%. Precursors were screened for charge states with a dynamic exclusion duration of 10 s and charge states of +2 to +6.

[0094] 3.3 Data Analysis

[0095] 3.3.1 DIA Data Analysis

[0096] Direct-DIA database search was performed using Spectronaut Pulsar X software (Biognosys, AG, Schlieren, Switzerland) with default settings. The database was: uniprot-human-81803-20230327.fasta. Based on the iRT calibration strategy, the optimal XIC extraction window was determined. Based on large-scale mass calibration, the mass tolerance strategy was set to dynamic. Cross-normalization was set to local normalization based on local regression. The total peak area of each fragment ion in MS2 was used to quantify peptide intensity. The search parameters were: protein database of the corresponding species, trypsin digestion, up to 2 missed cleavage sites, precursor ion and fragment ion mass errors of 10 ppm and 0.02 Da respectively, fixed modification of Carbamidomethyl (C), variable modifications of Oxidation (M), Acetyl (N-terminal). The false discovery rate (FDR) of polypeptides and proteins was <1.0%, and at least 1 specific peptide was identified for each protein.

[0097] Protein missing values were imputed using the KNN nearest neighbor method through the Wukong Cloud Platform (https: / / www.omicsolution.com / wkomics / main / ).

[0098] 3.3.2 Bioinformatics analysis

[0099] Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed using SIMCA software (version 14.1, Umetrics, Sweden) to visualize the grouping situation.

[0100] The mean ratio of all quantitative values of each protein in the compared sample pairs was defined as the fold change (FC). All protein quantitative values were Log2-transformed, and a t-test was used to evaluate the protein significance between groups, with the corresponding P-value as the significance index. The criteria for screening differentially expressed proteins (DEPs) were: FC ≥ 1.5 or FC ≤ 1 / 1.5, P < 0.05.

[0101] 3.3.3 Correlation analysis of differential proteins and clinical indicators

[0102] Correlation analysis was performed between the quantitative values of DEPs and the percentage change in the patient's VASI score. The percentage change in VASI score was calculated as (VASI score after treatment - VASI score before treatment) / VASI score before treatment. The criteria for screening for correlated proteins were a correlation coefficient r > 0.4 and P < 0.05. The predictive ability of the target protein was assessed using receiver operating characteristic (ROC) curve analysis. Correlation analysis, plotting, and ROC curve construction were performed using the R language (version 4.2.2).

[0103] 4. Biomarker Screening Phase Efficacy Test Results

[0104] 4.1 Analysis of plasma proteomics results before treatment identified 33 differentially expressed proteins

[0105] Compared with the slow response group, the expression of 12 proteins in the fast response group was up-regulated and the expression of 21 proteins was down-regulated, such as ​ As shown. The correlation analysis between the quantitative values of differential proteins before treatment and the percentage change of VASI scores of 16 patients was performed, and a total of 17 differential proteins correlated with the change values of VASI scores were screened out. Through further literature search, combined with the biological functions and AUC values of differential proteins, CCL5 and FGL1 were selected as plasma candidate efficacy prediction biomarkers. Compared with the slow-response group, CCL5 was upregulated in the fast-response group with a correlation coefficient of -0.61; FGL1 was downregulated in the fast-response group with a correlation coefficient of 0.5, as shown in Figure 2. ​ and ​ As shown in Figure 2, the AUC values of the proteins were 0.875 and 0.812, respectively. ​ and ​ shown.

[0106] 4.2 The urine proteomics results before treatment were analyzed, and a total of 44 differentially expressed proteins were identified.

[0107] Compared with the slow response group, the expression of 34 proteins in the fast response group was up-regulated, and the expression of 10 proteins was down-regulated. ​ As shown. The correlation analysis between the quantitative values of differential proteins before treatment and the percentage change of VASI scores of 14 patients was performed, and a total of 23 differential proteins correlated with VASI scores were screened out. Through further literature search, combined with the biological functions and AUC values of differential proteins, EZR and GAPDH were selected as candidate urine efficacy prediction biomarkers. Compared with the slow-response group, EZR and GAPDH were upregulated in the fast-response group, with correlation coefficients of -0.67 and -0.57, respectively. ​ and ​ As shown in Figure 2, the AUC values of the two proteins were 0.857 and 0.816, respectively. ​ and ​ shown.

[0108] ​

[0109] 1. Research objects

[0110] In this part, 16 patients in the combination treatment group in Example 1 were included, as well as 14 newly enrolled patients with progressive non-segmental vitiligo who visited the dermatology outpatient clinic of Peking Union Medical College Hospital. All these 14 patients adopted the treatment plan of the combination treatment group, that is, taking baricitinib orally at 2 mg / d, combined with NB-UVB three times a week.

[0111] 2. Main experimental reagents and equipment

[0112] 2.1 Main experimental instruments

[0113] Table 5

[0114] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

[0115] 2.2 Main reagents and consumables

[0116] Table 6

[0117] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Centrifuge tube Nest, Switzerland Cryogenic storage tube Corning, USA EP tube Axygen, USA Pipette tip Axygen, USA Sealing film for microplate Solarbio Science & Technology Co., Ltd., Beijing

[0118] 3. Experimental methods

[0119] 3.1 Sample and reagent preparation

[0120] ① Sample preparation: Take out the plasma and urine specimens from the -80°C refrigerator, thaw them on ice, centrifuge at 6000 - 10000 rpm for 1.5 min, and dilute the test samples to the corresponding concentrations according to the results of the preliminary experiment.

[0121] ② Serial dilution of standards:

[0122] Take out one standard from the kit and centrifuge it at 6000 - 10000 rpm for 30 seconds. Dissolve it with 1 ml of sample diluent, and repeatedly pipette against the bottom of the cryotube with a pipette tip to assist dissolution, and mix well to obtain standard S7, and set it aside for use;

[0123] Take 7 1.5-ml centrifuge tubes (S0 - S6) and arrange them in sequence. Add 250 μl of sample diluent to each. Pipette 250 μl of standard S7 into the first centrifuge tube (S6), and gently pipette to mix well. Pipette 250 μl from S6 into the second EP tube (S5), and gently pipette to mix well. And so on for the serial dilution of the standards. S0 is the sample diluent;

[0124] ③ Dilute the concentrated washing solution 1:25 with deionized water;

[0125] ④Dilute the biotin-labeled antibody solution 100-fold with the biotin-labeled antibody diluent and prepare it within 10 minutes before use;

[0126] ⑤Dilute the horseradish peroxidase-labeled avidin 100-fold with the horseradish peroxidase-labeled avidin diluent and prepare it within 10 minutes before use;

[0127] 3.2 Experimental procedures

[0128] The ELISA kit used in this part adopts the double antibody sandwich method. Coat the surface of the carrier with specific antibodies, add the plasma or urine samples to be tested that may contain the corresponding antigen, incubate and then wash, and then incubate with the enzyme-labeled specific antibody. The coated antibody, the antigen to be detected, and the enzyme-labeled antibody form a sandwich complex. Wash away the unbound substances, add the substrate for color development, and quantitatively detect the antigen according to the presence or absence of color or the depth of color.

[0129] ①Transfer various reagents to room temperature (18 - 25 °C) and equilibrate for at least 30 minutes, prepare the relevant reagents for standby;

[0130] ②Sample addition: Set up standard product wells and sample wells to be tested respectively. Add 100 μL of the standard product or the sample to be tested to each well, gently shake and mix evenly, cover with a plate sticker, and incubate at 37 °C for 2 hours;

[0131] ③Discard the liquid, centrifuge dry, no need to wash;

[0132] ④Add 100 μL of the biotin-labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37 °C for 1 hour;

[0133] ⑤Discard the liquid in the wells, centrifuge dry, wash the plate 3 times. Soak for 2 minutes each time, 200 μL per well, centrifuge dry;

[0134] ⑥Add 100 μL of the horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate sticker, and incubate at 37 °C for 1 hour;

[0135] ⑦Discard the liquid in the wells, centrifuge dry, wash the plate 5 times. Soak for 2 minutes each time, 200 μL per well, centrifuge dry;

[0136] ⑧Add 90 μL of the substrate solution to each well in sequence, and develop color at 37 °C in the dark for 15 - 30 minutes;

[0137] ⑨Add 50 μL of the termination solution to each well in sequence to terminate the reaction;

[0138] ⑩Measure the optical density (OD value) of each well in sequence with an enzyme-labeled instrument at a wavelength of 450 nm within 5 minutes after the reaction is terminated.

[0139] 3.3 Data analysis

[0140] With the standard substance concentration as the ordinate and the OD value as the abscissa, the standard curve regression equation was calculated using Curve Expert software (version 1.4). The OD value of the sample was substituted into the equation to calculate the concentration of each sample. If the sample was diluted before detection, the corresponding dilution factor needed to be multiplied. For the comparison of between-group differences before and after treatment, if the difference was normally or approximately normally distributed, a paired t-test was used; if the difference was severely skewed, a paired design Wilcoxon signed-rank test was used. For the comparison of between-group differences between the fast response group and the slow response group before treatment, if the two groups of data were normally distributed and had homogeneous variances, a t-test was used; if the two groups of data were not normally distributed or had inhomogeneous variances, a Wilcoxon Mann-Whitney test was used. A P value < 0.05 was considered statistically significant. Graphpad Prism software (version 9.0, San Diego, CA, USA) was used for data analysis and graphing.

[0141] The predictive ability of the target protein was analyzed by ROC curve analysis, and the ROC curve was drawn using R language (version 4.2.2).

[0142] 4. Experimental results and analysis in the biomarker validation stage

[0143] 4.1 In the validation cohort, the levels of CCL5 and FGL1 in the plasma samples of patients in the fast response group and the slow response group before treatment were detected. The results showed that compared with the slow response group, the level of CCL5 in the plasma samples of the fast response group was significantly increased, while the level of FGL1 was significantly decreased, which was consistent with the trend of the mass spectrometry results, as Figure 3A and Figure 3B shown.

[0144] 4.2 The levels of EZR and GAPDH in the urine samples of patients in the fast response group and the slow response group before treatment in the validation cohort were detected. The results showed that compared with the slow response group, the levels of EZR and GAPDH in the urine samples of the fast response group were both significantly increased, which was consistent with the trend of the mass spectrometry results, as Figure 3C and 3D shown.

[0145] The above results indicate that an increased level of CCL5 and a decreased level of FGL1 in the plasma before treatment, and increased levels of EZR and GAPDH in the urine before treatment can be used to predict the sensitivity to the baricitinib combined with NB-UVB therapy.

[0146] 4.3 The predictive model combining 4 proteins showed that the biomarker combination composed of differential proteins in plasma and urine showed a higher AUC value (AUC = 0.936), as Figure 4 shown, and could better predict the sensitivity of patients to the baricitinib combined with NB-UVB treatment regimen before treatment.

Claims

1. Use of a substance for a biomarker panel, characterized in that Comprising one or more of the following applications: A1) Application in the preparation of a product for predicting the sensitivity of baricitinib combined with narrow-band ultraviolet B for the treatment of vitiligo; A2) Application in the preparation of a product for screening the sensitivity of baricitinib combined with narrow-band ultraviolet B for the treatment of vitiligo; A3) Application in the preparation of a product for evaluating the sensitivity of baricitinib combined with narrow-band ultraviolet B for the treatment of vitiligo; The marker group is CCL5, FGL1, EZR and GAPDH.

2. The application according to claim 1, characterized in that: The substance is a reagent for detecting the expression level of the marker group, or a reagent that specifically recognizes and binds to the marker group, or a reagent for detecting the content of the marker group.

3. The application according to claim 2, characterized in that: The substance is a mass spectrometry identification reagent, an antibody or an antigen-binding fragment thereof.

4. The application according to claim 1, characterized in that The substance for detecting the marker group is one of the following a), b) or c): a) A reagent for detecting the expression level of the marker group; b) A reagent set containing the a); c) A kit containing the a) or the b).

5. The application according to any one of claims 1 to 4, characterized in that: The detection samples for the markers CCL5 and FGL1 are plasma, and the detection samples for the markers EZR and GAPDH are urine.

6. A kit for detecting a biomarker panel, characterized in that Comprising one or more of the following applications: A1) Application in the preparation of a product for predicting the sensitivity of baricitinib combined with narrow-band ultraviolet B for the treatment of vitiligo; A2) Application in the preparation of a product for screening the sensitivity of baricitinib combined with narrow-band ultraviolet B for the treatment of vitiligo; A3) Application in the preparation of a product for evaluating the sensitivity of baricitinib combined with narrow-band ultraviolet B for the treatment of vitiligo; The marker group is CCL5, FGL1, EZR and GAPDH, and the kit comprises a reagent for detecting the expression level of the marker group.

7. The kit according to claim 6, characterized in that: The detection samples for the markers CCL5 and FGL1 are plasma, and the detection samples for the markers EZR and GAPDH are urine.

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