A multi-target combined detection kit for detecting vitiligo-related factors and its preparation method

Abstract

This invention discloses a multi-target combined detection kit for detecting vitiligo-related factors and its preparation method. The kit includes multiple detection reagents, each comprising a solid-phase capture carrier, a labeled antibody solution, and an antibody gradient concentration calibrator. The antibodies in the labeled antibody solution and the antibody gradient concentration calibrator are CXCL9, CXCL10, CCL20, and HMGB1. This kit enables the simultaneous determination of the levels of CXCL9, CXCL10, CCL20, and HMGB1 in a sample using a unified detection platform and in a single detection experiment. The kit offers advantages such as high sensitivity, a wide linear range, and fast detection speed, accurately detecting target molecules at low levels in samples and meeting clinical requirements for detection accuracy and efficiency.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a multi-target combined detection kit for detecting vitiligo-related factors and its preparation method. Background Technology

[0002] Multiple studies have shown that chemokines play an indispensable role in the pathogenesis of vitiligo. Chemokines are small proteins that can guide the directional movement of cells and play a crucial role in the recruitment and activation of immune cells. Among them, CXCL9 and CXCL10 are highly expressed in the skin lesions and serum of vitiligo patients. IFN-γ can induce keratinocytes to produce CXCL9 and CXCL10. These two chemokines bind to the CXCR3 receptor on the surface of T cells, thereby recruiting T cells to the skin lesions and triggering an immune attack against melanocytes. Studies have shown that the expression levels of CXCL9 and CXCL10 in the skin lesions of vitiligo patients are positively correlated with the severity of the disease, further confirming their important role in the pathogenesis of vitiligo.

[0003] CCL20 also plays a crucial role in the pathogenesis of vitiligo. It can recruit immune cells expressing the CCR6 receptor, such as Th17 cells, to sites of inflammation, participating in inflammatory responses and immune regulation. In the skin of vitiligo patients, CCL20 expression is significantly increased, which may be closely related to disease activity and progression. Related studies have found that high CCL20 expression can promote Th17 cell infiltration, thereby exacerbating skin inflammation and further damaging melanocytes.

[0004] HMGB1, an important inflammation-related molecule, plays a crucial role in the pathogenesis of vitiligo. It is a non-histone chromosome-binding protein, normally located within the cell nucleus, but released extracellularly when cells are damaged or inflammatory, activating the immune system as a damage-associated molecular pattern (DAMP). In vitiligo patients, HMGB1 expression levels are significantly elevated. It can bind to cell surface receptors such as TLR4, activating downstream signaling pathways, promoting the release of inflammatory factors, and ultimately leading to melanocyte damage.

[0005] Currently, most detection of these factors uses enzyme-linked immunosorbent assay (ELISA). For example, the article "Expression of CXCL9 and IL-22 in Peripheral Blood of Patients with Vitiligo Vulgaris and Its Clinical Significance," by Ye Xiaoyan et al., *Chongqing Medical Journal*, Vol. 43, No. 31, November 30, 2014, published an article that used ELISA to detect serum CXCL9 and IL-22 levels and analyzed the correlation between CXCL9 and IL-22 and the age, course of the disease, and area of ​​skin lesions in vitiligo patients. The article "Expression and Significance of Serum Chemokine CXCL9, CXCL10, CCL3, and CCL4 in Vitiligo Patients," by Yu Jialin et al., *China Modern Drug Application*, Vol. 19, No. 8, April 30, 2025, published an article that used ELISA to detect the levels of CXCL9, CXCL10, CCL3, and CCL4 in plasma and cell supernatant and analyzed the correlation between chemokines and patient prognosis. However, the detection methods used in these two reports have drawbacks such as long detection time, low sensitivity, and narrow dynamic range. Furthermore, existing patent CN202410069024 discloses an HMGB1 chemiluminescence kit, which uses traditional enzyme-labeled technology to prepare enzyme-labeled anti-HMGB1 antibodies. This technology leads to random distribution of the binding sites of the labeled enzyme on the antibody molecule and modification of certain key sites, affecting antibody binding activity and enzyme catalytic efficiency. This makes the detection results highly susceptible to background values, and its accuracy and sensitivity need further improvement. Additionally, single-target detection may be interfered with by other inflammatory diseases (such as elevated CXCL9 / CXCL10 in SLE and RA). Since vitiligo is an autoimmune disease, its occurrence and development are closely related to the abnormal expression of various immune factors and inflammatory molecules. Studies have found that CXCL9 / CXCL10 recruits CD8+ T cells to the skin through the IFN-γ-CXCR3 axis, directly leading to melanocyte destruction. HMGB1 enhances CXCL9 / CXCL10 secretion through the TLR9-MyD88 pathway, forming a positive feedback loop. CCL20 may promote Th17 cell infiltration through the CCR6 receptor, jointly exacerbating the autoimmune response. CXCL9 / CXCL10 reflect Th1-dominant immunity, CCL20 is associated with the Th17 pathway, and HMGB1 indicates innate immune activation; combined detection can cover abnormalities in multiple pathways. However, there are currently no reports on the efficient and accurate combined detection of these four biomarkers.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] To address the problems existing in the prior art, the purpose of this invention is to provide a multi-target combined detection kit for detecting vitiligo-related factors and its preparation method, thereby solving the technical problem that the prior art cannot simultaneously and efficiently detect four biomarkers: vitiligo chemokines CXCL9, CXCL10, CCL20, and the related inflammatory molecule HMGB1.

[0008] A multi-target combined detection kit for detecting vitiligo-related factors, comprising multiple detection reagents. Each of the detection reagents includes a solid-phase capture carrier, a labeled antibody solution, and an antibody gradient concentration calibrator. The antibodies in the labeled antibody solution and antibody gradient concentration calibrator are: CXCL9, CXCL10, CCL20, and HMGB1.

[0009] Furthermore, the diluent for the antibody gradient concentration calibrator includes: MOPSO, NaCl, bovine serum albumin, trehalose, mannitol, Tween 20, EDTA-Na2, fish skin gelatin, L-arginine, and Proclin 300.

[0010] Furthermore, the diluent for the antibody gradient concentration calibrator comprises: 50 mM MOPSO, 150 mM NaCl, 2% bovine serum albumin, 5% trehalose, 2% mannitol, 0.02% Tween 20, 1 mM EDTA-Na2, 0.2% fish skin gelatin, 20 mM L-arginine, and 0.05% Proclin 300.

[0011] Furthermore, the pH of the diluent for the antibody gradient concentration calibrator is 7.2 ± 0.1.

[0012] Furthermore, the labeled antibody solution includes an enzyme-labeled detection antibody conjugate solution.

[0013] Furthermore, the labeled antibody solution includes an enzyme-labeled CXCL9 detection antibody conjugate solution, an enzyme-labeled CXCL10 detection antibody conjugate solution, an enzyme-labeled CCL20 detection antibody conjugate solution, and an enzyme-labeled HMGB1 detection antibody conjugate solution.

[0014] Furthermore, the labeled antibody solution also includes a biotin-labeled CXCL9 capture antibody solution.

[0015] Furthermore, the enzyme-labeled CXCL9 detection antibody-alkaline phosphatase conjugate solution comprises: 1%-5% 2-morpholinoethanesulfonic acid, 0.5%-1.5% potassium chloride, 0.01%-0.05% zinc chloride, 0.1%-0.5% magnesium chloride, 5%-15% trehalose, 0.1%-0.5% surfactant RPE1740, 0.05%-0.2% surfactant Triton X100, 0.1%-0.5% bovine serum albumin, 1%-3% arginine, 0.1%-0.5% PC300, pH 6.5, and 0.2-1 μg / mL of CXCL9 detection antibody-alkaline phosphatase conjugate.

[0016] Furthermore, the enzyme-labeled CXCL10 detection antibody-alkaline phosphatase conjugate solution comprises: 1%-5% 2-morpholinoethanesulfonic acid, 0.5%-1.5% potassium chloride, 0.01%-0.05% zinc chloride, 0.1%-0.5% magnesium chloride, 0.1%-0.5% bovine serum albumin, 2.5-5% trehalose, 0.1%-0.5% surfactant RPE1740, 0.1-0.5% lauryl alcohol polyoxyethylene ether, 0.1%-0.5% F-68 (polyoxyethylene-polyoxypropylene block copolymer), 1%-3% arginine, 0.1%-0.5% PC300, pH 6.5-7.0, and the concentration of the CXCL10 detection antibody-alkaline phosphatase conjugate is 0.2~1ug / mL.

[0017] Furthermore, the enzyme-labeled CCL20 detection antibody-alkaline phosphatase conjugate solution comprises: 1%-5% 2-morpholinoethanesulfonic acid, 0.5%-1.5% potassium chloride, 0.05%-0.2% Triton X100, 0.1%-0.5% RPE1740, 5%-15% trehalose, 0.01%-0.05% zinc chloride, 0.1%-0.5% magnesium chloride, 0.1%-0.5% bovine serum albumin, 1%-3% arginine, 0.1%-0.5% PC300, pH 6.5-7.0, and the concentration of the CCL20 detection antibody-alkaline phosphatase conjugate is 0.2-1 μg / mL.

[0018] Furthermore, the enzyme-labeled HMGB1 detection antibody-alkaline phosphatase conjugate solution comprises: 0.3% disodium hydrogen phosphate, 0.03% sodium dihydrogen phosphate, 0.5%-1.5% potassium chloride, 0.2%-0.5% Brij35, 0.05%-0.2% Triton X-100, 5%-15% trehalose, 5%-15% glycerol, 0.1%-0.5% bovine serum albumin, 1%-3% arginine, 0.01%-0.05% zinc chloride, 0.1%-0.5% magnesium chloride, 0.1%-0.5% PC300, pH 6.00-6.5, and the concentration of the HMGB1 detection antibody-alkaline phosphatase conjugate is 0.2-1 μg / mL.

[0019] Furthermore, the biotin-labeled CXCL9 capture antibody solution comprises: 0.2%-0.6% tris(hydroxymethyl)aminomethane, 0.2%-0.9% sodium chloride, 0.01%-0.05% Tween 20, 0.1%-0.5% surfactant RPE1740, 0.1%-0.5% bovine serum albumin, 0.1%-0.5% PC300, pH 7.0-7.5, and a biotin-labeled CXCL9 antibody concentration of 0.2-1 μg / mL.

[0020] Furthermore, the antibody-drug conjugate solutions for detecting enzyme-labeled CXCL9, CXCL10, CCL20, and HMGB1 all also contain 0.2% bovine serum albumin and 2.5% arginine.

[0021] Furthermore, the solid-phase capture carrier comprises a streptavidin-coated magnetic microparticle solution and an antibody-crosslinked magnetic microparticle solution.

[0022] Furthermore, the streptavidin-coated magnetic microparticle solution is used for the CXCL9 antibody.

[0023] Furthermore, the streptavidin-coated magnetic microparticle solution comprises 0.2%-0.6% tris(hydroxymethyl)aminomethane, 0.2%-0.9% sodium chloride, 0.01%-0.05% Tween 20, 0.1%-0.5% surfactant RPE1740, 0.1%-0.5% bovine serum albumin, 0.1%-0.5% PC300, pH 7.0-7.5, and 0.04%-0.1% streptavidin magnetic microparticles.

[0024] Furthermore, the antibody cross-linked magnetic microparticle solution includes a solution containing CXCL10 antibody cross-linked magnetic microparticles, a solution containing CCL20 antibody cross-linked magnetic microparticles, and a solution containing HMGB1 antibody cross-linked magnetic microparticles.

[0025] Furthermore, the solution containing CXCL10 antibody cross-linked magnetic microparticles comprises: 1%-5% 2-morpholine ethanesulfonic acid, 0.5%-1.5% potassium chloride, 0.01%-0.05% Tween-20, 0.1%-0.5% PC300, pH 6.5-7.0, and 0.04%-0.1% CXCL10 antibody cross-linked magnetic microparticles.

[0026] Furthermore, the solution containing CCL20 antibody-crosslinked magnetic microparticles comprises: 1%-5% MES, 0.5%-1.5% potassium chloride, 0.05%-0.2% Triton X100, 0.1%-0.5% RPE1740, 5-10% trehalose, 0.1%-0.5% bovine serum albumin, 0.1%-0.5% PC300, pH 6.5-7.0, and 0.04%-0.1% CCL20 crosslinked magnetic microparticles.

[0027] Furthermore, the solution containing HMGB1 antibody cross-linked magnetic microparticles comprises: 30mM-60mM Tris, 0.5%-1.5% sodium chloride, 0.1%-0.5% bovine serum albumin, 0.1%-0.5% PC300, pH 7.0-7.5, and 0.04%-0.1% HMGB1 cross-linked magnetic microparticles.

[0028] Furthermore, the antibody gradient concentration calibrators include CXCL9 calibrator, CXCL10 calibrator, CCL20 calibrator, and HMGB1 calibrator.

[0029] Furthermore, the gradient concentrations of the CXCL9 calibrator are 0, 31.25, 62.5, 125, 250, 500, 1000, and 2000 pg / mL.

[0030] Furthermore, the gradient concentrations of the CXCL10 calibrator are 0, 24.34, 42.93, 80.18, 157.8, 316.2, 565, and 1132 pg / mL.

[0031] Furthermore, the CCL20 calibrator has gradient concentrations of 0, 24.34, 42.93, 80.18, 157.8, 316.2, 565, and 1132 pg / mL.

[0032] Furthermore, the HMGB1 calibrator has gradient concentrations of 0, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, and 500 ng / mL.

[0033] Furthermore, the method for preparing the labeled antibody solution includes a pretreatment step: removing the Gal residue at the N-glycan terminus of the labeled antibody Fc fragment with β-1,4-galactosidase to expose the terminal N-acetylglucosamine (GlcNAc-), wherein the ratio of β-1,4-galactosidase to antibody is 3KU:1mg.

[0034] Furthermore, the β-1,4-galactosidase reacts with the antibody to be labeled at a temperature range of 30-37°C for 2-8 hours.

[0035] Furthermore, the preprocessing process includes the following steps: S1: Antibody modification: A non-natural nucleotide sugar derivative with a dibenzocyclooctylene (DBCO) functional group is linked to the N-glycan terminus of the antibody Fc fragment by a glycosyltransferase to obtain an antibody with a DBCO functional group. The amount of glycosyltransferase used is 6%-9% as a percentage of the number of moles of the receptor GlcNAc. S2: Alkaline phosphatase activation: Alkaline phosphatase is activated using a heterobifunctional cross-linking agent with an azide group to obtain alkaline phosphatase with an azide group. S3: Reaction cross-linking: The antibody modified in step S1 is mixed with the alkaline phosphatase activated in step S2, and a directional coupling reaction is performed. After purification, the alkaline phosphatase antibody marker is obtained.

[0036] Furthermore, in step S1, the non-natural nucleotide sugar derivative is UDP-Gal-DBCO.

[0037] Furthermore, the step S1 of linking a non-natural nucleotide sugar derivative with a dibenzocyclooctylene (DBCO) functional group to the N-glycan terminus of the antibody Fc fragment includes: adding UDP-DBCO-Gal and glycosyltransferase GalTY289L to an antibody solution in which the Gal residues at the N-glycan terminus of the Fc fragment have been removed; the molar ratio of UDP-DBCO-Gal to degalactosylated antibody is 5-15:1; the molar ratio of GalTY289L to degalactosylated antibody is 8:100; and reacting at a temperature of 22-30°C for 2-8 hours.

[0038] Furthermore, the activation reaction of alkaline phosphatase by the heterobifunctional cross-linking agent with azide groups in step S2 includes: adding the heterobifunctional cross-linking agent with azide groups dropwise to the alkaline phosphatase solution and reacting at a temperature of 25°C for 15-30 minutes, wherein the molar ratio of alkaline phosphatase to heterobifunctional cross-linking agent is 1:10~20.

[0039] Furthermore, the heterobifunctional crosslinking agent with azide groups is NHS-PEG. n -Azide, where n can be an integer from 4 to 12.

[0040] Furthermore, the directional coupling reaction in step S3 includes: reacting the modified antibody and the activated alkaline phosphatase mixture at room temperature for 2-4 hours or at 4°C for 12-16 hours, followed by centrifugation and elution after the reaction is complete.

[0041] Furthermore, in step S3, the molar ratio of the DBCO functional group-modified antibody to ALP-PEG4-N3 is 1:1.5~3.

[0042] Compared with the prior art, the beneficial technical effects of the present invention are as follows: 1. The detection kit provided by this invention integrates the detection reagents for key vitiligo-related factors CXCL9, CXCL10, CCL20 and HMGB1 for the first time, forming a dedicated multi-target detection combination. This enables simultaneous quantitative detection of multiple targets, saving samples, time and cost, improving the comparability and reliability of detection results, and facilitating multi-index correlation analysis.

[0043] 2. The detection reagent of this invention employs a newly designed site-directed cross-linking process between antibodies and labeled enzymes. The antibody is modified with UDP-Gal-DBCO, and alkaline phosphatase is activated with NHS-PEG4-Azide. The antibody's complementarity-determining region is fully exposed, resulting in highly specific click reactions between DBCO and azide groups. This process exhibits higher binding affinity and a faster binding rate, with only targeted coupling occurring. Non-specific cross-linking between antibodies and enzymes is eliminated, leading to improved homogeneity of the cross-linking products. Simultaneously, the inhibition of alkaline phosphatase catalytic activity by metal ions is avoided, maximizing the protection of the antibody's antigen-binding capacity and the enzyme's catalytic activity. This eliminates the non-specific adsorption and low reactivity caused by traditional random amine cross-linking, significantly reducing background noise and improving the signal-to-noise ratio and detection sensitivity.

[0044] 3. This invention optimizes the design of a universal calibrator matrix solution, enabling synergistic effects among its components, significantly outperforming comparative formulations and commercially available diluents. This provides a reliable calibrator stability solution for the vitiligo four-factor assay kit. Furthermore, the optimization of the universal dosage of glycosyltransferases for the four targets ensures consistent accessibility of Fc glycosylation sites for the four antibodies. The DBCO modification rate of all four antibodies reaches 1.9-2.3, balancing modification rate, reducing byproduct generation, and ensuring the modification quality of the four antibodies.

[0045] 4. This invention improves the thermal stability of enzyme-labeled detection antibodies by optimizing the dilution formulation of alkaline phosphatase-labeled antibodies. Simultaneously, due to the newly designed cross-linking process between the antibody and the labeling enzyme, the obtained enzyme-labeled detection antibody conjugate has a stable structure, resulting in a detection kit with excellent thermal stability and minimal batch-to-batch variation. Attached Figure Description

[0046] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 Graph of the linear equations for CXCL9; Figure 2The graph of the linear equations for CXCL10; Figure 3 The graph of the linear equations for CXCL20; Figure 4 The graph of the linear equation of HMGB1; Figure 5 A graph comparing the rate of thermal acceleration drop for four different formulations. Detailed Implementation

[0048] The core detection principle of the detection kit provided by this invention is as follows: 1. This invention provides a high-throughput detection reagent system based on magnetic microparticle separation technology and copper-free click chemistry. This system utilizes capture antibody-coated magnetic microparticles to specifically capture and enrich CXCL9, CXCL10, CCL20, and HMGB1 in samples; it uses glycosylation-mediated antibody-alkaline phosphatase (ALP) site-directed cross-links as detection probes to quantify target molecules via chemiluminescence signals. The detection antibodies and ALP site-directed conjugates in the CXCL9, CXCL10, CCL20, and HMGB1 detection reagents undergo a copper-free strain-promoted click chemistry (SPACC) reaction with the galactose (Gal) terminal of the antibody's Fc fragment N-glycan by removing the galactose (Gal) terminal and introducing a dibenzocyclooctylene (DBCO) functional group. This results in a uniform, directional, and highly active immune complex, enabling high-specificity and high-sensitivity simultaneous detection of the four targets.

[0049] 2. The CXCL9, CXCL10, CCL20 and HMGB1 detection reagent system provided by this invention takes into account the differences in molecular characteristics (including isoelectric point, glycosylation site, hydrophobicity and aggregation tendency) of vitiligo-related chemokines CXCL9, CXCL10, CCL20 and HMGB1 antibodies. In the process of preparing antibody-alkaline phosphatase conjugates for each target, the β-1,4-galactosidase treatment conditions are optimized, so that the accessibility of the Fc glycosylation site of the four antibodies tends to be consistent, thereby achieving efficient modification of the four antibodies with low by-products at the same enzyme dosage.

[0050] Example 1: Kit Composition 1. CXCL9 detection reagent: Reagent 1: Streptavidin-coated magnetic microparticle solution, formulation: 0.4% tris(hydroxymethyl)aminomethane, 0.55% sodium chloride, 0.03% Tween 20, 0.3% surfactant RPE1740, 0.3% bovine serum albumin, 0.3% PC300, pH 7.4, 0.05% streptavidin magnetic microparticles.

[0051] Reagent 2: Biotin-labeled CXCL9 capture antibody, formulation: 0.4% tris(hydroxymethyl)aminomethane, 0.55% sodium chloride, 0.03% Tween 20, 0.3% surfactant RPE1740, 0.3% bovine serum albumin, 0.3% PC300, pH 7.4, final concentration of biotin-labeled CXCL9 antibody is 1 μg / mL.

[0052] Reagent 3: Contains CXCL9 detection antibody-alkaline phosphatase conjugate. Formula: 3% 2-morpholinoethanesulfonic acid, 1% potassium chloride, 0.03% zinc chloride, 0.3% magnesium chloride, 0.2% bovine serum albumin, 2.5% arginine, 10% trehalose, 0.3% surfactant RPE1740, 0.1% surfactant Triton X100, 0.3% PC300, pH 6.5. The final concentration of the CXCL9 detection antibody-alkaline phosphatase conjugate is 1 μg / mL.

[0053] Calibrators: Calibrator diluent formulation: 50mM MOPSO, 150mM NaCl, 2% bovine serum albumin, 5% trehalose, 2% mannitol, 0.02% Tween 20, 1mM EDTA-Na2, 0.2% fish skin gelatin, 20mM L-arginine, 0.05% Proclin 300, pH 7.2±0.1. Calibrators S1-S8 were prepared at different concentrations: CXCL9 antigen was added sequentially to the calibrator diluent to final concentrations of 0, 31.25, 62.5, 125, 250, 500, 1000, and 2000 pg / mL, respectively.

[0054] 2. CXCL10 detection reagent: Reagent 1: CXCL10 antibody cross-linked magnetic microparticles, formulation: 3% 2-morpholine ethanesulfonic acid, 1% potassium chloride, 0.02% Tween-20, 0.3% PC300, pH 6.5, 0.05% CXCL10 antibody cross-linked magnetic microparticles; Reagent 2: CXCL10 detection antibody-alkaline phosphatase conjugate, formulation: 3% 2-morpholinoethanesulfonic acid, 1% potassium chloride, 0.03% zinc chloride, 0.3% magnesium chloride, 0.2% bovine serum albumin, 3.5% trehalose, 0.3% surfactant RPE1740, 0.3% lauryl alcohol polyoxyethylene ether, 0.3% F-68 (polyoxyethylene-polyoxypropylene block copolymer), 2.5% arginine, 0.3% PC300, pH 6.5, final concentration of CXCL10 detection antibody-alkaline phosphatase conjugate is 1 μg / mL.

[0055] Calibrators: Calibrator diluent formulation: 50mM MOPSO, 150mM NaCl, 2% bovine serum albumin, 5% trehalose, 2% mannitol, 0.02% Tween 20, 1mM EDTA-Na2, 0.2% fish skin gelatin, 20mM L-arginine, 0.05% Proclin 300, pH 7.2±0.1. Calibrators S1-S8 are prepared by sequentially adding CXCL10 antigen to the calibrator diluent to final concentrations of 0, 24.34, 42.93, 80.18, 157.8, 316.2, 565, and 1132 pg / mL, respectively.

[0056] 3. CCL20 detection reagent: Reagent 1: CCL20 antibody cross-linked magnetic microparticles, formulation: 2% MES, 1% potassium chloride, 0.1% Triton X100, 0.3% RPE1740, 7% trehalose, 0.3% bovine serum albumin, 0.3% PC300, pH 6.5, 0.05% CCL20 cross-linked magnetic microparticles; Reagent 2: CCL20 detection antibody-alkaline phosphatase conjugate, formulation: 2% MES, 1% potassium chloride, 0.1% Triton X100, 0.3% RPE1740, 10% trehalose, 0.2% bovine serum albumin, 0.03% zinc chloride, 0.3% magnesium chloride, 2.5% arginine, 0.3% PC300, pH 6.5, final concentration of CCL20 detection antibody-alkaline phosphatase conjugate is 1 μg / mL; Calibrators: Calibrator diluent formulation: 50mM MOPSO, 150mM NaCl, 2% bovine serum albumin, 5% trehalose, 2% mannitol, 0.02% Tween 20, 1mM EDTA-Na2, 0.2% fish skin gelatin, 20mM L-arginine, 0.05% Proclin 300, pH 7.2±0.1. Calibrators S1-S8 are prepared by sequentially adding CCL20 antigen to the calibrator diluent to final concentrations of 0, 24.34, 42.93, 80.18, 157.8, 316.2, 565, and 1132 pg / mL, respectively.

[0057] 4. HMGB1 test reagent Reagent 1: HMGB1 antibody-crosslinked magnetic microparticles, formulation: 40 mM Tris, 1.0% sodium chloride, 0.2% BSA, 0.3% PC300, pH 7.20, 0.05% HMGB1 crosslinked magnetic microparticles; Reagent 2: Contains HMGB1 detection antibody-alkaline phosphatase conjugate, formulation: 0.3% disodium hydrogen phosphate, 0.03% sodium dihydrogen phosphate, 1% potassium chloride, 0.4% Brij 35, 0.1% Triton X-100, 10% trehalose, 10% glycerol, 0.2% bovine serum albumin, 2.5% arginine, 0.03% zinc chloride, 0.3% magnesium chloride, 0.3% PC300, pH 6.00, final concentration of HMGB1 detection antibody-alkaline phosphatase conjugate is 1 μg / mL; Calibrator: HMGB1 calibrator diluent formulation: 50mM MOPSO, 150mM NaCl, 2% bovine serum albumin, 5% trehalose, 2% mannitol, 0.02% Tween 20, 1mM EDTA-Na2, 0.2% fish skin gelatin, 20mM L-arginine, 0.05% Proclin 300, pH 7.2±0.1. Calibrators S1-S10 are prepared by sequentially adding HMGB1 antigen to the calibrator diluent to final concentrations of 0, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, and 500 ng / mL.

[0058] Example 2: Preparation method of detection reagent 1. Preparation method of CXCL9 detection reagent: 1.1 The preparation method of biotin-labeled CXCL9 antibody includes: (1) Antibody desalting: Equilibrate the desalting column with equilibration buffer 1 (formulation: 0.3% sodium dihydrogen phosphate, 0.03% disodium hydrogen phosphate, 0.9% sodium chloride, pH 7.4), add CXCL9 to capture the antibody, centrifuge, and collect; (2) Coupling: Add biotin at a molar ratio of 1:20 to the desalted antibody, mix well, and place on a roller at 25°C for 40 minutes; (3) Desalting: Equilibrate the desalting column with equilibration buffer 1, add biotin-conjugated CXCL9 capture antibody, centrifuge, and collect.

[0059] 1.2 Preparation method of CXCL9 detection antibody-alkaline phosphatase conjugate 1.2.1 DBCO-modified CXCL9 detection antibody: (1) Pretreatment of the Fc-terminal N-glycan of the antibody—removal of terminal galactose (Gal) residues: Take 500 μg of purified CXCL9 monoclonal antibody, and use a centrifugal desalting column to replace the buffer three times by centrifugation (1500g, 1min) to replace it into reaction buffer A (50mM sodium phosphate buffer, pH 6.0). Add β-1,4-galactosidase to the antibody solution at a rate of 1500 units per 0.5 mg of antibody. After gently vortexing and mixing, place the reaction system in a constant temperature shaker at 37℃ and incubate at 300 rpm for 4 h.

[0060] (2) Enzymatic introduction of DBCO functional group: The reaction solution from step (1) was transferred to a new Zeba desalting column and pre-equilibrated with reaction buffer B (50mM HEPES buffer, containing 20mM MnCl2, pH 7.5). The column was desalted by centrifugation (1500g, 2min) to remove galactosidase and released Gal residues, and the CXCL9 detection antibody was simultaneously replaced in the optimal reaction buffer system for glycosyltransferase. The flow-through was collected as the treated antibody solution. An excess of UDP-Gal-DBCO (dissolved in a small amount of DMF, with the final DMSO concentration < 5% of the total volume) was added to the desalted antibody solution at a molar ratio of 5-15. Subsequently, glycosyltransferase GalTY289L was added, with the amount added being 8% of the molar number of the receptor GlcNAc. After gentle vortexing, the reaction system was placed in a constant temperature shaker at 30℃, protected from light, and reacted at 300rpm for 2-8h.

[0061] (3) Purification of DBCO-modified CXCL9 detection antibody: The reaction mixture was concentrated and purified using a desalting column. The mixture was washed at least 5 times with PBS buffer (pH 7.4) containing 1 mM EDTA, adding 500 μL of washing buffer each time to thoroughly remove unreacted UDP-Gal-DBCO, the byproduct UDP, and glycosyltransferase. Finally, DBCO-functionalized CXCL9 detection antibody was obtained.

[0062] 1.2.2 Activation of alkaline phosphatase (1) Buffer replacement of alkaline phosphatase (ALP): Take 0.5 mL (about 5 mg) of ALP stock solution and place it on a Zeba desalting column (pre-equilibrated with reaction buffer (PBS, pH 7.8)). Desalt and replace the buffer solution by centrifugation (1500 g, 2 min). Collect the flow-through and measure its absorbance at 280 nm. Calculate the concentration for later use.

[0063] (2) Preparation of NHS-PEG4-Azide working solution: Weigh 0.5 mg of NHS-PEG4-Azide powder and dissolve it in high-purity anhydrous DMSO to prepare a 10 mM stock solution. Prepare fresh before use, or dispense in small doses and store at -20°C in the dark to avoid repeated freeze-thaw cycles and moisture intrusion.

[0064] (3) Azide modification reaction: Place the ALP solution (5 mg, about 50 mM) treated in step (1) into a 1.5 mL centrifuge tube. Under continuous gentle vortexing, slowly add the calculated volume of NHS-PEG4-Azide to the ALP solution dropwise. The molar ratio of NHS-PEG4-Azide to ALP is 15:1. Mix the reaction mixture gently and in the dark for 1 hour at room temperature (25 °C) on a rotary mixer or shaker.

[0065] (4) Reaction termination and purification: After the reaction is completed, the reaction solution is immediately transferred to a new Zeba desalting column that has been pre-equilibrated with 0.1M sodium phosphate buffer (pH 7.4). Purification is performed by centrifugation (1500g, 2min). This step can effectively terminate the reaction and remove unreacted NHS-PEG4-Azide, its hydrolysis byproducts, and excess DMSO. The flow-through containing the protein is collected, which is the azide-modified alkaline phosphatase (ALP-PEG4-N3).

[0066] 1.2.3 Crosslinking of DBCO-modified CXCL9 detection antibody and activated alkaline phosphatase (1) Determine the molar ratio of DBCO-modified CXCL9 detection antibody to ALP-PEG4-N3: In order to achieve efficient quantitative coupling and avoid polymer formation, ALP-PEG4-N3 is slightly in excess of DBCO-antibody, with DBCO-antibody:ALP-PEG4-N3=1:2.5. In a light-protected centrifuge tube, add 0.5mg DBCO-antibody solution and 0.9mg ALP-PEG4-N3 in sequence, and add click reaction buffer (PBS, pH7.4) to the final reaction volume of 500μL. Mix gently by vortexing or pipetting.

[0067] (2) Click chemical reaction: Seal the reaction tube and place it in a rotary mixer or constant temperature shaker at room temperature (25℃), away from light, and mix continuously at 10-20 rpm for 2 hours.

[0068] (3) Purification of the conjugate: After the reaction, the reaction mixture was finely purified by liquid chromatography to separate the target conjugate, unreacted ALP-PEG4-N3 and unreacted DBCO-antibody. After the reaction solution was clarified by low-speed centrifugation, it was loaded onto a chromatographic column that had been pre-equilibrated with purification buffer (PBS, pH 7.4). Elution was performed with PBS at a constant flow rate (e.g., 0.5 mL / min). The UV absorption peak at 280 nm was monitored, and the elution fraction of the third peak (target conjugate) was collected.

[0069] 2. Preparation method of CXCL10 detection reagent: 2.1 The preparation method of CXCL10 cross-linked magnetic microparticles includes: (1) Washing: Take 100 μL of 10 mg (100 mg / ml) Tosyl magnetic bead stock solution and 900 μL of coupling buffer 1 (6‰ wt boric acid, pH 9.5), magnetically separate, and wash three times; (2) Coupling: After removing the supernatant, add 0.467 μL of coupling buffer 1 (formulation: 6‰wt boric acid, pH 9.5), mix well, add 200 μg of CXCL10 magnetic bead-terminated antibody, mix well, add 0.333 μL of coupling buffer 2 (formulation: 96.6%wt ammonium sulfate, 6.1% sodium hydroxide, pH 9.5), mix well, and place on a roller at 37°C for 16 to 24 hours; (3) Blocking: After magnetic adsorption, discard the supernatant and wash three times with blocking buffer 1 (formulation: 3‰wt sodium dihydrogen phosphate, 3%wt disodium hydrogen phosphate, 8.7%wt sodium chloride, 1% bovine serum albumin, 5‰ Tween-20, pH 9.5). Place it on a roller at 37℃ and rotate for 16 to 24 hours. (4) Preservation: After magnetic adsorption, discard the supernatant and wash three times with blocking buffer 1 (formulation: 3‰wt sodium dihydrogen phosphate, 3%wt disodium hydrogen phosphate, 8.7%wt sodium chloride, 1% bovine serum albumin, 5‰ Tween-20, pH 9.5).

[0070] 2.2 Preparation of CXCL10 detection antibody-alkaline phosphatase conjugate: The CXCL10 detection antibody-alkaline phosphatase conjugate was prepared using the same method as the CXCL9 detection antibody-alkaline phosphatase conjugate.

[0071] 3. Preparation method of CCL20 detection reagent: 3.1 Coating method for CCL20 cross-linked magnetic microparticles: The coating method is the same as that for CXCL10 cross-linked magnetic microparticles, except that the antibody added is CCL20.

[0072] 3.2 Preparation of CCL20 detection antibody-alkaline phosphatase conjugate: The CCL20 detection antibody-alkaline phosphatase conjugate was prepared using the same method as the CXCL9 detection antibody-alkaline phosphatase conjugate described above.

[0073] 4. Preparation method of HMGB1 detection reagent: 4.1 Preparation method of HMGB1 antibody cross-linked magnetic microparticles: The coating method is the same as that of CXCL10 cross-linked magnetic microparticles, except that the added antibody is HMGB1.

[0074] 4.2 Preparation method of HMGB1 detection antibody-alkaline phosphatase conjugate: The HMGB1 detection antibody-alkaline phosphatase conjugate was prepared using the same method as described above for the preparation of CXCL9 detection antibody-alkaline phosphatase conjugate.

[0075] Example 3: Multi-target joint detection method The detection of samples using the detection reagent system provided by this invention includes the following steps: S1: Sample preparation: Place the sample to be tested on the sample rack; S2: Reagent preparation: Place the CXCL10, CXCL9, CCL20, and HMGB1 detection reagents into the reagent tray; S3: Add samples: According to the detection order of CXCL10, CXCL9, CCL20, and HMGB1, take 30uL, 50uL, 50uL, and 50uL of the test samples in sequence and add them into 4 empty reaction cups, labeled C1, C2, C3, and C4 respectively. S4: Add detection reagents: Add 30uLCXCL10 reagent 1 and 50uLCXCL10 reagent 2 to C1; add 30uLCXCL9 reagent 1, 30uLCXCL9 reagent 2 and 50uLCXCL9 reagent 3 to C2; add 30uLCCL20 reagent 1 and 50uLCCL20 reagent 2 to C3; add 50uLHMGB1 reagent 1 and 50uLHMGB1 reagent 2 to C4. S5: Incubation: Mix the reaction solution in the above reaction cup and then place it in a 37℃ reaction dish for 20 min; S6: Magnetic cleaning: Place the reaction vessel in the magnetic separation plate, magnetically attract it, remove all supernatant, then add cleaning solution, mix well, magnetically attract it, remove supernatant, repeat the magnetic cleaning step 4 times; S7: Luminescence detection: Add 200 μL of fluorescent substrate AMPPD to each reaction vessel, incubate at 37°C for 5 minutes, and then measure the luminescence value (RLU). S8: Determination of calibrators: The calibrators of CXCL10, CXCL9, CCL20 and HMGB1 test reagents are measured in order of increasing labeled concentration using the reaction steps S1-S7 above. Based on the measured luminescence values, a standard curve is plotted with the luminescence value on the ordinate and the labeled concentration of the calibrator on the abscissa. S9: Result Calculation: Calculate the concentration of the target substance corresponding to the emission value measured in step S7 from the standard curve in step S8. The obtained concentration is the concentration of CXCL10, CXCL9, CCL20 and HMGB1 in the sample.

[0076] Example 4: Validation of the reagent kit's detection performance 1. Minimum detection limit Zero-concentration calibrators (calibrator dilutions) of CXCL10, CXCL9, CCL20, and HMGB1 were measured 20 times. The mean (M) and standard deviation (SD) of the relative light units (RLU) of the 20 measurements were calculated. The RLU value of the limit of detection (LOD) was calculated using the formula M + 2SD. The zero-concentration calibrator and adjacent calibrators were measured at least twice. A two-point regression was performed on the concentration-RLU values ​​of the zero-concentration calibrator and adjacent calibrators to derive a linear equation. The LOD RLU value was then substituted into the linear equation to obtain the corresponding concentration value, which is the LOD. Wherein: The calibrator dilution solution for CXCL10, CXCL9, CCL20, and HMGB1 is pH 7.2 and has the following formulation: 50 mM MOPSO, 150 mM NaCl, 2% bovine serum albumin, 5% trehalose, 2% mannitol, 0.02% Tween 20, 1 mM EDTA-Na2, 0.2% fish skin gelatin, 20 mM L-arginine, 0.05% Proclin 300, pH 7.2 ± 0.1.

[0077] Results analysis: As shown in Table 1, the limit of detection (LOD) for CXCL9 (CLIA) was 0.22 pg / mL, the LOD for CXCL10 (CLIA) was 1.84 pg / mL, the LOD for CCL20 (CLIA) was 0.02 pg / mL, and the LOD for HMGB1 (CLIA) was 1.14 ng / mL.

[0078] Table 1. Validation of the lowest detection limit of the detection kit

[0079] 2. Accuracy (Recovery Rate) Sample B, containing a higher concentration (>400 pg / ml), was added to sample A, containing a lower concentration (>10 pg / ml). B:V A =1:9, to obtain the accuracy sample C. Repeat the measurement of samples A, B and C three times each, and calculate the recovery rate according to formula (1). The acceptance criterion is: R should be between 85% and 115%.

[0080] (1) Where: R—recovery rate; V A —The volume of sample A; V B —The volume of sample B; C A —The concentration of sample A was determined; C B —The concentration measured in sample B; C C —The concentration of sample C was determined.

[0081] Results analysis: As shown in Table 2, the recovery rates of CXCL9 (CLIA) were 101.1%, CXCL10 (CLIA) were 106%, CCL20 (CLIA) were 98.7%, and HMGB1 (CLIA) were 103.9%, all of which met the acceptance criteria.

[0082] Table 2. Validation of the accuracy (recovery rate) of the four-item combination test kit

[0083] 3. Repeatability Using the detection kit provided by this invention, three samples of CXCL10, CXCL9, CCL20 and HMGB1 at different concentrations (low, medium and high) were repeatedly measured 10 times. The mean X and standard deviation SD of the measurement results were calculated respectively, and the coefficient of variation (CV) was calculated according to formula (2).

[0084] (2) In the formula: CV – Coefficient of Variation; SD – Standard deviation of the measurement result; X — The average value of the measurement results.

[0085] Results analysis: As shown in Table 3, the repeatability CV of the detection kit of the present invention for each of the three samples of CXCL10, CXCL9, CCL20 and HMGB1 is within 5%.

[0086] Table 3. Repeatability verification of the four-item combination test kit

[0087] 4. Linear range The linear sample L, covering the upper limit of the linear range, was diluted with calibrator diluent (the specific formulation is the same as the lowest detection limit detection in Example 4) according to the dilution ratios in Tables 4-7. Each sample (including sample L) was tested twice. Then, with the dilution concentration as the independent variable and the mean of the test results as the dependent variable, the average result and the dilution ratio were fitted to a straight line using the least squares method, and the linear correlation coefficient r was calculated.

[0088] Results analysis: As shown in Table 4 and Figure 1 As shown, using CXCL9 (CLIA) for linearity verification, the linear correlation coefficient r was 0.9997 in the concentration range of (2-2000) pg / mL; in the concentration range of (2-27) pg / mL, the absolute deviation of linearity did not exceed ±10 pg / mL; and in the concentration range of (27-2000) pg / mL, the relative deviation of linearity did not exceed ±10%. In summary, the linear range of CXCL9 (CLIA) is 2 pg / mL-2000 pg / mL.

[0089] Table 4. Linearity interval verification of CXCL9

[0090] As shown in Table 5 and Figure 2 As shown, linearity was verified using CXCL10 (CLIA). Within the concentration range of (3-1000) pg / mL, the linear correlation coefficient r was 0.9995; within the concentration range of (2-300) pg / mL, the absolute deviation of linearity did not exceed ±31 pg / mL; and within the concentration range of (300-1000) pg / mL, the relative deviation of linearity did not exceed ±10%. In summary, the linear range of CXCL10 (CLIA) is 3 pg / mL–1000 pg / mL.

[0091] Table 5. Linearity interval verification of CXCL10

[0092] As shown in Table 6 and Figure 3 As shown, linearity was validated using the CCL20 (CLIA) assay. Within the concentration range of (0.9-1000) pg / mL, the linear correlation coefficient r was 0.9999; within the concentration range of (0.9-115) pg / mL, the absolute linear deviation did not exceed ±10 pg / mL; and within the concentration range of (115-1000) pg / mL, the relative linear deviation did not exceed ±10%. In summary, the linear range for CCL20 (CLIA) is 0.9 pg / mL to 1000 pg / mL.

[0093] Table 6. Linearity interval verification of CCL20

[0094] As shown in Table 7 and Figure 4 As shown, using HMGB1 (CLIA) for linearity verification, the linear correlation coefficient r was 0.9999 in the concentration range of (5-4500) ng / mL; the absolute deviation of linearity did not exceed ±10 ng / mL in the concentration range of (5-500) ng / mL; and the relative deviation of linearity did not exceed ±10% in the concentration range of (500-4500) ng / mL. In summary, the linear range of HMGB1 (CLIA) is 5 ng / mL-4500 ng / mL.

[0095] Table 7. Linearity interval verification of HMGB1

[0096] Example 5: Using the reagent kit and detection method provided by this invention, samples from 35 healthy individuals and 35 vitiligo patients were tested and compared. As shown in Table 8, the median concentrations of CXCL9, CXCL10, and HMGB1 in the vitiligo patient samples were 179.57 pg / ml (minimum 63.86 pg / ml, maximum 2460.07 pg / ml), CXCL10, and HMGB1 were 178.66 pg / ml (minimum 61.04 pg / ml, maximum 815.20 pg / ml), respectively. The median concentrations of CXCL9, CXCL10, CCL20, and HMGB1 in the vitiligo patient samples were 61.48 pg / ml (minimum 24.15 pg / ml, maximum 87.12 pg / ml), 11.77 pg / ml (minimum 5.99 pg / ml, maximum 101.87 pg / ml), 21.21 pg / ml (minimum 2.09 pg / ml, maximum 38.85 pg / ml), and 148.35 pg / ml (minimum 85.49 pg / ml, maximum 252.51 pg / ml), respectively. These results indicate that the levels of all four related factors in the vitiligo patient samples showed an increased trend compared to the healthy controls.

[0097] Therefore, the detection kit provided by this invention can simultaneously detect four targets, CXCL9, CXCL10, CCL20 and HMGB1, in the same sample. It has high accuracy, precision and specificity, and the results are stable and reliable. It can provide auxiliary basis for assessing the body's immune status and is more convenient for clinical application.

[0098] Table 8. Detection results of the four-item combined test kit on samples from vitiligo patients and healthy individuals.

[0099] Example 6: Study on the dilution solution formulation of alkaline phosphatase-labeled antibody Due to the poor long-term stability of alkaline phosphatase-labeled antibodies, this example investigated the effects of adding bovine serum albumin and arginine to the enzyme-labeled diluents of CXCL9, CXCL10, CCL20, and HMGB1 detection reagents. Specific formulations are shown in Table 9. Alkaline phosphatase component reagents were prepared using the formulations in the table below. Each formulation was divided into two equal volumes and placed in incubators at 4℃ and 37℃ respectively. After 7 days of treatment, the reagents were removed and used in conjunction with other reagents for each target to detect luminescence values. The average relative deviation between the luminescence values ​​of the 37℃ reagent and the 4℃ reagent was calculated.

[0100] like Figure 5 As shown, adding bovine serum albumin and arginine to the enzyme-labeled diluent formulations for CXCL9, CXCL10, CCL20, and HMGB1 detection effectively improves the thermal stability of alkaline phosphatase-labeled antibodies. The thermal stability of CXCL9 was improved by 28.2%, CXCL10 by 33.86%, CCL20 by 27.99%, and HMGB1 by 27.99%. In conclusion, bovine serum albumin and arginine can act as protein protectants to enhance the thermal stability of enzyme-labeled antibodies.

[0101] Table 9. Study on enzyme-labeled formulations for four types of projects.

[0102] Example 7: Comparison of detection reagents with those prepared using traditional enzyme-labeled antibody processes 1. Traditional enzyme-labeled antibody process 1.1 Preparation for activating alkaline phosphatase (AP) (1) Dissolve AP in dilution buffer (formulation: 0.1 MTEA, 0.1 M sodium chloride, 0.1% magnesium chloride, 0.01% zinc chloride, pH 7.30 ± 0.05) to a concentration of 5-10 mg / mL; (2) Preparation of SMCC: Prepare fresh SMCC solution with anhydrous DMF (N,N-dimethylformamide) or DMSO at a concentration of 10-20 mM; (3) Activation reaction: Slowly add SMCC solution to AP solution. The molar ratio of SMCC to AP is 1:1. Excessive SMCC may cause AP to be over-modified and deactivated. (4) Gently mix: React at room temperature (25°C) in the dark for 30-60 minutes; (5) Termination and removal of excess cross-linking agent: The activated alkaline phosphatase was added to termination buffer 1 (pH 7.3) (formulation: 15% glycine) and allowed to stand for 5 min. Then, the activated alkaline phosphatase was collected by using a desalting column equilibrated with equilibration buffer 1 (pH 7.3) (formulation: 1.5% triethanolamine hydrochloride, 6‰ sodium chloride, 0.01% zinc chloride, 0.1% magnesium chloride) and the concentration was measured.

[0103] 1.2 Preparation of activated antibodies (1) Dissolve the detection antibody in 10mM PBS and set the final concentration at 5-10mg / ml; (2) Activation reaction: Add 2% of the total volume of activation buffer (pH 8.5) (formulation: 1.5% triethanolamine hydrochloride, 0.6% sodium chloride, 0.04% disodium ethylenediaminetetraacetate, 0.4% sodium hydroxide, 1.4% 2-IT), react at room temperature for 30-60 minutes, 2-IT reacts with primary amine NH2 to introduce thiol groups; (3) Termination and removal of excess activator: Add termination buffer 1 (pH 7.3) (formulation: 15% glycine) and let stand for 5 min. Then, use the desalting column equilibrated with equilibration buffer 2 (pH 7.3) (formulation: 1.5% triethanolamine hydrochloride, 0.6% sodium chloride, 0.04% disodium ethylenediaminetetraacetate) to remove 2IT and collect the activated antibody.

[0104] 1.3 Coupling Reaction Mixing (1) Mix the activated AP with the activated antibody at a molar ratio of 1:1; (2) Reaction: Mix gently and react overnight at 4°C; (3) Termination of reaction: Add 1% termination buffer 2 (pH 7.3) (formulation: 1.5% triethanolamine hydrochloride, 0.6% sodium chloride, 0.01% zinc chloride, 0.1% magnesium chloride, 1% N-ethylmaleimide) to the ligation product, let stand at room temperature for 10 min, then add 1% termination buffer 3 (pH 7.3) (formulation: 1.5% triethanolamine hydrochloride, 0.6% sodium chloride, 0.01% zinc chloride, 0.1% magnesium chloride, 1% ethanolamine), let stand at room temperature for 10 min.

[0105] (4) Product collection: Collect the cross-linked enzyme-labeled antibody conjugate by using a desalting column equilibrated with equilibration buffer 3 (pH 7.3) (formulation: 1.5% triethanolamine hydrochloride, 0.6% sodium chloride, 0.01% zinc chloride, 0.1% magnesium chloride) and determine the concentration.

[0106] 2. Detection reagents prepared using traditional enzyme-labeled antibody technology (1) The CXCL9 detection reagents provided in this embodiment include: reagent R1, reagent R2 and reagent R3.

[0107] Reagents R1 and R2: Same as CXCL9 detection reagents R1 and R2 in Example 1; Reagent R3: Prepared using the traditional enzyme-labeled antibody preparation process described in Example 7.

[0108] (2) The CXCL10, CCL20, and HMGB1 detection reagents provided in this embodiment include: reagent R1 and reagent R2. Reagent R1: Same as reagent R1 for CXCL10, CCL20, and HMGB1 detection in Example 1; Reagent R2: Prepared using the traditional enzyme-labeled antibody preparation process described in Example 7.

[0109] 3. Signal-to-noise ratio evaluation of detection reagents prepared by different processes Test samples: calibrators S1 and S8 of CXCL9, CXCL10, CCL20, and HMGB1 reagents in Example 1, and 10 healthy human samples.

[0110] Experimental method: The above samples were tested using the detection reagents of Example 7 and Example 1 according to the multi-target joint detection method described in Example 3, and the luminescence signal value (RLU) of each sample was recorded.

[0111] Results statistics: Calculate the average RLU value X for S1 and healthy individuals. A Calculate the signal-to-noise ratio: S8-RLU divided by X A The higher the signal-to-noise ratio, the better the reagent performance.

[0112] As shown in Table 10, the signal-to-noise ratio (SNR) test results of the four detection reagents CXCL9, CXCL10, CCL20, and HMGB1 show that the SNR of the detection reagent in Example 1 is 2.16 to 2.7 times higher than that of the detection reagent prepared using the traditional enzyme-labeled antibody process in Example 7. Its sensitivity is significantly higher than that of Example 7, and it has good positive and negative differentiation.

[0113] Table 10. Signal-to-noise ratio evaluation of the test reagents in Example 7 and Example 1

[0114] Example 8: Preparation of Universal Matrix Solution for Calibration This embodiment provides a universal matrix solution for calibrating a four-factor assay kit for vitiligo, wherein the four factors are CXCL9, CXCL10, CCL20 and HMGB1, and the beneficial effects of the invention are verified by comparative examples.

[0115] (1) Preparation of universal matrix solution for calibrators The universal calibrator matrix solution consists of components of the following concentrations: Table 11 General Diluent Formulations for Calibration Samples

[0116] The preparation method includes the following steps: (1) Weigh the above components and dissolve them in purified water in sequence, stirring until completely dissolved; (2) Adjust the pH to 7.2 using 5M NaOH or 5M HCl; (3) Use a 0.22μm filter membrane for sterilization filtration; (4) The filtered matrix solution should be stored at 2-8℃ for later use.

[0117] The universal calibrator diluent provided by this invention maintains pH stability through a MOPSO buffer system, uses trehalose and mannitol to synergistically maintain the native conformation of proteins, uses BSA, Tween20, and fish skin gelatin to synergistically reduce tube wall adsorption, uses L-arginine and fish skin gelatin to synergistically inhibit chemokine aggregation, and uses EDTA-Na2 and Proclin300 to synergistically inhibit enzymatic degradation. Through the synergistic effect of these components—anti-adsorption, conformational stabilization, anti-aggregation, and anti-degradation—the stability of four calibrators, CXCL9, CXCL10, CCL20, and HMGB1, is significantly improved. The formulation of this invention, through the hydrogen bond protection of trehalose, the osmotic pressure regulation of mannitol, and the anti-aggregation effect of L-arginine, ensures that the calibrators maintain conformational stability under high-temperature conditions. The synergistic anti-adsorption effect of BSA, Tween20, and fish skin gelatin effectively reduces the risk of tube wall adsorption and contamination during repeated bottle opening and use.

[0118] (2) Comparative experimental design Table 12 Comparative test formulations

[0119] (3) Evaluation of the performance of the calibrator diluent a. Take recombinant human CXCL9, CXCL10, CCL20 and HMGB1 standards respectively, and dilute them with the above universal calibrator matrix solution to four composite calibrators of 500 pg / mL, 250 pg / mL and 50 pg / mL. Place each calibrator in parallel at 4℃ and 37℃ for 14 days (each tube has parallel samples, and the results are averaged). After the expiration, the change rate (%) is measured. The results are shown in Table 13.

[0120] Table 13. Rate of change (%) after thermal acceleration for different calibrator formulations

[0121] Results Analysis: The universal calibrator diluent prepared according to the embodiments of the present invention showed the best protective effect on the four composite calibrators CXCL9, CXCL10, CCL20, and HMGB1. After storage at 4°C for 14 days, the change rate of the four calibrators at each concentration level was greater than -3.0% (i.e., the concentration decrease did not exceed 3.0%); after storage at 37°C under accelerated degradation conditions for 14 days, the change rate was greater than -9.2% (i.e., the concentration decrease did not exceed 9.2%).

[0122] The above data show that the formulation of this invention can effectively maintain the concentration stability of the four calibrators and significantly delay their degradation during storage. Comparative Example 1 (conventional BSA-PBS formulation): This formulation showed a change rate of approximately -16.5% after storage at 4°C for 14 days and approximately -40.0% after storage at 37°C for 14 days, the worst among all tested formulations, indicating that the conventional BSA-PBS formulation cannot meet the long-term stable storage requirements of the four calibrators. Comparative Example 2 (trehalose-deficient formulation): Compared with the embodiments of this invention, the change rate at 4°C decreased from approximately -2% to -10%, and the change rate at 37°C decreased from approximately -7% to -8% to -23%, indicating that trehalose is the core protective component of the formulation of this invention and plays an irreplaceable role in maintaining the stability of the calibrators. The absolute value of the change rate of Comparative Example 3 (lacking fish skin gelatin and L-arginine) was approximately 2.5 times that of the embodiments of the present invention; the absolute value of the change rate of Comparative Example 4 (lacking EDTA-Na2) was approximately 2 times that of the embodiments of the present invention; the absolute values ​​of the change rates of Comparative Example 5 (commercially available diluent A) and Comparative Example 6 (commercially available diluent B) at 37°C were 4 times and 2.5 times that of the embodiments of the present invention, respectively. The protective effects of each formulation, from best to worst, are as follows: Embodiment 8 of the present invention > Comparative Example 4 > Comparative Example 3 > Comparative Example 2 > Comparative Example 6 > Comparative Example 5 > Comparative Example 1.

[0123] b. Take recombinant human CXCL9, CXCL10, CCL20 and HMGB1 standards respectively, and dilute them to 500 pg / mL with comparative example 7 dilution buffer (adding conventional protein stability protectant fetal bovine serum). Place each calibrator in parallel at 4℃ and 37℃ for 14 days (each tube has parallel samples, and the results are averaged). After the expiration, use the change rate (%) to measure. The results are shown in Table 14.

[0124] Table 14. Rate of thermal acceleration (%) of Example 8 and Comparative Example 7 under different factors

[0125] As shown in Table 14, comparing Comparative Example 7 and Example 8, the absolute values ​​of the change rates of the four factors at 37°C generally increased after adding 5% fetal bovine serum to the formulation. Fetal bovine serum is rich in various proteins, growth factors, and protease inhibitors, and is easily thought to provide additional protein protection and inhibit protease degradation, thereby further improving the stability of the calibrator. However, the experimental results show that the addition of fetal bovine serum actually led to a significant decrease in the stability of the four factors. Bovine proteins in fetal bovine serum may non-specifically bind or cross-react with chemokines (CXCL9, CXCL10, CCL20), interfering with the dispersion of chemokines in solution and promoting their aggregation. HMGB1 itself is a nucleoprotein with chemokine activity, which easily binds non-specifically with albumin, globulin, etc. in serum to form high molecular weight complexes, resulting in a decrease in the concentration of free HMGB1.

[0126] The results in summary indicate that the universal calibrator matrix solution provided by this invention, through the synergistic effect of its components, is significantly superior to the comparative formulations and commercially available diluents, providing a reliable calibrator stability solution for the four-item detection kit for vitiligo-related factors.

[0127] Example 9: Universal optimization of glycosyltransferase dosage for four targets Due to the differences in molecular characteristics (including isoelectric point, glycosylation sites, hydrophobicity, and aggregation tendency) of antibodies against vitiligo-related chemokines CXCL9, CXCL10, CCL20, and HMGB1, the optimal dosage of the glycosyltransferase GalTY289L, which is independently optimized for a single target antibody, cannot be directly applied to other target antibodies. This embodiment optimizes the pretreatment steps (β-1,4-galactosidase treatment conditions) to make the accessibility of the Fc glycosylation sites of the four antibodies more consistent, thereby achieving efficient modification of the four antibodies with low byproducts at the same enzyme dosage, and screening for the optimal universal enzyme dosage.

[0128] 1. Take 500 μg of each of the four antibodies (CXCL9, CXCL10, CCL20, and HMGB1) and pretreat them under the following two conditions: Table 15 Optimization of Preprocessing Conditions

[0129] Pretreatment objective: To remove the terminal galactose residues of the Fc fragment, exposing the GlcNAc receptor site. Optimized pretreatment, by extending the incubation time and increasing the enzyme dosage, more thoroughly removes the terminal galactose and eliminates differences in site accessibility caused by differences in antibody spatial structure.

[0130] 2. Operating Instructions: (1) Take 100 μg of each of the four pretreated antibodies and add different amounts of glycosyltransferase GalTY289L. The amount of enzyme used is expressed as a percentage of the number of moles of receptor GlcNAc, and seven gradients are set: 2%, 5%, 8%, 10%, 12%, 15%, and 20%.

[0131] (2) Add UDP-Gal-DBCO (molar ratio of antibody 15:1) and the corresponding amount of glycosyltransferase to each group, and react at 30℃ in the dark for 20 hours. After the reaction, purify using a Zeba desalting column.

[0132] (3) The DBCO modification rate was determined by ultraviolet spectrophotometry. DBCO modification rate determination: The purified sample was used to measure the absorbance at 280 nm and 309 nm. The formula for calculating the DBCO modification rate is: C Ab = A 280 / ε 280(Ab) (ε) 280(Ab) = 2.1×10 5 M -1 ·cm -1 ) A 309(DBCO) = A 309 - R × A 280 (R = 0.065, correction factor) C DBCO = A 309(DBCO) / ε 309(DBCO) (ε) 309(DBCO) = 1.2×10 4 M -1 ·cm -1 ) DBCO DOL = C DBCO / C Ab (4) Determination of by-product formation: SEC-HPLC method was used. By-product formation (%) = (aggregate peak area + dimer peak area) / total protein peak area × 100%.

[0133] 3. Experimental Results: (1) DBCO modification rate in the unoptimized pretreatment group (control group) Table 16 DBCO modification rate of four antibodies under different enzyme dosages after unoptimized pretreatment (mol DBCO / mol antibody)

[0134] Under unoptimized pretreatment conditions, the modification rate of CCL20 antibody was significantly low. At 8% enzyme concentration, the CCL20 modification rate was only 1.3, far below the acceptable standard (≥1.9); at 10% enzyme concentration, the modification rate was only 1.7, still failing to meet the standard; and only at 12% enzyme concentration did the CCL20 modification rate reach 2.0. This indicates that without optimized pretreatment, a higher enzyme concentration is required to achieve the desired modification rate for CCL20, which is the most difficult to modify.

[0135] (2) Byproduct generation in the unoptimized pretreatment group (control group) Table 17. Byproduct generation (%) of four antibodies under different enzyme dosages after unoptimized pretreatment.

[0136] Under unoptimized pretreatment conditions, when the enzyme dosage reached 12% (the minimum dosage required to achieve CCL20 target), the HMGB1 byproduct reached 5.0% and the CXCL9 byproduct reached 4.2%, both of which were at relatively high levels.

[0137] (3) Optimize the DBCO modification rate of the pretreatment group (in this invention) Table 18 DBCO modification rate of four antibodies under different enzyme dosages after optimized pretreatment (mol DBCO / mol antibody)

[0138] After optimized pretreatment, the modification rate of CCL20 antibody was significantly improved. At an 8% enzyme concentration, the CCL20 modification rate increased from 1.3 to 1.9 (a 46% increase), just meeting the acceptable standard. The modification rates of all four antibodies reached 1.9-2.3 at an 8% enzyme concentration, all meeting the target.

[0139] (4) Optimize the amount of byproducts generated in the pretreatment group (in this invention). Table 19. Byproduct generation (%) of four antibodies under different enzyme dosages after optimized pretreatment

[0140] After optimized pretreatment, the byproduct content was controlled at 1.7%-2.3% (average 2.0%) with an 8% enzyme dosage, which is at a low level.

[0141] (5) Comparison of the minimum enzyme dosage required for the two groups to achieve all four criteria (modification rate ≥ 1.9) Table 20. Comparison of minimum enzyme dosage for two groups to achieve four criteria (modification rate ≥ 1.9)

[0142] The unoptimized pretreatment group required 12% more enzyme to achieve the CCL20 target (modification rate 2.0%), while other antibody byproducts were already high (HMGB1 reached 5.0%). The optimized pretreatment group only required 8% more enzyme to achieve all four targets simultaneously, reducing enzyme usage by 33% and significantly reducing byproducts.

[0143] (6) Comparison of byproducts between the two groups at their respective minimum enzyme dosages. Table 21. Comparison of byproducts between the unoptimized pretreatment group (12% enzyme) and the optimized pretreatment group (8% enzyme).

[0144] Under the minimum enzyme dosage required to achieve the four objectives, the byproducts of the optimized pretreatment group (8% enzyme) were reduced by an average of 52.4% compared to the unoptimized pretreatment group (12% enzyme), fully demonstrating the technical advantages of the optimized pretreatment of this invention.

[0145] 4. Experimental Results: (1) The unoptimized pretreatment group requires 12% enzyme dosage to make the CCL20 antibody meet the standard, at which point the average level of byproducts reaches 4.2%; and the CCL20 modification rate is only 1.3% with 8% enzyme dosage, which cannot meet the requirements.

[0146] (2) This invention optimizes the pretreatment (1500 units / 0.5 mg of β-1,4-galactosidase, incubated at 37°C for 4 hours) to make the accessibility of the Fc glycosylation sites of the four antibodies more consistent. After optimization of the pretreatment, the modification rate of CCL20 antibody at 8% enzyme dosage increased from 1.3 to 1.9 (an increase of 46.2%), and the range of modification rates of the four antibodies at 8% enzyme dosage decreased from 0.6 to 0.4.

[0147] (3) Through systematic screening at seven gradients of 2%, 5%, 8%, 10%, 12%, 15%, and 20%, this invention determined that the optimal universal enzyme dosage of glycosyltransferase GalT Y289L is 8% of the molar number of the receptor GlcNAc.

[0148] (4) Under optimized pretreatment conditions with 8% enzyme dosage, the DBCO modification rate of the four antibodies reached 1.9-2.3 (meeting the requirements for subsequent cross-linking), and the byproducts were controlled at 1.7%-2.3% (average 2.0%), achieving universal and efficient modification of the four antibodies. Compared with the unoptimized pretreatment group (requiring 12% enzyme and 4.2% byproducts), the enzyme dosage was reduced by 33%, and the byproducts were reduced by 52.4%.

[0149] (5) Deviations from the preferred dosage (≤5% or ≥10%) will result in insufficient modification rate or excessive byproducts, making it impossible to simultaneously ensure the modification quality of the four antibodies, which reflects the non-obviousness of the technical solution of the present invention.

[0150] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-target combined detection kit for detecting vitiligo-related factors, characterized in that, Includes multiple testing reagents, Each of the detection reagents includes a solid-phase capture carrier, a labeled antibody solution, and an antibody gradient concentration calibrator. The antibodies in the labeled antibody solution and antibody gradient concentration calibrator are: CXCL9, CXCL10, CCL20, and HMGB1.

2. The multi-target combined detection kit for detecting vitiligo-related factors as described in claim 1, characterized in that, The diluent for the antibody gradient concentration calibrator includes: MOPSO, NaCl, bovine serum albumin, trehalose, mannitol, Tween 20, EDTA-Na2, fish skin gelatin, L-arginine, and Proclin 300.

3. The multi-target combined detection kit for detecting vitiligo-related factors as described in claim 1, characterized in that, The labeled antibody solution includes enzyme-labeled antibody conjugates, which include enzyme-labeled CXCL9 detection antibody conjugate solution, enzyme-labeled CXCL10 detection antibody conjugate solution, enzyme-labeled CCL20 detection antibody conjugate solution and enzyme-labeled HMGB1 detection antibody conjugate solution; The enzyme-labeled antibody conjugate solution contains bovine serum albumin and arginine.

4. The multi-target combined detection kit for detecting vitiligo-related factors as described in claim 3, characterized in that, The labeled antibody solution also includes a biotin-labeled CXCL9 capture antibody solution; And / or, the enzyme-labeled CXCL9 detection antibody-alkaline phosphatase conjugate solution contains: 1%-5% 2-morpholinoethanesulfonic acid, 0.5%-1.5% potassium chloride, 0.01%-0.05% zinc chloride, 0.1%-0.5% magnesium chloride, 5%-15% trehalose, 0.1%-0.5% surfactant RPE1740, 0.05%-0.2% surfactant Triton X100, 0.1%-0.5% bovine serum albumin, 1%-3% arginine, 0.1%-0.5% PC300, pH 6.5, and 0.2-1 μg / mL of CXCL9 detection antibody-alkaline phosphatase conjugate; And / or, the enzyme-labeled CXCL10 detection antibody-alkaline phosphatase conjugate solution contains: 1%-5% 2-morpholinoethanesulfonic acid, 0.5%-1.5% potassium chloride, 0.01%-0.05% zinc chloride, 0.1%-0.5% magnesium chloride, 0.1%-0.5% bovine serum albumin, 2.5-5% trehalose, 0.1%-0.5% surfactant RPE1740, 0.1-0.5% lauryl alcohol polyoxyethylene ether, 0.1%-0.5% polyoxyethylene-polyoxypropylene block copolymer, 1%-3% arginine, 0.1%-0.5% PC300, pH 6.5-7.0, and the concentration of the CXCL10 detection antibody-alkaline phosphatase conjugate is 0.2~1ug / mL; And / or, the enzyme-labeled CCL20 detection antibody-alkaline phosphatase conjugate solution comprises: 1%-5% 2-morpholinoethanesulfonic acid, 0.5%-1.5% potassium chloride, 0.05%-0.2% Triton X100, 0.1%-0.5% RPE1740, 5%-15% trehalose, 0.01%-0.05% zinc chloride, 0.1%-0.5% magnesium chloride, 0.1%-0.5% bovine serum albumin, 1%-3% arginine, 0.1%-0.5% PC300, pH 6.5-7.0, and the concentration of the CCL20 detection antibody-alkaline phosphatase conjugate is 0.2-1 μg / mL; And / or, the enzyme-labeled HMGB1 detection antibody-alkaline phosphatase conjugate solution comprises: 0.3% disodium hydrogen phosphate, 0.03% sodium dihydrogen phosphate, 0.5%-1.5% potassium chloride, 0.2%-0.5% Brij35, 0.05%-0.2% Triton X-100, 5%-15% trehalose, 5%-15% glycerol, 0.1%-0.5% bovine serum albumin, 1%-3% arginine, 0.01%-0.05% zinc chloride, 0.1%-0.5% magnesium chloride, 0.1%-0.5% PC300, pH 6.00-6.5, and the concentration of the HMGB1 detection antibody-alkaline phosphatase conjugate is 0.2-1 μg / mL.

5. The multi-target combined detection kit for detecting vitiligo-related factors as described in claim 4, characterized in that, The biotin-labeled CXCL9 capture antibody solution comprises: 0.2%-0.6% tris(hydroxymethyl)aminomethane, 0.2%-0.9% sodium chloride, 0.01%-0.05% Tween 20, 0.1%-0.5% surfactant RPE1740, 0.1%-0.5% bovine serum albumin, 0.1%-0.5% PC300, pH 7.0-7.5, and a biotin-labeled CXCL9 antibody concentration of 0.2-1 μg / mL.

6. The multi-target combined detection kit for detecting vitiligo-related factors as described in claim 1, characterized in that... The solid-phase capture carrier comprises a streptavidin-coated magnetic microparticle solution and an antibody-crosslinked magnetic microparticle solution.

7. The multi-target combined detection kit for detecting vitiligo-related factors as described in claim 6, characterized in that... The streptavidin-coated magnetic microparticle solution was used for CXCL9 antibody; And / or the antibody cross-linked magnetic microparticle solution includes a solution containing CXCL10 antibody cross-linked magnetic microparticles, a solution containing CCL20 antibody cross-linked magnetic microparticles, and a solution containing HMGB1 antibody cross-linked magnetic microparticles.

8. The multi-target combined detection kit for detecting vitiligo-related factors as described in claim 1, characterized in that... The antibody gradient concentration calibrators include CXCL9 calibrator, CXCL10 calibrator, CCL20 calibrator, and HMGB1 calibrator.

9. A method for preparing a multi-target combined detection kit for detecting vitiligo-related factors as described in any one of claims 1-8, characterized in that, The method for preparing the labeled antibody solution includes a pretreatment step: removing the Gal residue at the end of the N-glycan of the Fc fragment of the labeled antibody with β-1,4-galactosidase to expose the terminal N-acetylglucosamine (GlcNAc-), and the ratio of β-1,4-galactosidase to antibody is 3KU:1mg.

10. The preparation method according to claim 9, characterized in that, The preprocessing process also includes the following steps: S1: Antibody modification: A non-natural nucleotide sugar derivative with a dibenzocyclooctylene (DBCO) functional group is linked to the N-glycan terminus of the antibody Fc fragment by a glycosyltransferase to obtain an antibody with a DBCO functional group. The amount of glycosyltransferase used is 6%-9% as a percentage of the number of moles of the receptor GlcNAc. S2: Alkaline phosphatase activation: Alkaline phosphatase is activated using a heterobifunctional cross-linking agent with an azide group to obtain alkaline phosphatase with an azide group. S3: Reaction cross-linking: The antibody modified in step S1 is mixed with the alkaline phosphatase activated in step S2, and a directional coupling reaction is carried out. After purification, the alkaline phosphatase antibody marker is obtained. And / or, the β-1,4-galactosidase reacts with the antibody at a temperature range of 30-37°C for 2-8 hours.

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