In-vitro diagnosis marker for gall-stone and application of in-vitro diagnosis marker

Abstract

The invention relates to the technical field of biomedicine, and particularly discloses an in-vitro diagnosis marker for gall-stone and application thereof.The in-vitro diagnosis marker is jointly composed of a metabolism marker and an inflammation marker, the metabolism marker is the concentration ratio of lithocholic acid to ursodesoxycholic acid (LCA / UDCA), and the inflammation marker is the concentration ratio of CD133 to lipopolysaccharide (CD133 / LPS); the invention further provides a kit containing the marker and a detection method of the kit. The kit integrates specific detection reagents for LCA, UDCA, CD133 and LPS, two specific value markers can be rapidly, sensitively and simultaneously detected through an optimized sample treatment and detection process, and result judgment is carried out based on a preset reference interval upper limit and a composite judgment rule.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to an in vitro diagnostic marker for gallstones and application thereof. BACKGROUND

[0002] Gallstones are a common digestive system disease, which is caused by multiple factors such as imbalance of bile components, deposition of cholesterol crystals, infection of biliary tract or metabolic disorders, and its incidence is increasing year by year worldwide. Gallstones have no obvious symptoms in the early stage, but as the stones grow or move, they are easy to cause severe complications such as biliary colic, acute cholecystitis and biliary obstruction.

[0003] At present, the clinical diagnosis of gallstones mainly relies on imaging examination and invasive detection methods. In imaging examination, abdominal ultrasound is the preferred method for gallstone screening due to its advantages of simple operation, no radiation and low cost, but its sensitivity for detecting small stones is low, and there is a certain rate of missed diagnosis. Although computer tomography and magnetic resonance cholangiopancreatography can improve the accuracy of stone detection, the high cost, radiation exposure risk or long examination time limit their application in large-scale screening and routine diagnosis. Invasive detection such as endoscopic retrograde cholangiopancreatography can achieve diagnosis and treatment at the same time, but it is an invasive operation, which has the risk of complications such as bleeding, infection and pancreatitis.

[0004] With the development of in vitro diagnostic technology, non-invasive diagnostic methods based on biomarkers have become a research hotspot in the field of disease diagnosis due to their advantages of convenience, speed, low cost and scalability. An ideal in vitro diagnostic marker for gallstones should have high specificity and high sensitivity, and can be detected in the early stage of the disease or even in the subclinical stage, providing a basis for early detection and early intervention of the disease. However, the research on specific in vitro diagnostic markers for gallstones is still in the exploratory stage, and the potential markers reported in existing studies (such as bile acid metabolites and inflammatory factors) have problems such as insufficient specificity, limited sensitivity or cross-reaction with other digestive system diseases, which makes it difficult to meet the actual needs of clinical diagnosis.

[0005] In addition, the pathogenesis of gallstones is complex, involving multiple pathophysiological processes such as cholesterol metabolism, bile stasis and inflammatory response, and a single marker is often difficult to fully reflect the occurrence and development of the disease. Based on the above statements, the present application provides an in vitro diagnostic marker for gallstones and application thereof. SUMMARY

[0006] To address the problems in current gallstone diagnosis, such as the high risk of missed diagnoses by imaging examinations, the unsuitability of invasive tests for routine screening, and the insufficient ability of existing potential in vitro diagnostic markers to differentiate gallstone types, thus failing to effectively guide the selection of individualized treatment plans, this application provides an in vitro diagnostic marker for gallstones and its application.

[0007] In a first aspect, this application provides an in vitro diagnostic biomarker for gallstones, employing the following technical solution:

[0008] An in vitro diagnostic biomarker for gallstones includes a metabolic biomarker and an inflammatory biomarker; the metabolic biomarker is the concentration ratio of lithocholic acid to ursodeoxycholic acid (LCA / UDCA); the inflammatory biomarker is the concentration ratio of CD133 to lipopolysaccharide (CD133 / LPS).

[0009] The aforementioned concentration ratio biomarkers characterize gallstone-related pathological states by reflecting the relative changes between two types of molecules with antagonistic or synergistic biological functions. Compared with single concentration indicators, concentration ratio biomarkers can eliminate the influence of sample volume differences, fluctuations in overall bile acid levels, and individual basal metabolic differences on test results, and more accurately reflect the relative changes between bile acid metabolic imbalance and inflammatory repair status.

[0010] Preferably, the upper limit of the reference interval for the metabolic marker is 2.3; and the upper limit of the reference interval for the inflammatory marker is 0.8.

[0011] Secondly, this application provides a gallstone detection kit, which adopts the following technical solution:

[0012] A gallstone detection kit includes a detection reagent for detecting the aforementioned in vitro diagnostic markers for gallstones, a sample processing solution, a calibrator, a positive control, and a negative control.

[0013] Preferably, the detection reagents include: lithocholic acid detection reagent, ursodeoxycholic acid detection reagent, CD133 detection reagent, and lipopolysaccharide detection reagent.

[0014] Preferably, the lithocholic acid detection reagent comprises: Tris-HCl, 3α-hydroxysteroid dehydrogenase (3α-HSD), thio-NAD (Thio-NAD), diaphorase, and nitrotetrazole blue chloride (NBT).

[0015] Preferably, the concentrations of each component in the lithocholic acid detection reagent are as follows: Tris-HCl (pH 8.5) 20-50 mmol / L, 3α-HSD 0.8-1.2 U / mL, Thio-NAD 0.2-0.4 mmol / L, flavoprotein enzyme 0.5-1.0 U / mL, and NBT 0.1-0.3 mmol / L.

[0016] Preferably, the ursodeoxycholic acid detection reagent comprises: Tris-HCl, 7β-hydroxysteroid dehydrogenase (7β-HSD), thio-NAD (Thio-NAD), diaphorase, and nitrotetrazole blue chloride (NBT).

[0017] Preferably, the concentrations of each component in the ursodeoxycholic acid detection reagent are as follows: Tris-HCl (pH 8.5) 20-50 mmol / L, 7β-HSD 1.0-1.5 U / mL, Thio-NAD 0.2-0.4 mmol / L, flavoprotein enzyme 0.5-1.0 U / mL, and NBT 0.1-0.3 mmol / L.

[0018] Preferably, the CD133 detection reagent is a CD133 sandwich HRP magnetic ball.

[0019] Preferably, the preparation method of the CD133 sandwich HRP magnetic sphere is as follows:

[0020] A1. Activation of magnetic microspheres: Take carboxylated magnetic microspheres with a particle size of 200 nm, adjust the concentration to 10 mg / mL with 0.05 mol / L MES buffer; add EDC and NHS to make the final concentrations 10 mmol / L and 5 mmol / L respectively, and activate by shaking at room temperature in the dark for 30 min to obtain magnetic microsphere suspension 1.

[0021] A2. Capture antibody conjugation: CD133 antibody was added to magnetic microsphere suspension 1. The mass ratio of CD133 antibody to carboxylated magnetic microspheres was 1:100. The mixture was conjugated by shaking at 4°C in the dark for 12 hours to obtain magnetic microsphere suspension 2.

[0022] A3. Detection of antibody binding: Add HRP-labeled CD133 detection antibody to magnetic microsphere suspension 2. The mass ratio of detection antibody to magnetic microsphere suspension 2 is 1:50. Incubate at 37°C in the dark with shaking for 30 min to obtain magnetic microsphere suspension 3.

[0023] A4. Blocking and Volume Adjustment: Add PBS buffer containing 2% BSA to the magnetic microsphere suspension 3 at a mass ratio of 1:20, and block the reaction at room temperature for 60 min; discard the supernatant after magnetic separation, resuspend in PBS buffer containing 0.02% ProClin 300, and adjust the final concentration to 5 mg / mL to obtain CD133 sandwich HRP magnetic spheres.

[0024] Preferably, the lipopolysaccharide detection reagent comprises: 0.1-0.3 EU / mL of Limulus amebocyte lysate (LAL) reagent activity unit, 0.2-0.5 mmol / L of chromogenic substrate (HD-Val-Leu-Arg-pNA), 10-20 mmol / L of Tris-HCl (pH 7.3), and 2-5 mmol / L of calcium chloride.

[0025] Preferably, the sample processing solution comprises: Tris-HCl 15-25 mmol / L, NaCl 120-180 mmol / L, EDTA 3-6 mmol / L, sucrose 5-10 g / L, 0.02-0.05% Tween-20, and pH 7.2-7.6.

[0026] Preferably, the calibrator is a serum matrix solution containing 0.5-8.0 μg / mL lithocholic acid, 0.3-4.0 μg / mL ursodeoxycholic acid, 0.2-0.3 ng / mL CD133 protein, and 0.1-10 EU / mL lipopolysaccharide, diluted to 5-7 concentration points using a geometric or arithmetic gradient to construct quantitative standard curves for each biomarker (LCA, UDCA, CD133, LPS).

[0027] Preferably, the positive control is a recombinant human serum matrix with LCA / UDCA ≥ 2.8 and CD133 / LPS ≥ 1.0, used to verify the positive detection capability of the detection system.

[0028] Preferably, the negative control is a recombinant human serum matrix.

[0029] Thirdly, this application provides a non-diagnostic detection method for a gallstone detection kit, employing the following technical solution:

[0030] The non-diagnostic testing method for gallstone detection kits specifically includes the following steps:

[0031] S1. Sample pretreatment: Take the sample to be tested, mix it with the sample processing solution, and let it stand at room temperature to obtain the pretreated sample.

[0032] S2. Biomarker detection: The concentrations of lithocholic acid, ursodeoxycholic acid, CD133 and lipopolysaccharide in the pretreated samples were determined using lithocholic acid detection reagent, ursodeoxycholic acid detection reagent, CD133 detection reagent and lipopolysaccharide detection reagent, respectively.

[0033] S3. Result determination: Calculate the concentration ratio of lithocholic acid to ursodeoxycholic acid and the concentration ratio of CD133 to lipopolysaccharide to obtain the quantitative detection value of the in vitro diagnostic marker.

[0034] Preferably, in step S1, the sample to be tested is a peripheral blood serum or plasma sample of the subject, the sample volume is 10-15 μL, and the sample needs to be refrigerated at 2-8℃ after collection and the test should be completed within 24 hours.

[0035] Preferably, in step S1, the volume ratio of the sample to be tested to the sample processing solution is 1:3-5, and the standing time at room temperature is 2-3 minutes.

[0036] Preferably, in step S2, the specific operation for determining the concentrations of lithocholic acid, ursodeoxycholic acid, CD133, and lipopolysaccharide in the pretreated sample is as follows:

[0037] (1) Determination of lithocholic acid concentration: Take 20 μL of pretreated sample and mix with 80 μL of lithocholic acid detection reagent, incubate at 37℃ for 10-15 min, read the absorbance value at 550 nm wavelength using an ELISA reader, and calculate the LCA concentration in the sample to be tested based on the LCA quantitative standard curve constructed by the calibrator.

[0038] (2) Determination of ursodeoxycholic acid concentration: Take 20 μL of pretreated sample and mix with 80 μL of ursodeoxycholic acid detection reagent, incubate at 37℃ for 10-15 min, read the absorbance value at 550 nm wavelength using an ELISA reader, and calculate the UDCA concentration in the sample to be tested based on the UDCA quantitative standard curve constructed by the calibrator.

[0039] (3) CD133 concentration determination: Take 50 μL of pretreated sample and mix with 30 μL of CD133 detection reagent, incubate at 37℃ with shaking for 5 min, separate CD133 sandwich HRP magnetic beads by magnetic rack adsorption, discard the supernatant; add 50 μL of TMB chromogenic substrate, incubate at room temperature in the dark for 5 min; add 25 μL of 2mol / L sulfuric acid stop solution; read the absorbance value at 450 nm wavelength using an enzyme-linked immunosorbent assay reader, and calculate the CD133 concentration according to the calibrator standard curve;

[0040] (4) Determination of lipopolysaccharide concentration: Take 20 μL of pretreated sample and mix with 80 μL of lipopolysaccharide detection reagent, incubate at 37℃ for 15-20 min, read the absorbance value at 405 nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the LPS concentration in the sample to be tested based on the LPS quantitative standard curve constructed by the calibrator.

[0041] Preferably, the result determination rules in step S3 are as follows:

[0042] Rule A: The detected values ​​of both metabolic markers (LCA / UDCA) and inflammatory markers (CD133 / LPS) exceed the upper limit of their reference range;

[0043] Rule B: The detected value of metabolic markers (LCA / UDCA) or inflammatory markers (CD133 / LPS) exceeds the upper limit of their reference interval, and the offset is ≥30%. Offset = (detected value - upper limit of reference interval) / upper limit of reference interval × 100%;

[0044] When either Rule A or Rule B is met, the test report will mark it as meeting the positive criteria.

[0045] The detection principle of lithocholic acid is as follows: lithocholic acid is converted into the corresponding ketosterol through an enzymatic reaction. Thio-NAD is reduced to Thio-NADH, which is then catalyzed by flavoprotein enzyme to generate blue-purple formazan through NBT. The absorbance of formazan (detection wavelength 550nm) is positively correlated with the concentration of lithocholic acid, and can be quantified by a standard curve.

[0046] The detection principle of ursodeoxycholic acid is as follows: ursodeoxycholic acid is converted into the corresponding ketosterone through an enzymatic reaction. Thio-NAD is reduced to Thio-NADH, which then catalyzes the formation of blue-violet formazan from NBT. The absorbance of formazan (detection wavelength 550 nm) is positively correlated with the concentration of ursodeoxycholic acid, and can be quantified by a standard curve.

[0047] The detection principle of CD133 is as follows: CD133 antibody (capture antibody) on the surface of HRP magnetic beads in the CD133 sandwich method specifically binds to CD133 protein in the sample, and then forms a double antibody sandwich complex with HRP-labeled CD133 detection antibody; HRP catalyzes TMB to generate a blue product, and after adding sulfuric acid to terminate the reaction, it turns yellow. The absorbance at 450nm is positively correlated with the CD133 concentration, and can be quantified by a standard curve.

[0048] The detection principle of the above-mentioned lipopolysaccharide is as follows: lipopolysaccharide activates the coagulating enzyme in the horseshoe crab reagent. The coagulating enzyme specifically cleaves the chromogenic substrate to release p-nitroaniline (pNA). The absorbance of pNA (detection wavelength 405nm) is positively correlated with the concentration of lipopolysaccharide, and can be quantified by a standard curve.

[0049] In the above sample processing solution, sucrose can maintain the stability of extracellular vesicle osmotic pressure, EDTA inhibits the activity of nucleases and proteases in the sample, and Tween-20 reduces non-specific adsorption, thus achieving overall structural stability and activity retention of various markers (LCA, UDCA, CD133, LPS) in the sample.

[0050] Compared with the prior art, this application has the following beneficial effects:

[0051] (1) This application is the first to propose the combined application of metabolic markers (lithocholic acid / ursodeoxycholic acid concentration ratio) and inflammatory markers (CD133 / lipopolysaccharide concentration ratio) to construct a novel combination of in vitro diagnostic markers for gallstones. This combination can simultaneously detect two key pathophysiological links: bile acid metabolism imbalance and inflammatory response related to intestinal flora translocation. It overcomes the problems of insufficient sensitivity and limited specificity of single markers and significantly improves the overall efficacy of gallstone diagnosis.

[0052] (2) The gallstone detection kit provided in this application integrates mature detection methods for four specific targets (enzyme cycling method for bile acid detection, sandwich immunoassay for CD133 detection, and chromogenic horseshoe crab assay for LPS detection), and optimizes the sample processing solution formulation and detection process. The kit is easy to operate, fast (can be completed within 1 hour), and highly sensitive, making it suitable for large-scale clinical sample screening and routine testing, providing a practical tool for the non-invasive diagnosis of gallstones.

[0053] (3) This application establishes a quantitative result judgment standard by setting a clear upper limit for the reference interval and a composite judgment rule (rule A and rule B) based on the ratio of two biomarkers. This method not only improves the objectivity and accuracy of diagnosis, but also effectively identifies potential patients in the early or subclinical stage of the disease, which helps to achieve early warning and risk stratification of gallstones.

[0054] (4) The detection method of this application only requires a small amount of peripheral blood sample (serum or plasma), which is a completely non-invasive operation. It avoids the risks of radiation exposure and contrast agent allergy in imaging examinations, and also avoids the complications of invasive examinations such as ERCP. At the same time, the method is relatively low in cost, easy to promote in medical institutions at all levels, and has good clinical application prospects and social and economic benefits.

[0055] (5) Unlike single biomarkers or simple parallel combinations, this application does not improve the AUC value through linear superposition. Instead, it constructs a dual biomarker system expressed in ratio form based on two independent but synergistic pathological pathways in the development of gallstones: the imbalance of bile acid metabolism and the imbalance of intestinal inflammation repair. A composite judgment rule is introduced to determine the results. This technical solution allows for accurate identification of samples where some single biomarkers do not exceed the upper limit of the reference interval, thereby significantly improving the detection capability of early and subclinical gallstones. Detailed Implementation

[0056] The present application will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the present application and are not intended to limit the scope of the present application.

[0057] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0058] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0059] Human Inflammation Array 1 was purchased from RayBiotech;

[0060] The horseshoe crab reagent was purchased from Haibo Biotechnology Co., Ltd.

[0061] Carboxylated magnetic microspheres were purchased from Beyotime Biotechnology.

[0062] CD133 antibody was purchased from Beyotime Biotechnology.

[0063] HRP-labeled CD133 detection antibody was purchased from Beijing Bio-Sen Biotechnology Co., Ltd.

[0064] PBS buffer was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0065] CD133 protein was purchased from MedChemExpress.

[0066] MES buffer was purchased from Maclean's reagents;

[0067] The recombinant human serum matrix was purchased from Xibao Biotechnology (Shanghai) Co., Ltd.

[0068] Example 1: Screening and Discovery of Diagnostic Markers for Gallstones

[0069] 1. Research cohort and sample collection

[0070] Case group: 60 patients with gallstones (diagnosed by abdominal ultrasound and magnetic resonance cholangiopancreatography), including 28 males and 32 females, aged 35-65 years. 35 cases contained cholesterol stones and 25 cases contained pigment stones. Patients with hepatitis, cirrhosis, pancreatitis, malignant tumors, or other diseases were excluded.

[0071] Control group: 60 healthy volunteers (normal abdominal ultrasound examination, no history of digestive system diseases, and normal liver and kidney function indicators), including 26 males and 34 females, aged 33-66 years;

[0072] Sample processing: Peripheral blood of all subjects was centrifuged within 2 hours after collection (3000 rpm, 10 min), aliquoted into 3 portions, and sealed and stored at -80℃ to avoid repeated freeze-thaw cycles.

[0073] 2. Non-targeted metabolomics and inflammatory factor screening

[0074] High-precision detection technology is used to screen for potential biomarkers, ensuring coverage of core pathological pathways:

[0075] (1) Metabolomics analysis (LC-MS / MS):

[0076] Instruments and Methods: Ultra-high performance liquid chromatography-tandem mass spectrometry was used with a C18 column (2.1 mm × 100 mm, 1.7 μm), and the mobile phase was acetonitrile-0.1% formic acid aqueous solution (gradient elution). Electrospray ionization positive ion mode was used for detection.

[0077] Detection range: The system detects bile acid-related metabolites, including cholic acid (CA), deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA).

[0078] (2) Detection of inflammatory factors:

[0079] Reagents and methods:

[0080] Inflammatory factor profile analysis: The Human Inflammation Array 1 protein chip was used to quantify soluble inflammatory mediators in the samples, including IL-6, TNF-α, and CRP.

[0081] Cell surface marker detection: The expression level of CD133 on the cell surface was detected by flow cytometry.

[0082] Endotoxin level determination: The content of lipopolysaccharide in the sample was quantitatively determined using the Limulus amebocyte lysate (LAL) reagent method.

[0083] 3. Data analysis and initial screening of biomarkers

[0084] Statistical analysis was performed using SPSS 26.0 and R-4.2.0 software. The screening logic was "single indicator difference screening - ratio model construction - diagnostic efficacy verification".

[0085] (1) Single marker difference analysis:

[0086] First, the concentrations of individual indicators in the case group and the control group were compared using an independent samples t-test, and the diagnostic efficacy parameters of each differential indicator were calculated. The results are shown in Table 1.

[0087] Table 1. Detection results and diagnostic efficacy analysis of individual biomarkers

[0088]

[0089] (2) Construction and optimization of ratio markers:

[0090] Based on the synergistic effect of metabolic imbalance and inflammatory response, the ratio between selected metabolic markers and inflammatory markers with significant differences was calculated; the diagnostic efficacy of each ratio was analyzed using a logistic regression model, and the combination with the highest AUC was selected. The results are shown in Table 2.

[0091] Table 2. Comparison of diagnostic efficacy of different biomarker combinations

[0092]

[0093] (3) Results verification and biological significance:

[0094] Advantages of the dual ratio model: LCA / UDCA (metabolic markers) reflects the imbalance between secondary bile acid production and hepatocellular protective function (LCA is toxic bile acid, UDCA is protective bile acid, and an elevated ratio indicates an increased risk of cholestasis and hepatocellular damage); CD133 / LPS (inflammatory markers) reflects the activation of intestinal endotoxin translocation and bile duct injury repair (LPS induces bile duct inflammation, CD133 participates in bile duct epithelial repair, and an elevated ratio indicates an imbalance between inflammation and repair).

[0095] Stability verification: 20 cases and 20 control samples were randomly selected and tested three times. The coefficients of variation (CV) of the LCA / UDCA ratio and the CD133 / LPS ratio were both <4%, indicating that the test results were stable and reliable.

[0096] Conclusion: The dual-indicator combination of LCA / UDCA and CD133 / LPS can simultaneously cover the core pathological processes of gallstone pathogenesis (metabolic imbalance + inflammatory response), and its diagnostic efficacy is significantly better than that of a single marker or other combinations. It can be used as a core marker for gallstone detection.

[0097] Example 2: Determination and Verification of the Upper Limit of the Reference Interval

[0098] 1. Research cohort and sample collection

[0099] Case group: 80 patients with gallstones (diagnosed by abdominal ultrasound and clinical symptoms, with no overlap with the cohort in Example 1), including 36 males and 44 females, aged 32-68 years, including 45 cases of cholesterol stones and 35 cases of pigment stones, excluding patients with other hepatobiliary and pancreatic diseases and malignant tumors.

[0100] Control group: 80 healthy controls (matched 1:1 in age and sex with the case group), with normal abdominal ultrasound examination and normal liver and kidney function, blood lipids and other indicators within the normal range;

[0101] Sample processing: Same as in Example 1. Separate serum within 2 hours after peripheral blood collection and store in a sealed container at -80°C to avoid repeated freeze-thaw cycles.

[0102] 2. ROC curve analysis and threshold determination

[0103] ROC curves were plotted using SPSS 26.0 software. The upper limit of the reference interval for each ratio marker was determined by maximizing the Youden index (sensitivity + specificity - 1). The diagnostic efficacy at different cutoff values ​​was also calculated. The results are shown in Table 3.

[0104] Table 3. Upper limits of reference intervals for metabolic and inflammatory markers and results of diagnostic efficacy validation.

[0105]

[0106] Conclusion: The upper limit of the reference interval for metabolic markers (LCA / UDCA) was 2.3 and the upper limit of the reference interval for inflammatory markers (CD133 / LPS) was 0.8. Both showed good diagnostic efficacy in the independent validation cohort (AUCs of 0.89 and 0.85, respectively), suggesting that the thresholds have broad applicability.

[0107] Example 3: Preparation of a gallstone detection kit

[0108] 1. Preparation of sample processing solution

[0109] Prepare 1L of sample processing solution: Weigh 2.42g Tris, 7.01g sodium chloride, 1.86g EDTA, 8g sucrose, and 0.3mL Tween-20, dissolve them in 800mL deionized water, adjust the pH to 7.4 with 1mol / L hydrochloric acid, and finally bring the volume to 1L with deionized water. Filter the solution through a 0.22μm filter membrane for sterilization and store at 4℃.

[0110] 2. Preparation of CD133 detection reagent (CD133 sandwich HRP magnetic beads)

[0111] A1. Activation of magnetic microspheres: Take carboxylated magnetic microspheres with a particle size of 200 nm, adjust the concentration to 10 mg / mL with 0.05 mol / L MES buffer; add EDC and NHS to make the final concentrations 10 mmol / L and 5 mmol / L respectively, and activate by shaking at room temperature in the dark for 30 min to obtain magnetic microsphere suspension 1.

[0112] A2. Capture antibody conjugation: CD133 antibody was added to magnetic microsphere suspension 1. The mass ratio of antibody to carboxylated magnetic microspheres was 1:100. The mixture was conjugated by shaking at 4°C in the dark for 12 hours to obtain magnetic microsphere suspension 2.

[0113] A3. Detection of antibody binding: Add HRP-labeled CD133 detection antibody to magnetic microsphere suspension 2. The mass ratio of detection antibody to magnetic microsphere suspension 2 is 1:50. Incubate at 37°C in the dark with shaking for 30 min to obtain magnetic microsphere suspension 3.

[0114] A4. Blocking and Volume Adjustment: Add PBS buffer containing 2% BSA to the magnetic microsphere suspension 3 at a mass ratio of 1:20, and block the reaction at room temperature for 60 min; discard the supernatant after magnetic separation, resuspend in PBS buffer containing 0.02% ProClin 300, adjust the final concentration to 5 mg / mL, and obtain CD133 sandwich HRP magnetic beads, which are stored at 4℃.

[0115] 3. Preparation of key testing reagents

[0116] (1) Lithocholic acid detection reagent: Add the following components to the final concentration: Tris-HCl (50 mmol / L, pH 8.5): 3α-HSD 1.0 U / mL, Thio-NAD 0.3 mmol / L, flavoprotein enzyme 0.8 U / mL, NBT 0.2 mmol / L; mix well and dispense into aliquots, and store at -20℃;

[0117] (2) Ursodeoxycholic acid assay reagent: Add the following components to the final concentration: Tris-HCl (50 mmol / L, pH 8.5): 7β-HSD 1.2 U / mL, Thio-NAD 0.3 mmol / L, flavoprotein enzyme 0.8 U / mL, NBT 0.2 mmol / L; mix well and dispense into aliquots, and store at -20℃;

[0118] (3) Lipopolysaccharide detection reagent: Add the following components to the final concentration: Tris-HCl (20 mmol / L, pH 7.3), Limulus amebocyte lysate (0.1 EU / mL), chromogenic substrate (HD-Val-Leu-Arg-pNA) 0.2 mmol / L, calcium chloride 5 mmol / L; mix well and dispense into aliquots, and store at -20℃.

[0119] 4. Preparation of calibrators and quality control samples

[0120] Calibrator: Add standard to recombinant human serum matrix to make it contain 8.0 μg / mL lithocholic acid, 4.0 μg / mL ursodeoxycholic acid, 0.3 ng / mL CD133 protein and 10 EU / mL lipopolysaccharide.

[0121] Positive control: Add standard to recombinant human serum matrix to achieve LCA / UDCA=3.2 and CD133 / LPS=1.2.

[0122] Negative control: Recombinant human serum matrix.

[0123] Example 4: Application of a non-diagnostic gallstone detection kit

[0124] The non-diagnostic testing method for gallstone detection kits specifically includes the following steps:

[0125] S1. Sample pretreatment: Collect fasting peripheral blood from the subject in the morning and separate the serum; take 12 μL of the serum sample to be tested and mix it gently with 48 μL of sample processing solution (volume ratio 1:4) in an EP tube, let it stand at room temperature for 3 min to obtain the pretreated sample.

[0126] S2, Marker Detection:

[0127] (1) Determination of lithocholic acid concentration: Take 20 μL of pretreated sample and mix with 80 μL of lithocholic acid detection reagent, incubate at 37℃ for 15 min, read the absorbance value at 550 nm wavelength using an ELISA reader, and calculate the LCA concentration in the sample to be tested based on the LCA quantitative standard curve constructed by the calibrator.

[0128] (2) Determination of ursodeoxycholic acid concentration: Take 20 μL of pretreated sample and mix with 80 μL of ursodeoxycholic acid detection reagent, incubate at 37℃ for 15 min, read the absorbance value at 550 nm wavelength using an ELISA reader, and calculate the UDCA concentration in the sample to be tested based on the UDCA quantitative standard curve constructed by the calibrator.

[0129] (3) CD133 concentration determination: Take 50 μL of pretreated sample and mix with 30 μL of CD133 detection reagent, incubate at 37℃ with shaking for 5 min, separate CD133 sandwich HRP magnetic beads by magnetic rack adsorption, discard the supernatant; add 50 μL of TMB chromogenic substrate, incubate at room temperature in the dark for 5 min; add 25 μL of 2mol / L sulfuric acid stop solution; read the absorbance value at 450 nm wavelength using an enzyme-linked immunosorbent assay reader, and calculate the CD133 concentration according to the calibrator standard curve;

[0130] (4) Determination of lipopolysaccharide concentration: Take 20 μL of pretreated sample and mix with 80 μL of lipopolysaccharide detection reagent, incubate at 37℃ for 15 min, read the absorbance value at 405 nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the LPS concentration in the sample to be tested based on the LPS quantitative standard curve constructed by the calibrator.

[0131] S3. Result determination: Calculate the concentration ratio of lithocholic acid to ursodeoxycholic acid and the concentration ratio of CD133 to lipopolysaccharide to obtain the quantitative detection value of in vitro diagnostic markers.

[0132] The rules for determining the results are as follows:

[0133] Rule A: The detected values ​​of both metabolic markers (LCA / UDCA) and inflammatory markers (CD133 / LPS) exceed the upper limit of their reference range;

[0134] Rule B: The detected value of metabolic markers (LCA / UDCA) or inflammatory markers (CD133 / LPS) exceeds the upper limit of their reference interval, and the offset is ≥30%. Offset = (detected value - upper limit of reference interval) / upper limit of reference interval × 100%;

[0135] When either Rule A or Rule B is met, the test report will mark it as meeting the positive criteria.

[0136] Fifty peripheral blood serum samples from different sources were tested using a non-diagnostic gallstone detection kit and method. Results showed significant differences in the concentration ratios of lithocholic acid to ursodeoxycholic acid (LCA / UDCA) and CD133 to lipopolysaccharide (CD133 / LPS) among the samples. Specifically, in 26 samples, both the metabolic marker ratio (LCA / UDCA) and the inflammatory marker ratio (CD133 / LPS) were above the upper limit of their respective reference intervals, satisfying Rule A; in another 24 samples, at least one of these two markers exceeded the upper limit of the reference interval, with a deviation of 30% or more, satisfying Rule B.

[0137] The above results demonstrate that the detection kit and detection method described in this application can simultaneously detect multiple gallstone-related biochemical markers under in vitro conditions, and can stratify and distinguish sample detection results through ratio analysis and judgment rules, reflecting the stability and feasibility of this method in scientific research, methodological research, risk assessment or health management-related research for non-diagnostic purposes.

[0138] The above specific embodiments are merely explanations of this application and are not intended to limit this application. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An in vitro diagnostic marker for gallstones, characterized in that, The in vitro diagnostic markers comprise metabolic markers and inflammatory markers; the metabolic markers are the concentration ratio of lithocholic acid to ursodeoxycholic acid; and the inflammatory markers are the concentration ratio of CD133 to lipopolysaccharide.

2. The in vitro diagnostic marker for gallstones according to claim 1, characterized in that, The upper limit of the reference interval of the metabolic markers is 2.3; and the upper limit of the reference interval of the inflammatory markers is 0.

8.

3. A kit for detecting gallstones, characterized by The kit comprises detection reagents for detecting the in vitro diagnostic markers of claim 1 or 2, sample processing fluid, calibrators, positive quality control and negative quality control.

4. The gallstone detection kit according to claim 3, characterized by, The detection reagents comprise lithocholic acid detection reagents, ursodeoxycholic acid detection reagents, CD133 detection reagents and lipopolysaccharide detection reagents.

5. The gallstone detection kit according to claim 4, characterized by The lithocholic acid detection reagents comprise Tris-HCl, 3α-hydroxysteroid dehydrogenase, thio-NAD, diaphorase and nitro blue tetrazolium chloride.

6. The gallstone detection kit according to claim 4, characterized by, The ursodeoxycholic acid detection reagents comprise Tris-HCl, 7β-hydroxysteroid dehydrogenase, thio-NAD, diaphorase and nitro blue tetrazolium chloride.

7. The gallstone detection kit according to claim 4, characterized by, The CD133 detection reagents are CD133 sandwich method HRP magnetic beads.

8. The gallstone detection kit according to claim 4, characterized by, The lipopolysaccharide detection reagents comprise limulus reagent, chromogenic substrate, Tris-HCl and calcium chloride.

9. A method for detecting non-diagnostic purposes of the gallstone detection kit according to any one of claims 3 to 8, characterized in that, Specifically comprising the following steps: S1, sample pretreatment: taking the sample to be tested, mixing with sample processing fluid, and obtaining pretreated sample after standing at room temperature; S2, marker detection: using lithocholic acid detection reagents, ursodeoxycholic acid detection reagents, CD133 detection reagents and lipopolysaccharide detection reagents to respectively determine the concentrations of lithocholic acid, ursodeoxycholic acid, CD133 and lipopolysaccharide in the pretreated sample; S3, result determination: calculating the concentration ratio of lithocholic acid to ursodeoxycholic acid and the concentration ratio of CD133 to lipopolysaccharide to obtain the quantitative detection values of metabolic markers and inflammatory markers.

10. The method of detecting for non-diagnostic purposes of a gallstone detection kit according to claim 9, wherein, In step S1, the sample to be tested is peripheral blood serum or plasma sample of a subject, the sample dosage is 10-15 μL, and the sample needs to be stored at 2-8℃ after collection and the detection needs to be completed within 24 h.

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