Method for detecting liver fibrosis-related protein, antibody screening method and detection kit

Abstract

The application discloses a kind of liver fibrosis related protein detection method, antibody screening method and detection kit.The detection method includes: corresponding different liver fibrosis related protein capture antibody is coupled to different solid phase carrier and is blocked;Phycoerythrin is labeled to detection antibody;The mixture of the measured object, capture carrier and detection antibody-phycoerythrin complex, forms immune complex;Fluorescence signal is generated by light source excitation, and the kind of measured object is determined according to the fluorescence signal of different solid phase carrier, and the concentration is calculated according to the fluorescence signal of phycoerythrin.The antibody screening method is screened from multiple groups of candidate antibody combinations with no endogenous interference with the standard of relative deviation ±10%.The kit comprising the screened paired antibody is also provided.The application can realize the synchronous detection of two liver fibrosis related proteins under low sample amount with single sample addition, and the correlation coefficient r of each item detection value and theoretical value is greater than 0.99, which can be used for the auxiliary diagnosis and condition assessment of chronic hepatitis B liver fibrosis.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostics, specifically to a method for detecting liver fibrosis-related proteins, an antibody screening method, and a detection kit. Background Technology

[0002] Chronic hepatitis B (CHB) is a common chronic liver disease in clinical practice. Liver fibrosis is a pathological feature of CHB, and without early intervention, it can gradually progress to cirrhosis and even liver cancer, seriously threatening the patient's quality of life. Currently, although liver biopsy is considered the "gold standard" for diagnosing CHB with liver fibrosis, it has limitations in dynamic assessment. Proteins associated with the progression of liver fibrosis are closely related to the pathological formation and disease progression of liver fibrosis and can serve as blood biomarkers to provide a non-invasive new approach for assessing liver fibrosis.

[0003] Liver fibrosis-related proteins (including Golgi protein 73 (GP73) and chitosanase 3-like protein 1 (CHI3L1)) play an important role in the auxiliary medical diagnosis of liver fibrosis progression in chronic hepatitis B. Literature indicates that serum GP73 levels in patients with chronic hepatitis B are significantly correlated with liver inflammation activity and the degree of fibrosis (P < 0.001); serum CHI3L1 levels in patients with chronic hepatitis B show an increasing trend across different stages of liver fibrosis; serum CHI3L1 and GP73 levels in the severe liver fibrosis group of chronic hepatitis B patients are higher than those in the mild and moderate groups (P < 0.05), and serum CHI3L1 and GP73 levels are positively correlated with the degree of liver fibrosis (both P < 0.05).

[0004] Immunoassays in medical diagnostics utilize highly specific antigen-antibody binding reactions to detect and analyze specific substances in biological samples. Current immunoassays for detecting liver fibrosis-related proteins primarily include enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay, both employing a double-antibody sandwich method. In ELISA, one antibody binds to a solid, forming an antibody-antigen-enzyme-labeled secondary antibody immune complex with the analyte and enzyme-labeled secondary antibody in the sample, which is then detected via enzymatic substrate activation. In chemiluminescence immunoassay, one antibody binds to a solid, while another antibody labels a luminescent substrate, forming an antibody-antigen-antibody-luminescent substrate immune complex with the analyte in the sample, which is then detected via enzymatic or electrochemical excitation of light intensity. ELISA's advantages, such as mature carriers, simple procedures, and low instrument dependence, have led to its rapid development and widespread application. Chemiluminescence immunoassay, with its high sensitivity, wide dynamic range, low background interference, and rapid detection capabilities, plays a crucial role in various fields, including diagnostics, food safety, environmental monitoring, and biomedical research.

[0005] Although ELISA and chemiluminescence immunoassay are relatively mature methods, they are still commonly used to determine single analytes. ELISA reaction times are relatively long, and for situations where sample acquisition is difficult, such as when sampling is challenging for specific populations or when the amount of sample that can be collected for a particular sample type is limited, combined detection techniques that can achieve single-sample addition with low sample volume requirements remain a challenge for immunoassay. Literature mentions ELISA and chemiluminescence immunoassay as methods for detecting liver fibrosis-related proteins. Both methods can only detect a single indicator, require large sample volumes, and ELISA reaction times are long. Furthermore, the precision and detectable range of ELISA are no longer sufficient for current medical diagnostic needs. Therefore, rapid multiplex detection methods capable of jointly detecting liver fibrosis-related proteins have high application value in assisting clinicians in assessing the formation and progression of liver fibrosis in patients with chronic hepatitis B. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting liver fibrosis-related proteins, an antibody screening method, and a reagent kit, thereby solving the problems of rapid multiplexing detection with low sample volume requirements that cannot be achieved with single sample loading in existing technologies, and simultaneously solving the problem of endogenous interfering substances affecting the accuracy of detection.

[0007] The first technical solution adopted in this invention is a method for detecting liver fibrosis-related proteins, comprising the following steps: coupling capture antibodies corresponding to different liver fibrosis-related proteins to different solid-phase carriers; blocking the solid-phase carriers to form capture carriers; labeling phycoerythrin onto detection antibodies to form detection antibody-phycoerythrin complexes; mixing the analyte with the capture carriers and the detection antibody-phycoerythrin complexes to form capture carrier-analyte-detection antibody-phycoerythrin immune complexes; exciting the immune complexes with a light source to generate corresponding fluorescence signals; determining the type of analyte and calculating the concentration of the analyte based on the different fluorescence signals.

[0008] Preferably, different solid-phase supports produce different fluorescence signals when excited by a light source, and the fluorescence signal of the phycoerythrin is related to the concentration of the analyte.

[0009] Preferably, the solid support is a magnetic microsphere.

[0010] Preferably, the liver fibrosis-related proteins include Golgi protein 73 and chitosanase 3-like protein 1.

[0011] The second technical solution adopted in this invention is an antibody screening method for detecting liver fibrosis-related proteins, comprising the following steps: providing multiple sets of candidate paired antibodies, each set of paired antibodies containing a capture antibody and a detection antibody corresponding to different liver fibrosis-related proteins; using each candidate paired antibody to detect interfering samples containing interfering substances; using a relative deviation of ±10% in the detection results of each item as the criterion for no interference; and screening out paired antibodies that do not interfere with the detection of all interfering samples.

[0012] Preferably, the interfering substance is selected from at least one of triglycerides, hemoglobin, and bilirubin.

[0013] Preferably, the liver fibrosis-related proteins include Golgi protein 73 and chitosanase 3-like protein 1.

[0014] The third technical solution adopted in this invention is a liver fibrosis-related protein detection kit, comprising the following components: a solid-phase carrier 1 coupled with a Golgi protein 73 capture antibody, and a solid-phase carrier 2 coupled with a chitosanase 3-like protein 1 capture antibody; a Golgi protein 73 detection antibody-phycoerythrin complex and a chitosanase 3-like protein 1 detection antibody-phycoerythrin complex.

[0015] Preferably, the solid support is a magnetic microsphere.

[0016] Preferably, the capture antibody is: Golgi protein 73 capture antibody NDX-GP73-01, or chitosanase 3-like protein 1 capture antibody NDX-CHI3L1-01.

[0017] Preferably, the detection antibody-phycoerythrin complex is: Golgi protein 73 detection antibody-phycoerythrin complex NDX-GP73-02 or chitosan polysaccharide enzyme 3-like protein 1 detection antibody-phycoerythrin complex NDX-CHI3L1-02.

[0018] The beneficial effects of this invention are as follows: 1. Achieving rapid, simultaneous multiplex detection with a single sample loading and low sample volume: Using solid-phase carriers with different fluorescent codes, different analytes in the biological sample react with their corresponding solid-phase carriers and antibodies in a one-step reaction to form immune complexes. Upon excitation by a light source, these complexes generate fluorescence signals. The type of analyte can be identified by the fluorescence signals of different solid-phase carriers, and the concentration of the analyte can be calculated using the fluorescence signal of phycoerythrin. Therefore, rapid multiplex detection with a single sample loading and low sample volume requirements can be achieved. A single sample loading can simultaneously obtain the detection results of two liver fibrosis-related proteins, solving the technical challenge of traditional ELISA and chemiluminescence methods that can only detect a single analyte and require large sample volumes.

[0019] 2. Systematic antibody screening effectively eliminates endogenous interference and ensures detection specificity: Endogenous interfering substances (such as hemoglobin, bilirubin, and triglycerides) present in human samples due to pathological reasons may interfere with detection results. To address this technical challenge, a systematic paired antibody screening method is proposed. By comparing and analyzing multiple groups of paired antibodies, and using a relative deviation of ±10% as the judgment criterion, the influence of endogenous interfering substances on the detection is eliminated. Experimental results showed that the relative deviations of the selected paired antibodies (Golgi protein 73 capture antibody NDX-GP73-01, chitosanase 3-like protein 1 capture antibody NDX-CHI3L1-01; Golgi protein 73 detection antibody-phycoerythrin complex NDX-GP73-02, chitosanase 3-like protein 1 detection antibody-phycoerythrin complex NDX-CHI3L1-02) in detecting interfering samples were all within ±10%, with no endogenous interference. In contrast, other unselected paired antibody sets showed deviations exceeding ±10%, demonstrating the effectiveness of the screening method and ensuring the accuracy and specificity of multiplex detection.

[0020] 3. Detection performance meets medical diagnostic standards: The detection results of two liver fibrosis-related protein composite gradient samples showed that the correlation coefficients (r values) between the detected values ​​and theoretical values ​​of chitosan polysaccharide enzyme 3-like protein 1 and Golgi protein 73 were 0.998 and 0.999, respectively, both greater than 0.99, meeting the requirements of generally accepted medical diagnostic standards. Based on the close association between GP73 and CHI3L1 and the progression of liver fibrosis in chronic hepatitis B, this invention can be used to assess the formation and progression of liver fibrosis in patients with chronic hepatitis B, and has clinical application value.

[0021] 4. High commercial maturity, enabling fully automated detection: All raw materials used are commercially available and mass-produced, requiring no special modifications. The experimental design is simple, requiring no complex or sophisticated equipment to complete item identification and concentration detection. Furthermore, the detection and analysis process is fully automated; a single sample requires only a single sample addition reaction, eliminating the need for multiple additions and repeated reactions, thus shortening reaction time, improving the clinical application efficiency of liver fibrosis-related protein detection, and reducing testing costs. Attached Figure Description

[0022] Figure 1 This is a flowchart of the liver fibrosis-related protein detection method of the present invention. Detailed Implementation

[0023] The following examples further illustrate the present invention, but are not intended to limit the invention. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments with the same designations. However, any modifications, equivalent changes, or alterations made to the following embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

[0024] I. Overall Procedure for Detection of Liver Fibrosis-Related Proteins like Figure 1 As shown, the method for detecting liver fibrosis-related proteins provided in this embodiment includes the following steps: Capture antibodies corresponding to different liver fibrosis-related proteins were conjugated to different solid-phase carriers; The solid-phase support is sealed to form a capture carrier; Phycoerythrin is labeled onto the detection antibody to form a detection antibody-phycoerythrin complex; The analyte is mixed with the capture carrier and the detection antibody-phycoerythrin complex to form a capture carrier-analyte-detection antibody-phycoerythrin immune complex. The immune complex is excited by a light source to generate a corresponding fluorescence signal; The type of analyte is determined and its concentration is calculated based on the different fluorescence signals.

[0025] Understandably, when detecting different analytes in a single sample with a single loading, different capture antibodies corresponding to different analytes need to be used based on the specificity of the binding between the analyte and the capture antibody. For example, when detecting two analytes (liver fibrosis-associated protein 1 and liver fibrosis-associated protein 2) in the same sample, the capture antibodies corresponding to these two analytes (capture antibody 1 and capture antibody 2) need to be used to specifically bind to the analytes in the sample, forming capture antibody-analyte complexes. Similarly, detection antibodies also need to be matched according to the type of analyte to form capture antibody-analyte-detection antibody complexes, i.e., double antibody sandwich complexes.

[0026] This embodiment employs different solid-phase carriers, each generating different fluorescence signals upon excitation by a light source. When different analytes in the same sample specifically bind to the capture antibodies on the surface of their respective capture carriers and the detection antibodies in the detection antibody-phycoerythrin complex, the light source excites all the capture carrier-analyte-detection antibody-phycoerythrin immune complexes to produce fluorescence signals. The different fluorescence signals from the solid-phase carriers can distinguish the types of analytes, and the concentration of the analyte can be calculated using the fluorescence signal of phycoerythrin. Therefore, rapid multiplex detection of liver fibrosis-related proteins in a single sample with a single sample loading can be achieved.

[0027] Based on the above method, this embodiment provides a liver fibrosis-related protein detection kit, comprising the following components: a solid-phase carrier 1 coupled with a Golgi protein 73 capture antibody, and a solid-phase carrier 2 coupled with a chitosanase 3-like protein 1 capture antibody; a Golgi protein 73 detection antibody-phycoerythrin complex and a chitosanase 3-like protein 1 detection antibody-phycoerythrin complex.

[0028] II. Screening of Paired Antibodies When establishing a detection system, interfering substances (such as hemoglobin, bilirubin, and triglycerides) present in human samples due to pathological reasons may interfere with the detection results. Specifically, this manifests as a non-specific reaction between endogenous interfering substances and paired antibodies, leading to deviations in the detection results. To eliminate the above interference, this embodiment collects and screens information on commercially available mass-produced raw materials, selecting three sets of paired antibodies for comparative analysis. The information on the three sets of paired antibodies is as follows.

[0029] Set 1: Golgi protein 73 capture antibody a, chitosanase 3-like protein 1 capture antibody a; Golgi protein 73 detection antibody-phycoerythrin complex a, chitosanase 3-like protein 1 detection antibody-phycoerythrin complex a Set 2: Golgi protein 73 capture antibody b, chitosanase 3-like protein 1 capture antibody b; Golgi protein 73 detection antibody-phycoerythrin complex b, chitosanase 3-like protein 1 detection antibody-phycoerythrin complex b Set 3: Golgi protein 73 capture antibody c, chitosanase 3-like protein 1 capture antibody c; Golgi protein 73 detection antibody-phycoerythrin complex c, chitosanase 3-like protein 1 detection antibody-phycoerythrin complex c The paired antibodies from kits 1, 2, and 3 were prepared according to the reagent preparation methods described in the examples below, resulting in reagent kits 1, 2, and 3, respectively. Interference samples were then tested using each of the three kits.

[0030] Interfering substances (bilirubin concentration of 257 μmol / L, triglyceride concentration of 10 mmol / L, and hemoglobin concentration of 5 mg / mL) were added to samples at two levels for each test item to prepare interfering samples. Samples without added interfering substances were used as control samples. The interfering samples and control samples were tested using three sets of reagents respectively. The sample information is as follows.

[0031] Project: Golgi protein 73

[0032] Project: Chitosan polysaccharide enzyme 3-like protein 1

[0033] The relative deviation between the interfering sample and the control sample should be within ±10% when using reagent testing. If the relative deviation exceeds ±10%, it indicates that the corresponding interfering substance interferes with the detection of the paired antibody.

[0034] 1. Test results and interference analysis of reagent group 1 The test results are as follows: Project: Golgi protein 73

[0035] Project: Chitosan polysaccharide enzyme 3-like protein 1

[0036] Relative deviation: Project: Golgi protein 73

[0037] Project: Chitosan polysaccharide enzyme 3-like protein 1

[0038] Interference analysis: No interference exists.

[0039] 2. Test results and interference analysis of reagent group 2 The test results are as follows: Project: Golgi protein 73

[0040] Project: Chitosan polysaccharide enzyme 3-like protein 1

[0041] Relative deviation: Project: Golgi protein 73

[0042] Project: Chitosan polysaccharide enzyme 3-like protein 1

[0043] Interference analysis: Triglycerides interfered with the detection of Golgi protein 73 paired antibody; hemoglobin interfered with the detection of chitosanase 3-like protein 1 paired antibody; bilirubin interfered with the detection of Golgi protein 73 paired antibody.

[0044] 3. Test results and interference analysis of reagent group 3 The test results are as follows: Project: Golgi protein 73

[0045] Project: Chitosan polysaccharide enzyme 3-like protein 1

[0046] Relative deviation: Project: Golgi protein 73

[0047] Project: Chitosan polysaccharide enzyme 3-like protein 1

[0048] Interference analysis: Triglycerides interfered with the detection of Golgi protein 73 paired antibody; hemoglobin interfered with the detection of Golgi protein 73 paired antibody; bilirubin interfered with the detection of chitosanase 3-like protein 1 paired antibody.

[0049] 4. Screening Conclusion The paired antibodies from kit 1 used in reagent group 1 and kit 3 used in reagent group 3 exhibit endogenous interference when detecting samples, which does not meet the requirements; the paired antibodies from kit 2 used in reagent group 2 are not affected by triglycerides, hemoglobin, and bilirubin in the samples when detecting samples, which meets the requirements.

[0050] The paired antibody information for Set 1 is as follows:

[0051] Subsequent embodiments all used the paired antibodies from Set 1, which were screened and confirmed to be free of endogenous interference, for reagent preparation and detection verification.

[0052] III. Detection of two liver fibrosis-related protein complex gradient samples 1. Experimental reagents and instruments 1.1. Experimental Reagents Two types of magnetic microspheres; Golgi protein 73 capture antibody (NDX-GP73-01), chitosanase 3-like protein 1 capture antibody (NDX-CHI3L1-01); Golgi protein 73 detection antibody-phycoerythrin complex (NDX-GP73-02), chitosanase 3-like protein 1 detection antibody-phycoerythrin complex (NDX-CHI3L1-02) were all purchased externally. All instrument consumables were provided by Nanjing Aituo Life Science Technology Co., Ltd. Unless otherwise specified, all other reagents and consumables were purchased externally.

[0053] 1.2. Experimental Apparatus AT2000 fully automated liquid suspension chip detector (Nanjing Aituo Life Technology Co., Ltd.), YFF-3 molecular hybridization instrument (Xinghua Analytical Instrument Factory), pipette (Thermo Fisher Scientific (China) Co., Ltd.).

[0054] 2. Experimental Methods 2.1. Preparation of the capture carrier mixture 550 μL of each of the two types of magnetic microspheres were measured into two 1.5 mL centrifuge tubes, and the corresponding microsphere information was marked on the centrifuge tubes. The microspheres were allowed to stand for 2 minutes after magnetic adsorption, and the supernatant was removed.

[0055] Add 1000 μL of 5 mM MES buffer to each centrifuge tube, magnetically adsorb and let stand for 2 minutes, then remove the supernatant.

[0056] Add 960 μL of 5 mM MES buffer, 20 μL of 50 mg / mL NHS and 20 μL of 50 mg / mL LEDC to each centrifuge tube and mix well.

[0057] Each centrifuge tube was placed in a molecular hybridization apparatus, the rotation speed was adjusted to 28 rpm, and incubated at 37°C for 30 minutes.

[0058] After incubation, remove each centrifuge tube, magnetically adsorb and let stand for 2 minutes, then remove the supernatant.

[0059] Add 1000 μL of 5 mM MES buffer to each centrifuge tube, magnetically adsorb and let stand for 2 minutes, then remove the supernatant.

[0060] Add 725 μL of 5 mM MES buffer and 275 μL of the corresponding capture antibody to each centrifuge tube and mix well.

[0061] Each centrifuge tube was placed in a molecular hybridization apparatus, the rotation speed was adjusted to 28 rpm, and incubated at 37°C for 3 hours.

[0062] After incubation, remove each centrifuge tube, magnetically adsorb and let stand for 2 minutes, then remove the supernatant.

[0063] Add 1000 μL of 100 mM phosphate buffer containing 2% BSA to each centrifuge tube and mix well.

[0064] Each centrifuge tube was placed in a molecular hybridization apparatus, the rotation speed was adjusted to 28 rpm, and incubated at 37°C for 1 hour.

[0065] After incubation, remove each centrifuge tube, magnetically adsorb and let stand for 2 minutes, then remove the supernatant.

[0066] Add 1000 μL of 100 mM phosphate buffer containing 2% BSA to each centrifuge tube and mix well.

[0067] Transfer the liquid from the two centrifuge tubes to a 300mL reagent bottle, add 273mL of 100mM phosphate buffer, and mix well.

[0068] 2.2. Preparation of the detection antibody-phycoerythrin working solution Measure 2750 μL of each of the Golgi protein 73 detection antibody-phycoerythrin complex and the chitosanase 3-like protein 1 detection antibody-phycoerythrin complex into a 300 mL reagent bottle, add 269.5 mL of 100 mM phosphate buffer, and mix well.

[0069] 2.3. Preparation of composite gradient samples of liver fibrosis-related proteins Take samples close to the lower limit of the linear range and dilute them with samples close to the upper limit of the linear range, diluting them into 6 concentrations according to the following ratios.

[0070]

[0071] 2.4. Measurement and Analysis Place the capture vector mixture, detection antibody-phycoerythrin working solution, and sample into the designated position on the AT2000 fully automated liquid suspension chip detector. Select two liver fibrosis-related protein parameters to begin the reaction. The specific reaction process is as follows: Add 25 μL of capture vector mixture, 25 μL of detection antibody-phycoerythrin working solution and 25 μL of sample to the reaction vessel.

[0072] The reaction vessel was kept at a constant temperature and oscillated at 800 rpm for 1 hour at 37°C.

[0073] After incubation, the reaction vessel was magnetically adsorbed and allowed to stand for 1 minute before the supernatant was removed.

[0074] Add 150 μL of 100 mM phosphate buffer to each reaction well, allow it to stand for 1 minute using magnetic adsorption, and then remove the supernatant.

[0075] Add 150 μL of 100 mM phosphate buffer to each reaction vessel.

[0076] After the reaction is complete, the reaction vessel is transferred to the detection area. The instrument's light source excites all the capture carrier-analyte-detection antibody-phycoerythrin immune complex to generate a fluorescent signal. The instrument collects the fluorescent signal, identifies the solid-phase carrier (i.e., the detection item), and analyzes the fluorescence signal of phycoerythrin to calculate the concentration of the analyte.

[0077] 3. Experimental Results The gradient sample detection results for each project are as follows: Chitosan polysaccharide enzyme 3-like protein 1:

[0078] Golgi protein 73:

[0079] 4. Results Analysis The two liver fibrosis-related protein composite gradient samples in this embodiment were tested, and both items were correctly identified. The test results showed that the correlation coefficient r between the detected values ​​of the composite gradient samples and the theoretical values ​​of the samples was greater than 0.99, which meets the requirements of the medical diagnostic standards.

[0080] This embodiment enables rapid multiplex detection of two liver fibrosis-related proteins. A single sample requires only a single loading reaction (25 μL sample volume) to simultaneously obtain results for both items. Compared to traditional ELISA methods that detect each protein individually, this invention saves sample volume, shortens reaction time, and improves detection efficiency.

[0081] IV. Summary of Technical Effects This embodiment demonstrates a high degree of commercial maturity, using commercially available, mass-produced raw materials with no special modification requirements. The experimental design is simple, requiring no complex or sophisticated equipment for project identification and concentration detection. This embodiment offers accurate identification and a simple principle in the rapid multiplex detection of different liver fibrosis-related proteins. The detection and analysis process is fully automated, requiring only a single sample addition reaction, eliminating the need for multiple additions and repeated reactions. This improves the clinical application efficiency of liver fibrosis-related protein detection and reduces testing costs.

[0082] Obviously, the above embodiments of the present invention are merely illustrative examples to illustrate the invention and are not intended to limit the implementation of the invention. Other obvious variations or modifications derived from the essential spirit of the invention still fall within the protection scope of the invention.

Claims

1. A method for detecting liver fibrosis-related proteins, characterized in that, Includes the following steps: Capture antibodies corresponding to different liver fibrosis-related proteins were conjugated to different solid-phase carriers; The solid-phase support is sealed to form a capture carrier; Phycoerythrin is labeled onto the detection antibody to form a detection antibody-phycoerythrin complex; The analyte is mixed with the capture carrier and the detection antibody-phycoerythrin complex to form a capture carrier-analyte-detection antibody-phycoerythrin immune complex. The immune complex is excited by a light source to generate a corresponding fluorescence signal; The type of analyte is determined and its concentration is calculated based on the different fluorescence signals.

2. The method for detecting liver fibrosis-related proteins according to claim 1, characterized in that, Different solid supports produce different fluorescence signals when excited by a light source, and the fluorescence signal of the phycoerythrin is related to the concentration of the analyte.

3. The method for detecting liver fibrosis-related proteins according to claim 1, characterized in that, The solid support is a magnetic microsphere.

4. The method for detecting liver fibrosis-related proteins according to claim 1, characterized in that, The liver fibrosis-related proteins include Golgi protein 73 and chitosanase 3-like protein 1.

5. An antibody screening method for detecting liver fibrosis-related proteins, characterized in that, Includes the following steps: Multiple candidate paired antibodies are provided, each pair of paired antibodies containing capture antibodies and detection antibodies corresponding to different liver fibrosis-related proteins; Each candidate paired antibody was used to detect the interfering sample containing the interfering substance. The relative deviation of the test results for each item within ±10% is used as the criterion for determining no interference; Paired antibodies that did not interfere with the detection of any interfering samples were selected.

6. The antibody screening method for detecting liver fibrosis-related proteins according to claim 5, characterized in that, The interfering substance is selected from at least one of triglycerides, hemoglobin, and bilirubin.

7. The antibody screening method for detecting liver fibrosis-related proteins according to claim 5, characterized in that, The liver fibrosis-related proteins include Golgi protein 73 and chitosanase 3-like protein 1.

8. A liver fibrosis-related protein detection kit, characterized in that, Includes the following components: Golgi protein 73 captures antibody-conjugated solid-phase carrier 1, and chitosanase 3-like protein 1 captures antibody-conjugated solid-phase carrier 2; Golgi protein 73 detection antibody-phycoerythrin complex, chitosanase 3-like protein 1 detection antibody-phycoerythrin complex.

9. The liver fibrosis-related protein detection kit according to claim 8, characterized in that, The solid support is a magnetic microsphere.

10. The liver fibrosis-related protein detection kit according to claim 8, characterized in that, The capture antibodies are: Golgi protein 73 capture antibody NDX-GP73-01 and chitosanase 3-like protein 1 capture antibody NDX-CHI3L1-01; the detection antibody-phycoerythrin complexes are: Golgi protein 73 detection antibody-phycoerythrin complex NDX-GP73-02 and chitosanase 3-like protein 1 detection antibody-phycoerythrin complex NDX-CHI3L1-02.

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