Kit for detecting liver fibrosis and liver cirrhosis

Abstract

The invention provides a kit for detecting hepatic fibrosis and cirrhosis, and belongs to the technical field of biology. According to the kit, by measuring the content of CHI3L1 in serum of a patient, normal people and patients with cirrhosis can be effectively distinguished, and patients with compensatory cirrhosis and patients with decompensatory cirrhosis can also be effectively distinguished, so that the detection result is more accurate, and the condition of the patient can be judged more accurately. And the kit provided by the invention has a good standard substance, so that the detection accuracy can be further improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a kit for detecting liver fibrosis and liver cirrhosis. Background Art

[0002] Cirrhosis refers to the middle and late stages of liver fibrosis. Its morphological definition is diffuse liver fibrosis accompanied by abnormal nodule formation. Diffuse liver fibrosis without nodule formation (such as congenital liver fibrosis) or nodule formation without fibrosis (such as nodular regenerative hyperplasia) cannot be called cirrhosis. From a clinical point of view, cirrhosis refers to the manifestations of liver failure and portal hypertension caused by the above-mentioned pathological changes in liver tissue. From a pathological point of view, chronic inflammatory necrosis first leads to the proliferation and deposition of liver fibrous connective tissue (fibrosis), which then leads to the destruction of the liver lobule structure and the formation of pseudolobules, and finally develops into cirrhosis.

[0003] There are no obvious clinical symptoms in the compensated stage of cirrhosis, while the decompensated stage is characterized by portal hypertension and severe liver damage. Patients often die from multiple organ failure due to complications such as ascites, gastrointestinal bleeding, sepsis, hepatic encephalopathy, hepatorenal syndrome, and cancer. The prognosis of decompensated cirrhosis is usually poor, with a 5-year survival rate of only 14-35% under conventional treatment. Patients with decompensated cirrhosis often develop acute-on-chronic liver failure (ACLF), which is associated with a high mortality rate.

[0004] Therefore, timely and accurate detection of liver fibrosis, cirrhosis, compensated cirrhosis and decompensated cirrhosis is of great significance for the diagnosis and treatment of cirrhosis. At present, there are many methods for the detection of cirrhosis. For example, Chinese patent CN103160575A discloses the application of SAA1β / β homozygous genotype in the prognosis diagnosis and diagnosis of cirrhosis. The patent detects human SAA1β / β homozygous genotype and non-SAA1β / β homozygous genotype by real-time fluorescence quantitative allele-specific PCR, thereby realizing the detection of people susceptible to cirrhosis; Chinese patent CN103160507A discloses miRNA serum markers for the detection of cirrhosis and their application. These methods are detected at the gene or mRNA level, the method is relatively cumbersome, and the detection cost is high. There are also some methods that use antigen-antibody reactions to detect at the protein level, but when these methods are converted into kits for practical application, poor standard samples will affect the detection specificity and sensitivity. In addition, in the course of liver fibrosis and cirrhosis, there are few studies and reports on methods or products that can simply and accurately distinguish the compensated stage from the decompensated stage. Therefore, a lot of research is still needed for the detection of liver fibrosis and cirrhosis. Summary of the invention

[0005] The purpose of the present invention is to provide a kit for liver fibrosis and liver cirrhosis. The kit of the present invention has good standard products and can effectively ensure the accuracy of the measurement results. In addition to being able to accurately distinguish whether a patient has liver fibrosis or liver cirrhosis, the kit of the present invention can also be used to accurately distinguish whether a patient with liver cirrhosis is in the compensated stage, decompensated stage or recompensated stage.

[0006] In order to achieve the above-mentioned purpose of the invention, the following technical scheme is adopted.

[0007] The invention discloses isotope-labeled CHI3L1, wherein the specific polypeptide fragment sequence of CHI3L1 is: ILGQQVPYATK; the isotope comprises 15 N and / or 13 C.

[0008] Preferably, the isotope-labeled CHI3L1 labeling site is at lysine.

[0009] The present invention also discloses a CHI3L1 standard, including CHI3L1 from cirrhotic serum of hepatitis B patients or chemically synthesized CHI3L1. Cirrhotic serum of hepatitis B patients is serum of patients with cirrhosis caused by hepatitis B virus, and contains all structural variants of chitin protein, especially chitin protein variants in hepatitis B-related fibrosis. Various protein modifications, epitopes, immunogenicity, etc. in cirrhotic serum of hepatitis B patients are more in line with actual protein characteristics, and are better standard products and value assignment sources.

[0010] Preferably, the use of the above-mentioned standard substances includes: obtaining calibrators and quality control substances suitable for liver fibrosis and cirrhosis kits through traceability chain assignment.

[0011] Preferably, the ratio of the traceability chain of chemically synthesized CHI3L1, isotope-labeled CHI3L1 and CHI3L1 in the serum of hepatitis B patients with cirrhosis is: Synthetic CHI3L1: isotope-labeled CHI3L1: CHI3L1 in the serum of cirrhotic hepatitis B patients = 150-250: 700-850: 150-250.

[0012] More preferably, the ratio of the traceability chain of chemically synthesized CHI3L1, heavy isotope labeled CHI3L1 and CHI3L1 in the serum of hepatitis B patients with cirrhosis is: Synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 in the serum of cirrhotic hepatitis B patients = 200-220: 770-780: 200-220.

[0013] More preferably, the ratio of the traceability chain of chemically synthesized CHI3L1, heavy isotope labeled CHI3L1 and CHI3L1 in the serum of hepatitis B patients with cirrhosis is: Synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 in the serum of cirrhotic hepatitis B patients = 218:780:220.

[0014] The present invention also discloses the use of isotope-labeled CHI3L1 as a reference material in the traceability and / or value assignment of standard products. The isotope-labeled CHI3L1 is labeled with a heavy isotope or a light isotope, and the labeling site is lysine.

[0015] Preferably, the isotopes include 15 N and / or 13 C.

[0016] Preferably, the CHI3L1 specific sequence is ILGQQVPYATK. This sequence is a CHI3L1 specific polypeptide sequence, has no crossover with other proteins, can well represent CHI3L1, and makes the detection more accurate.

[0017] Preferably, isotope-labeled CHI3L1 is used as a secondary reference material.

[0018] Preferably, the gravimetric method is the primary reference method.

[0019] Preferably, the secondary reference method is isotope dilution, and the heavy isotope labeled polypeptide is accurately assigned by comparing the heavy isotope labeled ILGQQVPYATK and the light isotope labeled ILGQQVPYATK using reaction monitoring and mass spectrometry.

[0020] Preferably, in the above use, the ratio of the traceability chain of chemically synthesized CHI3L1, heavy isotope labeled CHI3L1 and CHI3L1 in the serum of hepatitis B patients with cirrhosis is: Synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 in the serum of cirrhotic hepatitis B patients = 150-250: 700-850: 150-250.

[0021] More preferably, in the above use, the ratio of the traceability chain of chemically synthesized CHI3L1, heavy isotope labeled CHI3L1 and CHI3L1 in the serum of cirrhosis of hepatitis B patients is: Synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 in the serum of cirrhotic hepatitis B patients = 200-220: 770-780: 200-220.

[0022] More preferably, in the above use, the ratio of the traceability chain of chemically synthesized CHI3L1, heavy isotope labeled CHI3L1 and CHI3L1 in the serum of cirrhosis of hepatitis B patients is: Synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 in the serum of cirrhotic hepatitis B patients = 218:780:220.

[0023] The present invention also discloses the use of the above-mentioned isotope-labeled CHI3L1 as a reference material in the traceability or assignment of CHI3L1 standard protein, and the isotope-labeled CHI3L1 is used as a secondary reference material. The chemically synthesized CHI3L1 polypeptide is quantified by weighing method; the isotope-labeled CHI3L1 is a heavy isotope-labeled CHI3L1 polypeptide, and the heavy isotope-labeled CHI3L1 polypeptide is quantified by LC-MS; the CHI3L1 standard protein is quantified by LC-MS, and the heavy isotope-labeled CHI3L1 polypeptide is quantitatively traced back to the heavy isotope-labeled CHI3L1 polypeptide by LC-MS, and the heavy isotope-labeled CHI3L1 polypeptide is quantitatively traced back to the chemically synthesized CHI3L1 polypeptide by LC-MS, and the chemically synthesized CHI3L1 polypeptide is directly weighed to determine the mass, which can be accurately quantified to the mass unit of the International System of Units (SI).

[0024] Preferably, the chemically synthesized CHI3L1 polypeptide ILGQQVPYATK is the primary reference material, and the weighing method is the primary reference method.

[0025] Preferably, the isotope-labeled CHI3L1 is a heavy isotope-labeled CHI3L1 polypeptide.

[0026] More preferably, the heavy isotope-labeled CHI3L1 polypeptide is ILGQQVPYAT(K*)[K( 13 C6; 15 N2)].

[0027] Preferably, the secondary referencing method is by isotope dilution.

[0028] More preferably, the molar concentration ratio of the chemically synthesized CHI3L1 polypeptide and the heavy isotope-labeled CHI3L1 polypeptide is 1.1-1.6:1.

[0029] More preferably, the molar concentration ratio of the chemically synthesized CHI3L1 polypeptide and the heavy isotope-labeled CHI3L1 polypeptide is 1.2-1.5:1.

[0030] More preferably, the molar concentration ratio of the chemically synthesized CHI3L1 polypeptide and the heavy isotope-labeled CHI3L1 polypeptide is 1.38:1.

[0031] Preferably, the CHI3L1 standard protein is completely digested with trypsin to obtain a CHI3L1 standard polypeptide.

[0032] Preferably, the molar concentration ratio of the CHI3L1 standard polypeptide to the heavy isotope-labeled CHI3L1 polypeptide is 0.6-0.9:1.

[0033] More preferably, the molar concentration ratio of the CHI3L1 standard polypeptide to the heavy isotope-labeled CHI3L1 polypeptide is 0.7-0.9:1.

[0034] More preferably, the molar concentration ratio of the CHI3L1 standard polypeptide and the heavy isotope-labeled CHI3L1 polypeptide is 0.81:1.

[0035] The present invention also discloses a method for tracing the source of a CHI3L1 standard protein, comprising: The chemically synthesized CHI3L1 peptide ILGQQVPYATK was used as the primary reference material, and the chemically synthesized CHI3L1 peptide was quantified by the weighing method; Isotope-labeled CHI3L1 was used as a secondary reference material and isotope-labeled CHI3L1 was quantified by LC-MS; and, The CHI3L1 standard protein was completely digested by trypsin to obtain the CHI3L1 standard peptide, and the CHI3L1 standard peptide was quantified by LC-MS.

[0036] In the method of the present invention, the CHI3L1 standard protein is quantified by LC-MS, and isotope-labeled CHI3L1 is quantitatively traced back by LC-MS. The isotope-labeled CHI3L1 is quantitatively traced back to the chemically synthesized CHI3L1 polypeptide by LC-MS. The mass of the chemically synthesized CHI3L1 polypeptide is determined by direct weighing, and can be accurately quantified to the mass unit of the International System of Units (SI).

[0037] Preferably, the isotope-labeled CHI3L1 is a heavy isotope-labeled CHI3L1 polypeptide.

[0038] More preferably, the heavy isotope-labeled CHI3L1 polypeptide is ILGQQVPYAT(K*)[K( 13 C6; 15 N2)].

[0039] More preferably, the ratio of the traceability chain of chemically synthesized CHI3L1, heavy isotope-labeled CHI3L1 and CHI3L1 in the serum of hepatitis B patients with cirrhosis is: synthetic CHI3L1: heavy isotope-labeled CHI3L1: CHI3L1 in the serum of hepatitis B patients with cirrhosis = 200-220:770-780:200-220.

[0040] More preferably, the molar concentration ratio of the chemically synthesized CHI3L1 polypeptide to the heavy isotope-labeled CHI3L1 polypeptide is 1.1-1.6: 1. The molar concentration ratio of the CHI3L1 standard polypeptide to the heavy isotope-labeled CHI3L1 polypeptide is 0.6-0.9:1.

[0041] The present invention also discloses a kit for liver fibrosis and liver cirrhosis, the kit comprising standard products and / or quality control products; the standard products include CHI3L1 standard products assigned by the above-mentioned traceability method and / or next-level standard products prepared with reference to the CHI3L1 standard products.

[0042] Preferably, the quality control product includes the CHI3L1 standard product assigned a value by the above-mentioned traceability method.

[0043] The invention also discloses a kit for liver fibrosis and liver cirrhosis, comprising a standard product; the standard product is CHI3L1.

[0044] Preferably, the CHI3L1 used as the standard is derived from the serum of cirrhotic patients with hepatitis B or is chemically synthesized. When the CHI3L1 standard sample is taken from the serum of patients, especially the serum of patients in the Chinese population, the standard has a broad spectrum of applicability and the test results are accurate.

[0045] Preferably, CHI3L1 used as a standard is in various forms including solutions.

[0046] Preferably, the standard product derived from a patient is obtained by conventional protein separation and purification methods.

[0047] Preferably, the N-terminus or C-terminus of CHI3L1 used as a standard includes a tag.

[0048] Preferably, the above kit further comprises: The antibody that can bind to CHI3L1 is called the primary antibody; The antibody containing a marker that can bind to CHI3L1 is called a secondary antibody.

[0049] The present invention also discloses the use of CHI3L1 in preparing a kit for identifying and / or prognostic monitoring of liver fibrosis and liver cirrhosis.

[0050] Preferably, the above identification and / or prognostic monitoring of liver fibrosis and cirrhosis includes distinguishing the compensated stage, decompensated stage and recompensated stage of cirrhosis. By measuring the content of CHI3L1 in the patient's serum, while effectively distinguishing normal people from patients with cirrhosis, it is also possible to effectively distinguish patients with compensated cirrhosis and decompensated cirrhosis, making the test results more accurate and conducive to more accurate judgment of the patient's condition.

[0051] Preferably, the detection method applicable to the above kit includes at least one of the following: Colloidal gold method, enzyme-linked immunosorbent assay, magnetic particle chemiluminescence method, fluorescence method, microfluidics, latex turbidimetry.

[0052] More preferably, the luminescent substance of the magnetic particle chemiluminescence method includes at least one of the following: Acridinium ester, alkaline phosphatase, horseradish peroxidase, ABEI.

[0053] More preferably, the fluorescence method includes a dry method and a wet method.

[0054] Preferably, the above kit comprises: The antibody that can bind to CHI3L1 is called the primary antibody; The antibody containing a marker that can bind to CHI3L1 is called a secondary antibody.

[0055] More preferably, the source of the above-mentioned antibodies includes at least one of the following: Mice, rats, rabbits, sheep, chickens, camels, monkeys, humans.

[0056] More preferably, the method for preparing the above antibody comprises at least one of the following: Hybridoma technology, B cell cloning technology, phage display technology, recombinant expression technology, and humanization technology.

[0057] Preferably, the first CHI3L1 antibody can be immobilized on a solid support.

[0058] More preferably, the solid phase carrier includes various microplates with 96 wells, 384 wells or more, test tubes, sample cups, plastic microspheres, cellulose, paper or plastic test strips, latex particles, polymer particles, silica particles, magnetic particles, and nanoparticles.

[0059] More preferably, the nanoparticles have an average diameter of 0.1-10 μm.

[0060] More preferably, the first CHI3L1 antibody includes any antibody or antibody fragment capable of binding to CHI3L1, such as a recombinant antibody, a chimeric antibody, a humanized antibody and a murine antibody.

[0061] More preferably, the first antibody is a monoclonal antibody or a polyclonal antibody.

[0062] More preferably, the first antibody is a polyclonal antibody. Using a polyclonal antibody, various CHI3L1 variants can be captured.

[0063] More preferably, the variants of CHI3L1 include variants obtained by various modifications.

[0064] More preferably, the variants of CHI3L1 include variants obtained by glycosylation modification, and the sites of glycosylation modification include: amino acids 60 (Asn60) and 80 (Asn81).

[0065] More preferably, the variants of CHI3L1 include variants obtained by mucin-type O-glycosylation modification (O-GalNAc glycosylation modification), and the O-GalNAc glycosylation modification sites include 115, 220, 230, 270, 272, 279, and 288.

[0066] More preferably, the variants of CHI3L1 include variants obtained by phosphorylation modification, and the phosphorylation modification sites include: Y189, T383.

[0067] More preferably, the variants of CHI3L1 include variants obtained by acetylation modification, and the acetylation modification sites include: K321, K335, and K342.

[0068] Preferably, the label on the CHI3L1 secondary antibody can catalyze or activate a chemiluminescent compound or fluorescent dye, thereby rapidly converting a colorless substrate into a colored product or causing a light change; or converting a non-fluorescent fluorescent dye into a strong fluorescent product.

[0069] More preferably, the label is an activator, which can catalyze or activate chemiluminescent compounds or fluorescent dyes, induce chemical reactions, and thus quickly convert colorless substrates into colored products, or cause light changes, or convert non-fluorescent fluorescent dyes into strong fluorescent products.

[0070] More preferably, the labels include: transition metal salts, complexes and enzymes.

[0071] Still more preferably, the transition metals include elements from Groups 3 to 12 of the Periodic Table of Elements, especially iron, copper, cobalt, zinc, manganese and chromium.

[0072] Still more preferably, the complex comprises ferrous protoporphyrin and Mn-TPPS4.

[0073] Even more preferably, the enzyme comprises peroxidase.

[0074] Still further preferably, the peroxidase includes lactoperoxidase, microperoxidase, myeloperoxidase, haloperoxidase such as vanadium bromoperoxidase, horseradish peroxidase, lignin peroxidase and Mn-dependent peroxidase produced in white rot fungi, and soybean peroxidase.

[0075] Preferably, the second CHI3L1 antibody includes any labeled or tagged antibody or antibody fragment capable of binding to CHI3L1, and may be a recombinant, chimeric, humanized, or murine antibody.

[0076] The CHI3L1 secondary antibody can also be prepared by standard antibody preparation techniques, by expressing or synthesizing CHI3L1 protein or polypeptide, and screening and preparing specific monoclonal or polyclonal antibodies against CHI3L1. Antibody labeling or tagging can be performed according to conventional methods, or commercially available CHI3L1 antibodies can be used.

[0077] Preferably, the kit further comprises an antigen.

[0078] More preferably, the antigen includes purified, semi-purified or non-purified proteins obtained from human blood samples, tissue fluids, lymph fluids, etc.

[0079] More preferably, the antigen also includes a recombinant protein.

[0080] More preferably, the source of the recombinant protein includes at least one of the following: Prokaryotic expression system, eukaryotic expression system, wheat germ expression system.

[0081] Preferably, the kit further comprises a buffer.

[0082] More preferably, the buffer comprises at least one of: PBS, TBS, HEPES.

[0083] Preferably, the kit further comprises a surfactant.

[0084] More preferably, the surfactant comprises at least one of the following: Twain, Triton.

[0085] Preferably, the kit further comprises a preservative.

[0086] More preferably, the preservative is at least one of the following: Sodium azide, proclin300, thimerosal.

[0087] Preferably, the kit further comprises BSA, OVA, collagen, bovine serum and other animal sera.

[0088] Preferably, the kit further comprises a reaction carrier.

[0089] More preferably, the reaction carrier comprises at least one of the following: PVDF membrane, NC membrane, magnetic particles, enzyme-linked microplate, chemiluminescent microplate, latex, fluorescent microspheres, immune microspheres.

[0090] Preferably, the kit further comprises an initiator.

[0091] More preferably, the initiator is used to initiate a chemical reaction between the label on the second CHI3L1 antibody and the chemiluminescent compound or color development substrate or fluorescent dye that is a reaction target of the label.

[0092] Preferably, the above kit identifies and / or monitors the prognosis of liver fibrosis and cirrhosis by measuring the content of CHI3L1 in serum.

[0093] More preferably, the method for determining the content of CHI3L1 in serum includes enzyme-linked immunosorbent assay, colloidal gold assay and magnetic particle chemiluminescence assay.

[0094] More preferably, the ELISA assay step comprises: The anti-CHI3L1 antibody is used to coat the microplate to make a solid-phase antibody. When using, add the sample to be tested to the antibody-coated microplate. If the sample contains the analyte, it can bind to the solid-phase antibody to form an antibody-antigen complex. Then add the horseradish peroxidase (HRP)-labeled anti-CHI3L1 antibody to form an antibody-antigen-enzyme-labeled antibody complex. Finally, add the 3,3',5,5'-tetramethylbenzidine substrate system to produce a color reaction. After the color reaction is completed, use an enzyme reader to detect. A standard curve is made based on the concentration of CHI3L1 in the calibrator and its corresponding OD value, and the CHI3L1 concentration in the sample is calculated using the standard curve.

[0095] More preferably, the kit for identifying and / or prognostic monitoring of liver fibrosis and cirrhosis (enzyme-linked immunosorbent assay) comprises the following reagents: CHI3L1 microwell ELISA plate: The microwells are coated with rat (or mouse) anti-CHI3L1 monoclonal antibody; CHI3L1 detection antibody: horseradish peroxidase-labeled goat anti-(or non-goat, such as camel) CHI3L1 polyclonal antibody, stored in enzyme-linked diluent containing bovine serum albumin; CHI3L1 sample diluent: It is composed of phosphate buffer containing bovine serum albumin, used to dilute the sample before detection; Color developer: buffer containing 3,3',5,5'-tetramethylbenzidine; Stop solution: dilute sulfuric acid solution; CHI3L1 Calibrator: Phosphate buffered saline containing recombinant CHI3L1 and bovine serum albumin.

[0096] More preferably, the colloidal gold method determination step comprises: Using colloidal gold test paper, the test strip (T) on the test strip is coated with mouse anti-CHI3L1 antibody corresponding to the analyte, and the control strip (C) is coated with mouse anti-peptide antibody. The chemical coupling pad is coated with colloidal gold-labeled goat anti-CHI3L1 antibody (or non-goat, such as camel) and colloidal gold-labeled peptide. During the test, the test sample is added to the sample addition end. When the sample contains the analyte, the analyte in the sample forms an immune complex with the colloidal gold-labeled goat anti-chitinase 3-like protein 1 antibody. After chromatography, the immune complex is captured by the test strip (T) to form a strip. At the same time, the colloidal gold-labeled peptide on the chemical coupling pad is captured by the control strip (C) to form another strip.

[0097] More preferably, the kit for identifying and / or prognostic monitoring of liver fibrosis and liver cirrhosis (colloidal gold method) comprises the following reagents: Mouse anti-CHI3L1 antibody, colloidal gold-labeled goat anti-CHI3L1 antibody (or non-goat, such as camel), mouse anti-peptide antibody, colloidal gold-labeled peptide, nitrocellulose membrane, glass cellulose membrane, absorbent paper, tetrachloroauric acid, bovine serum albumin, trehalose, trisodium citrate, PVC bottom plate, plastic shell.

[0098] More preferably, the magnetic particle chemiluminescence determination step comprises: After reacting the sample with the labeled anti-CHI3L1 monoclonal antibody and the labeled anti-CHI3L1 polyclonal antibody, an antibody-antigen-antibody sandwich complex is formed; microparticles coated with streptavidin are added, and the complex forms a solid phase under the interaction of streptavidin and biotin; the reaction solution is placed in a magnetic field, and the magnetic particles in the test will be adsorbed, and the unbound substances are washed away by washing; then, the substrate solution for the fully automatic immunoassay system is injected, and the chemiluminescent photon intensity is detected. The light intensity generated is proportional to the CHI3L1 concentration in the sample. The amount of analyte in the sample is determined by the stored multi-point calibration curve.

[0099] More preferably, the magnetic particle chemiluminescence method includes acridinium ester magnetic particle chemiluminescence method and alkaline phosphatase magnetic particle chemiluminescence method.

[0100] More preferably, the magnetic particles include at least one of the following: Amino magnetic beads, carboxyl magnetic beads, streptavidin, avidin, aldehyde, thiol, tosyl.

[0101] More preferably, the kit for identifying and / or prognostic monitoring of liver fibrosis and cirrhosis (acridinium ester magnetic microparticle chemiluminescence method) comprises the following reagents: Reagent 1: containing an anti-CHI3L1 monoclonal antibody with a concentration of 0.2 μg / mL to 1.0 μg / mL and treated with a cross-linking agent, biotin or fluorescein; the monoclonal antibody includes at least one of mouse anti-human CHI3L1 monoclonal antibody, goat anti-human CHI3L1 monoclonal antibody and rabbit anti-human CHI3L1 monoclonal antibody; Reagent 2: containing an anti-CHI3L1 polyclonal antibody labeled with horseradish peroxidase, alkaline phosphatase or acridinium ester at a concentration of 0.2 μg / mL to 1.0 μg / mL; the polyclonal antibody includes at least one of mouse anti-human CHI3L1 polyclonal antibody, goat anti-human CHI3L1 polyclonal antibody and rabbit anti-human CHI3L1 polyclonal antibody; Reagent 3: Contains 0.5~2mg / mL magnetic particles; Calibrator: Contains different amounts of recombinant CHI3L1 antigen; Control 1 and Control 2: Contain different amounts of recombinant chitinase 3-like protein 1 antigen; Sample diluent: protein buffer, Proclin300; Coupling buffer: PBST and / or TBST buffer.

[0102] More preferably, the kit for identifying and / or prognostic monitoring of liver fibrosis and cirrhosis (alkaline phosphatase microparticle chemiluminescence method) comprises the following reagents: Magnetic bead reagent: immunomagnetic beads linked to goat anti-fluorescein isothiocyanate (FITC) antibody, magnetic bead concentration 1.0-3.0 mg / mL; Anti-A reagent: FITC-labeled CHI3L1 monoclonal antibody dilution; Anti-B reagent: dilution of CHI3L1 polyclonal antibody labeled with alkaline phosphatase; Calibrator solution: contains different amounts of recombinant CHI3L1 antigen; Sample diluent: buffer, surfactant and stabilizer.

[0103] More preferably, the above identification and / or prognostic monitoring of liver fibrosis and cirrhosis includes distinguishing the compensated stage, decompensated stage and recompensated stage of cirrhosis.

[0104] More preferably, the threshold for distinguishing the compensated stage, decompensated stage and recompensated stage of liver cirrhosis is 120-200 ng / mL.

[0105] More preferably, the sensitivity of distinguishing the compensated, decompensated and recompensated stages of liver cirrhosis is 70-97% and the specificity is 70-83%.

[0106] More preferably, the sensitivity and specificity are obtained by ROC analysis.

[0107] More preferably, the above-mentioned liver cirrhosis includes liver cirrhosis caused by hepatitis B.

[0108] Compared with the prior art, the present invention has the following beneficial effects: The kit for detecting liver fibrosis and cirrhosis of the present invention can effectively distinguish between compensated cirrhosis and decompensated cirrhosis of the liver by measuring the content of CHI3L1 in the patient's serum, while effectively distinguishing between normal people and patients with cirrhosis, so that the test results are more accurate, which is conducive to more accurately judging the patient's condition. Among them, the detection threshold for distinguishing between normal people and patients with cirrhosis is 79ng / mL, and the specificity and sensitivity can reach 94.6% and 92.4% respectively; the detection threshold for distinguishing between compensated cirrhosis and decompensated cirrhosis of the liver is 138.5 ng / mL or 196.7 ng / mL. If the threshold is 138.5ng / mL, the sensitivity is as high as 97% and the specificity is 71%. If the specificity is to be improved, the threshold can be 196.7ng / mL, with a specificity of about 83% and a sensitivity of 70%. In addition, the kit of the present invention can also be used to distinguish whether the cirrhosis patient after treatment is in the recompensation stage of cirrhosis, and the detection threshold of the recompensation stage is 196.67ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1 ROC curves for CHI3L1 detection in serum samples of patients with cirrhosis, serum samples of patients with cirrhosis of hepatitis B, and serum samples of normal people; Figure 2 The box plots of CHI3L1 test results in serum samples of normal people, compensated cirrhosis, and decompensated cirrhosis patients; Figure 3 ROC curves for CHI3L1 detection in serum samples of patients with compensated and decompensated cirrhosis; Figure 4 It is the traceability chain of CHI3L1 standard product; Figure 5 Selected ion reaction monitoring analysis for chemically synthesized CHI3L1 peptide and heavy isotope labeled CHI3L1 peptide; Figure 6 Selected ion reaction monitoring analysis of CHI3L1 standard peptide and heavy isotope labeled CHI3L1 peptide; Figure 7 Statistical analysis of product calibrators and working calibrators; Figure 8 Selected ion reaction monitoring of chemically synthesized non-(light) isotope labeled CHI3L1 peptide; Fig. 9 Selected ion reaction monitoring of heavy isotope-labeled CHI3L1 peptide; Fig.10 Selected ion reaction monitoring analysis of CHI3L1 standard peptide (from CHI3L1 standard protein); Fig.11 is the mass spectrum of CHI3L1 peptide P1; Fig.12 is the mass spectrum of CHI3L1 peptide P2; Fig.13 is the mass spectrum of CHI3L1 peptide P3; Fig.14 is the mass spectrum of CHI3L1 peptide P4; Fig.15 This is the mass spectrum of CHI3L1 peptide P5. DETAILED DESCRIPTION

[0110] Here, exemplary embodiments will be described in detail, and the embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Instead, they are only examples of methods consistent with some aspects of the present disclosure.

[0111] The experimental methods in the following examples, unless otherwise specified, are all conventional methods or conditions recommended by the manufacturer. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0112] Example 1 Verify the ability of the kit of the present invention to distinguish between normal people and patients with liver fibrosis and cirrhosis 1. Sample Collection A total of 725 serum samples from normal people, 314 patients with liver cirrhosis and 112 patients with hepatitis B were collected; the samples came from the First Affiliated Hospital of Zhejiang University School of Medicine (referred to as Center 01), the Second Affiliated Hospital of Zhejiang University School of Medicine (referred to as Center 02), and the Run Run Shaw Hospital of Zhejiang University School of Medicine (referred to as Center 03).

[0113] The sample collection situation is shown in Table 1.

[0114] Table 1 Sample distribution

[0115] 2. Detection of CHI3L1 concentration in serum Use the kit of the present invention to perform detection according to one of the following detection methods: 1. Enzyme-linked immunosorbent assay: (1) Equilibrate the components of the kit to room temperature.

[0116] (2) Prepare detergent at working concentration.

[0117] (3) Dilute the sample: Dilute the serum sample with CHI3L1 sample diluent.

[0118] (4) Select a certain amount of CHI3L1 enzyme-labeled strips, place the microwell strips to be used securely on the stand, and number them according to the needs of the experiment.

[0119] (5) Add calibrators and diluted samples at 100 μL / well. Repeat for calibrators. Tap the plate gently to mix. Cover the plate with a self-adhesive sheet and incubate at 37°C for 30 minutes.

[0120] (6) Take out the ELISA plate, remove the adhesive sheet, discard the liquid in the wells, add 200 μL / well of the working concentration washing solution prepared in step (2), let it stand for 30-60 seconds, discard the washing solution, pat dry on clean absorbent paper, and wash 4 times. If using a plate washer, select the 4-time wash program, add 300 μL / well, and let the working concentration washing solution stay in the microwells for 30-60 seconds each time, and aspirate it cleanly each time without any residue. After washing, pat dry on clean absorbent paper.

[0121] (7) Add the detection antibody at 100 μL / well, tap the ELISA plate to mix, cover the plate with a self-adhesive sheet, and incubate at 37°C for 30 minutes.

[0122] (8) Repeat step (6).

[0123] (9) Add chromogen, 100 μL / well, and incubate at room temperature in the dark for 10 minutes.

[0124] (10) Add 50 μL / well of stop solution and tap the ELISA plate to mix thoroughly.

[0125] (11) Use the microplate reader to read the OD value at a wavelength of 450 nm.

[0126] 2. Colloidal gold method: (1) Return the serum to be tested to room temperature, mix well, and then use a pipette to draw 100 µL of the sample into a tube pre-filled with 400 µL of sample diluent. Mix well and set aside as the test solution; (2) Turn on the detector; (3) Mark the sample number, add the test solution, and count down for 15 minutes; (4) After the countdown is completed, put the sample into the detector for testing and the test results will be automatically read.

[0127] 3. Magnetic particle chemiluminescence method: (1) Load the reagent into the reagent compartment of the instrument and determine the name of the loaded reagent on the software operating system; (2) The reagents should be gently mixed before loading; (3) Load the test sample into the sample bin and determine the sample test items on the software operating system; (4) Fill the reaction cups fully, or load a corresponding number of reaction cups according to the experimental conditions; (5) The sample volume for each test is 10µL. The instrument automatically draws the matching sample diluent and dilutes it 20 times before testing. The instrument automatically gives the test result.

[0128] The test results are shown in Table 2.

[0129] Table 2 CHI3L1 detection results

[0130] Spearman correlation analysis was performed on the correlation between CHI3L1 and cirrhosis. The results showed that the correlation coefficient was r=0.751, p<0.0001, indicating that there was a positive correlation between the CHI3L1 content in serum and cirrhosis, and the CHI3L1 content in cirrhosis samples was higher than that in normal population samples.

[0131] Further analysis of the results revealed that Figure 1 The ROC curve is shown in Figure 3. The ROC analysis results are shown in Table 3.

[0132] Table 3 ROC analysis results of serum CHI3L1 levels in the diagnosis of liver cirrhosis

[0133] As shown in Tables 2 and 3, CHI3L1 can be used to distinguish normal people from patients with liver fibrosis and cirrhosis, and has excellent specificity and sensitivity, reaching 94.6% and 92.4% respectively. The optimal diagnostic critical value for detecting liver cirrhosis is 79 ng / mL.

[0134] Example 2 Verification of the ability of the kit of the present invention to distinguish patients with compensated cirrhosis from those with decompensated cirrhosis 1. Sample collection A total of 97 normal people, 41 patients with compensated cirrhosis, and 33 patients with decompensated cirrhosis were collected; the diagnostic criteria for compensated cirrhosis and decompensated cirrhosis were based on the "Guidelines for the Diagnosis and Treatment of Cirrhosis" (2019 Chinese Medical Association Hepatology Branch).

[0135] 2. Detection of CHI3L1 concentration in serum The kit of the present invention was used to perform detection according to the detection method described in Example 1. The detection results are shown in Table 4.

[0136] Table 4 Serum concentrations of CHI3L1 in normal subjects, compensated cirrhosis, and decompensated cirrhosis patients

[0137]

[0138] The data in Table 4 are analyzed to obtain the statistical results shown in Table 5. Further analysis shows that the box plots of normal population, compensated cirrhosis, and decompensated cirrhosis are as follows: Figure 2 As shown in the figure. Figure 3 shown.

[0139] Table 5 Statistical results of serum concentrations of CHI3L1 in normal population, compensated cirrhosis, and decompensated cirrhosis patients

[0140] As shown in Table 5, the median value of the total serum CHI3L1 concentration in the normal group was 44.31 ng / mL, and the 95% CI was 41.864-48.338 ng / mL. The median value of the total serum CHI3L1 concentration in the compensated cirrhosis group was 110.463 ng / mL, and the 95% CI was 96.681-132.249 ng / mL. The median value of the total serum CHI3L1 concentration in the decompensated cirrhosis group was 221.864 ng / mL, and the 95% CI was 200.322-290.803 ng / mL.

[0141] Figure 2 Outliers in the data set can be intuitively identified. Compensated cirrhosis has more high outliers, which may represent errors in clinical diagnosis and may indicate that these patients have reached the stage of decompensated cirrhosis. Therefore, using the chitin protein (CHI3L1) can help clinicians pay special attention to these patients who have decompensated cirrhosis.

[0142] Depend on Figure 3 It can be seen that the ROC analysis for compensated cirrhosis and decompensated cirrhosis showed that the area under the ROC curve (AUC) of the chitinase protein for distinguishing compensated cirrhosis from decompensated cirrhosis was 0.830. The Youden index (correct index) was 0.677. If the threshold is 138.5 ng / mL, its sensitivity is as high as 97% and its specificity is 71%. If the specificity is to be improved, the threshold can be set to 196.7 ng / mL, with a specificity of about 83% and a sensitivity of 70%.

[0143] Example 3 Verify the ability of the kit of the present invention to determine whether patients with decompensated cirrhosis can be reversed to compensated cirrhosis (recompensated cirrhosis) after treatment The concentrations of CHI3L1 in the serum of the relevant cases before and after treatment were detected using the kit of the present invention according to the detection method described in Example 1, as shown in Table 6.

[0144] Table 6 Serum CHI3L1 concentrations in patients with decompensated cirrhosis before and after treatment

[0145] As shown in Table 6, the level of chitin protein decreased significantly after treatment. According to the clinical threshold of 196.67 ng / mL, all patients were converted to compensated cirrhosis after treatment, indicating that CHI3L1 can be used to distinguish the recompensation stage of cirrhosis. The kit of the present invention can effectively detect patients in the recompensation stage of cirrhosis.

[0146] Test Example 1 Traceability of kit standards Standard product traceability chain Figure 4 As shown. The isotope-labeled specific peptide was quantitatively traced back to the CHI3L1 specific peptide by LC-MS, and the mass of the CHI3L1 specific peptide was determined by direct weighing.

[0147] Among them, the amino acid sequences of the isotope-labeled specific polypeptide and the CHI3L1-specific polypeptide are the same, ILGQQVPYATK. The difference is that the former is labeled with an isotope at lysine, which is ILGQQVPYAT (K*). After querying the UniProtKB protein sequence database, this sequence is a CHI3L1-specific polypeptide sequence with no overlap with other proteins, and can well represent CHI3L1.

[0148] The primary reference method uses the weighing method, and the primary reference material is the artificially synthesized peptide ILGQQVPYATK; the secondary reference method uses the isotope dilution method, and the secondary reference material is the isotope-labeled ILGQQVPYATK specific peptide, and the labeling site is lysine; the determination method uses the conventional operation steps in this field, which will not be repeated here. After measurement, the ratio (molar concentration ratio) of the traceability chain of chemically synthesized CHI3L1, heavy isotope-labeled CHI3L1 and CHI3L1 in the serum of hepatitis B patients with cirrhosis is: Synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 in the serum of cirrhotic hepatitis B patients = 218:780:220.

[0149] Test Example 2 Clinical detection of cirrhosis Using the kit of the present invention, 1039 serum samples of patients who may have liver cirrhosis were tested according to a detection threshold of 79 ng / mL. The test results were compared with the clinical diagnosis results and statistically analyzed.

[0150] The test results are shown in Table 7.

[0151] Table 7 Liver cirrhosis test results

[0152] The above statistical data were further evaluated by Kappa test to evaluate the consistency between the test results of the test kit and the clinical diagnosis. If the Kappa value is ≥0.75, the consistency is good; if 0.75>Kappa value ≥0.40, the consistency is moderate; if the Kappa value is <0.40, the consistency is not ideal. The Kappa value for judging liver cirrhosis in healthy individuals is 0.8586, with a 95% confidence interval of (0.950, 0.991), P <0.0001, and the results of the two determination methods can be considered to be highly consistent.

[0153] Test Example 3: Method for tracing or assigning values ​​of isotope-labeled CHI3L1 peptides to CHI3L1 standards (AUC method) 1. Tracing method The traceability chain is: chemically synthesized CHI3L1 polypeptide (weighing method); heavy isotope-labeled CHI3L1 polypeptide (LC-MS quantification); CHI3L1 standard protein (LC-MS quantification). The heavy isotope-labeled CHI3L1 polypeptide is quantitatively traced back to the chemically synthesized CHI3L1 polypeptide by LC-MS quantification, and the heavy isotope-labeled CHI3L1 polypeptide is quantitatively traced back to the chemically synthesized CHI3L1 polypeptide by LC-MS, while the chemically synthesized CHI3L1 polypeptide is mass determined by direct weighing. The amino acid sequence of the heavy isotope-labeled CHI3L1 polypeptide is the same as that of the chemically synthesized CHI3L1 polypeptide, which is ILGQQVPYATK. The difference is that the former is labeled with an isotope at K, i.e., lysine, which is ILGQQVPYAT (K*). After querying the UniProtKB protein sequence database, the sequence is a CHI3L1-specific polypeptide sequence, which has no overlap with other proteins and can well represent CHI3L1. The specific traceability process is: 1) Chemically synthesized CHI3L1 peptide ILGQQVPYATK (with 1 H + The molecular weight after purification is 1218.4) as the primary reference material, the weighing method is the primary reference method, and the concentration of the chemically synthesized CHI3L1 polypeptide is 1 mg / ml (determined by weighing method).

[0154] 2) Heavy isotope labeled CHI3L1 peptide ILGQQVPYAT(K*)[K(13 C6; 15 N2)] (with 1 H + The molecular weight after addition was 1226.4) as the secondary reference material. The secondary reference method was to use the isotope dilution method to compare the heavy isotope labeled CHI3L1 peptide ILGQQVPYAT (K*) and the chemically synthesized CHI3L1 peptide ILGQQVPYATK by selected ion reaction monitoring (SRM) to accurately assign the heavy isotope labeled CHI3L1 peptide. 100 fmol was loaded, and selected ion reaction monitoring (SRM) detection was performed using an Agilent 6460A triple quadrupole mass spectrometer (MS) connected to a liquid chromatography (LC) (LC / MS / MS). The main parameters of the triple quadrupole mass spectrometer connected to the liquid chromatography are: m / z range: 5 to 3000 m / z; polarity switching: 30 ms; mass resolution: automatic tuning, half-maximum full width 0.7 Da; mass accuracy: 0.1 Da in the range of 5 to 1000 m / z, 0.01% in the range of 1000 to 2000 m / z, and 0.02% in the range of 2000 to 3000 m / z; mass stability: <0.1 Da within 24 hours; dynamic range: 6.0×10 6 ; Maximum scan rate: 12500Da / s; Minimum SRM dwell time: 1ms; SRM ion pairs: 450 ion pairs per time period, >13500 ion pairs per method; Dynamic SRM ion pairs: 4500 ion pairs per method; Triggered MRM ion pairs: Up to 10 MRM ion pairs (primary and secondary) for library search and compound confirmation; Collision cell ion clearance: <1ms.

[0155] The selected ion reaction monitoring analysis of chemically synthesized CHI3L1 peptide and heavy isotope labeled CHI3L1 peptide is shown in Figure 5 , a is a heavy isotope labeled CHI3L1 polypeptide, b is a chemically synthesized CHI3L1 polypeptide, the quantification of the chemically synthesized CHI3L1 polypeptide and the heavy isotope labeled CHI3L1 polypeptide is calculated by AUC (area under the curve), and the average ratio is 1.38:1. If the concentration of the chemically synthesized CHI3L1 polypeptide is 1 mg / mL (determined by the weighing method), the calculated concentration of the heavy isotope labeled CHI3L1 polypeptide = 1 / 1.38 × (1226.4 / 1218.4) = 0.73 mg / mL. If the concentration of the chemically synthesized CHI3L1 polypeptide is 5 μM, the calculated concentration of the heavy isotope labeled CHI3L1 polypeptide is = 5 / 1.38 = 3.62 μM.

[0156] 3) CHI3L1 standard protein (Recombinant Human Chitinase 3-like 1, produced by Hangzhou Puwang Biotechnology Co., Ltd., hereinafter referred to as "Puwang") was completely digested with trypsin to obtain CHI3L1 standard peptide. The digestion reaction conditions were as follows: 5 μg of CHI3L1 standard protein was dissolved in 5 μL 8M urea / 50mM Tris-HCl (pH 8), 1 μL 5mM DTT was added to the solution, and incubated at 37°C for 1 hour; iodoacetamide was added to a final concentration of 15mM, and incubated at room temperature for another 30 minutes in the dark; the reaction solution was diluted with three times the volume of 50mM ammonium bicarbonate (pH 7.8); 5 μL trypsin (100ng / mL) was added and incubated at 37°C overnight. The CHI3L1 standard protein was completely digested with trypsin to obtain the CHI3L1 standard peptide ILGQQVPYATK. The CHI3L1 standard was detected by selected ion reaction monitoring (SRM) with 100 fmol of sample loaded. The main parameters of the triple quadrupole mass spectrometer connected to the liquid chromatography were the same as in 2).

[0157] See SIM analysis of CHI3L1 standard peptide (from CHI3L1 standard protein) and heavy isotope labeled CHI3L1 peptide. Figure 6 , c is the heavy isotope labeled CHI3L1 polypeptide, d is the CHI3L1 standard polypeptide, and the AUC ratio of the CHI3L1 standard polypeptide and the heavy isotope labeled CHI3L1 polypeptide is 0.81:1. During the traceability process, the concentration of the chemically synthesized CHI3L1 polypeptide was 5μM, the concentration of the heavy isotope labeled CHI3L1 polypeptide was 3.62μM, and the concentration of the CHI3L1 standard polypeptide calculated according to the traceability chain = 3.62×0.81=2.94μM (i.e., the molar concentration ratio of the chemically synthesized CHI3L1 polypeptide, the heavy isotope labeled CHI3L1 polypeptide and the CHI3L1 standard protein: 5:3.62:2.94). According to the molecular weight of the CHI3L1 standard protein of 42625.47, the mass concentration of the CHI3L1 standard protein = 2.94×42625.47 / 1000=125 (μg / ml).

[0158] 2. Assignment method 2.1. Restore the CHI3L1 product calibrator to room temperature and set aside for later use; use the test kit to calibrate the reference material with the seven concentrations of the CHI3L1 product calibrator as the calibrator; operate the kit strictly in accordance with the kit instructions. The test result of the reference material is required to be between 108-132ng / mL, that is, within 10% of the nominal concentration of the reference material of 120ng / mL. The specific method is: 1) Preparation of working calibrants Use the calibration diluent to prepare seven concentrations of 0 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL and 800 ng / mL as working calibrators. Pack the working calibrators of each concentration according to the specifications and store them below -70°C.

[0159] 2) Assignment of working calibration materials The test was performed using the manufacturer's selected measurement procedure. The working calibrator was tested for 3 consecutive days, 2 times a day, 3 times each time, and the mean value of the 18 test results was calculated as the value assigned to the working calibrator.

[0160] 3) Preparation of product calibrants Because the measurement procedure selected by the manufacturer is the same as the manufacturer's regular measurement procedure, in order to reduce the transmission links of uncertainty in the traceability chain, the product calibrator is directly selected from the working calibrator, so there is no need to prepare product calibrators.

[0161] 4) Product calibration value assignment Because the measurement procedure selected by the manufacturer is the same as the manufacturer's permanent measurement procedure, in order to reduce the transmission links of uncertainty in the traceability chain, the product calibrator directly uses the working calibrator, so there is no need to assign values ​​to the product calibrators.

[0162] 2.2. Assignment of working calibrators and product calibrators The measurement procedure selected by Puwang Reagents is used to assign values ​​to Puwang Reagent working calibrators. The fixed-value Puwang Reagent working calibrators are used to assign values ​​to Puwang Reagent calibrators through Puwang Reagents' standing measurement procedures.

[0163] The data of the working calibrators are shown in Table 8. The data of the working calibrators’ value assignment verification and calibration product adjustment are shown in Table 9. The data of the product calibrators’ value assignment are shown in Tables 10-15. The statistical analysis results of the product calibrators and working calibrators are shown in Table 10-15. Figure 7 , within the detection concentration range, it is highly linear, R 2 =0.9999.

[0164] Table 8 Working Calibrator Assignment Data

[0165] Table 9 Assignment verification and calibration product adjustment

[0166] Table 10 Assignment data of product calibrator (concentration 1)

[0167] Table 11 Assignment data of product calibrator (concentration 2)

[0168] Table 12 Assignment data of product calibrator (concentration 3)

[0169] Table 13 Assignment data of product calibrator (concentration 4)

[0170] Table 14 Assignment data of product calibrator (concentration 5)

[0171] Table 15 Assignment data of product calibrator (concentration 6)

[0172] Test example 4: Method 2 for tracing or assigning values ​​of isotope-labeled CHI3L1 peptides to CHI3L1 standards (ionic strength method) 1. Tracing method The traceability chain is: chemically synthesized CHI3L1 polypeptide (weighing method); heavy isotope-labeled CHI3L1 polypeptide (LC-MS quantification); CHI3L1 standard protein (LC-MS quantification). The heavy isotope-labeled CHI3L1 polypeptide is quantitatively traced back to the chemically synthesized CHI3L1 polypeptide by LC-MS quantification, and the heavy isotope-labeled CHI3L1 polypeptide is quantitatively traced back to the chemically synthesized CHI3L1 polypeptide by LC-MS, while the chemically synthesized CHI3L1 polypeptide is mass determined by direct weighing. The amino acid sequence of the heavy isotope-labeled CHI3L1 polypeptide is the same as that of the chemically synthesized CHI3L1 polypeptide, which is ILGQQVPYATK. The difference is that the former is labeled with an isotope at K, i.e., lysine, which is ILGQQVPYAT (K*). After querying the UniProtKB protein sequence database, the sequence is a CHI3L1-specific polypeptide sequence, which has no overlap with other proteins and can well represent CHI3L1. The specific traceability process is: 1) Chemically synthesized CHI3L1 peptide ILGQQVPYATK (with 1 H + The molecular weight after purification is 1218.4) as the primary reference material, the weighing method is the primary reference method, and the concentration of the chemically synthesized CHI3L1 polypeptide is 1 mg / ml (determined by weighing method).

[0173] 2) Heavy isotope labeled CHI3L1 peptide ILGQQVPYAT(K*)[K( 13 C6; 15 N2)] (with 1 H +The molecular weight after addition was 1226.4) as the secondary reference material. The secondary reference method was to use the isotope dilution method to compare the heavy isotope labeled CHI3L1 peptide ILGQQVPYAT (K*) and the chemically synthesized CHI3L1 peptide ILGQQVPYATK by selected ion reaction monitoring (SRM) to accurately assign the heavy isotope labeled CHI3L1 peptide. 100 fmol was loaded, and selected ion reaction monitoring (SRM) detection was performed using an Agilent 6460A triple quadrupole mass spectrometer (MS) connected to a liquid chromatography (LC) (LC / MS / MS). The main parameters of the triple quadrupole mass spectrometer connected to the liquid chromatography are: m / z range: 5 to 3000 m / z; polarity switching: 30 ms; mass resolution: automatic tuning, half-maximum full width 0.7 Da; mass accuracy: 0.1 Da in the range of 5 to 1000 m / z, 0.01% in the range of 1000 to 2000 m / z, and 0.02% in the range of 2000 to 3000 m / z; mass stability: <0.1 Da within 24 hours; dynamic range: 6.0×10 6 ; Maximum scan rate: 12500Da / s; Minimum SRM dwell time: 1ms; SRM ion pairs: 450 ion pairs per time period, >13500 ion pairs per method; Dynamic SRM ion pairs: 4500 ion pairs per method; Triggered MRM ion pairs: Up to 10 MRM ion pairs (primary and secondary) for library search and compound confirmation; Collision cell ion clearance: <1ms.

[0174] See SIM analysis of chemically synthesized CHI3L1 peptides without (light) isotope labeling and CHI3L1 peptides with heavy isotope labeling. Figure 8 and Fig. 9 According to the selected ion reaction monitoring (SRM) analysis of light isotope labeled CHI3L1 peptide, the secondary ion of the peptide, such as y9, can be used for quantification. The intensity of the y9 ion of the chemically synthesized non-(light) isotope labeled CHI3L1 peptide is 218, and the intensity of the y9 ion of the heavy isotope labeled CHI3L1 peptide ILGQQVPYAT (K*) is 780. The amount of heavy isotope peptide is 3.5778 times that of light isotope peptide (780 / 218).

[0175] 3) CHI3L1 standard protein (Recombinant Human Chitinase 3-like 1, produced by Hangzhou Puwang Biotechnology Co., Ltd., hereinafter referred to as "Puwang") was completely digested with trypsin to obtain CHI3L1 standard peptide. The digestion reaction conditions were as follows: 5 μg of CHI3L1 standard protein was dissolved in 5 μL 8M urea / 50mM Tris-HCl (pH 8), 1 μL 5mM DTT was added to the solution, and incubated at 37°C for 1 hour; iodoacetamide was added to a final concentration of 15mM, and incubated at room temperature for another 30 minutes in the dark; the reaction solution was diluted with three times the volume of 50mM ammonium bicarbonate (pH 7.8); 5 μL trypsin (100ng / mL) was added and incubated at 37°C overnight. The CHI3L1 standard protein was completely digested with trypsin to obtain the CHI3L1 standard peptide ILGQQVPYATK. The CHI3L1 standard was detected by selected ion reaction monitoring (SRM) with 100 fmol of sample loaded. The main parameters of the triple quadrupole mass spectrometer connected to the liquid chromatography were the same as in 2).

[0176] Selected ion reaction monitoring analysis of CHI3L1 standard peptide (from CHI3L1 standard protein) is shown in Fig.10 , the intensity of the y9 ion of the ILGQQVPYATK peptide after trypsin digestion of the CHI3L1 standard is 220. Therefore, it can be obtained that the ratio of the traceability chain of the synthetic CHI3L1 peptide: heavy isotope labeled peptide: CHI3L1 standard protein is 218:780:220 (molar concentration ratio calculated according to ion intensity).

[0177] 2. Assignment method 2.1. Restore the CHI3L1 product calibrator to room temperature and set aside for later use; use the test kit to calibrate the reference material with the seven concentrations of the CHI3L1 product calibrator as the calibrator; operate the kit strictly in accordance with the kit instructions. The test result of the reference material is required to be between 108-132ng / mL, that is, within 10% of the nominal concentration of the reference material of 120ng / mL. The specific method is: 1) Preparation of working calibrants Use the calibration diluent to prepare seven concentrations of 0 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL and 800 ng / mL as working calibrators. Pack the working calibrators of each concentration according to the specifications and store them below -70°C.

[0178] 2) Assignment of working calibration materials The test was performed using the manufacturer's selected measurement procedure. The working calibrator was tested for 3 consecutive days, 2 times a day, 3 times each time, and the mean value of the 18 test results was calculated as the value assigned to the working calibrator.

[0179] 3) Preparation of product calibrants Because the measurement procedure selected by the manufacturer is the same as the manufacturer's regular measurement procedure, in order to reduce the transmission links of uncertainty in the traceability chain, the product calibrator is directly selected from the working calibrator, so there is no need to prepare product calibrators.

[0180] 4) Product calibration value assignment Because the measurement procedure selected by the manufacturer is the same as the manufacturer's permanent measurement procedure, in order to reduce the transmission links of uncertainty in the traceability chain, the product calibrator directly uses the working calibrator, so there is no need to assign values ​​to the product calibrators.

[0181] 2.2. Assignment of working calibrators and product calibrators The measurement procedure selected by Puwang Reagents is used to assign values ​​to Puwang Reagent working calibrators. The fixed-value Puwang Reagent working calibrators are used to assign values ​​to Puwang Reagent calibrators through Puwang Reagents' standing measurement procedures.

[0182] The data of the working calibrator assignment are shown in Table 16, and the verification of the working calibrator assignment and the calibration adjustment are shown in Table 17. The data of the product calibrator assignment are shown in Tables 18-23. The statistical analysis results of the product calibrator and the working calibrator are shown in Table 17. Within the detection concentration range, they show a highly linear relationship, R 2 =0.9996.

[0183] Table 16 Working Calibrator Assignment Data

[0184] Table 17 Assignment verification and calibration product adjustment

[0185] Table 18 Assignment data of product calibrator (concentration 1)

[0186] Table 19 Assignment data of product calibrator (concentration 2)

[0187] Table 20 Assignment data of product calibrator (concentration 3)

[0188] Table 21 Assignment data of product calibrator (concentration 4)

[0189] Table 22 Assignment data of product calibrator (concentration 5)

[0190] Table 23 Assignment data of product calibrator (concentration 6)

[0191] Test Example 5: Screening of SRM peptides Factors to consider when selecting a good SRM peptide include: 1) The specificity of the peptide represents the strength of a protein, that is, there is no peptide that may be produced by other homologous or similar proteins; 2) Peptides without sequence uncertainty (e.g., sequence conflicts between public protein databases) or amino acids that show variability due to single nucleotide polymorphisms (SNPs); 3) Amino acids without known post-translational modification sites; 4) Physicochemical properties suitable for reliable detection by mass spectrometers and significant features that generate characteristic MS / MS fragmentation patterns through collision-induced dissociation (CID), including the comprehensive performance and specificity of the peptide in chromatographic retention time, precursor ion mass, and product ion mass. Ultimately, the optimal signal-to-noise ratios can be achieved for selective detection and quantitative analysis of CHI3L1 peptides in complex matrices (such as serum completely digested into peptides with trypsin).

[0192] The SRM peptides were detected by selected ion reaction monitoring (SRM) with a sample load of 100 fmol. The main parameters of the triple quadrupole mass spectrometer connected to the liquid chromatography were the same as in 2). Fig.11 is the mass spectrum of CHI3L1 peptide P1 (ILGQQVPYATK, SEQ ID NO.1), Fig.12 The mass spectrum of CHI3L1 peptide P2 (EAGTLAYYEICDFLR, SEQ ID NO. 2) is shown in FIG. Fig.13 is the mass spectrum of CHI3L1 peptide P3 (SFTLASSETGVGAPISGPGIPGR, SEQ ID NO.3), Fig.14 is the mass spectrum of CHI3L1 peptide P4 (TLLSVGGWNFGSQR, SEQ ID NO.4), Fig.15The mass spectrum of CHI3L1 peptide P5 (TLLSVGGWNFGSQR, SEQ ID NO.5) shows that CHI3L1 peptide P3, CHI3L1 peptide P4 and CHI3L1 peptide P5 do not have good product ions for quantitative analysis, so CHI3L1 peptide P3, CHI3L1 peptide P4 and CHI3L1 peptide P5 are excluded as SRM peptides. In addition, in SRM analysis, the reduction and alkylation steps of cysteine ​​in proteins are intended to make the cleavage site of proteases more accessible to proteases and obtain complete enzymatic digestion. However, as with any chemical reaction, the sample preparation process may introduce unpredictable deviations due to incomplete reactions of the cysteine ​​reduction and alkylation steps. Therefore, peptides containing amino acids that are easily chemically modified, such as cysteine ​​(incomplete reduction / alkylation) and methionine (partial oxidation), should be avoided. CHI3L1 peptide P3 (SFTLASSETGVGAPISGPGIPGR) contains cysteine ​​(C) and is not the best choice. Therefore, CHI3L1 peptide P2 was excluded from use as an SRM polypeptide.

[0193] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0194] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for identifying and / or prognostic monitoring of liver fibrosis and cirrhosis, characterized in that: The kit includes a standard product and / or a quality control product; the standard product includes a CHI3L1 standard product polypeptide assigned by a traceability method and / or a next-level standard product prepared with the CHI3L1 standard product polypeptide as a reference; the traceability method uses an isotope-labeled CHI3L1 polypeptide, and the specific polypeptide fragment sequence of the isotope-labeled CHI3L1 polypeptide is: ILGQQVPYATK, and the isotope includes 15 N and / or 13 C, Labeling site is at lysine.

2. The kit according to claim 1, wherein the isotope-labeled CHI3L1 polypeptide is ILGQQVPYAT(K*)[K( 13 C6; 15 N2)].

3. The kit according to claim 1, characterized in that In the process of tracing and / or assigning values ​​of the standard substance, the standard substance includes CHI3L1 polypeptide from cirrhotic serum of hepatitis B patients and / or chemically synthesized CHI3L1 polypeptide.

4. The kit according to claim 3, characterized in that During the traceability and / or assignment process of the standard, the ratio of the traceability chains of the chemically synthesized CHI3L1 polypeptide, the heavy isotope-labeled CHI3L1 polypeptide and the CHI3L1 polypeptide in the cirrhotic serum of the hepatitis B patient is: chemically synthesized CHI3L1 polypeptide: heavy isotope-labeled CHI3L1 polypeptide: CHI3L1 polypeptide in the cirrhotic serum of the hepatitis B patient = 200-220:770-780:200-220.

5. The kit according to claim 1, characterized in that The quality control product includes a CHI3L1 standard polypeptide assigned a value by a traceability method.

6. The kit according to claim 1, characterized in that The kit identifies and / or monitors the prognosis of liver fibrosis and liver cirrhosis by measuring the content of CHI3L1 in serum.

7. The kit according to claim 6, characterized in that The identification and / or prognostic monitoring of liver fibrosis and cirrhosis includes distinguishing the compensated stage, decompensated stage and recompensated stage of cirrhosis.

8. The kit according to claim 7, characterized in that The threshold for distinguishing the compensated, decompensated, and recompensated stages of liver cirrhosis is 120-200 ng / mL.

9. The kit according to claim 8, characterized in that: The sensitivity of distinguishing the compensated stage, decompensated stage and recompensated stage of liver cirrhosis is 70-97%, and the specificity is 70-83%.

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