A kit for the detection of liver fibrosis and cirrhosis

Abstract

The application provides a kit for detecting liver fibrosis and cirrhosis, and belongs to the technical field of biotechnology.The kit can effectively distinguish normal people and cirrhosis patients, and can effectively distinguish compensated cirrhosis patients and decompensated cirrhosis patients, so that the detection result is more accurate, and the condition of the patient can be more accurately judged. Moreover, the kit has good standard products, and the accuracy of detection can be further improved.

Description

Technical Field

[0001] The present 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 mid-to-late stage of liver fibrosis. Its morphological definition is diffuse liver fibrosis accompanied by abnormal nodule formation. Diffuse fibrosis alone without nodule formation (such as in congenital hepatic fibrosis) or nodule formation without fibrosis (such as in nodular regenerative hyperplasia) does not qualify as cirrhosis. From a clinical perspective, cirrhosis refers to the manifestations of liver failure and portal hypertension caused by the aforementioned liver histopathological changes. Pathologically, chronic inflammatory necrosis first leads to the proliferation and deposition of fibrous connective tissue in the liver (fibrosis), which then leads to the destruction of the hepatic lobule architecture and the formation of pseudolobules, ultimately progressing to cirrhosis.

[0003] The compensated stage of cirrhosis is asymptomatic, 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. Cirrhosis is a common liver disease in China, with 3% of patients with compensated cirrhosis entering the decompensated stage each year. The prognosis for decompensated cirrhosis is generally poor, with a 5-year survival rate of only 14-35% with conventional treatment. Patients with decompensated cirrhosis frequently 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, as well as compensated and decompensated cirrhosis, is of great significance for the diagnosis and treatment of cirrhosis. Currently, there are many methods for detecting cirrhosis. For example, Chinese Patent CN103160575A discloses the application of the SAA1β / β homozygous genotype in the prognosis and diagnosis of cirrhosis. This patent uses real-time fluorescence quantitative allele-specific PCR to detect human SAA1β / β homozygous genotypes and non-SAA1β / β homozygous genotypes, thereby enabling detection of people susceptible to cirrhosis. Chinese Patent CN103160507A discloses miRNA serum markers for detecting cirrhosis and their applications. These methods detect at the gene or mRNA level, which is relatively cumbersome and has high testing costs. There are also methods that utilize antigen-antibody reactions to detect at the protein level, but when these methods are converted into practical kits, poor standards may affect detection specificity and sensitivity. Furthermore, there are few studies or reports on methods or products that can simply and accurately distinguish the compensated and decompensated stages of liver fibrosis and cirrhosis. Therefore, significant research is still needed in 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 cirrhosis. The kit of the present invention has good standard products and can effectively ensure the accuracy of the test results. In addition to being able to accurately distinguish whether a patient has liver fibrosis or 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 solution is adopted.

[0007] The present invention discloses isotope-labeled CHI3L1, wherein the specific polypeptide fragment sequence of CHI3L1 is: ILGQQVPYATK; the isotope includes 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 derived from the serum of patients with cirrhosis of the liver due to hepatitis B virus (HBV), or chemically synthesized CHI3L1. Serum derived from patients with cirrhosis of the liver due to HBV infection contains all structural variants of the chitinase protein, particularly those involved in HBV-related fibrosis. The various protein modifications, epitopes, and immunogenicity in serum from patients with HBV infection are more consistent with actual protein characteristics, making it a better standard and source for assigning values.

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

[0011] Preferably, the traceability ratio of chemically synthesized CHI3L1, isotope-labeled CHI3L1, and CHI3L1 in the serum of hepatitis B patients with cirrhosis is:

[0012] Synthetic CHI3L1: isotope-labeled CHI3L1: CHI3L1 in the serum of hepatitis B patients with cirrhosis = 150-250:700-850:150-250.

[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:

[0014] Synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 in cirrhotic serum of hepatitis B patients = 200-220:770-780:200-220.

[0015] More preferably, the traceability ratio of chemically synthesized CHI3L1, heavy isotope-labeled CHI3L1, and CHI3L1 in the serum of hepatitis B patients with cirrhosis is:

[0016] Synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 from cirrhotic serum of hepatitis B patients = 218:780:220.

[0017] The present invention also discloses the use of isotope-labeled CHI3L1 as a reference material in standard traceability and / or value assignment. The isotope-labeled CHI3L1 is labeled with a heavy isotope or a light isotope, and the labeling site is lysine.

[0018] Preferably, the isotopes include 15 N and / or 13 C.

[0019] Preferably, the CHI3L1-specific sequence is ILGQQVPYATK. This sequence is a CHI3L1-specific polypeptide sequence that has no cross-reactivity with other proteins and can well represent CHI3L1, making detection more accurate.

[0020] Preferably, isotope-labeled CHI3L1 is used as a secondary reference material.

[0021] Preferably, the gravimetric method is the primary reference method.

[0022] Preferably, the secondary reference method is isotope dilution, and the heavy isotope-labeled polypeptide is accurately assigned by comparing the heavy isotope-labeled ILGQQVPYATK with the light isotope-labeled ILGQQVPYATK using reaction monitoring and mass spectrometry.

[0023] Preferably, in the above use, the traceability ratio of chemically synthesized CHI3L1, heavy isotope-labeled CHI3L1, and CHI3L1 in the serum of hepatitis B patients with cirrhosis is:

[0024] Synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 in cirrhotic serum of hepatitis B patients = 150-250:700-850:150-250.

[0025] More preferably, in the above use, the traceability ratio of the chemically synthesized CHI3L1, the heavy isotope-labeled CHI3L1, and the CHI3L1 in the serum of hepatitis B patients with cirrhosis is:

[0026] Synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 in cirrhotic serum of hepatitis B patients = 200-220:770-780:200-220.

[0027] More preferably, in the above use, the traceability ratio of the chemically synthesized CHI3L1, the heavy isotope-labeled CHI3L1 and the CHI3L1 in the serum of hepatitis B patients with cirrhosis is:

[0028] The ratio of synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 in serum of hepatitis B patients with cirrhosis is 218:780:220.

[0029] The present invention also discloses the use of the aforementioned isotope-labeled CHI3L1 as a reference material in the traceability or assignment of CHI3L1 standard protein, with the isotope-labeled CHI3L1 serving as a secondary reference material. The chemically synthesized CHI3L1 polypeptide is quantified gravimetrically; the isotope-labeled CHI3L1 is a heavy isotope-labeled CHI3L1 polypeptide, which is quantified by liquid chromatography-mass spectrometry (LC-MS); the CHI3L1 standard protein is quantified by LC-MS, which is then traced back to the heavy isotope-labeled CHI3L1 polypeptide by LC-MS, which is then traced back to the chemically synthesized CHI3L1 polypeptide by LC-MS. The chemically synthesized CHI3L1 polypeptide is then mass-determined by direct gravimetric weighing, allowing for precise quantification to International System of Units (SI) units.

[0030] Preferably, the chemically synthesized CHI3L1 polypeptide ILGQQVPYATK is the primary reference material, and the gravimetric method is the primary reference method.

[0031] Preferably, the isotope-labeled CHI3L1 is a heavy isotope-labeled CHI3L1 polypeptide.

[0032] More preferably, the heavy isotope-labeled CHI3L1 polypeptide is ILGQQVPYAT(K*)[K( 13 C6; 15 N2)].

[0033] Preferably, the secondary referencing method is by isotope dilution.

[0034] 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.

[0035] More preferably, the molar concentration ratio of the chemically synthesized CHI3L1 polypeptide to the heavy isotope-labeled CHI3L1 polypeptide is 1.2-1.5:1.

[0036] More preferably, the molar concentration ratio of the chemically synthesized CHI3L1 polypeptide to the heavy isotope-labeled CHI3L1 polypeptide is 1.38:1.

[0037] Preferably, the CHI3L1 standard protein is completely digested with trypsin to form a CHI3L1 standard polypeptide.

[0038] Preferably, the molar concentration ratio of the CHI3L1 standard polypeptide to the heavy isotope-labeled CHI3L1 polypeptide is 0.6-0.9:1.

[0039] More preferably, the molar concentration ratio of the CHI3L1 standard polypeptide to the heavy isotope-labeled CHI3L1 polypeptide is 0.7-0.9:1.

[0040] More preferably, the molar concentration ratio of the CHI3L1 standard polypeptide to the heavy isotope-labeled CHI3L1 polypeptide is 0.81:1.

[0041] The present invention also discloses a method for tracing the source of a CHI3L1 standard protein, comprising:

[0042] The chemically synthesized CHI3L1 peptide ILGQQVPYATK was used as the primary reference material, and the chemically synthesized CHI3L1 peptide was quantified by the gravimetric method;

[0043] Isotope-labeled CHI3L1 was used as a secondary reference material and isotope-labeled CHI3L1 was quantified by LC-MS; and,

[0044] The CHI3L1 standard protein was completely digested with trypsin to produce CHI3L1 standard peptides, which were then quantified by LC-MS.

[0045] In the method of the present invention, the CHI3L1 standard protein is quantified by LC-MS, which is then traced back to isotope-labeled CHI3L1 by LC-MS. The isotope-labeled CHI3L1 is then 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 mass units of the International System of Units (SI).

[0046] Preferably, the isotope-labeled CHI3L1 is a heavy isotope-labeled CHI3L1 polypeptide.

[0047] More preferably, the heavy isotope-labeled CHI3L1 polypeptide is ILGQQVPYAT(K*)[K( 13 C6; 15 N2)].

[0048] More preferably, the traceability chain ratio 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.

[0049] 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.

[0050] The present invention also discloses a kit for liver fibrosis and cirrhosis, which includes a standard product and / or a quality control product; the standard product includes a CHI3L1 standard product assigned a value by the above-mentioned traceability method and / or a next-level standard product prepared with reference to the CHI3L1 standard product.

[0051] Preferably, the quality control product includes a CHI3L1 standard product assigned a value by the above-mentioned traceability method.

[0052] The present invention also discloses a kit for liver fibrosis and liver cirrhosis, comprising a standard product; the standard product is CHI3L1.

[0053] Preferably, the CHI3L1 used as a standard is derived from the serum of a cirrhotic hepatitis B patient or is chemically synthesized. When the CHI3L1 standard is obtained from the serum of a patient, especially a Chinese patient, the standard has broad applicability and the test results are accurate.

[0054] Preferably, CHI3L1 used as a standard is in various forms including solutions.

[0055] Preferably, the standard product derived from the patient is obtained by conventional protein separation and purification methods.

[0056] Preferably, the N-terminus or C-terminus of CHI3L1 used as a standard includes a tag.

[0057] Preferably, the above kit further comprises:

[0058] The antibody that can bind to CHI3L1 is called the primary antibody;

[0059] The antibody containing a label that can bind to CHI3L1 is called a secondary antibody.

[0060] The present invention also discloses the use of CHI3L1 in preparing a kit for identifying and / or prognostic monitoring of liver fibrosis and cirrhosis.

[0061] Preferably, the above-mentioned identification and / or prognostic monitoring of liver fibrosis and cirrhosis includes distinguishing between the compensated, decompensated, and recompensated stages of cirrhosis. By measuring the level of CHI3L1 in the patient's serum, it is possible to effectively distinguish between normal people and patients with cirrhosis, and also effectively distinguish between patients with compensated cirrhosis and patients with decompensated cirrhosis, making the test results more accurate and facilitating a more accurate diagnosis of the patient's condition.

[0062] Preferably, the detection method applicable to the above kit includes at least one of the following:

[0063] Colloidal gold method, enzyme-linked immunosorbent assay, magnetic particle chemiluminescence method, fluorescence method, microfluidics, latex turbidimetry.

[0064] More preferably, the luminescent substance of the magnetic particle chemiluminescence method includes at least one of the following:

[0065] Acridinium ester, alkaline phosphatase, horseradish peroxidase, ABEI.

[0066] More preferably, the fluorescence method includes a dry method and a wet method.

[0067] Preferably, the above kit comprises:

[0068] The antibody that can bind to CHI3L1 is called the primary antibody;

[0069] The antibody containing a label that can bind to CHI3L1 is called a secondary antibody.

[0070] More preferably, the sources of the above antibodies include at least one of the following:

[0071] Mice, rats, rabbits, sheep, chickens, camels, monkeys, and humans.

[0072] More preferably, the method for preparing the above-mentioned antibody comprises at least one of the following:

[0073] Hybridoma technology, B cell cloning technology, phage display technology, recombinant expression technology, and humanization technology.

[0074] Preferably, the first CHI3L1 antibody can be immobilized on a solid support.

[0075] 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.

[0076] More preferably, the nanoparticles have an average diameter of 0.1-10 μm.

[0077] 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.

[0078] More preferably, the first antibody is a monoclonal antibody or a polyclonal antibody.

[0079] More preferably, the first antibody is a polyclonal antibody. Using a polyclonal antibody, various CHI3L1 variants can be captured.

[0080] More preferably, the variants of CHI3L1 include variants obtained by various modifications.

[0081] 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).

[0082] 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.

[0083] More preferably, the variants of CHI3L1 include variants obtained by phosphorylation modification, and the phosphorylation modification sites include: Y189, T383.

[0084] More preferably, the variants of CHI3L1 include variants obtained by acetylation modification, and the acetylation modification sites include: K321, K335, and K342.

[0085] 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 strongly fluorescent product.

[0086] More preferably, the label is an activator, which can catalyze or activate a chemiluminescent compound or fluorescent dye, inducing a chemical reaction, thereby rapidly converting a colorless substrate into a colored product, or causing a light change, or converting a non-fluorescent fluorescent dye into a strongly fluorescent product.

[0087] More preferably, the labels include transition metal salts, complexes and enzymes.

[0088] 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.

[0089] Still more preferably, the complex comprises ferrous protoporphyrin and Mn-TPPS4.

[0090] Even more preferably, the enzyme comprises peroxidase.

[0091] 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.

[0092] Preferably, the second CHI3L1 antibody includes any labeled or tagged antibody or antibody fragment capable of binding to CHI3L1, and can be a recombinant, chimeric, humanized, or murine antibody.

[0093] CHI3L1 secondary antibodies can also be prepared by standard antibody preparation techniques, by expressing or synthesizing CHI3L1 proteins or polypeptides, and screening and preparing specific monoclonal or polyclonal antibodies against CHI3L1. Antibodies can be labeled or tagged according to conventional methods, or commercially available CHI3L1 antibodies can be used.

[0094] Preferably, the kit further comprises an antigen.

[0095] More preferably, the antigen includes purified, semi-purified or non-purified proteins obtained from human blood samples, tissue fluid, lymph fluid, etc.

[0096] More preferably, the antigen also includes a recombinant protein.

[0097] More preferably, the source of the recombinant protein includes at least one of the following:

[0098] Prokaryotic expression system, eukaryotic expression system, wheat germ expression system.

[0099] Preferably, the kit further comprises a buffer.

[0100] More preferably, the buffer comprises at least one of the following:

[0101] PBS, TBS, HEPES.

[0102] Preferably, the kit further comprises a surfactant.

[0103] More preferably, the surfactant comprises at least one of the following:

[0104] Twain, Triton.

[0105] Preferably, the kit further comprises a preservative.

[0106] More preferably, the preservative is at least one of the following:

[0107] Sodium azide, proclin300, thimerosal.

[0108] Preferably, the kit further comprises BSA, OVA, collagen, bovine serum and other animal serum.

[0109] Preferably, the kit further comprises a reaction carrier.

[0110] More preferably, the reaction carrier comprises at least one of the following:

[0111] PVDF membrane, NC membrane, magnetic particles, enzyme-linked microplate, chemiluminescent microplate, latex, fluorescent microspheres, immune microspheres.

[0112] Preferably, the kit further comprises an initiator.

[0113] More preferably, the initiator is used to initiate a chemical reaction between the label on the secondary CHI3L1 antibody and the chemiluminescent compound or colorimetric substrate or fluorescent dye that is a reaction target of the label.

[0114] Preferably, the above kit identifies and / or monitors the prognosis of liver fibrosis and cirrhosis by measuring the content of CHI3L1 in serum.

[0115] 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.

[0116] More preferably, the enzyme-linked immunosorbent assay step comprises:

[0117] A microplate is coated with an anti-CHI3L1 antibody to create a solid-phase antibody. The sample to be tested is added to the antibody-coated microplate. If the sample contains the analyte, it binds to the solid-phase antibody, forming an antibody-antigen complex. Horseradish peroxidase (HRP)-labeled anti-CHI3L1 antibody is then added to form an antibody-antigen-enzyme-labeled antibody complex. Finally, a 3,3',5,5'-tetramethylbenzidine substrate system is added to initiate a colorimetric reaction. After the colorimetric reaction, the sample is detected using a microplate reader. A standard curve is constructed based on the CHI3L1 concentration in the calibrator and its corresponding OD value. The CHI3L1 concentration in the sample is calculated using the standard curve.

[0118] More preferably, the kit for identifying and / or prognostic monitoring of liver fibrosis and cirrhosis (enzyme-linked immunosorbent assay) comprises the following reagents:

[0119] CHI3L1 microwell ELISA plate: The microwells are coated with rat (or mouse) anti-CHI3L1 monoclonal antibody;

[0120] CHI3L1 detection antibody: horseradish peroxidase-conjugated goat anti-CHI3L1 (or non-goat, such as camel) polyclonal antibody, stored in enzyme-linked diluent containing bovine serum albumin;

[0121] CHI3L1 sample diluent: phosphate buffered saline containing bovine serum albumin, used to dilute samples before testing;

[0122] Color development reagent: buffer containing 3,3',5,5'-tetramethylbenzidine;

[0123] Stop solution: dilute sulfuric acid solution;

[0124] CHI3L1 calibrator: phosphate-buffered saline containing recombinant CHI3L1 and bovine serum albumin.

[0125] More preferably, the colloidal gold method determination step includes:

[0126] Colloidal gold test paper is used. The test strip (T) is coated with a mouse anti-CHI3L1 antibody corresponding to the analyte, while the control strip (C) is coated with a mouse anti-peptide antibody. The chemical coupling pad is coated with colloidal gold-labeled goat anti-CHI3L1 (or non-goat, such as camel) anti-CHI3L1 antibody and a colloidal gold-labeled peptide. During testing, the sample is added to the sample loading port. When the sample contains the analyte, it 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 single band. Simultaneously, the colloidal gold-labeled peptide on the chemical coupling pad is captured by the control strip (C) to form another band.

[0127] More preferably, the kit for identifying and / or prognostic monitoring of liver fibrosis and cirrhosis (colloidal gold method) comprises the following reagents:

[0128] 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 housing.

[0129] More preferably, the magnetic particle chemiluminescence determination step includes:

[0130] The sample reacts with labeled anti-CHI3L1 monoclonal and polyclonal antibodies to form an antibody-antigen-antibody sandwich complex. Streptavidin-coated microparticles are added, and the complex forms a solid phase through the interaction of streptavidin and biotin. The reaction solution is placed in a magnetic field, which attracts the magnetic particles being tested. Unbound matter is removed by washing. The substrate solution for the automated immunoassay system is then injected, and the chemiluminescent photon intensity is measured. The generated light intensity is proportional to the CHI3L1 concentration in the sample. The amount of analyte in the sample is determined using a stored multi-point calibration curve.

[0131] More preferably, the magnetic particle chemiluminescence method includes acridinium ester magnetic particle chemiluminescence method and alkaline phosphatase magnetic particle chemiluminescence method.

[0132] More preferably, the magnetic particles include at least one of the following:

[0133] Amino magnetic beads, carboxyl magnetic beads, streptavidin, avidin, aldehyde, sulfhydryl, tosyl.

[0134] 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:

[0135] Reagent 1: Contains an anti-CHI3L1 monoclonal antibody at a concentration of 0.2 μg / mL to 1.0 μg / mL, which has been treated with a crosslinker, 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;

[0136] Reagent 2: Contains 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;

[0137] Reagent 3: Contains 0.5~2mg / mL magnetic particles;

[0138] Calibrator: Contains different amounts of recombinant CHI3L1 antigen;

[0139] Control 1 and Control 2: Contain different amounts of recombinant chitinase 3-like protein 1 antigen;

[0140] Sample diluent: protein buffer, Proclin300;

[0141] Coupling buffer: PBST and / or TBST buffer.

[0142] More preferably, the kit for identifying and / or prognostic monitoring of liver fibrosis and cirrhosis (alkaline phosphatase microparticle chemiluminescence method) comprises the following reagents:

[0143] Magnetic bead reagent: immunomagnetic beads linked to goat anti-fluorescein isothiocyanate (FITC) antibody, bead concentration 1.0-3.0 mg / mL;

[0144] Anti-A reagent: FITC-labeled CHI3L1 monoclonal antibody dilution solution;

[0145] Anti-B reagent: dilution of alkaline phosphatase-labeled CHI3L1 polyclonal antibody;

[0146] Calibrator solution: contains different amounts of recombinant CHI3L1 antigen;

[0147] Sample diluent: buffer, surfactant and stabilizer.

[0148] More preferably, the identification and / or prognostic monitoring of liver fibrosis and cirrhosis includes distinguishing between the compensated stage, the decompensated stage and the recompensated stage of cirrhosis.

[0149] More preferably, the threshold value for distinguishing the compensated stage, decompensated stage and recompensated stage of liver cirrhosis is 120-200 ng / mL.

[0150] More preferably, the sensitivity for distinguishing the compensated, decompensated, and recompensated stages of cirrhosis is 70-97%, and the specificity is 70-83%.

[0151] More preferably, the sensitivity and specificity are obtained by ROC analysis.

[0152] More preferably, the above-mentioned liver cirrhosis includes liver cirrhosis caused by hepatitis B.

[0153] Compared with the prior art, the present invention has the following beneficial effects:

[0154] The test kit for detecting liver fibrosis and cirrhosis of the present invention measures the level of CHI3L1 in the patient's serum. While effectively distinguishing between normal people and patients with cirrhosis, it can also effectively distinguish between patients with compensated cirrhosis and patients with decompensated cirrhosis, making the test results more accurate and facilitating a more accurate diagnosis of the patient's condition. Among them, the detection threshold for distinguishing between normal people and patients with cirrhosis is 79 ng / mL, with a specificity and sensitivity of up to 94.6% and 92.4%, respectively; the detection threshold for distinguishing between patients with compensated cirrhosis and patients with decompensated cirrhosis is 138.5 ng / mL or 196.7 ng / mL. If the threshold is 138.5 ng / mL, the sensitivity is as high as 97% and the specificity is 71%. If the specificity is to be improved, the threshold can be set to 196.7 ng / mL, with a specificity of approximately 83% and a sensitivity of 70%. In addition, the test kit of the present invention can also be used to distinguish whether a patient with cirrhosis after treatment is in the recompensation stage of cirrhosis. The detection threshold for the recompensation stage is 196.67 ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0155] Figure 1 ROC curves for CHI3L1 detection in serum samples from patients with cirrhosis, serum samples from patients with cirrhosis of the liver, and serum samples from normal people;

[0156] Figure 2 The box plots are for the CHI3L1 test results in serum samples of normal people, compensated cirrhosis, and decompensated cirrhosis patients;

[0157] Figure 3 ROC curves for CHI3L1 detection in serum samples of patients with compensated and decompensated cirrhosis;

[0158] Figure 4 It is the traceability chain of CHI3L1 standard product;

[0159] Figure 5 Selected ion reaction monitoring analysis of chemically synthesized CHI3L1 peptide and heavy isotope-labeled CHI3L1 peptide;

[0160] Figure 6 Selected ion reaction monitoring analysis of CHI3L1 standard peptide and heavy isotope-labeled CHI3L1 peptide;

[0161] Figure 7 Statistical analysis of product calibrators and working calibrators;

[0162] Figure 8 Selected ion reaction monitoring of chemically synthesized non-(light) isotope-labeled CHI3L1 peptide;

[0163] Figure 9Selected ion reaction monitoring of heavy isotope-labeled CHI3L1 peptide;

[0164] Figure 10 Selected ion reaction monitoring analysis of CHI3L1 standard peptide (derived from CHI3L1 standard protein);

[0165] Figure 11 is the mass spectrum of CHI3L1 peptide P1;

[0166] Figure 12 is the mass spectrum of CHI3L1 peptide P2;

[0167] Figure 13 is the mass spectrum of CHI3L1 peptide P3;

[0168] Figure 14 is the mass spectrum of CHI3L1 peptide P4;

[0169] Figure 15 This is the mass spectrum of CHI3L1 peptide P5. DETAILED DESCRIPTION

[0170] Here, exemplary embodiments will be described in detail. 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.

[0171] The experimental methods in the following examples, unless otherwise specified, are conventional methods or follow the conditions recommended by the manufacturer. The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0172] Example 1

[0173] Verify the ability of the kit of the present invention to distinguish normal people from patients with liver fibrosis and cirrhosis

[0174] 1. Sample Collection

[0175] A total of 725 serum samples from normal subjects, 314 patients with 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 Sir Run Run Shaw Hospital of Zhejiang University School of Medicine (referred to as Center 03).

[0176] The sample collection situation is shown in Table 1.

[0177] Table 1 Sample distribution

[0178]

[0179] 2. Detection of CHI3L1 Concentration in Serum

[0180] Use the kit of the present invention to perform detection according to one of the following detection methods:

[0181] 1. Enzyme-linked immunosorbent assay:

[0182] (1) Equilibrate all components of the kit to room temperature.

[0183] (2) Prepare working concentration detergent.

[0184] (3) Dilute the sample: Dilute the serum sample with CHI3L1 sample diluent.

[0185] (4) Select a certain amount of CHI3L1 enzyme-labeled strips, place the microwell strips used securely on the stand, and number them according to the needs of the experiment.

[0186] (5) Add calibrators and diluted samples at 100 μL / well. Repeat the wells for calibrators. Tap the plate gently to mix. Cover the plate with a self-adhesive sheet and incubate at 37°C for 30 minutes.

[0187] (6) Remove the ELISA plate, remove the adhesive film, discard the liquid in the wells, and add 200 μL / well of the working concentration wash solution prepared in step (2). Let it stand for 30-60 seconds, discard the wash solution, and pat dry on clean absorbent paper. Wash 4 times. If using a plate washer, select the 4-wash program, add 300 μL / well, and let the working concentration wash solution stay in the microwells for 30-60 seconds each time. Aspirate thoroughly each time to remove any residue. Pat dry on clean absorbent paper after washing.

[0188] (7) Add the detection antibody at 100 μL / well, tap the ELISA plate to mix, cover the ELISA plate with a self-adhesive sheet, and incubate at 37°C for 30 minutes.

[0189] (8) Repeat step (6).

[0190] (9) Add 100 μL / well of color developer and incubate at room temperature in the dark for 10 minutes.

[0191] (10) Add 50 μL / well of stop solution and tap the ELISA plate to mix thoroughly.

[0192] (11) Use a microplate reader at a wavelength of 450 nm to read the OD value.

[0193] 2. Colloidal gold method:

[0194] (1) Return the serum to be tested to room temperature, mix thoroughly, and then use a pipette to draw 100 μL of the sample into a tube pre-filled with 400 μL of sample diluent. Mix thoroughly and use it as the test solution.

[0195] (2) Turn on the detector;

[0196] (3) Mark the sample number, add the test solution, and count down for 15 minutes;

[0197] (4) After the countdown is completed, put it into the detector for testing and the test results will be automatically judged.

[0198] 3. Magnetic particle chemiluminescence method:

[0199] (1) Load the reagent into the reagent compartment of the instrument and determine the name of the loaded reagent in the software operating system;

[0200] (2) The reagents should be gently mixed before loading;

[0201] (3) Load the test sample into the sample bin and determine the sample test items on the software operating system;

[0202] (4) Fill the reaction cups completely, or load a corresponding number of reaction cups according to the experimental conditions;

[0203] (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 results.

[0204] The test results are shown in Table 2.

[0205] Table 2 CHI3L1 detection results

[0206]

[0207] 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.

[0208] Further analysis of the results revealed Figure 1 The ROC curve is shown in Figure 3, and the ROC analysis results are shown in Table 3.

[0209] Table 3 ROC analysis results of serum CHI3L1 levels in the diagnosis of liver cirrhosis

[0210]

[0211] As shown in Tables 2 and 3, CHI3L1 can be used to distinguish between normal people and patients with liver fibrosis and cirrhosis, with excellent specificity and sensitivity, reaching 94.6% and 92.4%, respectively. The optimal diagnostic cutoff value for detecting cirrhosis is 79 ng / mL.

[0212] Example 2

[0213] Verify the ability of the kit of the present invention to distinguish patients with compensated cirrhosis from patients with decompensated cirrhosis

[0214] 1. Sample collection

[0215] 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).

[0216] 2. Detection of CHI3L1 concentration in serum

[0217] The kit of the present invention was used to perform the test according to the test method described in Example 1. The test results are shown in Table 4.

[0218] Table 4 Serum concentrations of CHI3L1 in normal subjects, compensated cirrhosis, and decompensated cirrhosis patients

[0219]

[0220]

[0221] The data in Table 4 were analyzed to obtain the statistical results shown in Table 5. Further analysis showed that the box plots of normal population, compensated cirrhosis and decompensated cirrhosis were as follows: Figure 2 As shown in the ROC curve Figure 3 shown.

[0222] Table 5 Statistical results of serum CHI3L1 concentrations in normal subjects, compensated cirrhosis, and decompensated cirrhosis patients

[0223]

[0224] As shown in Table 5, the median serum total CHI3L1 concentration in the normal group was 44.31 ng / mL, with a 95% CI of 41.864-48.338 ng / mL. The median serum total CHI3L1 concentration in the compensated cirrhosis group was 110.463 ng / mL, with a 95% CI of 96.681-132.249 ng / mL. The median serum total CHI3L1 concentration in the decompensated cirrhosis group was 221.864 ng / mL, with a 95% CI of 200.322-290.803 ng / mL.

[0225] Figure 2Outliers in the dataset can be intuitively identified. The high number of high outliers in compensated cirrhosis may represent errors in clinical diagnosis and may indicate that these patients have reached the stage of decompensated cirrhosis. Therefore, using the chitinase protein (CHI3L1) can help clinicians pay special attention to these patients as they have decompensated cirrhosis.

[0226] Depend on Figure 3 Receiver operating characteristic (ROC) analysis of chitinase protein for distinguishing compensated and decompensated cirrhosis showed that the area under the ROC curve (AUC) was 0.830. The Youden index (accuracy index) was 0.677. A threshold of 138.5 ng / mL achieved a sensitivity of 97% and a specificity of 71%. To improve specificity, a threshold of 196.7 ng / mL could be used, yielding a specificity of approximately 83% and a sensitivity of 70%.

[0227] Example 3

[0228] 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

[0229] The concentrations of CHI3L1 in the serum of 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.

[0230] Table 6 Serum CHI3L1 concentrations in patients with decompensated cirrhosis before and after treatment

[0231]

[0232] 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 patients in the recompensated stage of cirrhosis. The kit of the present invention can effectively detect patients in the recompensated stage of cirrhosis.

[0233] Test Example 1

[0234] Traceability of kit standards

[0235] Standard product traceability chain Figure 4 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.

[0236] 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*). This sequence was queried in the UniProtKB protein sequence database and is a CHI3L1-specific polypeptide sequence with no overlap with other proteins, and can well represent CHI3L1.

[0237] The primary reference method used a weighing method, and the primary reference material was the synthetic peptide ILGQQVPYATK. The secondary reference method used an isotope dilution method, and the secondary reference material was an isotope-labeled ILGQQVPYATK-specific peptide, with the labeling site being lysine. The assays all followed conventional procedures in the field and are not detailed here. The ratio (molar concentration ratio) of the traceability chain between chemically synthesized CHI3L1, heavy isotope-labeled CHI3L1, and CHI3L1 in the serum of hepatitis B patients with cirrhosis was determined to be:

[0238] Synthetic CHI3L1: heavy isotope labeled CHI3L1: CHI3L1 from cirrhotic serum of hepatitis B patients = 218:780:220.

[0239] Test Example 2

[0240] Clinical detection of cirrhosis

[0241] Using the kit of the present invention, serum samples from 1039 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.

[0242] The test results are shown in Table 7.

[0243] Table 7 Liver cirrhosis test results

[0244]

[0245] The Kappa test was used to evaluate the consistency between the test kit's results and the clinical diagnosis. A Kappa value ≥ 0.75 indicated good consistency; a Kappa value greater than or equal to 0.40 indicated moderate consistency; and a Kappa value less than 0.40 indicated poor consistency. The Kappa value for cirrhosis in healthy individuals was 0.8586, with a 95% confidence interval of (0.950, 0.991) and a P value less than 0.0001, indicating high consistency between the two assays.

[0246] Test Example 3:

[0247] Method for tracing or assigning values ​​of isotope-labeled CHI3L1 peptides to CHI3L1 standards (AUC method)

[0248] 1. Traceability method

[0249] 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 via LC-MS quantification, and the heavy isotope-labeled CHI3L1 polypeptide is quantitatively traced back to the chemically synthesized CHI3L1 polypeptide via LC-MS quantification. The chemically synthesized CHI3L1 polypeptide has the same amino acid sequence as the chemically synthesized CHI3L1 polypeptide, ILGQQVPYATK. The difference is that the heavy isotope-labeled CHI3L1 polypeptide is labeled with an isotope at K, i.e., lysine, resulting in 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. The specific traceability process is as follows:

[0250] 1) Chemically synthesized CHI3L1 peptide ILGQQVPYATK (with 1 H + The molecular weight after purification was 1218.4) as the primary reference material, the gravimetric method was used as the primary reference method, and the concentration of the chemically synthesized CHI3L1 polypeptide was 1 mg / ml (determined by gravimetric method).

[0251] 2) Heavy isotope labeled CHI3L1 peptide ILGQQVPYAT(K*) [K( 13 C6; 15 N2)] (with 1 H +The molecular weight after the reaction was 1226.4) was used 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 in the selected ion reaction monitoring (SRM) mode 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 as follows: m / z range: 5 to 3000 m / z; polarity switching: 30 ms; mass resolution: automatic tuning, full width at half maximum 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: 1 millisecond; 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.

[0252] Selected ion reaction monitoring analysis of chemically synthesized CHI3L1 peptide and heavy isotope labeled CHI3L1 peptide can be found in Figure 5 Figure a shows a heavy isotope-labeled CHI3L1 peptide, and b shows a chemically synthesized CHI3L1 peptide. The chemically synthesized and heavy isotope-labeled CHI3L1 peptides were quantified by calculating the AUC (area under the curve), with an average ratio of 1.38:1. If the concentration of the chemically synthesized CHI3L1 peptide is 1 mg / mL (determined gravimetrically), the calculated concentration of the heavy isotope-labeled CHI3L1 peptide is 1 / 1.38 × (1226.4 / 1218.4) = 0.73 mg / mL. If the concentration of the chemically synthesized CHI3L1 peptide is 5 μM, the calculated concentration of the heavy isotope-labeled CHI3L1 peptide is 5 / 1.38 = 3.62 μM.

[0253] 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 produce CHI3L1 standard peptide. The digestion reaction conditions were as follows: 5 μg of CHI3L1 standard protein was dissolved in 5 μL of 8M urea / 50mM Tris-HCl (pH 8). 1 μL of 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 for an additional 30 minutes at room temperature in the dark. The reaction solution was diluted with three volumes of 50mM ammonium bicarbonate (pH 7.8). 5 μL of trypsin (100ng / mL) was added and incubated at 37°C overnight. The CHI3L1 standard protein was completely digested with trypsin to produce the CHI3L1 standard peptide ILGQQVPYATK. The CHI3L1 standard was detected by selected ion reaction monitoring (SRM) with a loading of 100 fmol. The main parameters of the triple quadrupole mass spectrometer connected to the liquid chromatography were the same as in 2).

[0254] See SIM analysis of CHI3L1 standard peptide (derived from CHI3L1 standard protein) and heavy isotope-labeled CHI3L1 peptide. Figure 6 Figure c represents a heavy isotope-labeled CHI3L1 peptide, and d represents a CHI3L1 standard peptide. The AUC ratio between the CHI3L1 standard and heavy isotope-labeled CHI3L1 peptides is 0.81:1. During the traceability process, the concentrations of the chemically synthesized CHI3L1 peptide were 5 μM, and the heavy isotope-labeled CHI3L1 peptide were 3.62 μM. The calculated concentration of the CHI3L1 standard peptide based on the traceability chain is 3.62 × 0.81 = 2.94 μM (i.e., the molar concentration ratio of the chemically synthesized CHI3L1 peptide, heavy isotope-labeled CHI3L1 peptide, and CHI3L1 standard protein is 5:3.62:2.94). Based on the molecular weight of the CHI3L1 standard protein, 42625.47, the mass concentration of the CHI3L1 standard protein is 2.94 × 42625.47 / 1000 = 125 μg / ml.

[0255] 2. Assignment method

[0256] 2.1. Bring the CHI3L1 product calibrator to room temperature and set aside. Use the test kit to calibrate the reference material using the seven concentrations of the CHI3L1 product calibrator as calibrants. Operate the kit strictly according to the kit instructions. The test result for the reference material should be between 108-132 ng / mL, that is, within 10% of the nominal concentration of 120 ng / mL. The specific method is as follows:

[0257] 1) Preparation of working calibrants

[0258] 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 using the calibrator diluent as working calibrators. Aliquot the working calibrators according to specifications and store below -70°C.

[0259] 2) Assignment of working calibrants

[0260] The working calibrator is tested using the manufacturer's selected measurement procedure. The working calibrator is tested for three consecutive days, twice a day, three times each day. The mean of the 18 test results is used as the assigned value for the working calibrator.

[0261] 3) Preparation of product calibrators

[0262] 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.

[0263] 4) Assignment of product calibration materials

[0264] 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 calibrator.

[0265] 2.2. Assignment of working calibrators and product calibrators

[0266] The measurement procedures selected by Puwang Reagents are 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 Reagent's established measurement procedures.

[0267] The data on the value assignment of working calibrators are shown in Table 8. The data on the value assignment of working calibrators and the calibration adjustment are shown in Table 9. The data on the value assignment of product calibrators are shown in Tables 10-15. The statistical analysis results of product calibrators and working calibrators are shown in Figure 7 , within the detection concentration range, it shows a highly linear relationship, R 2 =0.9999.

[0268] Table 8 Working calibrator assignment data

[0269]

[0270] Table 9 Assignment verification and calibration adjustment

[0271]

[0272] Table 10 Assignment data of product calibrator (concentration 1)

[0273]

[0274] Table 11 Assignment data of product calibrator (concentration 2)

[0275]

[0276] Table 12 Assignment data of product calibrator (concentration 3)

[0277]

[0278] Table 13 Assignment data of product calibrator (concentration 4)

[0279]

[0280] Table 14 Assignment data of product calibrator (concentration 5)

[0281]

[0282] Table 15 Assignment data of product calibrator (concentration 6)

[0283]

[0284] Test Example 4:

[0285] Method 2 for tracing or assigning values ​​of isotope-labeled CHI3L1 peptides to CHI3L1 standards (ionic strength method)

[0286] 1. Traceability method

[0287] 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 via LC-MS quantification, and the heavy isotope-labeled CHI3L1 polypeptide is quantitatively traced back to the chemically synthesized CHI3L1 polypeptide via LC-MS quantification. The chemically synthesized CHI3L1 polypeptide has the same amino acid sequence as the chemically synthesized CHI3L1 polypeptide, ILGQQVPYATK. The difference is that the heavy isotope-labeled CHI3L1 polypeptide is labeled with an isotope at K, i.e., lysine, resulting in 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. The specific traceability process is as follows:

[0288] 1) Chemically synthesized CHI3L1 peptide ILGQQVPYATK (with 1 H + The molecular weight after purification was 1218.4) as the primary reference material, the gravimetric method was used as the primary reference method, and the concentration of the chemically synthesized CHI3L1 polypeptide was 1 mg / ml (determined by gravimetric method).

[0289] 2) Heavy isotope labeled CHI3L1 peptide ILGQQVPYAT(K*) [K( 13 C6; 15 N2)] (with 1 H + The molecular weight after the reaction was 1226.4) was used 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 in the selected ion reaction monitoring (SRM) mode 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 as follows: m / z range: 5 to 3000 m / z; polarity switching: 30 ms; mass resolution: automatic tuning, full width at half maximum 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: 1 millisecond; 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.

[0290] The selected ion reaction monitoring analysis of chemically synthesized non-(light) isotope-labeled CHI3L1 peptide and heavy isotope-labeled CHI3L1 peptide can be found in Figure 8 and Figure 9According to the selected ion reaction monitoring (SRM) analysis of light isotope-labeled CHI3L1 peptides, the secondary ions of the peptides, 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, while the intensity of the y9 ion of the heavy isotope-labeled CHI3L1 peptide ILGQQVPYAT (K*) is 780. The amount of the heavy isotope peptide is 3.5778 times that of the light isotope peptide (780 / 218).

[0291] 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 produce CHI3L1 standard peptide. The digestion reaction conditions were as follows: 5 μg of CHI3L1 standard protein was dissolved in 5 μL of 8M urea / 50mM Tris-HCl (pH 8). 1 μL of 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 for an additional 30 minutes at room temperature in the dark. The reaction solution was diluted with three volumes of 50mM ammonium bicarbonate (pH 7.8). 5 μL of trypsin (100ng / mL) was added and incubated at 37°C overnight. The CHI3L1 standard protein was completely digested with trypsin to produce the CHI3L1 standard peptide ILGQQVPYATK. The CHI3L1 standard was detected by selected ion reaction monitoring (SRM) with a loading of 100 fmol. The main parameters of the triple quadrupole mass spectrometer connected to the liquid chromatography were the same as in 2).

[0292] Selected ion reaction monitoring analysis of CHI3L1 standard peptide (from CHI3L1 standard protein) is shown in Figure 10 The intensity of the y9 ion of the ILGQQVPYATK peptide after trypsin digestion of the CHI3L1 standard is 220. Therefore, the traceability chain ratio of the synthesized CHI3L1 peptide: heavy isotope-labeled peptide: CHI3L1 standard protein is 218:780:220 (molar concentration ratio calculated based on ionic strength).

[0293] 2. Assignment method

[0294] 2.1. Bring the CHI3L1 product calibrator to room temperature and set aside. Use the test kit to calibrate the reference material using the seven concentrations of the CHI3L1 product calibrator as calibrants. Operate the kit strictly according to the kit instructions. The test result for the reference material should be between 108-132 ng / mL, that is, within 10% of the nominal concentration of 120 ng / mL. The specific method is as follows:

[0295] 1) Preparation of working calibrants

[0296] 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 using the calibrator diluent as working calibrators. Aliquot the working calibrators according to specifications and store below -70°C.

[0297] 2) Assignment of working calibrants

[0298] The working calibrator is tested using the manufacturer's selected measurement procedure. The working calibrator is tested for three consecutive days, twice a day, three times each day. The mean of the 18 test results is used as the assigned value for the working calibrator.

[0299] 3) Preparation of product calibrators

[0300] 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.

[0301] 4) Assignment of product calibration materials

[0302] 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 calibrator.

[0303] 2.2. Assignment of working calibrators and product calibrators

[0304] The measurement procedures selected by Puwang Reagents are 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 Reagent's established measurement procedures.

[0305] The value assignment data of the working calibrator are shown in Table 16, and the value assignment verification and calibration adjustment of the working calibrator are shown in Table 17. The value assignment data of the product calibrator 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, there is a highly linear relationship, R 2 =0.9996.

[0306] Table 16 Working Calibrator Assignment Data

[0307]

[0308] Table 17 Assignment verification and calibration adjustment

[0309]

[0310] Table 18 Assignment data of product calibrator (concentration 1)

[0311]

[0312] Table 19 Assignment data of product calibrator (concentration 2)

[0313]

[0314] Table 20 Assignment data of product calibrator (concentration 3)

[0315]

[0316] Table 21 Assignment data of product calibrator (concentration 4)

[0317]

[0318] Table 22 Assignment data of product calibrator (concentration 5)

[0319]

[0320] Table 23 Assignment data of product calibrator (concentration 6)

[0321]

[0322] Test Example 5:

[0323] Screening of SRM peptides

[0324] Factors to consider when selecting a good SRM peptide include: 1) peptide specificity, representing the strength of a protein, i.e., the absence of peptides that may be derived from other homologous or similar proteins; 2) peptides without sequence uncertainty (e.g., sequence conflicts between public protein databases) or amino acid variability due to single nucleotide polymorphisms (SNPs); 3) amino acids without known post-translational modification sites; and 4) physicochemical properties suitable for reliable detection by mass spectrometry and the generation of a characteristic MS / MS fragmentation pattern via collision-induced dissociation (CID). This includes a comprehensive performance and specificity of the peptide in terms of chromatographic retention time, precursor ion mass, and product ion mass. Ultimately, the goal is to achieve selective detection and quantification of CHI3L1 peptides with optimal signal-to-noise ratios in complex matrices (e.g., serum completely digested into peptides with trypsin).

[0325] Selected ion reaction monitoring (SRM) was used to detect SRM peptides, 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). Figure 11 is the mass spectrum of CHI3L1 peptide P1 (ILGQQVPYATK, SEQ ID NO.1), Figure 12 This is the mass spectrum of CHI3L1 peptide P2 (EAGTLAYYEICDFLR, SEQ ID NO. 2), Figure 13 This is the mass spectrum of CHI3L1 peptide P3 (SFTLASSETGVGAPISGPGIPGR, SEQ ID NO.3), Figure 14 is the mass spectrum of CHI3L1 peptide P4 (TLLSVGGWNFGSQR, SEQ ID NO.4), Figure 15 The mass spectrum of CHI3L1 peptide P5 (TLLSVGGWNFGSQR, SEQ ID NO. 5) revealed that CHI3L1 peptides P3, P4, and P5 did not produce good product ions for quantitative analysis, thus eliminating them as peptides for SRM analysis. Furthermore, in SRM analysis, the reduction and alkylation steps of cysteine ​​residues in proteins are intended to make the protease cleavage site more accessible and allow for complete enzymatic digestion. However, as with any chemical reaction, this sample preparation process can introduce unpredictable bias due to incomplete cysteine ​​reduction and alkylation. Therefore, peptides containing amino acids susceptible to chemical modification, such as cysteine ​​(incomplete reduction / alkylation) and methionine (partial oxidation), should be avoided. CHI3L1 peptide P3 (SFTLASSETGVGAPISGPGIPGR), which contains cysteine ​​(C), is not an optimal choice. Therefore, the CHI3L1 peptide P2 was excluded from use as an SRM polypeptide.

[0326] 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.

[0327] 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 scope of protection of the present invention.

Claims

1. Use of an isotope-labeled CHI3L1 polypeptide in the preparation of a kit for identifying and / or prognostic monitoring of liver fibrosis and cirrhosis, comprising: The isotope-labeled CHI3L1 polypeptide is ILGQQVPYAT (K*) [K ( 13 C6; 15 N2)], isotope-labeled CHI3L1 peptides were used to trace the origin and assign values ​​to CHI3L1 standards; The traceability method includes: The chemically synthesized CHI3L1 peptide ILGQQVPYATK was used as the primary reference material, and the chemically synthesized CHI3L1 peptide was quantified by the gravimetric method; The isotope-labeled CHI3L1 polypeptide is used as a secondary reference material. The isotope-labeled CHI3L1 polypeptide is quantified by LC-MS. The specific polypeptide fragment sequence of the isotope-labeled CHI3L1 polypeptide is: ILGQQVPYATK. The isotope includes 15 N and / or 13 C, labeling site is at lysine; and, The CHI3L1 standard protein was completely digested with trypsin to produce CHI3L1 standard peptides, which were then quantified by LC-MS. During the standard traceability and / or value assignment process, the ratio of the traceability chains of the chemically synthesized CHI3L1 polypeptide, the heavy isotope-labeled CHI3L1 polypeptide, and the CHI3L1 polypeptide from the serum of a hepatitis B patient with cirrhosis is: chemically synthesized CHI3L1 polypeptide: heavy isotope-labeled CHI3L1 polypeptide: CHI3L1 polypeptide from the serum of a hepatitis B patient with cirrhosis = 200-220:770-780:200-220; The molar concentration ratio of the chemically synthesized CHI3L1 polypeptide and the heavy isotope-labeled CHI3L1 polypeptide is 1.1-1.6:1; the molar concentration ratio of the CHI3L1 standard polypeptide and the heavy isotope-labeled CHI3L1 polypeptide is 0.6-0.9:1; The kit includes a first antibody and a second antibody, wherein the first antibody is an antibody that binds to CHI3L1; and the second antibody is an antibody that binds to CHI3L1 and contains a marker.

2. The use according to claim 1, characterized in that The CHI3L1 standard polypeptide includes a CHI3L1 polypeptide from the sera of hepatitis B patients with cirrhosis and / or a chemically synthesized CHI3L1 polypeptide.

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