CN114478785A

CN114478785A - Mouse-derived monoclonal antibody of anti-human CHI3L1 and application

Info

Publication number: CN114478785A
Application number: CN202210023663.XA
Authority: CN
Inventors: 芮兵
Original assignee: Nanjing Baikang Biotechnology Co ltd
Current assignee: Nanjing Baikang Biotechnology Co ltd
Priority date: 2021-11-22
Filing date: 2022-01-11
Publication date: 2022-05-13
Also published as: CN113817064A

Abstract

The invention discloses a mouse-derived monoclonal antibody of anti-human CHI3L1, which comprises CHI-Ab1 and CHI-Ab2, and the antibody has strong affinity and specificity to CHI3L1 protein. In addition, the invention also discloses a chemiluminescence kit prepared by using the antibody, which has high sensitivity and precision and can meet the application requirement. The method can be used for quantitatively detecting the content of CHI3L1 in human serum, judging the process and the severity of hepatic fibrosis, and can be used as an early screening index of liver cirrhosis and liver cancer.

Description

Anti-human CHI3L1 mouse-derived monoclonal antibody and application thereof

Technical Field

The invention belongs to the field of antibody preparation and sequence determination, and particularly relates to a mouse-derived monoclonal antibody of anti-human CHI3L1 and application thereof.

Background

Chitinase-3-like protein 1(CHI 3L1), a YKL-40 protein, is a heparin-binding glycoprotein of the secreted chitinase protein family, having a molecular weight of about 40 kDa. CHI3L1 was first found in the milk secretion of non-lactating cows and was secreted by a variety of cells, including mainly: chondrocytes and fibroblast-like synoviocytes, activated macrophages and macrophages of late differentiated stages, neutrophils from arthritic patients; other cell types, such as human osteosarcoma cells (MG63), differentiated vascular smooth muscle cells, mammary epithelial cells, subpopulations of macrophages, smooth muscle cells of different inflammatory tissues, and a variety of cells, also detected expression of CHI3L 1.

Hepatic fibrosis is a pathophysiological process, which refers to abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. There are many causes of hepatic fibrosis, and viral hepatitis, alcoholic liver, fatty liver, autoimmune diseases, etc. are common in clinic. Any liver injury has liver fibrosis in the process of liver repair and healing, and if the injury factor cannot be removed for a long time, the fibrosis process can be continuously developed into liver cirrhosis for a long time. Hepatic fibrosis can cause a series of symptoms such as fatigue, weakness, anorexia, nausea, vomiting, chronic dyspepsia, abdominal flatulence, constipation or diarrhea, liver area vague pain and the like; many chronic hepatitis patients have gastric symptoms such as acid regurgitation, belching, hiccup, epigastric dull pain and epigastric fullness; chronic hepatitis affects the synthesis of prothrombin and other blood coagulation factors due to hypohepatia, the clinical manifestations of hepatic fibrosis often show spider nevus, epistaxis and gingival bleeding, skin and mucous membrane have purpura or bleeding spots, women often have menorrhagia symptoms.

Four examinations of liver fibrosis are generally required for diagnosing liver fibrosis. The examination is mainly used for examining and diagnosing the disease development condition and the treatment effect of patients with chronic liver diseases and measuring the important basis of inflammation activity and fibrosis degree. The four liver fibrosis items mainly comprise the following four inspection indexes:

PCIII (type III procollagen), which reflects collagen type III synthesis in the liver, and the serum content is consistent with the degree of hepatic fibrosis and is obviously related to the level of serum gamma-globulin. PC III is closely related to the activity degree of hepatic fibrosis formation, but has no specificity. PC iii is also elevated when other organs are fibrotic. Chronic hepatitis patients with persistent elevation of PC III suggest development of the disease towards cirrhosis, and reduction of PC III to normal may indicate remission of the disease.

2. IV-C (type IV collagen) is a main component forming basement membrane, reflects the collagen renewal rate of the basement membrane, can more sensitively reflect the hepatic fibrosis process when the content is increased, and is one of the early markers of the hepatic fibrosis. (1) The appearance of the liver fibrosis is the earliest, and the method is suitable for early diagnosis of the liver fibrosis. (2) Can reflect the degree of hepatic fibrosis, and the content of IV-C collagen in serum is gradually increased along with the evolution of chronic hepatitis → liver cirrhosis → liver cancer course.

LN (laminin), a specific non-collagenous structural protein in basement membrane, positively correlated with the degree of hepatic fibrosis activity and portal vein pressure, and markedly increased in slow-living liver and cirrhosis and primary liver cancer. LN may also reflect the progression and severity of liver fibrosis. In addition, higher LN levels are more pronounced in patients with cirrhosis. Reflecting that the liver fibrosis contains a small amount of LN in normal liver interstitium, and in hepatic fibrosis and cirrhosis, myofibroblasts increase → a large amount of interstitial components such as collagen and LN are synthesized and secreted → an intact basement membrane (liver sinus capillarity) is formed. Sinusoidal liver capillary sclerosis is a characteristic pathological change of cirrhosis. LN is positively correlated with the degree of fibrosis and portal hypertension, and the later stage of fibrosis is particularly obviously raised.

HA (hyaluronidase) is one of matrix components, is synthesized by mesenchymal cells, can accurately and sensitively reflect the amount of fibers generated in the liver and the damage condition of the hepatic cells, and is considered to be a sensitive index for hepatic fibrosis and cirrhosis, which can completely reflect the overall appearance of the diseased liver compared with hepatic biopsy. However, the four examinations of liver fibrosis are not specific because they are greatly affected by the inflammation of the liver, and are only used for clinical reference.

The expression level of chitinase 3-like protein 1(CHI 3L1) in the liver is particularly active, the content of the chitinase 3-like protein 1 is 15-227 times of that of other tissues and organs (such as brain, heart, mammary gland and the like) of a human body, the specificity of CHI3L1 in the liver is highest, and meanwhile, the absolute expression level of the chitinase 3-like protein 1 in the liver is also higher, and the chitinase 3-like protein 1 is a gene with high liver enrichment. The concentration of CHI3L1 in serum was shown to be increased in patients with chronic liver disease, and the serum test results of most patients with alcoholic cirrhosis or hepatitis showed a significant increase in the concentration of CHI3L 1. Serum CHI3L1 was closely related to the degree of fibrosis, as determined by the highest level of moderate to severe fibrosis in the pathological patients. Slightly fibrotic patients had elevated serum CHI3L1 to a lesser extent, but this elevation was still significantly greater than in patients without fibrosis. Therefore, the CHI3L1 level can reflect the degree of hepatic fibrosis and can be used as a diagnostic marker of hepatic fibrosis.

Hepatic fibrosis is a key stage of chronic liver diseases, can diagnose the hepatic fibrosis timely and accurately, and has important significance for preventing and treating the progress of diseases such as cirrhosis or liver cancer. Currently, the detection methods on the market are Westernblot, Elisa and the like. The methods are complex in operation, long in time consumption, narrow in detection range, poor in precision, and incapable of realizing automatic detection due to the fact that operation of professionals is needed.

The chemiluminescence immunoassay method has been developed to the present and become a mature and advanced ultramicro active substance detection technology, the application range is wide, and the chemiluminescence immunoassay technology as a main means of disease diagnosis has been widely used in vitro diagnosis experiments in the aspects of organism immune function, infectious diseases, endocrine function, tumor markers, sex hormone, thyroid function and the like. Chemiluminescence was used as a test for measuring CHI3L1 levels in serum.

Disclosure of Invention

In order to solve the problem of the demand of antibody raw materials in the market, the invention provides a mouse-derived monoclonal antibody of anti-human CHI3L1 with high affinity and specificity.

Meanwhile, the kit for detecting the human CHI3L1 is provided, is based on a magnetic particle chemiluminescence method, is combined with the existing luminescence detection instrument in the market, and can realize convenient operation and accurate result.

The invention provides the following technical scheme:

mouse-derived monoclonal antibodies against human CHI3L1, comprising CHI-Ab1 and CHI-Ab2,

the amino acid sequence of the heavy chain variable region of the CHI-Ab1 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2;

the amino acid sequence of the heavy chain variable region of the CHI-Ab2 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.

The application of the anti-human CHI3L1 murine monoclonal antibody in preparing a diagnostic reagent for the early auxiliary detection of liver cirrhosis and liver cancer.

The anti-human CHI3L1 murine monoclonal antibody was used in an in vitro diagnostic test kit.

The kit is a magnetic particle chemiluminescence detection kit, and CHI-Ab2 is used as a detection antibody, and CHI-Ab1 is used as a coating.

In the application, CHI3L1 specific epitope is taken as a target antigen, in order to enhance the screening of specific and strong affinity antibodies and improve the expression quantity of the CHI3L1 recombinant protein, the preferred codon of escherichia coli is used for optimization, and a new nucleotide sequence is used. And (3) synthesizing the optimized nucleotide sequence, performing enzyme digestion and connection by using restriction enzymes, and introducing a target nucleotide fragment into a vector pET-28b to complete the construction of the CHI3L1 protein expression vector. Then, pET-28b was transformed into E.coli BL21(DE3) competent cells, and an expression strain containing the plasmid of interest was obtained by antibiotic selection. And (3) carrying out large-scale culture on the recombinant strain, carrying out ultrahigh-pressure physical bacterium breaking and low-temperature centrifugation, taking the supernatant, purifying the supernatant by an affinity chromatography column through a nickel column, and eluting to obtain the reconstructed CHI3L1 protein.

After a mouse is immunized by the recombinant CHI3L1 protein, spleen cells of the mouse are taken and fused with sp2/0 myeloma cells. Preparing Balb/c mouse ascites from the hybridoma cell strain, purifying the monoclonal antibody by a protein A affinity chromatography, and respectively measuring and calculating the affinity and the antibody epitope pairing. The mateable antibody is prepared into a chemiluminescence reagent, and a detection standard substance shows that 2E4-2 (CHI-Ab 1) monoclonal antibody coating is matched with 1G11-14 (CHI-Ab 2) label to be the best combination for detecting CHI3L 1.

The magnetic particles used in the invention take magnetic metal or metal oxide as an inner core, the outer surface of the inner core is coated with a shell modified with carboxyl, and the functional groups are coupled with some biological targeting molecules through a coupling agent to form a magnetic microsphere compound with biological activity. The suspension magnetic particles as a carrier have higher specific surface area, can react with a sample more fully, and have the advantages of higher sensitivity, higher detection speed, better repeatability and the like compared with an enzyme-labeled plate carrier by the flexible application of an external magnetic field, and are widely applied to the fields of biological and medical detection and the like at present.

The kit is prepared based on an acridinium ester magnetic particle direct chemiluminescence method, is matched with a SMART500S series full-automatic chemiluminescence determinator for use, can realize automatic detection, and is simple and convenient to operate and high in detection speed.

The kit quantitatively measures the content of CHI3L1 in human serum by adopting a double-antibody sandwich method combined with a magnetic particle chemiluminescence immunoassay method.

Heterophily antibodies are a class of multi-specific immunoglobulins which are produced by stimulating a human body by known or unknown antigens and can be weakly combined with various animal immunoglobulins, and can interfere the detection result to show false positives or miss detection. Aiming at the problem, a blocking agent is added into a sample diluent of the kit, and is specially used for eliminating the interference of heterophilic antibodies on the detection result, so that the accuracy of the detection result of CHI3L1 is improved.

The detection principle of the kit is disclosed. And reacting 15 mu L of serum sample, a strain of CHI3L1 monoclonal antibody coated with magnetic particles and another strain of CHI3L1 monoclonal antibody marked by azathidine under an incubation condition, and capturing the antigen in the sample by the antibody to form an antigen-antibody sandwich complex. After the incubation is finished, the magnetic field is used for precipitation, supernatant is removed, a washing solution is used for washing a precipitation compound, and waste liquid is sucked dry to remove substances which are not combined with the magnetic particles. Finally, the reaction cup is introduced into the measuring chamber. The instrument automatically pumps in two excitation liquids to make the compound generate a chemiluminescence signal, and the light intensity is measured by a photomultiplier. The instrument automatically calculates the detection result according to the working curve.

Compared with the prior art, the invention has the beneficial effects that: the invention discloses expression and purification of recombinant CHI3L1 protein, mouse immunization, preparation of hybridoma cells, screening of monoclonal antibodies, and expression and purification of the antibodies. The CHI3L1 monoclonal antibody is applied to a magnetic particle chemiluminescence method to prepare a detection kit, the content of CHI3L1 in human serum is quantitatively detected, the progress and the severity of hepatic fibrosis are judged, and the detection kit can be used as an early screening index for cirrhosis and liver cancer. Compared with the CHI3L1 detection kit on the market, the kit provided by the invention has higher sensitivity and wider detection range, the linear range is 0.1-400ng/mL, the detection requirements of all samples can be met, and the samples do not need to be diluted.

Drawings

FIG. 1 shows the correlation between the linearity of the kit of the present invention and the detection result of a kit.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: optimization of recombinant CHI3L1 nucleotide sequence

CHI3L1 nucleotide and protein sequences were queried and downloaded at the NCBI database. In order to improve the expression amount of the recombinant CHI3L1 protein in Escherichia coli, the nucleotide is optimized by using an Escherichia coli preferred codon, the sequence is further replaced to optimize the secondary structure of mRNA, and the nucleotide sequences are cut by respectively adding enzyme cutting sites BamHI and EcoRI at the upper and lower positions of the optimized nucleotide sequence. The synthesized target gene is connected to pMD19-T vector.

Example 2: construction of recombinant CHI3L1 protein expression vector

The pMD19-T vector containing the target gene and the pET-28b vector were digested simultaneously with BamHI and EcoRI for 10 hours at 37 ℃ by two restriction enzymes. And (3) carrying out 1.2% agarose gel electrophoresis on the enzyme digestion product, and respectively cutting the gel to recover the target gene and the pET-28b vector. After the recovered target gene and pET-28b vector were ligated at 4 ℃ for 20 hours using T4 ligase in a certain ratio, the ligation product was transformed into DH 5. alpha. competent cells and plated on LB plates containing kanamycin resistance (55. mu.g/mL). After being cultured at 37 ℃ for overnight, the monoclonal strains are picked on a plate to LB liquid culture medium containing kanamycin resistance (50 mu g/mL), after being cultured for 16 hours at 37 ℃ for a constant temperature shaking table, plasmids are extracted and identified by BamHI and EcoRI double enzyme digestion, and the correct recombinant expression vector is obtained.

Example 3: construction of recombinant CHI3L1 protein expression Strain

E.coli BL21(DE3) competent cells were transformed with the constructed recombinant expression vector, plated on a kanamycin-resistant (55. mu.g/mL) LB plate, and cultured overnight at 37 ℃. Then, the monoclonal strains on the plates are picked to LB liquid culture medium containing kanamycin resistance (50 mu g/mL), and after the strains are cultured for 6 hours in a constant temperature shaking table at 37 ℃, an inducer IPTG (final concentration of 1.05mmol/L) is added for induction expression for 6 hours, and then protein electrophoresis samples are prepared. And (3) the result of 15% polyacrylamide gel electrophoresis shows that the recombinant protein is successfully expressed, and the recombinant CHI3L1 protein expression strain is obtained.

Example 4: purification of recombinant CHI3L1 protein

Inoculating a recombinant protein expression strain to an LB liquid culture medium, adding kanamycin to a final concentration of 55 mu g/mL, carrying out shake culture at a constant temperature of 37 ℃ for 12 hours, and then, adding the strain into the LB liquid culture medium containing 50 mu g/mL kanamycin according to the weight ratio of 1: 65 diluting, dispensing into a bacteria culture bottle, placing into a constant temperature shaking table at 37 ℃ to culture until OD600 is 1.8, adding inducer isopropylthio-beta-D-galactoside to the final concentration of 1.05mmol/L, and continuing culturing and inducing for 8 hours. And (3) centrifugally collecting thalli, breaking the thalli at ultrahigh pressure, centrifuging at low temperature, taking the supernatant, performing affinity chromatography on the supernatant through a nickel column, and washing and eluting to finally obtain the purified recombinant CHI3L1 protein.

Example 5: hybridoma cell line construction

The CHI3L1 protein immunogen was mixed well with an equal volume of Sigma-Aldrich Freund's complete adjuvant in an emulsifier at a concentration of 1.1mg/ml and emulsified to give an oily emulsified antigen. The emulsified antigen was injected into ALB/c mice, each mouse was injected with a 0.2ml dose in the lower part of the body. Emulsifying 1.5mg/ml CHI3L1 protein immunogen and Sigma-Aldrich Freund incomplete adjuvant in an emulsifier in an equal volume 10 days after the first immunization, administering 0.15ml dose immune enhancing injection to each mouse from the tarsal joint after emulsification, collecting tail vein blood when enhancing immunity reaches three needles, and detecting serum titer by indirect ELISA method, wherein the titer reaches the fusion requirement. Satisfactory mice were sacrificed to take lymphocytes. Using 50% PEG4000 as a fusogenic agent, sp2/0 myeloma cells and lymphocytes were fused using conventional methods, at a ratio of 1:3 to 1: 7. The fused cells were selectively cultured in HAT conditioned medium. The cells were cultured in a CO2 incubator at 37 ℃ for 8 to 12 days, and the fusion state was observed. Screening was performed using indirect ELISA plates starting at 15 days. And selecting cell strains with strong positive and large thin numbers from the preliminarily screened positive clones, and carrying out subcloning for 4 times by using a limiting dilution method. Through the screening process, two cell strains which have high affinity and stably secrete the CHI3L1 antibody are obtained, and the corresponding two monoclonal antibodies are named as CHI-Ab1 and CHI-Ab2 respectively.

Example 6: monoclonal antibody preparation and purification

Taking 6-8 weeks old healthy Balb/c male mouse, injecting liquid paraffin into abdominal cavity, 450 μ L/mouse, injecting monoclonal cell (about 1.2 × 10) into abdominal cavity after 5 days⁶One/one), 8-9 days later, the abdomen of the mouse was swollen, and ascites was collected. The agarose affinity medium ProteinA column was equilibrated with 50mL of equilibration buffer PBS (pH7.4) to a computerized nucleic acid protein detector showing an absorbance of 0. The ascites was centrifuged at 12500rpm for 5 minutes, the supernatant was collected and passed through a 0.45 μ M filter and then washed with PBS until the absorbance became 0, followed by elution with 0.1M glycine (pH3.0), and the effluent was collected and neutralized to about pH7.0 with 500mM Tris-HCl (pH8.5) buffer to obtain a purified monoclonal antibody.

Example 7: preparation of CHI3L1 chemiluminescence detection kit

The preparation of the CHI3L1 chemiluminescence detection kit is based on the principles of a double-antibody sandwich method and an acridinium ester luminescence method, and the antibodies in the application are CHI-Ab1 and CHI-Ab 2.

1. Magnetic particle coating CHI-Ab1 was prepared. 50ul of magnetic beads (5 mg,100 mg/ml) were pipetted, 1.8ml of MES coating solution was added, sonicated for 20 seconds, and magnetically separated for 2 minutes. Removing supernatant, adding 2ml MES coating solution, adding 250ul EDC solution (mass ratio of 2: 1, concentration of 10mg/ml), ultrasonic treating for 20 s, reacting at 37 deg.C, and shaking table rotating speed of 220r/min for 30 min. Magnetic separation for 3 minutes, washing 2 times with 2ml MES. 50ug of antibody (100: 1 by mass) was added at 37 ℃ and a shaker rotation speed of 220r/min for 3 hours. And finishing coating after cleaning. R1 reagent was prepared by adding R1 storage solution. The formula of the R1 preparation solution is 30mM Tris, 60Mm Kcl, 1.2% BSA, 0.6% Tween20 and 0.13% proclin 300.

2. Acridinium ester marker CHI-Ab2

Acridine ester and CHI-Ab2 were mixed at a molecular molar ratio of 15: 1 in CB for 2.5 hours; after the reaction is finished, carrying out ultrafiltration centrifugation by using a 25kDa ultrafiltration centrifugal tube to remove free acridinium ester; after centrifugation is finished, collecting the marker in the ultrafiltration tube and detecting the concentration of the acridinium ester-antibody by using a BCA method; diluting the acridinium ester-antibody to 1.5ug/ml by using a R2 preparation solution, wherein the diluted solution is the R2 reagent, and the formula of the R2 preparation solution is 20Mm Tris, 100mM KCl, 1.3% BSA, 0.16% Tween20 and 0.16% proclin 300.

3. Preparing calibrator and quality control product

Using the antigen diluent to respectively dilute the CHI3L1 antigen to 0.2, 0.78, 3.12, 12.5, 50 and 200ng/ml, and subpackaging into calibrator tubes. Diluting CHI3L1 antigen with antigen diluent to 2 ng/ml and 80 ng/ml respectively, and packaging into quality control tubes.

The antigen diluent formula comprises 50mMPB, 1500mMNacl, 3% BSA, 5% Glycerin, 0.3% procin300, and the pH = 7.8.

The performance evaluation of the kit of the invention:

the precision evaluation of the kit provided by the invention is realized by taking two batches of the kit provided by the invention to carry out precision experiments, respectively detecting high, medium and low value samples for 20 times, calculating the variation of the measured concentration, and measuring results are shown in table 1.

TABLE 1 precision test Table for the kit of the present invention

The kit of the invention is compared with the mainstream kits on the market:

the kit of the invention and the enzyme-linked immunosorbent assay kit with high acceptance in the market are used for detecting serum samples of 75 clinical patients, and linear correlation analysis is carried out on two groups of results, and the results show that the kit shows good linear correlation (shown in figure 1).

The invention discloses expression and purification of recombinant CHI3L1 protein, mouse immunization, preparation of hybridoma cells, screening of monoclonal antibodies, and expression and purification of the antibodies. The invention applies the CHI3L1 monoclonal antibody to a magnetic particle chemiluminescence method to prepare a detection kit, quantitatively detects the content of CHI3L1 in human serum, judges the progress and severity of hepatic fibrosis, and can be used as an early screening index of cirrhosis and liver cancer.

Sequence listing

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Claims

1. A murine monoclonal antibody directed against human CHI3L1, characterized by: comprises CHI-Ab1 and CHI-Ab2,

2. The use of the murine monoclonal antibody against human CHI3L1 of claim 1 for the preparation of a diagnostic reagent for the early auxiliary detection of liver cirrhosis and liver cancer.

3. The use of the murine monoclonal antibody against human CHI3L1 as claimed in claim 2 for the preparation of diagnostic reagents for the early auxiliary detection of liver cirrhosis and liver cancer, wherein: the anti-human CHI3L1 murine monoclonal antibody was used in an in vitro diagnostic test kit.

4. A detection kit for detecting human CHI3L1, comprising: comprising the antibody of claim 1.

5. The test kit for human CHI3L1 of claim 4, wherein: the kit is a magnetic particle chemiluminescence detection kit, and CHI-Ab2 is used as a detection antibody, and CHI-Ab1 is used as a coating.