Antibodies to CHI3L1, antibody compositions and uses thereof

Abstract

The present invention relates to the field of antibody technology, and specifically to antibodies, antibody compositions and applications of CHI3L1. The present invention provides an antibody that can specifically bind to human CHI3L1, which has high specificity and high affinity for human CHI3L1, and can achieve specific and sensitive detection of human CHI3L1. The human CHI3L1 detection kit developed based on the antibody has high specificity, sensitivity and accuracy, a wide linear range, is also applicable to low-concentration CHI3L1 samples, and has high stability, can meet detection requirements, can be used for the detection of human CHI3L1 in practice, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to antibodies, antibody compositions and applications of CHI3L1. Background Art

[0002] Chitinase-3-like-1 protein (CHI3L1) is a member of the glycosyl hydrolase family 18, with a relative molecular mass of about 40 kDa and composed of 383 amino acids. It is also called YKL-40 because the first three amino acids at the N-terminus of its G functional form are tyrosine (Y), lysine (K), and leucine (L). Although CHI3L1 belongs to the glycosyl hydrolase family 18 composed of chitinase and chitinase-like protein (CLP), it lacks the activity of hydrolyzing chitin due to the mutation of glutamic acid at position 140 to leucine.

[0003] CHI3L1 can be secreted by many types of cells, including chondrocytes, synoviocytes, activated monocyte-derived macrophages, neutrophils, endothelial cells, vascular smooth muscle cells, and some tumor cells, and is stimulated by a variety of mediators including IL-13, IL-6, IL-1β, and IFN-γ. Studies have shown that CHI3L1 can regulate cell proliferation, apoptosis, cell differentiation, cell invasion, and participate in embryonic development, inflammation, tissue remodeling, angiogenesis, and tumor metastasis. Studies have found that circulating levels of CHI3L1 are elevated in many malignant tumors, including prostate cancer, colon cancer, lung cancer, ovarian cancer, renal cancer, and breast cancer, as well as glioblastoma and malignant melanoma. In these diseases, the level of CHI3L1 is often directly correlated with disease progression and negatively correlated with disease-free period and survival.

[0004] CHI3L1 has been considered a biomarker for early neuroinflammation detection and disease diagnosis in Alzheimer's disease (AD). In the brain, CHI3L1 is mainly provided by astrocytes, indicating a reactive neurotoxic state caused by inflammation and other stress signals. At the same time, CHI3L1 is also considered to be a valuable biomarker in the diagnosis of liver fibrosis. Under normal circumstances, CHI3L1 is expressed in liver tissue at a higher level than other tissues, and its expression level increases abnormally when liver tissue fibrosis occurs. Liver fibrosis is a chronic repair process of damaged liver cells caused by viral infection, toxins, alcohol, etc. Cirrhosis is the late stage of liver fibrosis, which can eventually lead to the occurrence of hepatocellular carcinoma. Therefore, it is very important to accurately judge the occurrence and development stage of the disease through medical testing, so as to take appropriate treatment measures in time to control the occurrence and development of the disease.

[0005] At present, biopsy is the gold standard for the determination and staging of liver fibrosis. However, biopsy is an invasive procedure, and there may be problems such as bleeding, sampling errors, and inconsistent judgments by different pathologists. In addition, the commonly used detection methods in clinical practice also include ultrasound detection and serum index detection. Among them, serological detection is a more convenient detection method. However, the content of CHI3L1 in normal human serum is low, which puts high demands on the sensitivity of the corresponding detection kit. Therefore, it is still necessary to develop antibodies and kits that can achieve high-sensitivity detection of CHI3L1. Summary of the invention

[0006] The present invention provides antibodies, antibody compositions and applications of CHI3L1.

[0007] The present invention uses CHI3L1 antigen to immunize mice, and after the serum titer reaches a certain height, fusion screening is performed to obtain two monoclonal antibodies with good affinity, specificity and stability. Based on these two monoclonal antibodies, the present invention develops an enzyme-linked immunosorbent assay kit based on the double antibody sandwich method, which can be used to detect the presence and content of human CHI3L1, has the characteristics of high sensitivity, and can realize the detection of samples with low CHI3L1 content.

[0008] Specifically, the present invention provides the technical solution described below.

[0009] In the first aspect, the present invention provides an antibody or an antigen-binding fragment thereof against CHI3L1, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or the antigen-binding fragment thereof are shown in SEQ ID NO.1, 2, and 3, respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is YAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.5.

[0010] The above-mentioned CHI3L1 antibody or antigen-binding fragment thereof can specifically bind to CHI3L1, has high affinity and high stability, and can significantly improve the sensitivity of detection when used for CHI3L1 detection.

[0011] In the present invention, the CHI3L1 is preferably human CHI3L1.

[0012] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.11 or has at least 80% similarity to the sequence shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.12 or has at least 80% similarity to the sequence shown in SEQ ID NO.12.

[0013] The above sequence similarity is preferably at least 85%, more preferably at least 88%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 98.5%, more preferably at least 99%, more preferably at least 99.5%, more preferably at least 99.8%, more preferably at least 99.9%.

[0014] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.12.

[0015] The above-mentioned antibody or antigen-binding fragment thereof may be a monoclonal antibody, Fab, Fab', F(ab')2, Fd, Fv or a single-chain antibody.

[0016] In some embodiments of the invention, the antibody is a monoclonal antibody.

[0017] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0018] According to the amino acid sequence of the antibody or its antigen-binding fragment provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the above antibody or its antigen-binding fragment. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding an antibody or its antigen-binding fragment is not unique, and all nucleic acid molecules capable of encoding the above antibody or its antigen-binding fragment are within the scope of protection of the present invention.

[0019] In some specific embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment is shown as SEQ ID NO.15, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment is shown as SEQ ID NO.16.

[0020] In a third aspect, the present invention provides a biological material, wherein the biological material contains the nucleic acid molecule described above; the biological material is an expression cassette, a vector or a host cell.

[0021] The above-mentioned expression cassette can be obtained by connecting a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and / or connecting a transcription or translation regulatory element such as a terminator downstream thereof.

[0022] The above-mentioned vectors include but are not limited to plasmid vectors, phage vectors, virus vectors, artificial chromosome vectors and the like.

[0023] The host cells include microbial cells, insect cells or other mammalian cells. The microbial cells may be bacteria or fungi, bacteria include but are not limited to Escherichia coli, and fungi include but are not limited to yeast. The mammalian cells include but are not limited to Chinese hamster ovary cells (CHO), baby hamster embryonic kidney cells (BHK), mouse myeloma cells (SP0 / 2), African green monkey kidney cells (Vero) and human embryonic kidney 293 cells (HEK293).

[0024] In a fourth aspect, the present invention provides an antibody conjugate, which is obtained by conjugating the above-mentioned antibody or antigen-binding fragment thereof with a marker, and the marker is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, colloidal gold labeling, and radioactive labeling.

[0025] In a fifth aspect, the present invention provides an antibody composition of CHI3L1, wherein the antibody composition comprises the antibodies of (1) and (2) below:

[0026] (1) The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3 respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is YAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.5;

[0027] (2) The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs. 6, 7 and 8, respectively. The amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO. 9, the amino acid sequence of CDR2 is YTS, and the amino acid sequence of CDR3 is shown in SEQ ID NO. 10.

[0028] Preferably, the amino acid sequence of the heavy chain variable region of the antibody described in (1) above is as shown in SEQ ID NO.11 or has at least 80% similarity to the sequence shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.12 or has at least 80% similarity to the sequence shown in SEQ ID NO.12. The amino acid sequence of the heavy chain variable region of the antibody described in (2) above is as shown in SEQ ID NO.13 or has at least 80% similarity to the sequence shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14 or has at least 80% similarity to the sequence shown in SEQ ID NO.14.

[0029] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody described in (1) above is shown as SEQ ID NO.11, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.12. The amino acid sequence of the heavy chain variable region of the antibody described in (2) above is shown as SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.14.

[0030] The antibody composition described above can be used as a paired antibody for detecting CHI3L1 by double antibody sandwich ELISA method, wherein the two antibodies in the antibody composition serve as a coating antibody and a labeled antibody, respectively (preferably, the antibody described in (1) serves as a coating antibody, and the antibody described in (2) serves as a labeled antibody). The double antibody sandwich ELISA method for detecting CHI3L1 using the antibody composition described above has high specificity, sensitivity and accuracy.

[0031] In a sixth aspect, the present invention provides any of the following uses of the above-mentioned antibody or antigen-binding fragment thereof, the nucleic acid molecule, the biomaterial, the antibody conjugate, or the antibody composition:

[0032] (1) Use in the preparation of a product for detecting the presence or level of CHI3L1 in a sample;

[0033] (2) Application in the preparation of products for detecting liver fibrosis;

[0034] (3) Application in the preparation of products for detecting liver cirrhosis;

[0035] (4) Use in the preparation of products for detecting Alzheimer's disease;

[0036] (5) Use in the preparation of products for malignant tumor detection and / or prognosis assessment.

[0037] In the above (1), the sample may be a body fluid sample (such as blood, serum, tissue fluid, etc.), a cell sample or a tissue sample derived from a human, or a cell or protein sample prepared by in vitro culture. The presence in the sample refers to whether the sample contains CHI3L1, and the level refers to the content of CHI3L1 in the sample.

[0038] In (2)-(5) above, it is known that CHI3L1 can be used as a biomarker for liver fibrosis, Alzheimer's disease and malignant tumors. Therefore, the antibody or its antigen-binding fragment can be used to detect the above diseases or for the diagnosis or auxiliary diagnosis of the above diseases. It is known that the content of CHI3L1 in malignant tumors is negatively correlated with the disease-free period and survival period. Therefore, the antibody or its antigen-binding fragment can be used for the prognosis assessment of malignant tumors. Among them, the malignant tumors include prostate cancer, colon cancer, lung cancer, ovarian cancer, kidney cancer, breast cancer, glioblastoma or malignant melanoma.

[0039] In the above application, the product is a detection reagent or a detection kit.

[0040] In a seventh aspect, the present invention provides use of the above-mentioned antibody or antigen-binding fragment thereof, the antibody conjugate or the antibody composition in detecting the presence or level of CHI3L1 in a sample for purposes other than disease diagnosis and treatment.

[0041] For detection purposes other than disease diagnosis and treatment, the sample does not include samples directly derived from living humans or animals, but may be cell or protein samples prepared by in vitro culture.

[0042] In the present invention, methods for detecting CHI3L1 include, but are not limited to, immunological detection methods such as ELISA, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, and immunochromatography.

[0043] In an eighth aspect, the present invention provides a kit comprising the above-described antibody or antigen-binding fragment thereof, or the antibody conjugate, or the antibody composition.

[0044] The present invention does not impose any particular limitation on the types of the kit, including but not limited to ELISA detection kits, chemiluminescence detection kits, radioimmunoassay kits, fluorescent immunoassay kits, etc.

[0045] In some embodiments of the present invention, the kit is a double antibody sandwich ELISA detection kit. The double antibody sandwich ELISA detection kit comprises a coated antibody and a labeled antibody, wherein the coated antibody is the antibody described in (1) of the fifth aspect above, and the labeled antibody is the antibody described in (2) of the fifth aspect above. The coated antibody is coated on an ELISA plate. The labeled antibody is labeled with biotin. The kit may also include a second antibody carrying a detectable label and other reagents for ELISA detection, including but not limited to one or more selected from an ELISA plate, a coating buffer, a diluent, a human CHI3L1 standard, a PBST washing solution, a blocking solution, a color developing solution, and a stop solution.

[0046] In a ninth aspect, the present invention provides a method for detecting CHI3L1, comprising: using the above-mentioned antibody or antigen-binding fragment thereof or the antibody conjugate or the antibody composition or the kit to detect the content of CHI3L1 in a sample to be tested.

[0047] The above detection method can be selected from immunological detection methods such as ELISA, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, and immunochromatography.

[0048] In a tenth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof.

[0049] The pharmaceutical composition described above is preferably used to treat malignant tumors, hepatotoxicity, non-alcoholic steatohepatitis or non-alcoholic fatty liver disease.

[0050] Preferably, the malignant tumor is a solid tumor, such as lung cancer, brain cancer, skin cancer, head and neck cancer, liver cancer, pancreatic cancer, stomach cancer, bladder cancer, colon cancer, testicular cancer, cervical cancer, breast cancer or uterine cancer; or the malignant tumor is a malignant blood disease, such as myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelomonocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute myeloid leukemia or polycythemia vera.

[0051] In an eleventh aspect, the present invention also provides a bispecific antibody comprising the antibody or antigen-binding fragment thereof.

[0052] Preferably, the bispecific antibody comprises the above-mentioned antibody or antigen-binding fragment thereof and an anti-PD1 antibody.

[0053] The beneficial effects of the present invention include at least: the present invention provides an antibody that can specifically bind to human CHI3L1, the antibody is highly specific to human CHI3L1, does not recognize human serum albumin (ALB), human IgG, human IgE, human IgM, human IgA, human CRP, human Insulin (INS), human HSP27, human IL-1β, human IFN-α, human IL-8, human TNF-α, human IL-12, human TNF-β, GAPDH and other similar proteins, has a high affinity for human CHI3L1, and can achieve specific and sensitive detection of human CHI3L1. The human CHI3L1 detection kit developed based on the antibody has high specificity, sensitivity and accuracy, a wide linear range (15.6-1000pg / mL), is also applicable to low-concentration CHI3L1 samples, and has high stability (accelerated stability test at 37°C, no significant change in the kit within 13 days), can meet the detection requirements, can be used for the detection of human CHI3L1 in practice, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0055] Figure 1 This is the SDS-PAGE test result of the monoclonal antibody in Example 1 of the present invention.

[0056] Figure 2 This is the standard curve for detecting human CHI3L1 in Example 3 of the present invention. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] Example 1 Preparation of anti-human CHI3L1 monoclonal antibody

[0059] 1. Animal immunization

[0060] Female Balb / c mice aged 6-8 weeks were selected and immunized with commercial CHI3L1 antigen emulsified with an equal volume of Freund's adjuvant. The immunization cycle was two weeks. Blood was collected after three immunizations to measure the titer, and the immunization was boosted again three days before fusion.

[0061] 2. Cell fusion

[0062] The mice were killed by cervical dislocation, and the spleen was removed aseptically, squeezed and ground in a plate to prepare a spleen cell suspension. The prepared syngeneic myeloma cells were mixed with mouse spleen cells in a certain proportion, and the fusion promoter polyethylene glycol (PEG) was added. Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells. The specific operation is described as follows.

[0063] (1) Myeloma (SP2 / 0) cell activation

[0064] Commercial SP2 / 0 cells were thawed and revived, then resuspended in nutrient solution (RPMI-1640 basal culture medium supplemented with 20% fetal bovine serum), and cultured in an incubator at 37°C and 5% CO2; passaged after 3-5 days;

[0065] The cells were collected and suspended in RPMI-1640 basal culture medium, and 0.5-1×10 6 The cells were injected subcutaneously into the back of Balb / c mice and cultured for 9-10 days. When the back tumor volume increased to about 0.8 cm in diameter, the mice were killed by pulling the neck, and the tumors were removed by aseptic operation after soaking in 75% alcohol for 5 minutes.

[0066] The tumor pieces were cut off and placed in a sterilized homogenizer. After adding RPMI-1640 basal culture medium and grinding thoroughly, 10 mL of RPMI-1640 basal culture medium was added and allowed to stand for 2 min. The cell suspension on the upper layer was aspirated and placed in another centrifuge tube. 10 mL of RPMI-1640 basal culture medium was added and grinding was repeated twice. The cell suspension obtained above was centrifuged at 1000 r / min for 10 min to remove the supernatant, and then resuspended in 30 mL of RPMI-1640 basal culture medium.

[0067] Add 15 mL of lymphocyte separation fluid to another centrifuge tube, and carefully place the above cell suspension on the separation fluid; then centrifuge at 1200 r / min for 15 min, aspirate the dense white cell layer at the interface with a pipette, wash the cells twice with RPMI-1640 basal culture fluid, and resuspend them in 10 mL of RPMI-1640 basal culture fluid, count and set aside.

[0068] (2) Preparation of immune spleen cells

[0069] Take a Balb / c mouse that has been boosted with immunization, bleed it from its orbits (collect serum, i.e., positive serum), and immerse it in 75% alcohol for 5-10 minutes for disinfection. Then fix it on a dissecting board for dissection, remove the spleen, cut it, and place it in a sterile homogenizer. The grinding and cell suspension preparation methods are the same as those described in SP2 / 0, and the cells are counted and set aside.

[0070] (3) Preparation of feeder cells

[0071] Take an unimmunized Balb / c mouse, bleed from its orbit, and collect the negative serum. Inject 2-3 mL of RPMI-1640 basal culture medium into the mouse's peritoneal cavity, aspirate it after blowing, and place it in another centrifuge tube for standby use. The liquid contains peritoneal macrophages. Centrifuge at 1000 r / min for 10 minutes to remove the supernatant, suspend the cells with HAT medium, and place them in a 37°C, 5% CO2 incubator for standby use.

[0072] (4) Integration

[0073] 1-2×10 7 SP2 / 0 and 10 8 Mix the immune cells in a 50mL centrifuge tube, centrifuge at 1000r / min for 8min. After discarding the supernatant, place the centrifuge tube containing the cell mixture in a 37℃ water bath, then add 0.8mL of 50% PEG (Sigma) preheated to 37℃, stir and let stand for 30s. After standing, add 10mL of RPMI-1640 basal culture medium preheated at 37℃. After mixing, centrifuge at 1000r / min for 5min, discard the supernatant and let stand at 37℃ for 5-8min. Then mix with the feeder cell suspension, seed in a 96-well culture plate, 250μL / well, and culture in a 37℃, 5% CO2 incubator. On the 4th day after fusion, change to HT medium and continue to culture. When the fused cell colony grows to 1 / 4 of the culture well and the culture medium turns slightly yellow, perform antibody detection.

[0074] 3. Screening of hybridoma positive clones and cell cloning

[0075] The purpose of selective culture is to screen fused hybridoma cells, using HAT selective culture medium. In HAT culture medium, unfused myeloma cells die because they lack hypoxanthine-guanine-phosphoribosyltransferase and cannot synthesize DNA using the salvage pathway. Although unfused lymphocytes have hypoxanthine-guanine-phosphoribosyltransferase, they themselves cannot survive in vitro for a long time and gradually die. Only fused hybridoma cells can survive and proliferate in HAT culture medium because they have obtained hypoxanthine-guanine-phosphoribosyltransferase from spleen cells and have the characteristics of unlimited proliferation of myeloma cells. The specific operation is described as follows.

[0076] (1) Screening positive hybridoma cells using indirect ELISA, the steps are as follows:

[0077] Coating with known antigens: dilute the purified coating antigen (human CHI3L1 recombinant protein) to 1-10 mg / mL with coating buffer; add 100 μL to each microwell, shake gently, and place in a 4°C refrigerator overnight or at 37°C for 1 hour; shake off the liquid in the well (tap dry as much as possible); wash 3 times, 2-3 minutes each time.

[0078] Block the areas in the ELISA wells that are not coated with antigens: add 200 μL of blocking solution (5% skim milk powder or 0.1% BSA) to each microwell, shake gently, and place at 37°C for 1 hour; shake off the liquid in the wells; fill each well with washing buffer, let it stand for 2-3 minutes, shake off the liquid in the wells, pat dry, and wash three times with washing buffer using this method.

[0079] Sample addition: Take 50 μL of supernatant from each well of the hybridoma to be tested and add it to the enzyme-labeled wells in order, shake gently, place at 37°C for 1 hour, wash, and pat dry.

[0080] Add enzyme-labeled antibody: first dilute the enzyme-labeled secondary antibody to an appropriate working concentration using diluent according to the instructions, add 100 μL to each well, shake gently, and place at 37°C for 1 hour; then wash and pat dry.

[0081] Add color developing solution: Add 100 μL of freshly prepared color developing solution to each well, shake gently, and incubate at 37°C for 10 min.

[0082] Stop reaction: add 50 μL of stop solution to each well.

[0083] Determination result: OD of microplate reader 450 The reading was performed at 3 times that of the negative well, which was considered positive.

[0084] (2) Cloning of hybridoma cells (limiting dilution method)

[0085] Prepare a mouse feeder cell layer before cloning; gently blow the hybridoma cells to be cloned from the culture well and count the number of live cells using a hemocytometer; dilute the cells to 10 cells / mL with complete culture medium;

[0086] Add the cell suspension of the above concentration to the prepared 96-well culture plate of feeder cells, 100 μL / well, so that each well contains 1 cell. Add one drop of liquid on the 4th day of culture, and carefully observe the growth of cells in each well on the 5th-6th day and record it;

[0087] (3) Detection of specific antibodies

[0088] On the 7th to 9th day after cloning, when the cell clones cover 1 / 3-1 / 2 of the visual field, they can be detected; the cells in the positive wells can be moved to a 24-well culture plate, and when the cells in the 24-well plate grow well, they can be intraperitoneally inoculated into mice to collect ascites.

[0089] Two monoclonal antibodies that specifically bind to human CHI3L1 were obtained through the above hybridoma cell screening and were named monoclonal antibodies 5E10 and 1E3, respectively.

[0090] 4. Sequence determination of the variable regions of monoclonal antibodies 5E10 and 1E3

[0091] The number of hybridoma cells collected was greater than 10 6 , using the total RNA extraction kit (Solebao Technology Co., Ltd.), and according to the instructions, the total RNA of the two cell lines was extracted respectively; the reverse transcription kit (Solebao Technology Co., Ltd.) was used to synthesize the first chain of cDNA and perform PCR amplification, and the obtained target DNA fragment was connected to the T vector and sent to Qingke Bio for subsequent sequencing. The gene sequencing results were as follows: the heavy chain variable region sequence of the monoclonal antibody 5E10 was 357 bp long, encoding 119 amino acids, the DNA sequence was shown in SEQ ID NO.15, and the protein sequence was shown in SEQ ID NO.11, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region were shown in SEQ ID NO.1, 2, and 3, respectively; the light chain variable region sequence was 321 bp long, encoding 107 amino acids, the DNA sequence was shown in SEQ ID NO.16, and the protein sequence was shown in SEQ ID NO.12; the amino acid sequence of the complementary determining region CDR1 of the light chain variable region was shown in SEQ ID NO.4, the amino acid sequence of CDR2 was YAS, and the amino acid sequence of CDR3 was shown in SEQ ID NO.5. The heavy chain variable region sequence of monoclonal antibody 1E3 is 357 bp long, encoding 119 amino acids, the DNA sequence is shown in SEQ ID NO.17, the protein sequence is shown in SEQ ID NO.13, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO.6, 7, and 8, respectively; the light chain variable region sequence is 321 bp long, encoding 107 amino acids, the DNA sequence is shown in SEQ ID NO.18, and the protein sequence is shown in SEQ ID NO.14; the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.9, the amino acid sequence of CDR2 is YTS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.10.

[0092] 5. Large-scale preparation of monoclonal antibodies 5E10 and 1E3

[0093] The established hybridoma cells were injected into the peritoneal cavity of mice, and the ascites was collected about 7 days later. The antibodies were purified by Protein G affinity chromatography and detected by SDS-PAGE. The results are as follows: Figure 1 shown.

[0094] Example 2 Affinity determination of monoclonal antibodies 5E10 and 1E3

[0095] The relative affinity constants of monoclonal antibodies 5E10 and 1E3 were determined as follows.

[0096] The antigen was coated on the ELISA plate and blocked. After washing the plate with PBST, the monoclonal antibodies 5E10 and 1E3 were diluted to saturation concentration and added to the ELISA plate, 100 μL / well, and incubated at room temperature for 2 hours. After washing the plate with PBST, 0, 0.5, 1.5, 2.5, 3.5, and 4.5 mol / L NaSCN solutions were added in sequence at 60 μL / well, and incubated at room temperature for 15 minutes. After washing the plate with PBST, HRP-labeled goat anti-mouse IgG was added, and the plate was incubated at room temperature for 45 minutes for color development detection. The sodium thiocyanate concentration corresponding to the OD value at 450nm after elution dropped to 50% of that without elution is the relative affinity constant of the antibody, expressed in mol / L. The results show (Table 1) that the relative affinity constants of monoclonal antibodies 5E10 and 1E3 are 1 mol / L and 2 mol / L, respectively, and both have good affinity.

[0097] Table 1

[0098]

[0099] Example 3 Establishment and evaluation of double antibody sandwich ELISA detection kit and method

[0100] A human CHI3L1 double antibody sandwich ELISA detection kit and detection method were constructed based on monoclonal antibodies 5E10 and 1E3, and their performance was evaluated as described below.

[0101] 1. Biotin labeling of monoclonal antibody 1E3

[0102] Add the monoclonal antibody 1E3 purified by Protein G affinity chromatography to the dialysis bag and immerse it in 0.1M CB buffer for overnight dialyzing at 4°C. Dissolve NHS-D-Biotin in DMSO to prepare a 2.2mg / mL solution. Add the prepared NHS-D-Biotin solution to the antibody solution that has been dialyzed overnight, and stir gently for 4 hours at room temperature in the dark. The reacted solution is fully dialyzed with 0.01M PBS (pH 7.2~7.4) at 4°C. Remove the labeled monoclonal antibody 1E3 from the dialysis bag, add an equal volume of glycerol, preservative, and BSA (final concentration 10mg / mL), and store it in an antibody tube at -20°C.

[0103] 2. Preparation of ELISA plate coated with monoclonal antibody 5E10

[0104] Using a 96-well flat-bottom polystyrene ELISA plate as a solid phase carrier, the monoclonal antibody 5E10 purified by Protein G affinity chromatography was diluted to 2 μg / mL with antibody coating solution, and the diluted antibody was added to the microwells of the ELISA plate, 100 μL / well, and the plate was sealed with a sealing film and placed at 4°C for overnight coating. The coated ELISA plate was taken out, the liquid in the well was discarded, and the plate was washed 3 times with ELISA washing solution, and then a blocking solution containing 2% BSA was added, 250 μL / well, and the plate was sealed with a sealing film and placed at room temperature for 2 hours, and the liquid in the plate well was discarded, and it was placed in a dry room for drying for 16-18 hours. It was placed in an aluminum foil bag containing a desiccant, vacuum-sealed, and stored at 4°C.

[0105] 3. Establishment of double antibody sandwich ELISA method

[0106] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 5E10, return to room temperature, wash the plate 3 times and spin dry. After making multiple dilutions of human CHI3L1 standard (1000, 500, 250, 125, 62.5, 31.25, 15.625 pg / mL), add it to the ELISA plate, 100 μL / well, and set up a blank control well; incubate at room temperature (25±2℃) for 2 hours, then wash, dilute the biotin-labeled monoclonal antibody 1E3 to the working concentration, add it to each reaction well of the ELISA plate, 100 μL / well; incubate at room temperature (25±2℃) for 1 hour, then wash, dilute the enzyme conjugate to the working concentration, add it to the ELISA plate, In each reaction well of the plate, 100 μL / well; incubate at room temperature (25±2℃) with shaking for 30 minutes, then wash and add TMB color substrate to each reaction well, 100 μL / well; let stand at room temperature (25±2℃) for 10~20 minutes to develop color; finally, add stop solution to each reaction well, 50 μL / well, terminate the reaction, and use an ELISA reader to perform dual wavelength detection within 5 minutes, measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and subtract the OD value at 630nm from the OD value at 450nm. With the concentration of the standard as the horizontal axis and the absorbance OD value as the vertical axis, the standard curve was drawn using ELISA Calc software (using four-parameter fitting), and the results showed that ( Figure 2 ), its detection range is 15.6-1000pg / mL, R 2 is 0.99996.

[0107] 4. Specificity detection of double antibody sandwich ELISA method

[0108] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 5E10, return to room temperature, wash the plate three times and spin dry. The human CHI3L1 standard was diluted in multiples (1000, 500, 250, 125, 62.5, 31.25, 15.625 pg / mL) and added to the ELISA plate at 100 μL / well. A blank control well was set up at the same time. The following human recombinant proteins (ALB 40 ng / mL, IgG 80 ng / mL, IgE 50 ng / mL, IgM 500 ng / mL, IgA 20 ng / mL, INS 11.6 ng / mL, HSP27 10 ng / mL, GAPDH 100 ng / mL, IL-1β 1000 pg / mL, IFN-α 5000 pg / mL, IL-8 2000 pg / mL, TNF-α 1000 pg / mL, IL-12 4000 pg / mL, TNF-β 2000 pg / mL, OPN 2000pg / mL, CRP 1000pg / mL) were added to other wells of the ELISA plate at 100μL / well, incubated at room temperature (25±2℃) for 2h, then washed, biotinylated monoclonal antibody 1E3 was diluted to the working concentration and added to each well of the ELISA plate at 100μL / well; incubated at room temperature (25±2℃) for 1h, then washed, enzyme conjugate was diluted to the working concentration and added to each well of the ELISA plate at 100μL / well; incubated at room temperature (25±2℃) for 3 ... 0min, then wash, add TMB color substrate to each reaction well, 100μL / well; let stand at room temperature (25±2℃) for 10~20min; finally add stop solution to each reaction well, 50μL / well, stop the reaction, use a microplate reader to perform dual wavelength detection within 5min, measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and subtract the OD value at 630nm from the OD value at 450nm. The results show (Table 2) that the cross-reaction rate with the above proteins is less than 3%.

[0109] Table 2

[0110]

[0111] 5. Stability test of double antibody sandwich ELISA test kit

[0112] The ELISA plate coated with monoclonal antibody 5E10, biotin-labeled monoclonal antibody 1E3 and standard human CHI3L1 were placed at 37°C for accelerated stability test for 13 days (equivalent to -20°C for one and a half years). Then the plate was taken out for testing. The testing method was as follows: the ELISA plate was washed 3 times and dried; the human CHI3L1 standard was diluted in multiples (1000, 500, 250, 125, 62.5, 31.25, 15.625 pg / mL) and added to the ELISA plate at 100 μL / well. A blank control well was set up at the same time. The plate was incubated at room temperature (25±2°C) for 2 hours with shaking, and then washed. The biotin-labeled monoclonal antibody 1E3 was diluted to the working concentration and added to each reaction well of the ELISA plate at 100 μL / well; the plate was incubated at room temperature (25±2°C) for 1 hour with shaking, and then washed. The enzyme conjugate was diluted to the working concentration and added to each reaction well of the ELISA plate at 100 μL / well. Dissolve to working concentration, add to each reaction well of the ELISA plate, 100μL / well; incubate at room temperature (25±2℃) for 30min, then wash, add TMB color substrate to each reaction well, 100μL / well; let stand at room temperature (25±2℃) for 10~20min; finally add stop solution to each reaction well, 50μL / well, stop the reaction, use an ELISA reader for dual wavelength detection within 5min, measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and subtract the OD value at 630nm from the OD value at 450nm. With the concentration of the standard as the horizontal axis and the absorbance OD value as the vertical axis, the standard curve was drawn using ELISA Calc software (using four-parameter fitting), and the results showed (Table 3) that the stability of the kit was good.

[0113] Table 3

[0114]

[0115] 6. Recovery rate and linearity detection of double antibody sandwich ELISA method

[0116] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 5E10, return to room temperature, wash the plate 3 times and spin dry. After diluting the human CHI3L1 standard in multiples (1000, 500, 250, 125, 62.5, 31.25, 15.625 pg / mL), add it to the ELISA plate, 100 μL / well, and set up a blank control well; select RPMI-1640 basal culture medium and serum of healthy volunteers, add different concentrations of human CHI3L1 standard, 100 μL / well; incubate at room temperature (25±2℃) for 2 hours, then wash, dilute the biotin-labeled monoclonal antibody 1E3 to the working concentration, add it to each reaction well of the ELISA plate, 100 μL / well; incubate at room temperature (25±2℃) for 2 hours, then wash, dilute the biotin-labeled monoclonal antibody 1E3 to the working concentration, add it to each reaction well of the ELISA plate, 100 μL / well; incubate at room temperature (25±2℃) Incubate for 1 hour, then wash, dilute the enzyme conjugate to the working concentration, add it to each reaction well of the ELISA plate, 100μL / well; incubate at room temperature (25±2℃) for 30min, then wash, add TMB color substrate to each reaction well, 100μL / well; let it stand for 10~20min at room temperature (25±2℃); finally, add stop solution to each reaction well, 50μL / well, stop the reaction, use the ELISA reader for dual wavelength detection within 5min, measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and subtract the OD value at 630nm from the OD value at 450nm. With the concentration of the standard as the horizontal axis and the absorbance OD value as the vertical axis, use ELISA Calc software to draw the standard curve (using four-parameter fitting), establish the equation, and calculate the spike recovery rate. The results show (Table 4) that the recovery rate is between 80% and 120%, and the spike recovery rate is good.

[0117] Table 4

[0118]

[0119] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 5E10, return to room temperature, wash the plate 3 times and spin dry. After diluting the human CHI3L1 standard in multiples (1000, 500, 250, 125, 62.5, 31.25, 15.625 pg / mL), add it to the ELISA plate at 100 μL / well, and set up blank control wells; select RPMI-1640 basal culture medium and serum of healthy volunteers, add human CHI3L1 standard, dilute it in multiples, and then add it to the ELISA plate at 100 μL / well; set up blank control wells and negative control wells at the same time; incubate at room temperature (25±2℃) for 2 hours, then wash and dilute the biotin-labeled monoclonal antibody 1E3 to the working concentration, add it to each reaction well of the ELISA plate at 100 μL / well ; Incubate at room temperature (25±2℃) for 1h, then wash, dilute the enzyme conjugate to the working concentration, add to each reaction well of the ELISA plate, 100μL / well; Incubate at room temperature (25±2℃) for 30min, then wash, add TMB color substrate to each reaction well, 100μL / well; Stand at room temperature (25±2℃) for 10~20min; Finally, add stop solution to each reaction well, 50μL / well, stop the reaction, use the microplate reader for dual wavelength detection within 5min, measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and subtract the OD value at 630nm from the OD value at 450nm. With the concentration of the standard as the horizontal axis and the absorbance OD value as the vertical axis, the standard curve was drawn using ELISACalc software (using four-parameter fitting), and the equation was established to calculate the linear dilution recovery rate. The results show (Table 5) that the recovery rate is between 80% and 120%, and the linear dilution recovery rate is good.

[0120] Table 5

[0121]

[0122] 7. Sensitivity test of double antibody sandwich ELISA method

[0123] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 5E10, return to room temperature, wash the plate 3 times and spin dry. After diluting the human CHI3L1 standard in multiples (1000, 500, 250, 125, 62.5, 31.25, 15.625 pg / mL), add it to the ELISA plate, 100 μL / well, and set up multiple blank control wells (generally ≥20), 100 μL / well; incubate at room temperature (25±2℃) for 2 hours, then wash, dilute the biotin-labeled monoclonal antibody 1E3 to the working concentration, add it to each reaction well of the ELISA plate, 100 μL / well; incubate at room temperature (25±2℃) for 1 hour, then wash, dilute the enzyme conjugate to the working concentration, add it to each reaction well of the ELISA plate, 100 μL / well; incubate at room temperature (25±2℃) for 1 hour, then wash, dilute the enzyme conjugate to the working concentration, add it to each reaction well of the ELISA plate, 100 μL / well; As concentration, add to each reaction well of the ELISA plate, 100μL / well; incubate at room temperature (25±2℃) for 30min, then wash, add TMB color substrate to each reaction well, 100μL / well; let stand at room temperature (25±2℃) for 10~20min; finally, add stop solution to each reaction well, 50μL / well, stop the reaction, use the ELISA reader for dual wavelength detection within 5min, measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and subtract the OD value at 630nm from the OD value at 450nm. With the concentration of the standard as the horizontal axis and the absorbance OD value as the vertical axis, use ELISA Calc software to draw the standard curve (using four-parameter fitting), establish an equation, and calculate the sensitivity. The calculation formula for sensitivity is: the average value of the blank well OD value + 2×the calculated value of the standard deviation, the sensitivity obtained is 0.98pg / mL, and the blank well OD value is shown in Table 6.

[0124] Table 6

[0125]

[0126] Example 4 Comparison of test results of the same type of kit

[0127] The commercially available human CHI3L1 double antibody sandwich ELISA detection kit A (RND, item number DY2599) and the human CHI3L1 double antibody sandwich ELISA detection kit of Example 3 of the present invention were selected for analogy testing, wherein the kit A was tested according to the method in its instructions, and the kit of the present invention was tested according to the detection method of Example 3. The performance of the kits was compared in the following three aspects.

[0128] 1. Comparison of standard curve

[0129] The results show (Table 7) that the R 2 All of them are greater than 0.999, and the fitting degree is good; however, the background performance of the kit of the present invention is better than that of kit A.

[0130] Table 7

[0131]

[0132] 2. Comparison with standard products

[0133] The results show (Table 8) that the kit of the present invention has a good gradient when detecting the SZ standard (the standard of kit A), and the recovery rate is similar, while the kit A measures a higher concentration value at a low concentration when detecting the standard of the kit of the present invention, and the recovery rate is very different. Therefore, for the content detection of recombinant human CHI3L1, the accuracy of the present kit is better than that of the A kit.

[0134] Table 8

[0135]

[0136] 3. Comparison of sample measurement results

[0137] 16 human serum samples were randomly selected and measured simultaneously (unit: ng / mL). The samples were diluted to be within the measurement range of the standard curve. The measurement results (Table 9) showed that the sample values ​​measured by kit A were generally higher than those measured by the kit of the present invention, which was consistent with the comparison results of the above 2 standards, indicating that the accuracy of kit A for sample detection was not as good as that of the antibody pair of the present invention.

[0138] Table 9

[0139]

[0140] Example 5 Determination of CHI3L1 Content in Human Serum

[0141] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 5E10, return to room temperature, wash the plate 3 times and spin dry. After the human CHI3L1 standard was diluted in multiples (5000, 2500, 1250, 625, 312.5, 156.25, 78.125 pg / mL), it was added to the ELISA plate at 100 μL / well, and a blank control well was set up at the same time; 28 human serum samples and 20 human plasma samples were selected, diluted, added to the ELISA plate at 100 μL / well, incubated at room temperature (25±2°C) for 2 hours, then washed, and the biotin-labeled monoclonal antibody 1E3 was diluted to the working concentration and added to each reaction well of the ELISA plate at 100 μL / well; incubated at room temperature (25±2°C) for 1 hour, Then wash, dilute the enzyme conjugate to the working concentration, add it to each reaction well of the ELISA plate, 100 μL / well; incubate at room temperature (25±2°C) for 30 minutes, then wash, add TMB color substrate to each reaction well, 100 μL / well; incubate at room temperature (25±2°C) for 10-20 minutes; finally, add stop solution to each reaction well, 50 μL / well, terminate the reaction, use an ELISA reader to perform dual wavelength detection within 5 minutes, measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and subtract the OD value at 630nm from the OD value at 450nm. With the concentration of the standard as the horizontal axis and the absorbance OD value as the vertical axis, use ELISA Calc software to draw a standard curve (using four-parameter fitting), establish an equation, and calculate the sample concentration. The results show (Table 10 and Table 11) that the detection results obtained using the kit of the present invention are consistent with the content of CHI3L1 in human serum / plasma reported in the literature.

[0142] Table 10

[0143]

[0144] Table 11

[0145]

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against CHI3L1, characterized in that: The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody or its antigen-binding fragment are shown in SEQ ID NO.1, 2, and 3, respectively; the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is YAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

5.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO.11 or has at least 80% similarity to the sequence shown in SEQ ID NO.11, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.12 or has at least 80% similarity to the sequence shown in SEQ ID NO.

12.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof is a monoclonal antibody, Fab, Fab', F(ab')2, Fv or a single-chain antibody.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. Biomaterial, characterized in that The biological material contains the nucleic acid molecule according to claim 4; The biological material is an expression cassette, a vector or a host cell.

6. An antibody conjugate, characterized in that: The antibody conjugate is obtained by conjugating the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 with a label, and the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, colloidal gold labeling, and radioactive labeling.

7. An antibody composition of CHI3L1, characterized in that The antibody composition comprises the following antibodies (1) and (2): (1) The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO.1, 2 and 3 respectively, the amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO.4, the amino acid sequence of CDR2 is YAS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.5; (2) The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NOs. 6, 7 and 8, respectively. The amino acid sequence of the complementary determining region CDR1 of the light chain variable region is shown in SEQ ID NO. 9, the amino acid sequence of CDR2 is YTS, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

10.

8. Any of the following uses of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the biomaterial according to claim 5, the antibody conjugate according to claim 6, or the antibody composition according to claim 7: (1) Use in the preparation of a product for detecting the presence or level of CHI3L1 in a sample; (2) Application in the preparation of products for detecting liver fibrosis; (3) Application in the preparation of products for detecting liver cirrhosis; (4) Use in the preparation of products for detecting Alzheimer's disease; (5) Use in the preparation of products for malignant tumor detection and / or prognosis assessment.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the antibody conjugate according to claim 6, or the antibody composition according to claim 7 in detecting the presence or level of CHI3L1 in a sample for purposes other than disease diagnosis and treatment.

10. A kit, characterized in that: It comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the antibody conjugate according to claim 6, or the antibody composition according to claim 7.

Images