CN118812717B

CN118812717B - Antibodies that specifically bind to human CHI3L1 and kits for detecting human CHI3L1

Info

Publication number: CN118812717B
Application number: CN202411313251.5A
Authority: CN
Inventors: 刘星; 陈新新; 马玉岭; 魏彦辉; 潘悦; 苑晓松; 赵海龙; 路轲; 王芳; 刘碧霞
Original assignee: Beijing Solarbio Technology Co ltd
Current assignee: Beijing Solarbio Technology Co ltd
Priority date: 2024-09-20
Filing date: 2024-09-20
Publication date: 2024-11-22
Anticipated expiration: 2044-09-20
Also published as: CN118812717A

Abstract

The invention relates to the technical field of antibodies, in particular to an antibody specifically combined with human CHI3L1 and a kit for detecting human CHI3L 1. The antibody provided by the invention can be specifically combined with human CHI3L1, has higher affinity to human CHI3L1, can be used for detecting human CHI3L1, has higher specificity and sensitivity, and is also suitable for low-concentration CHI3L1 samples. The human CHI3L1 detection kit developed based on the antibody has higher specificity, sensitivity and accuracy, wider linear range and higher stability, can meet detection requirements, can be used for detecting human CHI3L1 in practice, and has better application prospect.

Description

Antibodies that specifically bind to human CHI3L1 and kits for detecting human CHI3L1

Technical Field

The invention relates to the technical field of antibodies, in particular to an antibody specifically combined with human CHI3L1 and a kit for detecting human CHI3L 1.

Background

Chitinase 3-like protein 1 (Chitinase-3-like-1 protein, CHI3L 1) is a member of the glycosyl hydrolase 18 family, having a relative molecular mass of about 40 kDa, consisting of 383 amino acids, and being characterized by the first three amino acids at the N-terminus of the G functional form being tyrosine (Y), lysine (K), leucine (L), also known as YKL-40. Although CHI3L1 belongs to glycosyl hydrolase family 18 consisting of chitinase and chitinase-like protein (CLP), it lacks chitin hydrolyzing activity due to the mutation of glutamic acid to leucine at position 140.

CHI3L1 is secreted by a variety of cell types 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-1 beta, and IFN-gamma. Studies have now shown that CHI3L1 can regulate cell proliferation, apoptosis, cell differentiation, cell invasion and is involved in embryonic development, inflammation, tissue remodeling, angiogenesis and tumor metastasis. Circulating levels of CHI3L1 have been found to be elevated in a number of malignancies, including prostate, colon, lung, ovarian, renal and breast cancers, as well as glioblastomas and malignant melanoma. In these diseases, the levels of CHI3L1 are generally directly related to disease progression, and inversely related to disease-free and survival.

CHI3L1 has been considered as a biomarker for early neuroinflammation detection and disease diagnosis in Alzheimer's Disease (AD). In the brain, CHI3L1 is predominantly provided by astrocytes, suggesting a reactive neurotoxic state triggered by inflammation and other stress signals. Meanwhile, CHI3L1 is also considered as a relatively valuable biomarker in diagnosis of hepatic fibrosis, and CHI3L1 is normally expressed in liver tissue in higher amounts than other tissues, and the expression level of the CHI3L1 is abnormally increased when hepatic fibrosis occurs. Liver fibrosis is a chronic repair process of damaged hepatocytes caused by virus infection, toxins, alcohol, etc., and cirrhosis is a later stage of fibrosis of the liver, which can ultimately lead to the occurrence of hepatocellular carcinoma. Therefore, the occurrence and development stages of the diseases are accurately judged through medical detection, and the method is very important for timely adopting the adaptive treatment means to control the occurrence and development of the diseases.

Currently, biopsies are the gold standard of detection for the determination and staging of liver fibrosis. However, biopsy is an invasive procedure and may present problems such as bleeding, sampling errors and inconsistent judgment by different pathologists. In addition, the detection method commonly used in clinic also comprises two methods of ultrasonic detection and serum index detection. Among them, serological detection is a more convenient detection method. However, the CHI3L1 content in serum of normal people is low, and high requirements are put on the sensitivity of the corresponding detection kit. Therefore, there remains a need to develop antibodies and kits that enable highly sensitive detection of CHI3L 1.

Disclosure of Invention

The present invention provides antibodies that specifically bind to human CHI3L1 and kits for detecting human CHI3L 1.

According to the invention, the CHI3L1 antigen is used for immunizing a mouse, and fusion screening is carried out after the serum titer reaches a certain height, so that two monoclonal antibodies with good affinity, specificity and stability are obtained. Based on the two monoclonal antibodies, the invention develops an ELISA kit based on a double-antibody sandwich method, can be used for detecting the existence and the content of human CHI3L1, has the characteristic of high sensitivity, and can realize the detection of samples with low CHI3L1 content.

Specifically, the present invention provides the following technical solutions.

In a first aspect, the present invention provides an antibody or antigen binding fragment thereof of CHI3L1, wherein the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of said antibody or antigen binding fragment thereof are shown in SEQ ID No.6, 7, 8, respectively, the amino acid sequence of complementarity 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.

The antibody or antigen binding fragment thereof of the CHI3L1 can specifically bind to the CHI3L1, has higher affinity and higher stability, and can remarkably improve the detection sensitivity when being used for detecting the CHI3L 1.

In the present invention, the CHI3L1 is preferably human CHI3L1.

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.13 or has at least 80% similarity to the sequence as 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 as shown in SEQ ID NO. 14.

The sequence similarity described above 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%.

In some embodiments of the 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.13 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 14.

The antibody or antigen binding fragment thereof described above may be a monoclonal antibody, fab ', F (ab') ₂, fd, fv or single chain antibody.

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

In a second aspect, the invention provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof as described above.

According to the above provided amino acid sequences of antibodies or antigen binding fragments thereof, the skilled person can obtain nucleotide sequences of nucleic acid molecules encoding the above antibodies or antigen binding fragments thereof. Because of the degeneracy of the codons, the nucleotide sequence of a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof is not unique and all nucleic acid molecules capable of encoding the production of such antibodies or antigen-binding fragments thereof are within the scope of the invention.

In some embodiments of the invention, the nucleotide sequence of a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO.17 and the nucleotide sequence of a nucleic acid molecule encoding the light chain variable region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO. 18.

In a third aspect, the present invention provides a biological material comprising a nucleic acid molecule as described above; the biological material is an expression cassette, a vector or a host cell.

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

Such vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, and the like.

The host cells include microbial cells, insect cells, or other mammalian cells. Wherein the microbial cells may be bacteria, including but not limited to E.coli, or fungi, including but not limited to yeast. Such mammalian cells include, but are not limited to, chinese hamster ovary Cells (CHO), baby hamster embryo kidney cells (BHK), mouse myeloma cells (SP 0/2), african green monkey kidney cells (Vero), and human embryo kidney 293 cells (HEK 293), among others.

In a fourth aspect, the present invention provides an antibody conjugate obtained by coupling an antibody or antigen binding fragment thereof as described above with a label selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, a colloidal gold label, and a radioactive label.

In a fifth aspect, the present invention provides an antibody composition for CHI3L1 comprising the antibodies in the following (1) and (2):

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

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

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

In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody described in (1) above 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. The amino acid sequence of the heavy chain variable region of the antibody described in the above (2) 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.

The antibody composition described above can be used as a pair antibody for detecting CHI3L1 by a double-antibody sandwich ELISA method, and the two antibodies in the antibody composition are respectively used as a coated antibody and a labeled antibody (preferably, the antibody described in (1) is used as a coated antibody, and the antibody described in (2) is used as a labeled antibody). The detection of CHI3L1 by using the antibody composition and adopting a double-antibody sandwich ELISA method has higher specificity, sensitivity and accuracy.

In a sixth aspect, the invention provides the use of any of the antibodies or antigen binding fragments thereof or the nucleic acid molecules or the biological material or the antibody conjugates or the antibody compositions described above:

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

(2) Use in the manufacture of a product for detecting liver fibrosis;

(3) Use in the preparation of a product for detecting cirrhosis;

(4) Use in the manufacture of a product for detecting alzheimer's disease;

(5) Use in the preparation of a product for malignancy detection and/or prognosis evaluation.

In the above (1), the sample may be a body fluid sample (e.g., blood, serum, tissue fluid, etc.), a cell sample or a tissue sample derived from a human, or may be 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 CHI3L1 content in the sample.

In the above (2) - (5), CHI3L1 is known as a biomarker for liver fibrosis, alzheimer's disease and malignant tumor, and thus, the antibody or antigen-binding fragment thereof can be used for detecting the above-mentioned diseases or for diagnosis or auxiliary diagnosis of the above-mentioned diseases. The amount of CHI3L1 in a malignancy is known to be inversely related to disease-free and survival, and therefore, the antibodies or antigen-binding fragments thereof can be used for prognostic evaluation of malignancy. Wherein the malignant tumor comprises prostate cancer, colon cancer, lung cancer, ovarian cancer, renal cancer, breast cancer, glioblastoma or malignant melanoma.

In the application, the product is a detection reagent or a detection kit.

In a seventh aspect, the invention provides the use of an antibody or antigen binding fragment thereof or said antibody conjugate or said antibody composition as described above for detecting the presence or level of CHI3L1 in a sample for non-disease diagnostic and therapeutic purposes.

For detection of non-disease diagnostic and therapeutic purposes, the sample does not include samples derived directly from a living human or animal, and may be cell or protein samples prepared by in vitro culture.

In the present invention, the method for detecting CHI3L1 includes, but is not limited to, ELISA, chemiluminescent immunoassay, radioimmunoassay, fluoroimmunoassay, immunochromatography, and other immunological detection methods.

In an eighth aspect, the invention provides a kit comprising an antibody or antigen binding fragment thereof as described above, or comprising the antibody conjugate, or comprising the antibody composition.

The type of the kit is not particularly limited, and includes, but is not limited to, ELISA detection kit, chemiluminescent detection kit, radioimmunoassay kit, fluorescent immunodetection kit, etc.

In some embodiments of the 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 in the (1) of the fifth aspect, and the labeled antibody is the antibody in the (2) of the fifth aspect. The coated antibody is coated on an ELISA plate. The labeled antibody is labeled by biotin. The kit may also include a secondary antibody carrying a detectable label and other reagents for ELISA detection, including, but not limited to, one or more selected from the group consisting of an ELISA plate, coating buffer, diluent, human CHI3L1 standard, PBST wash, blocking solution, chromogenic solution, stop solution.

In a ninth aspect, the present invention provides a method for detecting CHI3L1, the method comprising: detecting the content of CHI3L1 in the sample to be tested using the above-described antibodies or antigen binding fragments thereof or the antibody conjugates or the antibody compositions or the kit.

The detection method can be selected from ELISA, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, etc.

In a tenth aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described above.

The pharmaceutical composition described above is preferably used for the treatment of malignant tumors, liver toxicity, non-alcoholic steatohepatitis or non-alcoholic fatty liver disease.

Preferably, the malignancy 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 malignancy is a hematological malignancy, such as myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, chronic myelogenous leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute myelogenous leukemia, or polycythemia vera.

In an eleventh aspect, the invention also provides bispecific antibodies comprising the antibodies or antigen binding fragments thereof.

Preferably, the bispecific antibody comprises the above-described antibody or antigen-binding fragment thereof, and an anti-PD 1 antibody.

The beneficial effects of the invention at least comprise: the invention provides an antibody capable of specifically binding to human CHI3L1, which has high specificity 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-1 beta, human IFN-alpha, human IL-8, human TNF-alpha, human IL-12, human TNF-beta, GAPDH and other similar proteins, has higher affinity to human CHI3L1, and can realize specific and sensitive detection of human CHI3L 1. The human CHI3L1 detection kit developed based on the antibody has higher specificity, sensitivity and accuracy, has wider linear range (15.6-1000 pg/mL), is applicable to low-concentration CHI3L1 samples as well, has higher stability (37 ℃ accelerated stability experiment, no obvious change of the kit in 13 days), can meet detection requirements, can be used for detecting human CHI3L1 in practice, and has better application prospect.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the SDS-PAGE detection of the monoclonal antibody of example 1 of the present invention.

FIG. 2 is a standard curve of human CHI3L1 detection in example 3 of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 preparation of anti-human CHI3L1 monoclonal antibodies

1. Immunization of animals

Female Balb/c mice of 6-8 weeks old are selected, emulsified with commercial CHI3L1 antigen and an equal volume of Freund's adjuvant for immunization, the immunization period is two weeks, blood is taken for measuring the titer after 3 times of immunization, and the immunization is boosted again three days before fusion.

2. Cell fusion

Mice were sacrificed by cervical scission, spleens were removed by aseptic manipulation, and spleen cell suspensions were prepared by squeeze milling in a plate. The prepared syngeneic myeloma cells and the spleen cells of the mice are mixed according to a certain proportion, and a fusogenic agent polyethylene glycol (PEG) is added. Under the action of polyethylene glycol, various lymphocytes can be fused with myeloma cells to form hybridoma cells. The specific operation is as follows.

(1) Myeloma (SP 2/0) cell activation

Thawing and resuscitating commercial SP2/0 cells, then re-suspending in nutrient solution (RPMI-1640 basal medium, adding 20% fetal bovine serum), and culturing in incubator under the condition of 37deg.C and 5% CO ₂; passaging is carried out after 3-5 d;

And collecting cells and suspending the cells in RPMI-1640 basal culture solution, counting, taking 0.5-1 multiplied by 10 ⁶ cells to be injected into the back of a Balb/c mouse subcutaneously, and continuously culturing for 9-10 d. After the tumor volume of the back is increased to about 0.8cm in diameter, the mice are killed by pulling the neck, and the tumor is taken out after 75% alcohol soaking for 5 min.

Cutting off tumor blocks, placing the cut tumor blocks in a sterilized homogenizer, adding the RPMI-1640 basic culture solution, fully grinding, adding 10mL of the RPMI-1640 basic culture solution, standing for 2min, sucking the cell suspension at the upper layer, placing in another centrifuge tube, adding 10mL of the RPMI-1640 basic culture solution, and repeatedly grinding for two times; the cell suspension obtained above was centrifuged at 1000r/min for 10min to remove the supernatant, followed by resuspension in 30mL of RPMI-1640 basal medium.

Adding 15mL of lymphocyte separation liquid into another centrifuge tube, and carefully placing the cell suspension on the separation liquid; and then centrifuging at 1200r/min for 15min, sucking a white cell layer positioned at the interface by a suction tube, cleaning the cells by using RPMI-1640 basal culture solution for 2 times, then re-suspending the cells in 10mL of RPMI-1640 basal culture solution, and counting for later use.

(2) Preparation of immune spleen cells

Taking one Balb/c mouse with enhanced immunity, performing orbital exsanguination and sacrifice (collecting serum, namely positive serum), soaking in 75% alcohol for 5-10min for sterilization, then fixing the Balb/c mouse on an dissecting plate for dissection, taking out spleen, shearing the spleen, and placing the spleen in a sterilized homogenizer; grinding and cell suspension preparation method are as described in SP2/0, and counting for later use.

(3) Preparation of feeder cells

One non-immunized Balb/c mouse was taken, the orbit was exsanguinated, and serum was collected as negative serum. Injecting 2-3 mL of RPMI-1640 basic culture solution into the abdominal cavity of the mouse, sucking out the solution after blowing and placing the solution into another centrifuge tube for standby, wherein the solution contains abdominal macrophages. Centrifuging at 1000r/min for 10min to remove supernatant, suspending cells in HAT medium, and placing in a 5% CO ₂ incubator at 37deg.C for use.

(4) Fusion of

1-2X 10 ⁷ SP2/0 and 10 ⁸ immunocytes were mixed in a 50mL centrifuge tube, centrifuged at 1000r/min for 8min. After discarding the supernatant, the centrifuge tube containing the cell mixture was placed in a 37℃water bath, followed by adding 0.8mL of 50% PEG (Sigma) pre-warmed to 37℃and allowed to stand for 30s after stirring. After standing, 10mL of RPMI-1640 basal medium was added at 37 ℃. Centrifuging at 1000r/min for 5min, discarding supernatant, and standing at 37deg.C for 5-8min. Subsequently mixed with feeder cell suspension, seeded in 96-well plates, 250. Mu.L/well, and incubated in a 5% CO ₂ incubator at 37 ℃. HT medium was changed for continued culture on day 4 after fusion. And (4) when the colony of the fused cells grows to 1/4 of the culture hole and the culture medium turns yellow slightly, detecting the antibody.

3. Selection of hybridoma positive clones and cloning of cells

The purpose of the selective culture is to screen the fused hybridoma cells using HAT selective medium. In HAT medium, unfused myeloma cells lack hypoxanthine-guanine-phosphoribosyl transferase and cannot synthesize DNA by salvage pathways to die. Unfused lymphocytes have hypoxanthine-guanine-phosphoribosyl transferase, but do not survive in vitro for long periods and die. Only fused hybridoma cells survive and proliferate in HAT medium due to the hypoxanthine-guanine-phosphoribosyl transferase obtained from spleen cells and the immortalized nature of myeloma cells. The specific operation is as follows.

(1) Positive hybridoma cells were screened by indirect ELISA as follows:

Coating known antigens: diluting the purified coating antigen (human CHI3L1 recombinant protein) to 1-10mg/mL with coating buffer; adding 100 μl of the solution into each of the microwells, shaking gently, standing overnight at 4deg.C in a refrigerator or standing at 37deg.C for 1 hr; the liquid in the hole is thrown away (the liquid in the hole is beaten as much as possible); washing for 2-3 min for 3 times.

Blocking the positions of the enzyme-labeled wells not coated with antigen: 200. Mu.L of blocking solution (5% skimmed milk powder or 0.1% BSA) was added to each well, gently shaken and left at 37℃for 1h; throwing away the liquid in the hole; washing buffer solution by Kong Jiaman, standing for 2-3 min, throwing away liquid in the hole, beating to dry, and washing 3 times by using the washing buffer solution.

Sample adding: 50 mu L of supernatant liquid is taken from each hole of the hybridoma to be detected, sequentially added into the enzyme-labeled holes, gently shaken, placed at 37 ℃ for 1h, washed and patted dry.

Adding enzyme-labeled antibody: diluting the enzyme-labeled secondary antibody to a proper working concentration according to instructions by using a diluent, adding 100 mu L of the secondary antibody into each hole, gently shaking the secondary antibody, and standing the secondary antibody at 37 ℃ for 1h; then washing and beating to dry.

Adding a color development liquid: each well was added with 100. Mu.L of freshly prepared color development solution, gently shaken well, at 37℃for 10min.

Terminating the reaction: 50. Mu.L of stop solution was added to each well.

Determination result: the enzyme label instrument OD ₄₅₀ is used for reading, and the detection result is 3 times larger than that of the negative hole, so that the detection result can be judged positive.

(2) Cloning of hybridoma cells (limiting dilution method)

Preparing a mouse feeder cell layer before cloning; gently blowing the hybridoma cells to be cloned from the culture well, and counting the number of living cells by using a blood cell counting plate; diluting cells to 10 cells/mL with complete medium;

The cell suspensions at the above concentrations were added to 96-well plates of prepared feeder cells, 100. Mu.L/well, respectively, so that 1 cell was contained in each well. Culturing until 4 days of fluid infusion is one drop, carefully observing the growth condition of cells in each hole on 5-6 days, and recording;

(3) Detection of specific antibodies

The cell clone can be detected when 1/3-1/2 field of vision is full of the cell clone on the 7 th to 9 th days after the cloning; cells in the positive holes can be transferred to a 24-hole culture plate, and when the cells in the 24-hole plate grow well, the mice can be inoculated in the abdominal cavity to collect ascites.

Two monoclonal antibodies which specifically bind to human CHI3L1 are obtained through the hybridoma cell screening, and are named as monoclonal antibodies 5E10 and 1E3 respectively.

4. Monoclonal antibodies 5E10 and 1E3 variable region sequencing

Collecting hybridoma cells with the number of more than 10 ⁶, adopting a total RNA extraction kit (Soy Bao technology Co., ltd.) and extracting total RNA of the two strains of cells respectively according to the operation of a specification; the first strand of cDNA was synthesized using a reverse transcription kit (Soy technologies Co., ltd.) and PCR amplified, and the resulting DNA fragment of interest was ligated to a T vector and sent to a Praeparata for subsequent sequencing. The gene sequencing results were obtained as follows: the heavy chain variable region sequence of the monoclonal antibody 5E10 is 357bp in length, 119 amino acids are coded, the DNA sequence is shown as SEQ ID NO.15, the protein sequence is shown as SEQ ID NO.11, wherein the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID NO.1, 2 and 3 respectively; the variable region sequence of the light chain is 321bp long, codes 107 amino acids, the DNA sequence is shown as SEQ ID NO.16, and the protein sequence is shown as SEQ ID NO. 12; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown as SEQ ID NO.4, the amino acid sequence of CDR2 is YAS, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 5. The heavy chain variable region sequence of the monoclonal antibody 1E3 is 357bp in length, 119 amino acids are coded, the DNA sequence is shown as SEQ ID NO.17, the protein sequence is shown as SEQ ID NO.13, and the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID NO.6, 7 and 8 respectively; the variable region sequence of the light chain is 321bp long, codes 107 amino acids, the DNA sequence is shown as SEQ ID NO.18, and the protein sequence is shown as SEQ ID NO. 14; the amino acid sequence of the complementarity determining region CDR1 of the light chain variable region is shown as SEQ ID NO.9, the amino acid sequence of the CDR2 is YTS, and the amino acid sequence of the CDR3 is shown as SEQ ID NO. 10.

5. Mass production of monoclonal antibodies 5E10 and 1E3

The hybridoma cells after the strain establishment were injected into the abdominal cavity of the mouse, ascites were collected for about 7 days, antibodies were purified by Protein G affinity chromatography, and the antibodies were detected by SDS-PAGE, and the results are shown in FIG. 1.

Example 2 affinity assay of monoclonal antibodies 5E10 and 1E3

The relative affinity constants of monoclonal antibodies 5E10 and 1E3 were determined as described below.

Antigen was coated onto the elisa plate and blocked. After washing the plates with PBST, monoclonal antibodies 5E10 and 1E3 were diluted to saturation concentration and added to the ELISA plate at 100. Mu.L/well, and incubated at room temperature for 2h. After PBST plate washing, naSCN solution of 0, 0.5, 1.5, 2.5, 3.5 and 4.5mol/L was added in sequence at 60. Mu.L/well, and incubated at room temperature for 15min. After PBST plate washing, HRP-labeled goat anti-mouse IgG is added, and the plate is incubated at room temperature for 45min for chromogenic detection. The concentration of sodium thiocyanate corresponding to the decrease of OD value at 450nm after elution to 50% without elution is the relative affinity constant of the antibody, which is expressed in mol/L. The results show (Table 1) that the monoclonal antibodies 5E10 and 1E3 have relative affinity constants of 1mol/L and 2mol/L, respectively, with good affinity.

TABLE 1

Example 3 establishment and evaluation of double antibody sandwich ELISA detection kit and method

Human CHI3L1 double-antibody sandwich ELISA detection kits and detection methods were constructed based on monoclonal antibodies 5E10 and 1E3 and their performance was evaluated as described in detail below.

1. Biotin labelling of monoclonal antibody 1E3

The Protein G affinity chromatography purified monoclonal antibody 1E3 was added to a dialysis bag and dialyzed overnight at 4℃in 0.1M CB buffer. NHS-D-Biotin was dissolved in DMSO to prepare a 2.2mg/mL solution. The prepared NHS-D-Biotin solution was added to the antibody solution dialyzed overnight, protected from light at room temperature, and gently stirred for 4 hours. The reacted solution was thoroughly dialyzed against 0.01M PBS (pH 7.2 to 7.4) at 4 ℃. The labeled monoclonal antibody 1E3 was removed from the dialysis bag, added with equal volumes of glycerol, preservative and BSA (final concentration 10 mg/mL), and stored in an antibody tube at-20 ℃.

2. Preparation of monoclonal antibody 5E10 coated ELISA plate

Using 96-well flat-bottom polystyrene ELISA plate as solid phase carrier, diluting Protein G affinity chromatography purified monoclonal antibody 5E10 to 2 mug/mL with antibody coating liquid, adding diluted antibody into micropores of ELISA plate, 100 mug/hole, sealing with sealing plate membrane, coating at 4deg.C overnight, taking out coated ELISA plate, discarding liquid in the hole, washing the plate 3 times with ELISA washing liquid, adding sealing liquid containing 2% BSA, 250 mug/hole, sealing with sealing plate membrane, sealing for 2h at room temperature, discarding liquid in the plate hole, and drying for 16-18 h in dry room. Placing into aluminum foil bag containing drying agent, vacuum sealing, and preserving at 4deg.C.

3. Establishment of double antibody sandwich ELISA method

And taking out the ELISA plate coated with the monoclonal antibody 5E10 30min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. Human CHI3L1 standard is diluted by multiple ratio (1000, 500, 250, 125, 62.5, 31.25, 15.625 pg/mL) and then added into an ELISA plate, 100 mu L/well, and a blank control well is arranged at the same time; incubating for 2h at room temperature (25+ -2deg.C) with shaking, washing, diluting biotin-labeled monoclonal antibody 1E3 to working concentration, adding into each reaction well of the ELISA plate, and adding 100 μL/well; incubating for 1h at room temperature (25+ -2deg.C) with shaking, washing, diluting enzyme conjugate to working concentration, adding into each reaction well of the ELISA plate, and adding into 100 μL/well; incubating for 30min at room temperature (25+ -2deg.C) with shaking, and washing, adding TMB chromogenic substrate, 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min; finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well to stop the reaction, and the reaction was subjected to dual wavelength detection using an enzyme-labeled instrument within 5 minutes to determine the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and the OD measurement value of 630nm was subtracted from the OD measurement value of 450 nm. The standard curve (using four parameter fitting) was plotted with the standard concentration on the abscissa and the absorbance OD on the ordinate using ELISA CALC software, and the results indicated (fig. 2) that the detection range was 15.6-1000pg/mL and R ² was 0.99996.

4. Specific detection of double-antibody sandwich ELISA method

And taking out the ELISA plate coated with the monoclonal antibody 5E10 30min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. Human CHI3L1 standard is subjected to multiple dilution (1000, 500, 250, 125, 62.5, 31.25 and 15.625 pg/mL), added into an ELISA plate, 100 mu L/hole is formed, blank control holes are formed at the same time, the following human recombinant protein （ALB 40ng/mL、IgG 80ng/mL、IgE 50ng/mL、IgM 500ng/mL、IgA 20ng/mL、INS 11.6ng/mL、HSP27 10ng/mL、GAPDH 100ng/mL、IL-1β 1000pg/mL、IFN-α 5000pg/mL、IL-8 2000pg/mL、TNF-α 1000pg/mL、IL-12 4000pg/mL、TNF-β 2000g/mL、OPN 2000pg/mL、CRP 1000pg/mL） is added into other holes of the ELISA plate, 100 mu L/hole is formed, the mixture is subjected to shaking incubation at room temperature (25+/-2 ℃) for 2 hours, then washing is carried out, biotin-labeled monoclonal antibody 1E3 is diluted to a working concentration, and 100 mu L/hole is added into each reaction hole of the ELISA plate; incubating for 1h at room temperature (25+ -2deg.C) with shaking, washing, diluting enzyme conjugate to working concentration, adding into each reaction well of the ELISA plate, and adding 100 μL/well; incubating for 30min at room temperature (25+ -2deg.C) with shaking, washing, adding TMB chromogenic substrate, and measuring 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min; finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well to stop the reaction, and the reaction was subjected to dual wavelength detection using an enzyme-labeled instrument within 5 minutes to determine the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and the OD measurement value of 630nm was subtracted from the OD measurement value of 450 nm. The results show (Table 2) that the cross-reactivity with the above proteins was < 3%.

TABLE 2

5. Stability detection of double-antibody sandwich ELISA detection kit

The ELISA plate coated with the monoclonal antibody 5E10, the biotin-labeled monoclonal antibody 1E3 and the standard human CHI3L1 were subjected to an accelerated stability test at 37℃for 13 days (equivalent to half a year at-20 ℃). Then taking out for detection, wherein the detection method comprises the following steps: washing the ELISA plate for 3 times and spin-drying; human CHI3L1 standard is subjected to multiple dilution (1000, 500, 250, 125, 62.5, 31.25 and 15.625 pg/mL), added into an ELISA plate, 100 mu L/hole is formed, blank control holes are formed at the same time, shaking and incubation are carried out for 2 hours at room temperature (25+/-2 ℃), then washing is carried out, biotin-labeled monoclonal antibody 1E3 is diluted to a working concentration, and added into each reaction hole of the ELISA plate, and 100 mu L/hole is formed; incubating for 1h at room temperature (25+ -2deg.C) with shaking, washing, diluting enzyme conjugate to working concentration, adding into each reaction well of the ELISA plate, and adding 100 μL/well; incubating for 30min at room temperature (25+ -2deg.C) with shaking, washing, adding TMB chromogenic substrate, and measuring 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min; finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well to stop the reaction, and the reaction was subjected to dual wavelength detection using an enzyme-labeled instrument within 5 minutes to determine the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and the OD measurement value of 630nm was subtracted from the OD measurement value of 450 nm. Standard curves were plotted with ELISA CALC software (using four parameter fitting) on the abscissa for standard concentration and on the ordinate for absorbance OD, and the results indicated (table 3) that the stability of the kit was good.

TABLE 3 Table 3

6. Recovery rate and linear detection of double antibody sandwich ELISA method

And taking out the ELISA plate coated with the monoclonal antibody 5E10 30min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. Human CHI3L1 standard is diluted by multiple ratio (1000, 500, 250, 125, 62.5, 31.25, 15.625 pg/mL) and then added into an ELISA plate, 100 mu L/well, and a blank control well is arranged at the same time; selecting RPMI-1640 basic culture solution and serum of healthy volunteers respectively, and adding human CHI3L1 standard substances with different concentrations into the culture solution and the serum of healthy volunteers, wherein the concentration is 100 mu L/hole; incubating for 2h at room temperature (25+ -2deg.C) with shaking, washing, diluting biotin-labeled monoclonal antibody 1E3 to working concentration, adding into each reaction well of the ELISA plate, and adding 100 μL/well; incubating for 1h at room temperature (25+ -2deg.C) with shaking, washing, diluting enzyme conjugate to working concentration, adding into each reaction well of the ELISA plate, and adding 100 μL/well; incubating for 30min at room temperature (25+ -2deg.C) with shaking, washing, adding TMB chromogenic substrate, and measuring 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min; finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well to stop the reaction, and the reaction was subjected to dual wavelength detection using an enzyme-labeled instrument within 5 minutes to determine the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and the OD measurement value of 630nm was subtracted from the OD measurement value of 450 nm. And drawing a standard curve (using four-parameter fitting) by using ELISA CALC software with the standard substance concentration as an abscissa and the absorbance OD value as an ordinate, establishing an equation, and calculating the standard adding recovery rate. The results show (Table 4) that the recovery rate is 80% -120%, and the standard recovery rate is good.

TABLE 4 Table 4

And taking out the ELISA plate coated with the monoclonal antibody 5E10 30min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. Human CHI3L1 standard is diluted by multiple ratio (1000, 500, 250, 125, 62.5, 31.25, 15.625 pg/mL) and then added into an ELISA plate, 100 mu L/well, and a blank control well is arranged at the same time; respectively selecting RPMI-1640 basal culture solution and serum of healthy volunteers, adding a human CHI3L1 standard substance, performing multiple dilution, and then adding the diluted product into an ELISA plate at a concentration of 100 mu L/hole; setting blank control holes and negative control holes at the same time; incubating for 2h at room temperature (25+ -2deg.C) with shaking, then washing to dilute biotin-labeled monoclonal antibody 1E3 to working concentration, adding into each reaction well of the ELISA plate, and adding 100 μL/well; incubating for 1h at room temperature (25+ -2deg.C) with shaking, washing, diluting enzyme conjugate to working concentration, adding into each reaction well of the ELISA plate, and adding 100 μL/well; incubating for 30min at room temperature (25+ -2deg.C) with shaking, washing, adding TMB chromogenic substrate, and measuring 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min; finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well to stop the reaction, and the reaction was subjected to dual wavelength detection using an enzyme-labeled instrument within 5 minutes to determine the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and the OD measurement value of 630nm was subtracted from the OD measurement value of 450 nm. And drawing a standard curve (using four-parameter fitting) by using ELISA CALC software with the standard substance concentration as an abscissa and the absorbance OD value as an ordinate, establishing an equation, and calculating the linear dilution recovery rate. The results showed (Table 5) that the recovery rate was 80% -120%, and the linear dilution recovery rate was good.

TABLE 5

7. Sensitivity detection of double-antibody sandwich ELISA method

And taking out the ELISA plate coated with the monoclonal antibody 5E10 30min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. Human CHI3L1 standard is diluted by multiple times (1000, 500, 250, 125, 62.5, 31.25, 15.625 pg/mL), and then added into an ELISA plate, wherein 100 mu L/hole is provided, and a plurality of blank control holes (generally more than or equal to 20) and 100 mu L/hole are arranged at the same time; incubating for 2h at room temperature (25+ -2deg.C) with shaking, washing, diluting biotin-labeled monoclonal antibody 1E3 to working concentration, adding into each reaction well of the ELISA plate, and adding 100 μL/well; incubating for 1h at room temperature (25+ -2deg.C) with shaking, washing, diluting enzyme conjugate to working concentration, adding into each reaction well of the ELISA plate, and adding 100 μL/well; incubating for 30min at room temperature (25+ -2deg.C) with shaking, washing, adding TMB chromogenic substrate, and measuring 100 μl/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min; finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well to stop the reaction, and the reaction was subjected to dual wavelength detection using an enzyme-labeled instrument within 5 minutes to determine the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and the OD measurement value of 630nm was subtracted from the OD measurement value of 450 nm. And drawing a standard curve (using four-parameter fitting) by using ELISA CALC software with the standard substance concentration as an abscissa and the absorbance OD value as an ordinate, and establishing an equation to calculate the sensitivity. The calculation formula of the sensitivity is: the calculated mean +2×standard deviation of the blank OD values gave a sensitivity of 0.98pg/mL, and the blank OD values are shown in Table 6.

TABLE 6

Example 4 comparison of test results with isotype kit

A commercially available human CHI3L1 double-antibody sandwich ELISA test kit A (RND, product number DY 2599) was selected for an analog test with the human CHI3L1 double-antibody sandwich ELISA test kit of example 3 of the present invention, wherein the test kit A was tested according to the method in the specification thereof, and the test kit of the present invention was tested according to the test method of example 3. The performance of the kit was compared by the following 3 aspects.

1. Standard curve comparison

The results show (Table 7) that R ² of the standard curves of the kit and the kit A are both larger than 0.999, and the fitting degree is good; however, the background performance of the kit of the invention is better than that of the kit A.

TABLE 7

2. Standard substance comparison

The results show (Table 8) that the kit of the invention has good gradient and close recovery rate when detecting SZ standard (standard of kit A), while the kit A has higher concentration value measured at low concentration and has a large difference in recovery rate when detecting the standard of the kit of the invention. Therefore, for the content detection of recombinant human CHI3L1, the accuracy of the kit is better than that of the kit A.

TABLE 8

3. Sample measurement result comparison

16 Human serum samples were randomly selected for simultaneous assay (unit ng/mL). The measurement is performed by diluting the sample so that the sample is within the measurement range of the standard curve. The measurement results show (Table 9) that the sample value measured by the kit A is generally higher than that measured by the kit of the invention, and is consistent with the comparison result of the standard substance in the step 2, so that the accuracy of the kit A for sample detection is lower than that of the antibody pair of the invention.

TABLE 9

EXAMPLE 5 human serum CHI3L1 assay

And taking out the ELISA plate coated with the monoclonal antibody 5E10 30min before the experiment, recovering to room temperature, washing the plate for 3 times, and spin-drying. Human CHI3L1 standard is diluted by multiple ratio (5000, 2500, 1250, 625, 312.5, 156.25, 78.125 pg/mL) and then added into an ELISA plate, 100 mu L/well, and a blank control well is arranged at the same time; respectively selecting 28 human serum samples and 20 human plasma samples, diluting, adding into an ELISA plate, standing at room temperature (25+/-2 ℃) for incubation for 2 hours, washing, diluting biotin-labeled monoclonal antibody 1E3 to a working concentration, and adding into each reaction hole of the ELISA plate to 100 mu L/hole; standing at room temperature (25+/-2 ℃) for incubation for 1h, then washing, diluting the enzyme conjugate to the working concentration, adding the enzyme conjugate into each reaction well of the ELISA plate, and adding the enzyme conjugate into 100 mu L/well; standing at room temperature (25+/-2 ℃) for 30min, washing, adding TMB chromogenic substrate into each reaction well, and adding 100 mu L/well; standing at room temperature (25+/-2 ℃) for color development for 10-20 min; finally, a stop solution was added to each reaction well at a concentration of 50. Mu.L/well to stop the reaction, and the reaction was subjected to dual wavelength detection using an enzyme-labeled instrument within 5 minutes to determine the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 630nm, and the OD measurement value of 630nm was subtracted from the OD measurement value of 450 nm. And drawing a standard curve (using four-parameter fitting) by using ELISA CALC software with the standard substance concentration as an abscissa and the absorbance OD value as an ordinate, establishing an equation, and calculating the sample concentration. The results show (tables 10 and 11) that the detection results obtained using the kit of the invention are consistent with the levels of CHI3L1 in human serum/plasma reported in the literature.

Table 10

TABLE 11

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart 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 that binds to human 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 antigen-binding fragment thereof are shown in SEQ ID NO.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.

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.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.

3 . The antibody or antigen-binding fragment thereof according to claim 1 or 2 , wherein 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. A biological material, 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 antibody conjugate according to claim 6, or the antibody composition according to claim 7:

(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 the detection and/or prognosis assessment of malignant tumors; the malignant tumors include prostate cancer, colon cancer, lung cancer, ovarian cancer, kidney cancer, breast cancer, glioblastoma or malignant melanoma.

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.