Highly sensitive liver disease detection kit based on chitinase 3-like protein 1

Abstract

The present invention relates to a high-sensitivity liver disease detection kit based on chitinase 3-like protein 1. The kit comprises the following parts: a pre-coated plate, each well of which is pre-coated with a monoclonal antibody made from one of the following CHI3L1 antigen epitope peptides (1) or peptides (2): Tyr-Arg-Lys-Ser-Val-Pro-Pro-Phe-Leu-Arg-Thr, (2) Tyr-Arg-Ser-Ala-Ala-Leu-Ser-Ala-Gly-Lys-Val-Thr-Ile-Asp; a CHI3L1 calibrator; a binding antibody, which is a solution of a polyclonal antibody made from one of the antigen epitope peptides (1) or peptides (2); (D) an enzyme conjugate, which is a horseradish enzyme-labeled goat anti-rabbit IgG antibody solution; a washing buffer; a color developer A; a color developer B; and a stop solution. The antigen epitope peptides used in the pre-coated plate and the binding antibody are different. It also relates to a method for determining the concentration of CHI3L1 in serum using the in vitro diagnostic kit. The CHI3L1 antigen epitope peptide of the present invention has good antigenicity and can be applied to the in vitro detection of human CHI3L1, for example, for the detection and diagnosis of liver diseases.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide chemistry and immunology, and relates to a chitinase 3-like protein 1 (CHI3L1) antigen epitope peptide. Using the antigen epitope peptide, by using a carrier protein KLH for modification, a CHI3L1-specific antigen can be prepared, and then a corresponding monoclonal antibody or polyclonal antibody can be prepared. In addition, such antibodies can be used to prepare a human CHI3L1 in vitro diagnostic kit, and such a kit can be used, for example, to detect the CHI3L1 concentration in a serum sample of a patient with liver disease, providing an effective scientific basis for liver disease diagnosis. The present invention thus relates to a method for preparing an antigen by using a carrier protein to modify a chitinase 3-like protein 1 antigen epitope peptide, and a high-sensitivity liver disease detection kit based on chitinase 3-like protein 1 designed thereby. Background Art

[0002] Chitinase-3-like protein 1 (CHI3L1), also known as YKL-40 protein, is a glycoprotein of the chitinase protein family, about 40 kDa in size. CHI3L1 was first discovered in the milk secretions of non-lactating cows and can be secreted by a variety of cells, mainly including: ① chondrocytes and fibroblast-like synoviocytes of arthritis patients; ② activated macrophages and macrophages in the late stage of differentiation; ③ neutrophils; ④ others, human osteosarcoma cells (MG-63), differentiated vascular smooth muscle cells, mammary epithelial cells, macrophage subsets, smooth muscle cells of different inflammatory tissues and other cells can also detect CHI3L1 expression.

[0003] CHI3L1 protein is encoded by the CHI3L1 gene, and CHI3L1 mRNA is strongly expressed in chondrocytes and liver, and weakly or not expressed in the brain, kidney, heart, etc.

[0004] Current research data show that the biological functions of CHI3L1 mainly include: ① promoting the growth of connective tissue cells and playing an important role in the proliferation and differentiation of chondrocytes and osteoblasts; ② regulating the morphology of vascular endothelial cells and playing an important role in the formation of new blood vessels; ③ regulating matrix remodeling; ④ helping cells to adapt to changes in the growth environment and repair pathological damage such as hypoxia, promoting cell proliferation and survival and protecting cells from apoptosis; ⑤ initiating signal cascade reactions in connective tissue cells, leading to cell proliferation and promoting tissue fibrosis.

[0005] In patients with chronic liver disease, serum CHI3L1 concentration increases, and most patients with alcoholic cirrhosis or post-hepatitis cirrhosis have elevated serum CHI3L1. The range of serum CHI3L1 in patients with non-cirrhotic fibrosis gradually increases from the normal starting range. Serum CHI3L1 is closely related to the degree of fibrosis, which is determined by the highest level of moderate to severe fibrosis in pathological patients. Serum CHI3L1 is elevated to a lesser extent in patients with mild fibrosis, but this increase is still significantly greater than that in patients without fibrosis. Therefore, CHI3L1 levels can reflect the degree of liver fibrosis and can serve as a diagnostic marker for liver fibrosis.

[0006] The most ideal method for detecting the level of CHI3L1 in serum is immunoassay. Therefore, finding suitable immunogenic CHI3L1 epitope peptides and preparing specific CHI3L1 antigens and antibodies have become the key points.

[0007] CN111197040A (application number 2020100721960, Yitong Bio) discloses a chitinase 3-like protein 1 (CHI3L1) antigen epitope peptide, antigen, antibody, use and kit; the amino acid sequence of the CHI3L1 antigen epitope peptide of the invention, the amino acid chain fragment of the CHI3L1 antigen epitope peptide is as follows Tyr-Arg-Lys-Ser-Val-Pro-Pro-Phe-Leu-Arg-Thr and Tyr-Arg-Ser-Ala-Ala-Leu-Ser-Ala-Gly-Lys-Val- Thr-Ile-Asp; the CHI3L1 antigen of the invention is prepared by coupling the CHI3L1 antigen epitope peptide with a protein carrier; the CHI3L1 monoclonal antibody or polyclonal antibody of the invention is prepared by the CHI3L1 antigen of the invention; the CHI3L1 monoclonal antibody or polyclonal antibody of the invention is used to prepare a CHI3L1 in vitro diagnostic kit; the CHI3L1 antigen epitope peptide of the invention exhibits good antigenicity, and the antigen (immunogen) prepared therefrom can produce highly specific monoclonal antibodies and polyclonal antibodies in animals immunized with it, thereby being applicable to the in vitro detection of human CHI3L1.

[0008] However, the art still expects a method for preparing the above-mentioned chitinase 3-like protein 1 antigen, for example, by modifying it with a carrier protein KLH to prepare a CHI3L1-specific antigen; or, the art still expects a high-sensitivity liver disease detection kit based on chitinase 3-like protein 1 designed by such CHI3L1-specific antigen and corresponding monoclonal antibodies or polyclonal antibodies. Summary of the invention

[0009] The object of the present invention is to provide a CHI3L1 antigenic epitope peptide, a CHI3L1 specific antigen prepared with the antigenic epitope peptide and a corresponding monoclonal antibody or polyclonal antibody, its use in preparing a CHI3L1 kit, and a CHI3L1 in vitro diagnostic kit. For example, the object of the present invention is to provide a method for preparing a high-purity CHI3L1 antigenic epitope peptide, for example, the object of the present invention is to provide a high-purity CHI3L1 antigenic epitope peptide. For example, the object of the present invention is to provide a high-sensitivity liver disease detection kit based on chitinase 3-like protein 1. It has been unexpectedly found that a high-sensitivity liver disease detection kit based on chitinase 3-like protein 1 can be obtained by the method of the present invention. The present invention is completed based on such a discovery.

[0010] To this end, the first aspect of the present invention provides a CHI3L1 antigen epitope peptide, which is an amino acid chain fragment selected from the following peptide (1) or peptide (2):

[0011] (1)Tyr-Arg-Lys-Ser-Val-Pro-Pro-Phe-Leu-Arg-Thr,

[0012] (2)Tyr-Arg-Ser-Ala-Ala-Leu-Ser-Ala-Gly-Lys-Val-Thr-Ile-Asp.

[0013] In the present invention, the above-mentioned amino acid chain fragments (1) and (2) may be referred to as peptide (1) and peptide (2), or may be referred to as antigen epitope peptide (1) and antigen epitope peptide (2), or may be referred to as CHI3L1 antigen epitope peptide (1) and CHI3L1 antigen epitope peptide (2) or similar names.

[0014] According to the first aspect of the present invention, when preparing the CHI3L1 antigen epitope peptide (1) or (2), Tyr is introduced in the following manner:

[0015] To the prepared Fmoc-Arg(Tos)-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin, add 20% hexahydropyridine / dimethylformamide solution, shake at room temperature for decapping reaction to remove the Fmoc protecting group at the nitrogen end, filter out the solvent, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and filter out the solvent, and add Fmoc-Tr(tBu)-OH, HBTU, and HOBT to dimethylformamide to make Dissolve, add DIEA and tromethamine, stir well, transfer into the reactor containing the treated resin, react at room temperature, filter out the reaction solution, then wash the resin twice with dimethylformamide, methanol and dichloromethane alternately, filter out the solvent, and after the peptide connection reaction is completed, filter out the resin and dry it in a vacuum dryer to obtain Fmoc-Tyr(tBu)-Arg(Tos)-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin, i.e., protected 11-peptide resin; and

[0016] To the prepared Fmoc-Arg(Tos)-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin, add 20% hexahydropyridine / dimethylformamide solution, shake at room temperature for decapping reaction to remove the Fmoc protecting group at the nitrogen end, filter out the solvent, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and filter out the solvent, and add Fmoc-Tyr(tBu)-OH, HBTU, and HOBT to dimethylformamide. The mixture was dissolved, DIEA and tromethamine were added, stirred well, and transferred into a reactor containing the treated resin. The mixture was shaken at room temperature for reaction, and the reaction solution was removed by suction. The resin was then washed twice with dimethylformamide, methanol, and dichloromethane, respectively, and the solvent was removed by suction. After the peptide-linking reaction was completed, the resin was filtered out and dried in a vacuum dryer to obtain Fmoc-Tyr(tBu)-Arg(Tos)-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin, i.e., the protected tetradecapeptide resin.

[0017] According to the first aspect of the present invention, in the process of preparing the CHI3L1 antigen epitope peptide (1) or peptide (2) for inserting Tyr, the molar ratio of the Fmoc-protected amino acid: HBTU: HOBT: DIEA used is 1:1:1:4.

[0018] According to the first aspect of the present invention, in the process of preparing the CHI3L1 antigen epitope peptide (1) or peptide (2) to which Tyr is introduced, the amount of tromethamine is 6% of DIEA, calculated as a molar percentage.

[0019] According to the first aspect of the present invention, the CHI3L1 antigen epitope peptide, wherein the peptide (1) is prepared by the following method:

[0020] Step 1: Preparation of Fmoc-Thr(tBu)-resin

[0021] Place 10 g of Rink Amide-MBHA resin in a reactor, add dichloromethane, shake and soak, wash twice with dichloromethane, methanol and dimethylformamide respectively, and remove the solvent by suction filtration.

[0022] Add 20% hexahydropyridine / dimethylformamide solution to the above-treated resin, shake at room temperature for decapping reaction to remove the Fmoc protecting group at the nitrogen end, remove the solvent by filtration, then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and remove the solvent by filtration.

[0023] 30 mmol of Fmoc-Thr(tBu)-OH, HBTU and HOBT were added to dimethylformamide to dissolve, DIEA was added, stirred, and transferred into the reactor containing the treated resin, and reacted at room temperature by shaking, and the reaction liquid was removed by suction filtration, and the resin was then washed twice with dimethylformamide, methanol and dichloromethane alternately, and the solvent was removed by suction filtration to obtain Fmoc-Thr(tBu)-resin;

[0024] Step 2: Add 20% hexahydropyridine / dimethylformamide solution to the resin obtained in the previous step, shake at room temperature for decapping reaction to remove the Fmoc protecting group at the nitrogen end, filter out the solvent, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and filter out the solvent, add 30 mmol of Fmoc-Arg(Tos)-OH, HBTU, and HOBT to dimethylformamide to dissolve, add DIEA, stir well, and transfer to the reactor containing the treated resin, shake at room temperature for reaction, filter out the reaction solution, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and filter out the solvent to obtain Fmoc-Arg(Tos)-Thr(tBu)-resin;

[0025] Step 3: Take the resin obtained in the previous step and 30 mmol of Fmoc-Leu-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Leu-Arg(Tos)-Thr(tBu)-resin;

[0026] Step 4: Take the resin obtained in the previous step and 30 mmol of Fmoc-Phe-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Phe-Leu-Arg(Tos)-Thr(tBu)-resin;

[0027] Step 5: Take the resin obtained in the previous step and 30 mmol of Fmoc-Pro-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin;

[0028] Step 6: Take the resin obtained in the previous step and 30 mmol of Fmoc-Pro-OH, and refer to the operation method of step 2 to prepare Fmoc-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin;

[0029] Step 7: Take the resin obtained in the previous step and 30 mmol of Fmoc-Val-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin;

[0030] Step 8: Take the resin obtained in the previous step and 30 mmol of Fmoc-Ser(tBu)-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin;

[0031] Step 9: Take the resin obtained in the previous step and 30 mmol of Fmoc-Lys(Boc)-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin;

[0032] Step 10: Take the resin obtained in the previous step and 30 mmol of Fmoc-Arg(Tos)-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Arg(Tos)-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin;

[0033] Step 11: Add 20% hexahydropyridine / dimethylformamide solution to the resin obtained in the previous step, shake at room temperature for decapping reaction to remove the Fmoc protecting group at the nitrogen end, filter out the solvent, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and filter out the solvent, add 30 mmol of Fmoc-Tyr(tBu)-OH, HBTU, and HOBT to dimethylformamide to dissolve, add DIEA and tromethamine, stir well, and transfer the resin containing the above-treated The resin was placed in a reactor and shaken at room temperature for reaction. The reaction solution was removed by suction filtration. The resin was then washed twice with dimethylformamide, methanol and dichloromethane alternately. The solvent was removed by suction filtration. After the peptide-attaching reaction was completed, the resin was filtered out and dried in a vacuum dryer to obtain Fmoc-Tyr(tBu)-Arg(Tos)-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin, i.e., the protected 11-peptide resin;

[0034] Step 12: Cleavage of the peptide chain:

[0035] The resin obtained in the previous step is transferred to a round-bottom flask, and a pre-cooled cutting solution (e.g., 95% trifluoroacetic acid / 2% TIS / 2% EDT / 1% water) is added. The reaction is stirred at room temperature, and the filtrate is separated by suction filtration. The resin is washed with trifluoroacetic acid, and the filtrate and the washing solution are combined. Ice-cold ether is added to precipitate, and the precipitate is filtered to obtain a crude product of the 11-peptide;

[0036] Step 13: Separate and purify the crude peptide obtained in the previous step using an ion exchange chromatography system and high performance liquid chromatography in sequence to obtain a refined peptide, namely, the CHI3L1 antigen epitope peptide (1).

[0037] According to the first aspect of the present invention, the separation and purification of the CHI3L1 antigen epitope peptide in step 13 of preparing the peptide (1) can be carried out according to the examples described herein or in a manner known in the prior art.

[0038] According to the first aspect of the present invention, in the steps 1 to 11 of preparing the peptide (1), the molar ratio of Fmoc-protected amino acid: HBTU: HOBT: DIEA used in each step is 1:1:1:4.

[0039] According to the CHI3L1 antigen epitope peptide of the first aspect of the present invention, in step 1 of preparing peptide (1), when treating RinkAmide-MBHA resin, 80 ml of dichloromethane is added, shaken and soaked for 60 minutes, and washed alternately twice with dichloromethane, methanol, and dimethylformamide, each time with 80 ml, and filtered to remove the solvent.

[0040] According to the first aspect of the present invention, the CHI3L1 antigen epitope peptide, in step 1 of preparing peptide (1), is added to the treated resin with 100 ml of 20% hexahydropyridine / dimethylformamide solution, and the uncapping reaction is carried out at room temperature with shaking for 60 minutes to remove the nitrogen-terminal Fmoc protecting group, and the solvent is removed by filtration, and then the resin is washed twice with dimethylformamide, methanol, and dichloromethane alternately, each time with 80 ml, and the solvent is removed by filtration.

[0041] According to the first aspect of the present invention, the CHI3L1 antigen epitope peptide, in step 1 of preparing peptide (1), 30mmol of Fmoc-Thr(tBu)-OH, HBTU, HOBT are added to 100ml of dimethylformamide to dissolve, DIEA is added, stirred evenly, and transferred into a reactor containing the treated resin, and the reaction is oscillated at room temperature for 1 hour, the reaction solution is filtered out, and the resin is then washed twice with dimethylformamide, methanol, and dichloromethane alternately, each time with 80ml, and the solvent is removed by filtration.

[0042] According to the first aspect of the present invention, in the step 2 of preparing the peptide (1), 100 ml of 20% hexahydropyridine / dimethylformamide solution is added, and the uncapping reaction is carried out at room temperature with shaking for 60 minutes to remove the nitrogen-terminal Fmoc protecting group, and the solvent is removed by filtration. The resin is then washed twice with dimethylformamide, methanol, and dichloromethane alternately, each time with 80 ml, and the solvent is removed by filtration.

[0043] According to the first aspect of the present invention, the CHI3L1 antigen epitope peptide, in step 2 of preparing peptide (1), 30mmol of Fmoc-Arg(Tos)-OH, HBTU, HOBT are added to 100ml of dimethylformamide to dissolve, DIEA is added, stirred evenly, and transferred into a reactor containing the treated resin, and the reaction is oscillated at room temperature for 1 hour, the reaction solution is filtered out, and the resin is then washed twice with dimethylformamide, methanol, and dichloromethane alternately, each time with 80ml, and the solvent is removed by filtration.

[0044] According to the first aspect of the present invention, in the step 11 of preparing the peptide (1), a 20% hexahydropyridine / dimethylformamide solution, for example 100 ml, is added, and the uncapping reaction is carried out by shaking at room temperature, for example 60 minutes, to remove the Fmoc protecting group at the nitrogen end, and the solvent is removed by filtration. The resin is then washed twice with dimethylformamide, methanol, and dichloromethane, for example 80 ml each time, and the solvent is removed by filtration.

[0045] According to the first aspect of the present invention, in the step 11 of preparing the peptide (1), 30 mmol of Fmoc-Tyr(tBu)-OH, HBTU and HOBT are added to, for example, 100 ml of dimethylformamide to dissolve, DIEA and tromethamine are added, stirred evenly, and transferred into a reactor containing the treated resin, and reacted at room temperature for, for example, 1 hour, and the reaction solution is filtered off by suction, and the resin is then washed twice with dimethylformamide, methanol and dichloromethane, for example, 80 ml each time, and the solvent is removed by suction.

[0046] According to the CHI3L1 antigen epitope peptide of the first aspect of the present invention, in step 11 of preparing peptide (1), the amount of tromethamine is 6% of DIEA, calculated in molar percentage.

[0047] According to the first aspect of the present invention, the CHI3L1 antigen epitope peptide, wherein the peptide (2) is prepared by the following method:

[0048] Step 1: Preparation of Fmoc-Asp(OtBu)-resin

[0049] Place 10 g of Rink Amide-MBHA resin in a reactor, add dichloromethane, shake and soak, wash twice with dichloromethane, methanol and dimethylformamide respectively, and remove the solvent by suction filtration.

[0050] Add 20% hexahydropyridine / dimethylformamide solution to the above-treated resin, shake at room temperature for decapping reaction to remove the Fmoc protecting group at the nitrogen end, remove the solvent by filtration, then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and remove the solvent by filtration.

[0051] 30 mmol of Fmoc-Asp(OtBu)-OH, HBTU and HOBT were added to dimethylformamide to dissolve, DIEA was added, stirred evenly, and transferred into the reactor containing the treated resin, and reacted at room temperature by shaking, and the reaction liquid was removed by suction filtration, and the resin was then washed twice with dimethylformamide, methanol and dichloromethane alternately, and the solvent was removed by suction filtration to obtain Fmoc-Asp(OtBu)-resin;

[0052] Step 2: Add 20% hexahydropyridine / dimethylformamide solution to the resin obtained in the previous step, shake at room temperature for decapping reaction to remove the Fmoc protecting group at the nitrogen end, filter out the solvent, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and filter out the solvent, add 30 mmol of Fmoc-Ile-OH, HBTU, and HOBT to dimethylformamide to dissolve, add DIEA, stir well, and transfer to the reactor containing the treated resin, shake at room temperature for reaction, filter out the reaction solution, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and filter out the solvent to obtain Fmoc-Ile-Asp(OtBu)-resin;

[0053] Step 3: Take the resin obtained in the previous step and 30 mmol of Fmoc-Thr(tBu)-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Thr(tBu)-Ile-Asp(OtBu)-resin;

[0054] Step 4: Take the resin obtained in the previous step and 30 mmol of Fmoc-Val-OH and add them, refer to the operation method of step 2 to obtain Fmoc-Val-Thr(tBu)-Ile-Asp(OtBu)-resin.

[0055] Step 5: Take the resin obtained in the previous step and 30 mmol of Fmoc-Lys(Boc)-OH and add them. Refer to the operation method of step 2 to prepare Fmoc-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin.

[0056] Step 6: Take the resin obtained in the previous step and 30 mmol of Fmoc-Gly-OH and add them, refer to the operation method of step 2 to obtain Fmoc-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin.

[0057] Step 7: Take the resin obtained in the previous step and 30 mmol of Fmoc-Ala-OH and add them, refer to the operation method of step 2 to obtain Fmoc-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin.

[0058] Step 8: Take the resin obtained in the previous step and 30 mmol of Fmoc-Ser(tBu)-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin.

[0059] Step 9: Take the resin obtained in the previous step and 30 mmol of Fmoc-Leu-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin;

[0060] Step 10: Take the resin obtained in the previous step and 30 mmol of Fmoc-Ala-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin;

[0061] Step 11: Take the resin obtained in the previous step and 30 mmol of Fmoc-Ala-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin;

[0062] Step 12: Take the resin obtained in the previous step and 30 mmol of Fmoc-Ser(tBu)-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin;

[0063] Step 13: Take the resin obtained in the previous step and 30 mmol of Fmoc-Arg(Tos)-OH and add them, refer to the operation method of step 2 to prepare Fmoc-Arg(Tos)-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin;

[0064] Step 14: Add 20% hexahydropyridine / dimethylformamide solution to the resin obtained in the previous step, shake at room temperature for decapping reaction to remove the Fmoc protecting group at the nitrogen end, filter out the solvent, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and filter out the solvent, add 30 mmol of Fmoc-Tyr(tBu)-OH, HBTU, and HOBT to dimethylformamide to dissolve, add DIEA and tromethamine, stir well, and transfer to the reactor containing the treated resin, and heat at room temperature. The reaction was shaken at low temperature, and the reaction solution was removed by suction filtration. The resin was then washed twice with dimethylformamide, methanol, and dichloromethane alternately, and the solvent was removed by suction filtration. After the peptide-linking reaction was completed, the resin was filtered out and dried in a vacuum dryer to obtain Fmoc-Tyr(tBu)-Arg(Tos)-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin, i.e., the protected tetradecapeptide resin;

[0065] Step 15: Peptide chain cleavage

[0066] The resin obtained in the previous step is transferred to a round-bottom flask, and a pre-cooled cutting solution (e.g., 95% trifluoroacetic acid / 2% TIS / 2% EDT / 1% water) is added. The reaction is stirred at room temperature, and the filtrate is separated by suction filtration. The resin is washed twice with trifluoroacetic acid, and the filtrate and the washing solution are combined. Ice-cold ether, e.g., 1200 ml, is added to precipitate for e.g., 5 hours, and the precipitate is filtered to obtain a crude product of tetradecapeptide;

[0067] Step 16: Separate and purify the crude peptide obtained in the previous step using an ion exchange chromatography system and high performance liquid chromatography in sequence to obtain a refined peptide, namely, the CHI3L1 antigen epitope peptide (2).

[0068] According to the first aspect of the present invention, the separation and purification of the CHI3L1 antigen epitope peptide in step 16 of preparing the peptide (2) can be carried out according to the methods described in the examples herein or known in the prior art.

[0069] According to the CHI3L1 antigen epitope peptide of the first aspect of the present invention, in steps 1 to 14 of preparing peptide (2), the molar ratio of Fmoc-protected amino acid: HBTU: HOBT: DIEA used in each step is 1:1:1:4.

[0070] According to the CHI3L1 antigen epitope peptide of the first aspect of the present invention, in step 1 of preparing peptide (2), when treating RinkAmide-MBHA resin, dichloromethane, for example 80 ml, is added, shaken and soaked for, for example, 60 minutes, and washed alternately twice with dichloromethane, methanol, and dimethylformamide, for example 80 ml each time, and filtered to remove the solvent.

[0071] According to the first aspect of the present invention, the CHI3L1 antigen epitope peptide, in step 1 of preparing peptide (2), is added to the treated resin with 100 ml of 20% hexahydropyridine / dimethylformamide solution, and the uncapping reaction is carried out at room temperature with shaking for 60 minutes to remove the Fmoc protecting group at the nitrogen end; after the solvent is removed by filtration, the resin is washed twice with dimethylformamide, methanol and dichloromethane alternately, each time with 80 ml, and the solvent is removed by filtration.

[0072] According to the first aspect of the present invention, in the step 1 of preparing the peptide (2), 30 mmol of Fmoc-Asp(OtBu)-OH, HBTU and HOBT are added to 100 ml of dimethylformamide to dissolve, DIEA is added, stirred evenly, and transferred into a reactor containing the treated resin, and reacted at room temperature for 1 hour by shaking; the reaction solution is removed by suction filtration, and the resin is then washed twice with dimethylformamide, methanol and dichloromethane, 80 ml each time, and the solvent is removed by suction filtration.

[0073] According to the first aspect of the present invention, in the step 2 of preparing the peptide (2), 100 ml of a 20% hexahydropyridine / dimethylformamide solution is added, and the uncapping reaction is carried out at room temperature with shaking for 60 minutes to remove the Fmoc protecting group at the nitrogen end; after the solvent is removed by filtration, the resin is washed twice with dimethylformamide, methanol and dichloromethane alternately, each time with 80 ml, and the solvent is removed by filtration.

[0074] According to the first aspect of the present invention, in the step 2 of preparing the peptide (2), 30 mmol of Fmoc-Ile-OH, HBTU and HOBT are added to 100 ml of dimethylformamide to dissolve, DIEA is added, stirred evenly, and transferred into a reactor containing the treated resin, and reacted at room temperature for 1 hour by shaking; the reaction solution is removed by suction filtration, and the resin is then washed twice with dimethylformamide, methanol and dichloromethane alternately, 80 ml each time, and the solvent is removed by suction filtration.

[0075] According to the first aspect of the present invention, in the step 14 of preparing the peptide (2), a 20% hexahydropyridine / dimethylformamide solution, for example 100 ml, is added, and the uncapping reaction is carried out by shaking at room temperature, for example 60 minutes, to remove the Fmoc protecting group at the nitrogen end; after the solvent is removed by filtration, the resin is washed twice alternately with dimethylformamide, methanol, and dichloromethane, for example 80 ml each time, and the solvent is removed by filtration.

[0076] According to the first aspect of the present invention, in the step 14 of preparing the peptide (2), 30 mmol of Fmoc-Tyr(tBu)-OH, HBTU and HOBT are added to, for example, 100 ml of dimethylformamide to dissolve, DIEA and tromethamine are added, stirred evenly, and transferred into a reactor containing the treated resin, and reacted at room temperature for, for example, 1 hour by shaking; the reaction solution is removed by suction filtration, and the resin is then washed twice with dimethylformamide, methanol and dichloromethane, for example, 80 ml each time, and the solvent is removed by suction filtration.

[0077] According to the CHI3L1 antigen epitope peptide of the first aspect of the present invention, in step 14 of preparing peptide (2), the amount of tromethamine is 6% of DIEA, calculated in molar percentage.

[0078] Furthermore, the second aspect of the present invention provides a method for preparing a CHI3L1 antigen epitope peptide (1) or peptide (2), wherein the peptide (1) or (2) has the following amino acid sequence:

[0079] (1)Tyr-Arg-Lys-Ser-Val-Pro-Pro-Phe-Leu-Arg-Thr,

[0080] (2)Tyr-Arg-Ser-Ala-Ala-Leu-Ser-Ala-Gly-Lys-Val-Thr-Ile-Asp;

[0081] Among them, the method is to access Tyr in the following way:

[0082] To the prepared Fmoc-Arg(Tos)-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin, add 20% hexahydropyridine / dimethylformamide solution, shake at room temperature for decapping reaction to remove the Fmoc protecting group at the nitrogen end, filter out the solvent, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and filter out the solvent, and add Fmoc-Tr(tBu)-OH, HBTU, and HOBT to dimethylformamide to make Dissolve, add DIEA and tromethamine, stir well, transfer into the reactor containing the treated resin, react at room temperature, filter out the reaction solution, then wash the resin twice with dimethylformamide, methanol and dichloromethane alternately, filter out the solvent, and after the peptide connection reaction is completed, filter out the resin and dry it in a vacuum dryer to obtain Fmoc-Tyr(tBu)-Arg(Tos)-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin, i.e., protected 11-peptide resin; and

[0083] To the prepared Fmoc-Arg(Tos)-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin, add 20% hexahydropyridine / dimethylformamide solution, shake at room temperature for decapping reaction to remove the Fmoc protecting group at the nitrogen end, filter out the solvent, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, and filter out the solvent, and add Fmoc-Tyr(tBu)-OH, HBTU, and HOBT to dimethylformamide. The mixture was dissolved, DIEA and tromethamine were added, stirred well, and transferred into a reactor containing the treated resin. The mixture was shaken at room temperature for reaction, and the reaction solution was removed by suction. The resin was then washed twice with dimethylformamide, methanol, and dichloromethane, respectively, and the solvent was removed by suction. After the peptide-linking reaction was completed, the resin was filtered out and dried in a vacuum dryer to obtain Fmoc-Tyr(tBu)-Arg(Tos)-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin, i.e., the protected tetradecapeptide resin.

[0084] According to the method of the second aspect of the present invention, in the process of preparing peptide (1) or peptide (2) for accession of Tyr, the molar ratio of Fmoc-protected amino acid: HBTU: HOBT: DIEA used is 1:1:1:4.

[0085] According to the method of the second aspect of the present invention, in the process of preparing peptide (1) or peptide (2) to which Tyr is introduced, the amount of tromethamine is 6% of DIEA, calculated as a molar percentage.

[0086] According to the method of the second aspect of the present invention, the steps for preparing peptide (1) or peptide (2) are as described in any one of the first aspect of the present invention.

[0087] Furthermore, the third aspect of the present invention provides a CHI3L1 antigen, which is prepared by coupling a CHI3L1 antigen epitope peptide (1) with a carrier protein, or by coupling a CHI3L1 antigen epitope peptide (2) with a carrier protein.

[0088] As described above and below, the amino acid chain fragments of the CHI3L1 antigen epitope peptide (1) and the CHI3L1 antigen epitope peptide (2) are respectively:

[0089] (1)Tyr-Arg-Lys-Ser-Val-Pro-Pro-Phe-Leu-Arg-Thr;

[0090] (2)Tyr-Arg-Ser-Ala-Ala-Leu-Ser-Ala-Gly-Lys-Val-Thr-Ile-Asp.

[0091] According to the third aspect of the present invention, the CHI3L1 antigen is prepared by coupling the CHI3L1 antigen epitope peptide (1) with the carrier protein KLH using the bis-nitrogenated benzidine dichloride method, or by coupling the CHI3L1 antigen epitope peptide (2) with the carrier protein KLH.

[0092] The CHI3L1 antigen according to the third aspect of the present invention is prepared by using the following method: dissolving the CHI3L1 peptide (1) or (2) in PBS buffer, dissolving KLH in borate buffer, then mixing the two, cooling to 0°C, adding 110 μL of bis(nitrogenated benzidine) dichloride, reacting at room temperature for 1.5 hours, and dialyzing for 12 to 15 hours.

[0093] According to the CHI3L1 antigen of the third aspect of the present invention, the formula of the PBS buffer is: 81 ml of 0.2 mol / L Na2HPO4 and 19 ml of 0.2 mol / L NaH2PO4.

[0094] According to the CHI3L1 antigen of the third aspect of the present invention, the formula of the borate buffer is: 80 ml of 0.05 mol / L borax and 20 ml of 0.2 mol / L boric acid.

[0095] The CHI3L1 antigen according to the third aspect of the present invention is prepared by the following method: 10.0 mg of CHI3L1 peptide (1) or (2) is dissolved in 1 ml of 0.1M PBS buffer (pH 7.4); 10 mg of KLH is dissolved in 20 ml of 0.2M borate buffer (pH 9.0); the two are then mixed, cooled to 0°C, 110 μL of BDBCl2 is added, the mixture is reacted at room temperature for 1.5 hours, and dialyzed for 12 to 15 hours.

[0096] According to the CHI3L1 antigen of the third aspect of the present invention, the borate buffer is replaced by an equal volume of carbonate buffer, and the carbonate buffer is prepared as follows: weigh 2.94g of NaHCO3, 1.58g of Na2CO3, 0.25g of ammonium pyruvate, and 0.12g of sodium nitrite, dissolve them in water, and make up to 1000ml with water to obtain a carbonate buffer with a concentration of 50mM and pH 9.6.

[0097] According to the CHI3L1 antigen of the third aspect of the present invention, the CHI3L1 peptide (1) or (2) is the CHI3L1 antigen epitope peptide (1) or CHI3L1 antigen epitope peptide (2) according to any one of the first aspect of the present invention.

[0098] Furthermore, the fourth aspect of the present invention provides a method for preparing CHI3L1 antigen, which is prepared by coupling CHI3L1 antigen epitope peptide (1) with carrier protein KLH using the bis-nitrogenated benzidine dichloride method, or by coupling CHI3L1 antigen epitope peptide (2) with carrier protein KLH.

[0099] The method according to the fourth aspect of the present invention comprises the following steps: dissolving CHI3L1 peptide (1) or (2) with PBS buffer, dissolving KLH with borate buffer, then mixing the two, cooling to 0°C, adding 110 μL of bis(nitrogenated benzidine) dichloride, reacting at room temperature for 1.5 hours, and dialyzing for 12 to 15 hours to obtain CHI3L1 antigen.

[0100] According to the method of the fourth aspect of the present invention, the formula of the PBS buffer is: 81 ml of 0.2 mol / L Na2HPO4 and 19 ml of 0.2 mol / L NaH2PO4.

[0101] According to the method of the fourth aspect of the present invention, the formula of the borate buffer is: 80 ml of 0.05 mol / L borax and 20 ml of 0.2 mol / L boric acid.

[0102] The method according to the fourth aspect of the present invention comprises the following steps: taking 10.0 mg of CHI3L1 peptide (1) or (2), dissolving it in 1 ml of 0.1M PBS buffer (pH 7.4); dissolving 10 mg of KLH in 20 ml of 0.2M borate buffer (pH 9.0); then mixing the two, cooling to 0°C, adding 110 μL of BDBCl2, reacting at room temperature for 1.5 hours, and dialyzing for 12 to 15 hours to obtain CHI3L1 antigen.

[0103] According to the method of the fourth aspect of the present invention, the borate buffer is replaced by an equal volume of carbonate buffer, and the carbonate buffer is prepared in the following manner: weigh 2.94g of NaHCO3, 1.58g of Na2CO3, 0.25g of ammonium pyruvate, and 0.12g of sodium nitrite, dissolve them in water, and make up to 1000ml with water to obtain a carbonate buffer with a concentration of 50mM and pH 9.6.

[0104] According to the method of the fourth aspect of the present invention, the CHI3L1 peptide (1) or (2) is obtained according to the method for preparing the CHI3L1 antigen epitope peptide (1) or CHI3L1 antigen epitope peptide (2) described in any one of the first aspect of the present invention, or is prepared according to the method described in any one of the second aspect of the present invention.

[0105] A fifth aspect of the present invention further provides a CHI3L1 in vitro diagnostic kit, which comprises the following parts:

[0106] (A) A pre-coated plate, each well of which is pre-coated with a monoclonal antibody made from one of the following CHI3L1 antigen epitope peptides (1) or peptides (2):

[0107] (1)Tyr-Arg-Lys-Ser-Val-Pro-Pro-Phe-Leu-Arg-Thr,

[0108] (2)Tyr-Arg-Ser-Ala-Ala-Leu-Ser-Ala-Gly-Lys-Val-Thr-Ile-Asp;

[0109] (B) CHI3L1 calibrator, which is a recombinant human YKL-40 / CHI3L1 protein solution with a series of concentrations;

[0110] (C) a binding antibody, which is a solution of a polyclonal antibody prepared from one of the CHI3L1 antigen epitope peptide (1) or peptide (2) described in (A);

[0111] (D) Enzyme conjugate, which is a solution of goat anti-rabbit IgG antibody labeled with horseradish enzyme;

[0112] (E) 10× washing buffer, which is 10× PBS-Tween 20 solution;

[0113] (F) Color developer A, prepared by: 35.5 g citric acid, 10 g urea peroxide, 10 ml Tween 20, dissolved in distilled water to 1000 ml;

[0114] (G) Color developer B, prepared by dissolving 120 g citric acid, 1 g EDTA-2Na, 2 g TMB·2HCl in distilled water to 1000 ml;

[0115] (H) stop solution, which is 2 M sulfuric acid solution;

[0116] The antigen epitope peptides used for the pre-coated plate and the bound antibody are different.

[0117] According to the fifth aspect of the CHI3L1 in vitro diagnostic kit of the present invention, each well of the pre-coated plate is pre-coated with 0.1 μg of CHI3L1 monoclonal antibody.

[0118] According to the fifth aspect of the CHI3L1 in vitro diagnostic kit of the present invention, the pre-coated plate is a 48 / 96-well plate.

[0119] According to the fifth aspect of the CHI3L1 in vitro diagnostic kit of the present invention, the method for preparing the pre-coated plate is: dissolving the CHI3L1 monoclonal antibody in a 0.05M carbonate buffer at pH 9.6 to prepare a pre-coating solution; adding 100 μl of the pre-coating solution to each well of the ELISA plate at 0.1 μg / well, placing it at 4°C for 18-24 hours, taking it out, discarding the coating solution, washing, blocking with BSA for 16 hours, drying, placing it in an aluminum foil bag and vacuum sealing, and obtaining the pre-coated plate.

[0120] According to the CHI3L1 in vitro diagnostic kit of the fifth aspect of the present invention, the preparation method / formula of the 0.05M carbonate buffer at pH 9.6 is: 16.0 grams of Na2CO3, 29.0 grams of NaHCO3, and distilled water dissolved to 1000 ml.

[0121] According to the CHI3L1 in vitro diagnostic kit of the fifth aspect of the present invention, the CHI3L1 calibrator is a calibrator solution of 7 concentration series, each with 1.0 ml, and the concentrations are 25 ng / ml, 10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1 ng / ml, 0.5 ng / ml, and 0.25 ng / ml respectively.

[0122] According to the CHI3L1 in vitro diagnostic kit of the fifth aspect of the present invention, the concentration of the (C) CHI3L1 polyclonal antibody solution bound to the antibody is 0.1-1 μg / 100 μl or adjusted to a wider concentration or range as needed or determined by a square array titration experiment.

[0123] According to the fifth aspect of the CHI3L1 in vitro diagnostic kit of the present invention, (C) the antibody-bound CHI3L1 polyclonal antibody solution is dissolved and / or diluted using an enzyme marker diluent as a solvent.

[0124] According to the fifth aspect of the CHI3L1 in vitro diagnostic kit of the present invention, 10 ml of a solution containing binding antibodies is provided in each kit.

[0125] According to the CHI3L1 in vitro diagnostic kit of the fifth aspect of the present invention, the preparation method / formula of the enzyme marker diluent is: 10 ml of 10×PBS-Tween 20 solution, 20 ml of calf serum, 1 gram of enzyme stabilizer DCE0061A, 1 ml of biological preservative Proclin 300, and distilled water dissolved to 1000 ml.

[0126] According to the CHI3L1 in vitro diagnostic kit of the fifth aspect of the present invention, the (D) enzyme conjugate is prepared by diluting ZB-2301 horseradish enzyme-labeled goat anti-rabbit IgG antibody with an enzyme labeling diluent at a ratio of 1:5000.

[0127] According to the fifth aspect of the CHI3L1 in vitro diagnostic kit of the present invention, 10 ml of enzyme conjugate solution is provided in each kit.

[0128] According to the CHI3L1 in vitro diagnostic kit of the fifth aspect of the present invention, the preparation method / formula of (E) 10× washing buffer is: 58 grams of Na2HPO4·12H2O, 4 grams of KH2PO4, 100 grams of NaCl, 4 grams of KCl, 20 ml of Tween 20, and distilled water dissolved to 1000 ml, with a pH of 7.2.

[0129] According to the fifth aspect of the CHI3L1 in vitro diagnostic kit of the present invention, each kit provides 20 ml of 10× washing buffer.

[0130] According to the fifth aspect of the CHI3L1 in vitro diagnostic kit of the present invention, 6 ml of the color developing agent A is provided in each kit.

[0131] According to the fifth aspect of the CHI3L1 in vitro diagnostic kit of the present invention, 6 ml of the color developing agent B is provided in each kit.

[0132] According to the fifth aspect of the CHI3L1 in vitro diagnostic kit of the present invention, 6 ml of stop solution is provided in each kit.

[0133] According to the CHI3L1 in vitro diagnostic kit of the fifth aspect of the present invention, the preparation method / formula of the color developer B provided in each kit is: 120 grams of citric acid, 1 gram of EDTA-2Na, 2 grams of TMB·2HCl, 1.5g of magnesium chloride, 2.5g of serine, dissolved in distilled water to 1000ml.

[0134] The sixth aspect of the present invention provides a method for determining the concentration of CHI3L1 in serum, which is performed using the CHI3L1 in vitro diagnostic kit according to any one of the fifth aspects of the present invention, and is performed by the following steps:

[0135] (a) Add 100 μl / well of the blood sample to be tested and CHI3L1 calibrator to each well of the pre-coated plate, in duplicate, incubate at 37°C for 60 min, wash five times with 1× wash buffer, and pat dry;

[0136] (b) Add 100 μl / well of CHI3L1-binding antibody to each well, incubate at 37°C for 30 min, wash five times with 1× wash buffer, and pat dry;

[0137] (c) Add 100 μl / well of enzyme conjugate to each well, incubate at 37°C for 30 min, wash five times with 1× wash buffer, and pat dry;

[0138] (d) Add 50 μl of color developing reagent A and B to each well, mix well, and incubate at 37°C for 15 min;

[0139] (e) Add 50 μl / well of stop solution to terminate the reaction and measure the absorbance at dual wavelengths of 450 nm and 620 nm using an enzyme-linked detector.

[0140] (f) Using calibrators of various concentrations to determine their average absorbance, a standard curve is drawn using the logarithmic values ​​of the calibrator concentrations and the corresponding absorbances, and the CHI3L1 concentration in the sample being tested is calculated based on the standard curve.

[0141] The present invention also provides a CHI3L1 antibody, which is a monoclonal antibody or a polyclonal antibody prepared from the CHI3L1 antigen.

[0142] The present invention also provides use of the CHI3L1 antibody in preparing a CHI3L1 in vitro diagnostic kit.

[0143] The present invention also provides a CHI3L1 in vitro diagnostic kit, which comprises the CHI3L1 antibody as a coating antibody.

[0144] Preferably, a CHI3L1 in vitro diagnostic kit further comprises a binding antibody, wherein the binding antibody is the CHI3L1 antibody, and when the binding antibody is derived from one of the CHI3L1 antigen epitope peptides (1) or (2), the coating antibody is derived from the other of the CHI3L1 antigen epitope peptides (1) or (2). Preferably, the kit further comprises an enzyme-labeled second antibody. Preferably, the coating antibody is a monoclonal antibody. Preferably, the binding antibody is a polyclonal antibody.

[0145] Compared with the prior art, the present invention has the following advantages and positive effects:

[0146] 1. The CHI3L1 antigen epitope peptide of the present invention has good antigenicity, and the antigen (immunogen) prepared therefrom can produce highly specific monoclonal antibodies and polyclonal antibodies in animals immunized with the antigen (immunogen).

[0147] 2. The CHI3L1 monoclonal antibody and polyclonal antibody prepared by the present invention can bind to CHI3L1 in blood samples with high specificity.

[0148] 3. The CHI3L1 in vitro diagnostic kit of the present invention can effectively detect the level of chitinase 3-like protein 1 (CHI3L1) in the blood, and can be used to determine the degree of liver fibrosis.

[0149] In the steps of the above-mentioned preparation method of the present invention, although the specific steps described therein are different from the steps described in the preparation examples in the specific implementation mode part below in some details or language descriptions, those skilled in the art can fully summarize the above-mentioned method steps based on the detailed disclosure of the full text of the present invention.

[0150] Any embodiment of any aspect of the present invention can be combined with other embodiments, as long as they do not conflict. In addition, in any embodiment of any aspect of the present invention, any technical feature can be applied to the technical features in other embodiments, as long as they do not conflict. The present invention is further described below.

[0151] All documents cited in the present invention are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with the present invention, the description of the present invention shall prevail. In addition, the various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still hopes to provide a more detailed description and explanation of these terms and phrases. If the terms and phrases mentioned are inconsistent with the known meanings, the meanings expressed in the present invention shall prevail. DETAILED DESCRIPTION OF THE INVENTION

[0153] 1. CHI3L1 epitope peptide

[0154] The CHI3L1 protein described in the present invention is known in the art, and its amino acid sequence is known in the art and can be found in professional databases such as NCBI.

[0155] After extensive theoretical research and experimental exploration, the inventors of the present invention finally screened two CHI3L1 antigen epitope peptides (1) and (2) with good antigenicity from the amino acid sequence of the CHI3L1 protein. The amino acid chain fragments of the CHI3L1 antigen epitope peptides (1) and (2) are shown below:

[0156] (1)Tyr-Arg-Lys-Ser-Val-Pro-Pro-Phe-Leu-Arg-Thr;

[0157] (2)Tyr-Arg-Ser-Ala-Ala-Leu-Ser-Ala-Gly-Lys-Val-Thr-Ile-Asp;

[0158] Among them, the CHI3L1 antigen epitope peptide (1) is composed of a 9-amino acid peptide segment at positions 121 to 129 of the N-terminus of the CHI3L1 protein plus two hydrophilic amino acids Y (i.e. Tyr) and R (i.e. Arg); the CHI3L1 antigen epitope peptide (2) is composed of a 12-amino acid peptide segment at positions 175 to 186 of the N-terminus of the CHI3L1 protein plus two hydrophilic amino acids Y and R; the above two peptide segments are both hydrophilic, highly antigenic and easy to synthesize.

[0159] At present, the present invention has found that the CHI3L1 antigen epitope peptide of the present invention has the following functions:

[0160] 1. It is antigenic; 2. After being connected to a carrier protein, it can serve as an immunogen to stimulate animals to produce specific antibodies; 3. Antibodies prepared using antigen epitope peptides can specifically bind to CHI3L1.

[0161] The preparation method of the CHI3L1 antigen epitope peptide of the present invention can be prepared by chemical synthesis: using the American ABI431A type polypeptide automatic synthesizer, the antigen epitope peptide is synthesized by solid phase method. The molecular weights of the antigen epitope peptides (1) and (2) of the present invention are 1363.61 and 1451.62 respectively, and can be determined by mass spectrometry. The synthesized antigen epitope peptide sequence can be identified by polypeptide sequence determination. The purity of the peptide segment is evaluated by high performance liquid chromatography, and the concentration of the antigen epitope peptide is determined.

[0162] 2. CHI3L1 Antigen

[0163] The present invention also provides a CHI3L1 antigen, which is prepared by coupling a CHI3L1 antigen epitope peptide (1) with a carrier protein, or by coupling a CHI3L1 antigen epitope peptide (2) with a carrier protein.

[0164] Specifically, the present invention provides CHI3L1 antigens (1) and (2); the CHI3L1 antigen (1) is prepared by coupling the CHI3L1 antigen epitope peptide (1) of the present invention to a carrier protein; the CHI3L1 antigen (2) is prepared by coupling the CHI3L1 antigen epitope peptide (2) of the present invention to a carrier protein; the CHI3L1 antigen (1) and the CHI3L1 antigen (2) of the present invention have immunogenicity and specificity, are immunogens, and can be used to immunize animals to prepare specific CHI3L1 antibodies; in the present invention, examples of carrier proteins that can be used include KLH (keyhole limpet hemocyanin), bovine serum albumin (BSA), ovalbumin OVA, etc.; since KLH (keyhole limpet hemocyanin) has strong immunogenicity, many binding sites, good immune effect, and is distantly related to the immunized animal, it is not easy to cause cross-reaction when used as a carrier protein, and therefore is preferred.

[0165] 3. CHI3L1 monoclonal antibody, CHI3L1 polyclonal antibody and CHI3L1 in vitro diagnostic kit

[0166] The present invention also provides CHI3L1 monoclonal antibodies and CHI3L1 polyclonal antibodies, which are monoclonal antibodies or polyclonal antibodies prepared from the CHI3L1 antigen (1) or CHI3L1 antigen (2) (immunogen). These monoclonal antibodies and polyclonal antibodies can be prepared by conventional techniques in the art, and exemplary methods can be found in Example 2.

[0167] The CHI3L1 antibody of the present invention can be used to prepare a CHI3L1 in vitro diagnostic kit, which can detect CHI3L1 in human tissues, cells or body fluids based on an immunological method, preferably detecting CHI3L1 in a blood sample.

[0168] Therefore, the present invention provides a CHI3L1 in vitro diagnostic kit, which comprises the CHI3L1 monoclonal antibody or polyclonal antibody.

[0169] Currently known immunoassay methods that can be used for clinical testing mainly include the following: ELISA, chemiluminescence, fluorescence chromatography, colloidal gold immunoassay, etc.

[0170] The ELISA method includes the following types: double antibody sandwich method for detecting antigens, double antigen sandwich method for detecting antibodies, indirect method for detecting antibodies, competitive method for detecting antibodies, competitive method for detecting antigens, capture coating method for detecting antibodies, etc.

[0171] The CHI3L1 in vitro diagnostic kit of the present invention preferably uses the ELISA double antibody sandwich method to detect the CHI3L1 protein. The kit may include a coating antibody, a binding antibody, an enzyme-labeled second antibody and / or necessary tools and reagents.

[0172] Preferably, the CHI3L1 in vitro diagnostic kit uses the CHI3L1 monoclonal antibody of the present invention as a coating antibody. Here, the term "coating antibody" refers to an antibody coated on a solid-phase ELISA plate; in addition, the CHI3L1 in vitro diagnostic kit also preferably comprises a CHI3L1 polyclonal antibody as a binding antibody, wherein, when the binding antibody is derived from one of the CHI3L1 antigen epitope peptides (1) and (2) of the present invention, the coating antibody is derived from the other of the antigen epitope peptides (1) and (2); here, the term "binding antibody" refers to a specific antibody in the kit that can bind to the antigen to be detected and the enzyme-labeled second antibody; the kit may also comprise an enzyme-labeled second antibody (also referred to as an enzyme conjugate), the second antibody may be a goat anti-rabbit IgG antibody, and the enzyme label may be horseradish peroxidase, alkaline phosphatase, etc.

[0173] The kit of the present invention may also include any reagents or tools required for detection, such as a pre-coated plate, a washing solution, a color developer, a stop solution, and the like.

[0174] The results of testing serum samples with the CHI3L1 in vitro diagnostic kit of the present invention showed that there was a significant difference in serum CHI3L1 concentration between patients with liver disease and healthy controls, and there was also a significant difference in serum CHI3L1 concentration between patients with liver fibrosis, cirrhosis, and liver cancer, indicating that serum CHI3L1 levels can be used for liver disease diagnosis and are positively correlated with the severity of liver disease. DETAILED DESCRIPTION

[0175] The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. The present invention provides general and / or specific descriptions of the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible herein. The following examples further illustrate the present invention, rather than limiting the present invention.

[0176] The present invention is further explained or illustrated by various embodiments below: unless otherwise specified, the solutions described below are all aqueous solutions; when it comes to percentages, the percentages of mixed materials prepared as liquid / liquid are all volume / volume percentages, the percentages of mixed materials prepared as solid / liquid are all mass / volume percentages, and the percentages of mixed materials prepared as solid / solid are all mass / mass percentages.

[0177] Some of the following typical raw materials used in CN111197040A (application number 2020100721960, Yitong Bio) can also be used in the present invention when necessary: ​​HMP resin (HMP resin, P-hydroxymethylphenoxymethyl polyethylene resin, purchased from Sigma-Aldrich); Fmoc-AA (9-fluorenylmethoxycarbonyl protected amino acid, provided according to the needs of polypeptide synthesis, purchased from Merck); NMP (nitromethylpyrrolidone, purchased from Sigma-Aldrich); DCM (dichloromethane, purchased from Zhongyuan Chemical Company); MeOH (methanol, purchased from Zhongyuan Chemical Company); Piperidine (piperidine, purchased from Sigma-Aldrich); DMAP (dimethylaminopyrrolidone) pyridine, purchased from Sigma-Aldrich Company); HOBT (hydroxybenzotriazole, purchased from Sigma-Aldrich Company); DCC (dicyclohexylcarbodiimide, purchased from Sigma-Aldrich Company); TFA (trifluoroacetic acid, purchased from Sigma-Aldrich Company); EDT (1,2-ethanedithiol, purchased from Sigma-Aldrich Company); thioanisole, purchased from Guangzhou Wei Bo Chemical Co., Ltd.; crystalline phenol, purchased from Sinopharm Chemical Reagent Co., Ltd.; acetonitrile, purchased from Sinopharm Chemical Reagent Co., Ltd. The raw materials not mentioned are also easily purchased from the market.

[0178] Some of the following typical instruments used in CN111197040A (application number 2020100721960, Yitong Bio) can also be used in the present invention when necessary: ​​automatic peptide synthesizer, model 431A, purchased from ABI; rotary evaporator, model R-201, purchased from Shanghai Shenshun Company; high performance liquid chromatograph, Waters600, purchased from Waters Company, USA; freeze dryer, model VFD-2000, purchased from Beijing Boyikang Company. Instruments and equipment not mentioned in the present invention are also easily available from the market.

[0179] Example 1: Preparation of CHI3L1 antigen epitope peptides (1) and (2)

[0180] As described in Example 1 of CN111197040A (application number 2020100721960, Yitong Bio), the process of preparing CHI3L1 antigen epitope peptides (1) and (2) is recorded, and two target peptides of corresponding purity are obtained.

[0181] Example 11: Preparation of CHI3L1 antigen epitope peptide (1)

[0182] The Rink Amide-MBHA resin and Fmoc-AA-OH used in the present invention, especially in Example 11 and Example 12, are purchased from Gill Biochemical Company, and reagents such as HBTU, HOBT, and DIEA are purchased from Aladdin Reagent Company. Other reagents can also be easily obtained through commercial channels, such as described in Example 1 of CN111197040A (Application No. 2020100721960, Yitong Bio).

[0183] This Example 11 uses a classical method to prepare the following CHI3L1 antigen epitope peptide (1) of the present invention:

[0184] (1)Tyr-Arg-Lys-Ser-Val-Pro-Pro-Phe-Leu-Arg-Thr.

[0185] In each of the following preparation steps, the molar ratio of amino acid (Fmoc-AA-OH): peptide coupling agent HBTU: amide bond formation promoter HOBT: organic base DIEA is 1:1:1:4;

[0186] The amount of solvent used is determined based on experience and specific operations. For example, the amount of solvent used in cleaning, decapping reaction, and coupling reaction of 10g of resin is 50-150ml. In particular, use as little solvent as possible each time during cleaning.

[0187] Step 1: Preparation of Fmoc-Thr(tBu)-resin

[0188] In this step, 10 g of Rink Amide-MBHA resin (0.79 mmol / g) and 30 mmol (11.93 g) of Fmoc-Thr(tBu)-OH were added.

[0189] Place Rink Amide-MBHA resin in a reactor, add 80 ml of dichloromethane, shake and soak for 60 minutes, wash twice with dichloromethane, methanol and dimethylformamide alternately, each time with 80 ml, and filter to remove the solvent.

[0190] Add 20% hexahydropyridine / dimethylformamide solution (100 ml) to the resin obtained in the previous step, and shake at room temperature for 60 minutes to remove the Fmoc protecting group at the nitrogen end. After filtering off the solvent, wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, 80 ml each time, and filter off the solvent. The ninhydrin test should show blue. If it does not show blue, repeat this step. (The above-mentioned ninhydrin test is also called KT test, and a trace amount of 1 to 2 mg of resin can be used for testing, the same below). Add the specified proportion of Fmoc-Thr(tBu)-OH, HBTU, and HOBT to dimethylformamide (100 ml) to dissolve, add DIEA, stir well, transfer to the reactor containing the above-treated resin, and shake at room temperature for 1 hour. The reaction solution was removed by suction filtration, and the resin was washed alternately twice with dimethylformamide, methanol, and dichloromethane, 80 ml each time, and the solvent was removed by suction filtration; the KT test should show yellow, if not, the reaction time should be extended. The title resin obtained in this step 1, i.e., Fmoc-Thr(tBu)-resin, was tested to have a coupling rate of 0.91. The resin was used in all subsequent reaction steps (the same below).

[0191] Step 2: Preparation of Fmoc-Arg(Tos)-Thr(tBu)-resin

[0192] Referring to the operation method of step 1, the resin obtained in the previous step and 30 mmol of Fmoc-Arg(Tos)-OH were added;

[0193] Add 20% hexahydropyridine / dimethylformamide solution (100 ml) to the resin obtained in the previous step, and shake at room temperature for 60 minutes to remove the Fmoc protecting group at the nitrogen end. After filtering to remove the solvent, wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, 80 ml each time, and filter to remove the solvent. The ninhydrin test should show blue, if not blue, repeat this step. Add the specified proportion of Fmoc-protected amino acids, HBTU, and HOBT to dimethylformamide (100 ml) to dissolve, add DIEA, stir well, transfer to the reactor containing the treated resin, and shake at room temperature for 1 hour. Filter to remove the reaction solution, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, 80 ml each time, and filter to remove the solvent; KT test should show yellow, if not yellow, extend the reaction time. The title resin obtained in this step has a coupling rate of 0.89 after testing.

[0194] Step 3: Preparation of Fmoc-Leu-Arg(Tos)-Thr(tBu)-resin

[0195] The resin obtained in the previous step and 30 mmol of Fmoc-Leu-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.92.

[0196] Step 4: Preparation of Fmoc-Phe-Leu-Arg(Tos)-Thr(tBu)-resin

[0197] The resin obtained in the previous step and 30 mmol of Fmoc-Phe-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.90.

[0198] Step 5: Preparation of Fmoc-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin

[0199] The resin obtained in the previous step and 30 mmol of Fmoc-Pro-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.89.

[0200] Step 6: Preparation of Fmoc-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin

[0201] The resin obtained in the previous step and 30 mmol of Fmoc-Pro-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.91.

[0202] Step 7: Preparation of Fmoc-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin

[0203] The resin obtained in the previous step and 30 mmol of Fmoc-Val-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.90.

[0204] Step 8: Preparation of Fmoc-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin

[0205] The resin obtained in the previous step and 30 mmol of Fmoc-Ser(tBu)-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.92.

[0206] Step 9: Preparation of Fmoc-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin

[0207] The resin obtained in the previous step and 30 mmol of Fmoc-Lys(Boc)-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.89.

[0208] Step 10: Preparation of Fmoc-Arg(Tos)-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin

[0209] The resin obtained in the previous step and 30 mmol of Fmoc-Arg(Tos)-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.90.

[0210] Step 11: Preparation of Fmoc-Tyr(tBu)-Arg(Tos)-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin

[0211] Referring to the operation method of step 2, the resin obtained in the previous step and 30 mmol of Fmoc-Tyr(tBu)-OH were added;

[0212] Add 20% hexahydropyridine / dimethylformamide solution (100 ml) to the resin obtained in the previous step, and shake at room temperature for 60 minutes to remove the Fmoc protecting group at the nitrogen end. After filtering to remove the solvent, wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, 80 ml each time, and filter to remove the solvent. The ninhydrin test should show blue. If it does not show blue, repeat this step. Add the specified proportion of Fmoc-protected amino acids, HBTU, and HOBT to dimethylformamide (100 ml) to dissolve, add DIEA and tromethamine (the amount is 6% of DIEA, in molar percentage), stir well, transfer to the reactor containing the treated resin, and shake at room temperature for 1 hour. Filter to remove the reaction solution, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, 80 ml each time, and filter to remove the solvent; KT test should show yellow. If it does not show yellow, extend the reaction time. The coupling rate of the title resin obtained in this step was 0.91 after detection.

[0213] After the peptide-linking reaction in step 11 is completed, the resin is filtered out, placed in a vacuum desiccator and dried overnight, and weighed to obtain the protected 11-peptide resin.

[0214] Step 12: Peptide chain cleavage

[0215] Transfer the resin obtained in the previous step to a 500 ml round-bottom flask, add 100 ml of pre-cooled cutting solution (95% trifluoroacetic acid / 2% TIS / 2% EDT / 1% water), stir and react at room temperature for 2 hours, filter to separate the filtrate, wash the resin twice with trifluoroacetic acid, 20 ml each time, combine the filtrate and the washing solution; add 1200 ml of ice-cold ether to precipitate for 4 hours, filter to obtain the precipitate, which is the crude product of 11-peptide.

[0216] Step 13: Isolation and purification

[0217] (1) The crude peptide obtained in the previous step was dissolved in 70% acetonitrile (containing 0.1% trifluoroacetic acid), and eluted using an ion exchange chromatography system Shodex IEC SP-420N (Beijing Spectron) with 70% acetonitrile (containing 0.1% trifluoroacetic acid) as solvent to collect the main peak fraction of the peptide;

[0218] (2) Separate and purify the main peak of the peptide using the following high performance liquid chromatography method:

[0219] Chromatographic column: C8, 10×100 mm, purchased from Waters, USA; Chromatography: YMC industrial preparative liquid chromatography; Mobile phase: Mobile phase A is 0.1% TFA (trifluoroacetic acid) aqueous solution, mobile phase B is 70% acetonitrile with 0.1% TFA (trifluoroacetic acid), elution gradient is 15% B-60% B during 0 to 45 minutes; Flow rate: 4 ml / min; Detection wavelength: 214 nm;

[0220] (3) The main peak mobile phase is collected, concentrated, and freeze-dried using a freeze dryer to obtain a refined peptide, namely, the CHI3L1 antigen epitope peptide (1), or peptide (1).

[0221] Purity and content were determined by HPLC purity determination method. From the 10 g resin in step 1 to the final product, the 13 steps of the refined peptide, the total yield was calculated to be 31.1%, and the purity of the refined peptide was 98.3%.

[0222] According to the above results, compared with CN111197040A which can only synthesize a small batch of peptides at a time using an automatic peptide synthesizer, this Example 11 can prepare a large batch of peptides at one time, and its yield is satisfactory, the purity of the polypeptide is high, the molecular weight is the same as the theoretical value, and the sequence is the same as the target value.

[0223]

HPLC purity determination method

[0224] This method uses HPLC to determine the purity of CHI3L1 peptide, and the main determination conditions are as follows: chromatographic column: C18, 4.6×150mm, Waters Company, chromatograph: Agilent 1260 high performance liquid chromatograph, Agilent; mobile phase: 0.1% TFA aqueous solution as mobile phase A, acetonitrile containing 0.1% TFA as mobile phase B, elution gradient 0-60% B during 30 minutes; flow rate: 1 ml / min; detection wavelength: 214 nm.

[0225]

Mass spectrometry determination of peptide molecular weight

[0226] This method uses mass spectrometry to determine the molecular weight of the polypeptide obtained in the present invention, and the main determination conditions are as follows:

[0227] (1) Reagent materials: TFA (trifluoroacetic acid, purchased from Sigma-Aldrich), HCCA (α-cyano-4-hydroxycinnamic acid, purchased from Sigma-Aldrich), acetonitrile (purchased from Sinopharm Chemical Reagent Co., Ltd.);

[0228] (2) Instrument: Matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) (model: REFLEXIII, Bruker, Germany);

[0229] (3) Matrix solution: α-CCA was dissolved in 50% ACN solution containing 0.1% TFA to make a saturated solution, centrifuged, and the supernatant was collected;

[0230] (4) Instrument detection conditions: reflection detection method; flight tube length 3 m; nitrogen laser: wavelength 337 nm, acceleration voltage 20 KV; reflection voltage 23 KV;

[0231] (5) Operation steps: Take 10 μL of purified peptide sample (dissolved in acetonitrile to a concentration of 50 mg / ml), mix it with 10 μL of matrix solution, take 1 μL and spot it on the sample target, and send it into the ion source for detection.

[0232] Results: The molecular weight of the CHI3L1 antigen epitope peptide (1) obtained in Example 11 was 1361.34, and the molecular weight of the CHI3L1 antigen epitope peptide (2) obtained in Example 12 was 1451.93, which were consistent with their theoretical molecular weights of 1361.76 and 1451.62, respectively, proving that the synthetic polypeptides were the target products.

[0233]

Peptide Sequencing Method

[0234] This method uses a polypeptide amino acid sequence analyzer to determine the amino acid sequence of the polypeptide obtained in the present invention. The main determination conditions / operations are as follows:

[0235] (1) Principle: The basic principle of peptide amino acid sequence analysis is Edman degradation, which is a cyclic chemical reaction process including three main chemical steps:

[0236] Coupling: Phenyl isothiocyanate reacts with the N-terminal residues of proteins and peptides to form phenylthiocarbamoyl (PTC) derivatives, namely PTC-peptides;

[0237] Cyclization cleavage: PTC-peptide cyclization cleavage;

[0238] Conversion: ATZ is converted to PTH-amino acid, and the peptide with one amino acid residue reduced in the solution is repeated. The entire sequencing process is now performed automatically by a sequencer.

[0239] (2) Instrument: Model 491 protein / peptide N-terminal amino acid sequence analyzer from ABI, USA;

[0240] (3) Reagent materials: phenyl isothiocyanate PITC (Sigma-Aldrich), n-heptane (Sinopharm Chemical Reagent Co., Ltd.), trimethylamine TMA aqueous solution (Sinopharm Chemical Reagent Co., Ltd.), trifluoroacetic acid (TFA, Sigma-Aldrich), ethyl acetate (Sinopharm Chemical Reagent Co., Ltd.), chlorobutane (Sigma-Aldrich), acetonitrile (Sinopharm Chemical Reagent Co., Ltd.);

[0241] (4) Measurement: Perform according to the instrument manual.

[0242] As a result, it was identified that the sequences of the CHI3L1 antigen epitope peptides (1) and (2) obtained in Example 11 and Example 12 were respectively:

[0243] (1) YRKSVPPFLRT; (2) YRSAALSAGKVTID.

[0244] The above results are consistent with the target synthetic peptides prepared in the two examples.

[0245] Example 11a: Various peptide resins were prepared by referring to the operations of steps 1 to 11 of Example 11 herein, except that tromethamine was not added in step 11, and 11 kinds of title resins were obtained. Upon testing, the coupling rates of the title resins of steps 1 to 10 were all in the range of 0.88 to 0.92, for example, the coupling rate of the title resin of step 10 = 0.89, and the coupling rate of the title resin of step 11 = 0.65.

[0246] Example 11b: Various peptide resins were prepared by referring to the operations of steps 1 to 11 of Example 11 herein, with the only difference being that in step 11, the Fmoc-Tyr(tBu)-OH used was replaced with an equimolar amount of Fmoc-Tyr(PO3H2)-OH to obtain 11 kinds of title resins. Upon testing, the coupling rates of the title resins of steps 1 to 10 were all within the range of 0.88 to 0.91, for example, the coupling rate of the title resin of step 10 = 0.90, while the coupling rate of the title resin of step 11 = 0.61.

[0247] Example 11c: Various peptide resins were prepared by referring to the operations of steps 1 to 11 of Example 11 herein, with the only difference being that in step 11, the Fmoc-Tyr(tBu)-OH used was replaced with an equimolar amount of Fmoc-Tyr(Bzl)-OH to obtain 11 kinds of title resins. Upon testing, the coupling rates of the title resins of steps 1 to 10 were all in the range of 0.89 to 0.92, for example, the coupling rate of the title resin of step 10 = 0.90, while the coupling rate of the title resin of step 11 = 0.70.

[0248] Example 11d: Various peptide resins were prepared by referring to the operations of steps 1 to 11 of Example 11 herein, with the only difference being that in step 11, the Fmoc-Tyr(tBu)-OH used was replaced with an equimolar amount of Fmoc-Tyr-OH to obtain 11 kinds of title resins. Upon testing, the coupling rates of the title resins of steps 1 to 10 were all within the range of 0.88 to 0.91, for example, the coupling rate of the title resin of step 10 = 0.88, while the coupling rate of the title resin of step 11 = 0.73.

[0249] Example 11e: Various peptide resins were prepared by referring to the operations of steps 1 to 11 of Example 11 herein, with the only difference being that in step 11, the Fmoc-Tyr(tBu)-OH used was replaced with an equimolar amount of Fmoc-Tyr(Me)-OH to obtain 11 kinds of title resins. Upon testing, the coupling rates of the title resins of steps 1 to 10 were all within the range of 0.89 to 0.92, for example, the coupling rate of the title resin of step 10 = 0.89, while the coupling rate of the title resin of step 11 = 0.64.

[0250] Example 11f: Various peptide resins were prepared by referring to the operation of step 11 of Example 11b, Example 11c, Example 11d, and Example 11e herein, with the only difference being that in step 11, tromethamine was not added, and four kinds of title resins were obtained; upon testing, the coupling rates of the four kinds of title resins obtained in this step 11 were all within the range of 0.64 to 0.69. For example, the coupling rate of the title resin of step 11 obtained by referring to Example 11b = 0.67.

[0251] According to the results of Examples 11a to 11f and Example 11, it was unexpectedly found that when the amino acid Tyr was introduced into the peptide chain, the use of Fmoc-Tyr(tBu)-OH and the simultaneous addition of 6% tromethamine to the reaction solvent significantly increased the coupling rate, while the coupling rate was significantly lower when tromethamine was not used or other protected forms of Tyr were used instead.

[0252] Example 12: Preparation of CHI3L1 antigen epitope peptide (2)

[0253] In this Example 12, the following CHI3L1 antigen epitope peptide (2) of the present invention was prepared using a classical method:

[0254] (2)Tyr-Arg-Ser-Ala-Ala-Leu-Ser-Ala-Gly-Lys-Val-Thr-Ile-Asp.

[0255] In the following preparation steps, the molar ratio of amino acid (Fmoc-AA-OH): peptide coupling agent HBTU: amide bond formation promoter HOBT: organic base DIEA is 1:1:1:4; the amount of solvent is determined based on experience and specific operations. For example, the amount of solvent used in washing, decapping reaction, and coupling reaction for 10 g of resin is 50 to 150 ml, especially when washing, use as little solvent as possible each time.

[0256] Step 1: Preparation of Fmoc-Asp(OtBu)-resin

[0257] In this step, 10 g of Rink Amide-MBHA resin (0.79 mmol / g) and 30 mmol (12.34 g) of Fmoc-Asp(OtBu)-OH were added.

[0258] Place Rink Amide-MBHA resin in a reactor, add 80 ml of dichloromethane, shake and soak for 60 minutes, wash twice with dichloromethane, methanol and dimethylformamide alternately, each time with 80 ml, and filter to remove the solvent.

[0259] Add 20% hexahydropyridine / dimethylformamide solution (100 ml) to the resin obtained in the previous step, and shake at room temperature for 60 minutes to remove the Fmoc protecting group at the nitrogen end. After filtering off the solvent, wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, 80 ml each time, and filter off the solvent. The ninhydrin test should show blue. If it does not show blue, repeat this step. (The above ninhydrin test is also called KT test, and a trace amount of 1 to 2 mg of resin can be used for testing, the same below). Add the specified proportion of Fmoc-Asp(OtBu)-OH, HBTU, and HOBT to dimethylformamide (100 ml) to dissolve, add DIEA, stir well, transfer to the reactor containing the above treated resin, and shake at room temperature for 1 hour. The reaction solution was removed by suction filtration, and the resin was washed alternately twice with dimethylformamide, methanol, and dichloromethane, 80 ml each time, and the solvent was removed by suction filtration; the KT test should show yellow, if not, the reaction time should be extended. The title resin obtained in step 1, i.e., Fmoc-Asp(OtBu)-resin, was tested to have a coupling rate of 0.90. The resin was used in all subsequent reaction steps (the same below).

[0260] Step 2: Preparation of Fmoc-Ile-Asp(OtBu)-resin

[0261] Referring to the operation method of step 1, the resin obtained in the previous step and 30 mmol of Fmoc-Ile-OH were added;

[0262] Add 20% hexahydropyridine / dimethylformamide solution (100 ml) to the resin obtained in the previous step, and shake at room temperature for 60 minutes to remove the Fmoc protecting group at the nitrogen end. After filtering to remove the solvent, wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, 80 ml each time, and filter to remove the solvent. The ninhydrin test should show blue, if not blue, repeat this step. Add the specified proportion of Fmoc-protected amino acids, HBTU, and HOBT to dimethylformamide (100 ml) to dissolve, add DIEA, stir well, transfer to the reactor containing the treated resin, and shake at room temperature for 1 hour. Filter to remove the reaction solution, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, 80 ml each time, and filter to remove the solvent; KT test should show yellow, if not yellow, extend the reaction time. The title resin obtained in this step has a coupling rate of 0.91 after testing.

[0263] Step 3: Preparation of Fmoc-Thr(tBu)-Ile-Asp(OtBu)-resin

[0264] The resin obtained in the previous step and 30 mmol of Fmoc-Thr(tBu)-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.90.

[0265] Step 4: Preparation of Fmoc-Val-Thr(tBu)-Ile-Asp(OtBu)-resin

[0266] The resin obtained in the previous step and 30 mmol of Fmoc-Val-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.88.

[0267] Step 5: Preparation of Fmoc-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin

[0268] The resin obtained in the previous step and 30 mmol of Fmoc-Lys(Boc)-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling ratio was tested to be 0.91.

[0269] Step 6: Preparation of Fmoc-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin

[0270] The resin obtained in the previous step and 30 mmol of Fmoc-Gly-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.90.

[0271] Step 7: Preparation of Fmoc-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin

[0272] The resin obtained in the previous step and 30 mmol of Fmoc-Ala-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.89.

[0273] Step 8: Preparation of Fmoc-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin

[0274] The resin obtained in the previous step and 30 mmol of Fmoc-Ser(tBu)-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.90.

[0275] Step 9: Preparation of Fmoc-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin

[0276] The resin obtained in the previous step and 30 mmol of Fmoc-Leu-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.90.

[0277] Step 10: Preparation of Fmoc-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin

[0278] The resin obtained in the previous step and 30 mmol of Fmoc-Ala-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.92.

[0279] Step 11: Preparation of Fmoc-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin

[0280] The resin obtained in the previous step and 30 mmol of Fmoc-Ala-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.89.

[0281] Step 12: Preparation of Fmoc-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin

[0282] The resin obtained in the previous step and 30 mmol of Fmoc-Ser(tBu)-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.90.

[0283] Step 13: Preparation of Fmoc-Arg(Tos)-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin

[0284] The resin obtained in the previous step and 30 mmol of Fmoc-Arg(Tos)-OH were added, and the operation method of step 2 was referred to obtain the title resin. The coupling rate was tested to be 0.89.

[0285] Step 14: Preparation of Fmoc-Tyr(tBu)-Arg(Tos)-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin

[0286] Referring to the operation method of step 2, the resin obtained in the previous step and 30 mmol of Fmoc-Tyr(tBu)-OH were added;

[0287] Add 20% hexahydropyridine / dimethylformamide solution (100 ml) to the resin obtained in the previous step, and shake at room temperature for 60 minutes to remove the Fmoc protecting group at the nitrogen end. After filtering to remove the solvent, wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, 80 ml each time, and filter to remove the solvent. The ninhydrin test should show blue. If it does not show blue, repeat this step. Add the specified proportion of Fmoc-protected amino acids, HBTU, and HOBT to dimethylformamide (100 ml) to dissolve, add DIEA and tromethamine (the amount is 6% of DIEA, in molar percentage), stir well, transfer to the reactor containing the treated resin, and shake at room temperature for 1 hour. Filter to remove the reaction solution, and then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, 80 ml each time, and filter to remove the solvent; KT test should show yellow. If it does not show yellow, extend the reaction time. The coupling rate of the title resin obtained in this step was detected to be 0.90.

[0288] After the peptide-linking reaction in step 14 is completed, the resin is filtered out, placed in a vacuum desiccator and dried overnight, and weighed to obtain the protected 14-peptide resin.

[0289] Step 15: Peptide chain cleavage

[0290] Transfer the resin obtained in the previous step to a 500 ml round-bottom flask, add 120 ml of pre-cooled cutting solution (95% trifluoroacetic acid / 2% TIS / 2% EDT / 1% water), stir and react at room temperature for 2 hours, filter to separate the filtrate, wash the resin twice with trifluoroacetic acid, 20 ml each time, combine the filtrate and the washing solution; add 1200 ml of ice-cold ether to precipitate for 5 hours, filter to obtain the precipitate, which is the crude product of tetradecapeptide.

[0291] Step 16: Isolation and purification

[0292] (1) The crude peptide obtained in the previous step was dissolved in 70% acetonitrile (containing 0.1% trifluoroacetic acid), and eluted using an ion exchange chromatography system Shodex IEC SP-420N (Beijing Spectron) with 70% acetonitrile (containing 0.1% trifluoroacetic acid) as solvent to collect the main peak fraction of the peptide;

[0293] (2) Separate and purify the main peak of the peptide using the following high performance liquid chromatography method:

[0294] Chromatographic column: C8, 10×100 mm, purchased from Waters, USA; Chromatography: YMC industrial preparative liquid chromatography; Mobile phase: Mobile phase A is 0.1% TFA (trifluoroacetic acid) aqueous solution, mobile phase B is 0.1% TFA (trifluoroacetic acid) in 70% acetonitrile, elution gradient is 15% B-60% B during 0 to 45 minutes; Flow rate: 4 ml / min; Detection wavelength: 214 nm;

[0295] (3) The main peak mobile phase is collected, concentrated, and freeze-dried using a freeze dryer to obtain a refined peptide, namely, CHI3L1 antigen epitope peptide (2), or peptide (2).

[0296] Purity and content were determined by HPLC purity determination method. From the 10 g resin in step 1 to the 16 steps of the final product, the total yield was calculated to be 28.3%. The purity of the peptide product was 98.7%.

[0297] Example 12a: Various peptide resins were prepared by referring to the operations of steps 1 to 14 of Example 12 herein, except that tromethamine was not added in step 14, and 14 kinds of title resins were obtained. Upon testing, the coupling rates of the title resins of steps 1 to 13 were all in the range of 0.89 to 0.91, for example, the coupling rate of the title resin of step 13 = 0.90, and the coupling rate of the title resin of step 14 = 0.67.

[0298] Example 12b: Various peptide resins were prepared by referring to the operations of steps 1 to 14 of Example 12 of this article, with the only difference being that in step 14, the Fmoc-Tyr(tBu)-OH used was replaced with an equimolar amount of Fmoc-Tyr(PO3H2)-OH to obtain 14 kinds of title resins; upon testing, the coupling rates of the title resins of steps 1 to 13 were all in the range of 0.88 to 0.92, for example, the coupling rate of the title resin of step 13 = 0.91, while the coupling rate of the title resin of step 14 = 0.60.

[0299] Example 12c: Various peptide resins were prepared by referring to the operations of steps 1 to 14 of Example 12 herein, with the only difference being that in step 14, the Fmoc-Tyr(tBu)-OH used was replaced with an equimolar amount of Fmoc-Tyr(Bzl)-OH to obtain 14 kinds of title resins. Upon testing, the coupling rates of the title resins of steps 1 to 13 were all within the range of 0.88 to 0.90, for example, the coupling rate of the title resin of step 13 = 0.90, while the coupling rate of the title resin of step 14 = 0.65.

[0300] Example 12d: Various peptide resins were prepared by referring to the operations of steps 1 to 14 of Example 12 of this article, with the only difference being that in step 14, the Fmoc-Tyr(tBu)-OH used was replaced with an equimolar amount of Fmoc-Tyr-OH to obtain 14 kinds of title resins; upon testing, the coupling rates of the title resins of steps 1 to 13 were all in the range of 0.89 to 0.92, for example, the coupling rate of the title resin of step 13 = 0.91, while the coupling rate of the title resin of step 14 = 0.69.

[0301] Example 12e: Various peptide resins were prepared by referring to the operations of steps 1 to 14 of Example 12 herein, with the only difference being that in step 14, the Fmoc-Tyr(tBu)-OH used was replaced with an equimolar amount of Fmoc-Tyr(Me)-OH to obtain 14 kinds of title resins. Upon testing, the coupling rates of the title resins of steps 1 to 13 were all in the range of 0.89 to 0.91, for example, the coupling rate of the title resin of step 13 = 0.90, while the coupling rate of the title resin of step 14 = 0.62.

[0302] Example 12f: Various peptide resins were prepared by referring to the operation of step 14 of Example 12b, Example 12c, Example 12d, and Example 12e herein, except that tromethamine was not added in step 14, and four kinds of title resins were obtained. Upon testing, the coupling rates of the four kinds of title resins obtained in step 14 were all within the range of 0.61 to 0.68. For example, the coupling rate of the title resin of step 14 obtained by referring to Example 12b = 0.65.

[0303] According to the results of Examples 12a to 12f and Example 12, it was unexpectedly found that when the amino acid Tyr was introduced into the peptide chain, the coupling rate could be significantly increased by using Fmoc-Tyr(tBu)-OH and adding 6% tromethamine to the reaction solvent, while the coupling rate was significantly lower when tromethamine was not used or other protected forms of Tyr were used.

[0304] In the specific experiments below, unless otherwise stated, the antigen epitope peptides (1) and (2) used were prepared from Examples 11 and 12, respectively.

[0305] Example 21: Preparation of CHI3L1 antigens (1) and (2)

[0306] In this example, the CHI3L1 antigen epitope peptides (1) and (2) obtained in Example 11 and Example 12 were connected to carrier proteins to prepare CHI3L1 antigens (1) and (2). The preparation process is as follows:

[0307] CHI3L1 peptides (1) and (2) were respectively linked to the carrier protein KLH (keyhole limpet haemocyanin) using the BDB (Bis-diazotized benzidine dichloride) method to prepare CHI3L1 antigens (1) and (2);

[0308] Take 10.0 mg of CHI3L1 peptide (1) or (2) and dissolve it in 1 ml of 0.1M PBS buffer (pH 7.4); dissolve 10 mg of KLH in 20 ml of 0.2M borate buffer (pH 9.0); then mix the two, cool to 0°C, add 110 μL of BDBCl2, react at room temperature for 1.5 hours, dialyze overnight (12 to 15 hours), then divide and store at -20°C (freeze-dried if necessary), thereby preparing peptide (1)-KLH coupled protein and peptide (2)-KLH coupled protein, which are CHI3L1 antigen (1) and CHI3L1 antigen (2), respectively.

[0309] In this embodiment, the formula of PBS buffer is: 81 ml of 0.2 mol / L Na2HPO4 and 19 ml of 0.2 mol / L NaH2PO4; the formula of borate buffer is: 80 ml of 0.05 mol / L borax and 20 ml of 0.2 mol / L boric acid.

[0310] [Bradford method]: Dilute the coupled antigen into different concentrations (according to the preliminary test, the A595 absorbance is within the absorbance range of the standard curve series concentration); take 0.1ml of each sample and KLH standard protein solution and add 5ml of Bradford dye solution, react at room temperature for 5-30min, and measure the A595 value; the CHI3L1 antigen epitope peptide is a small peptide, which is connected to KLH to become a part of the protein and increase the protein concentration; the excess A595 is the concentration of the CHI3L1 antigen epitope peptide, and the coupled protein binding ratio (peptide / KLH value) is calculated from it. In the above method, Bradford stain: 100 mg Coomassie Brilliant Blue G-250 is dissolved in 50 ml of 96% ethanol solution, 100 ml of phosphoric acid is added, and water is added to 200 ml; KLH standard curve: an appropriate amount of KLH is accurately weighed and dissolved in PBS (10 mM, pH 7.4), and diluted with PBS to prepare a series of solutions with final concentrations of 50, 100, 200, 300, 400, 500, and 600 g / L. The Bradford method described in the present invention can be used to determine the coupling protein binding ratio. It is known that the relative molecular weight of KLH is not easy to determine. The present invention uses the Bradford method to determine and calculate the binding ratio of the above two coupled proteins; the Bradford method (Coomassie Brilliant Blue method) is a classic protein quantification method in this field. The method was established by Bradford in 1976. The reagent preparation is simple, the operation is simple and fast, the reaction is very sensitive, and the sensitivity is 4 times higher than that of the Lowry method. It can determine the microgram protein content, and the protein concentration range is 0-1000μg / mL. The minimum detectable protein is 2.5μg / mL. It is a commonly used trace protein rapid determination method; the principle of the Bradford method is that Coomassie Brilliant Blue G-250 has two different colors of red and blue. Under a certain concentration of ethanol and acidic conditions, it can be prepared into a light red solution. After combining with the protein, a blue compound is formed. The compound has a maximum absorption value at 595nm, and the color depth of the compound is proportional to the concentration of the protein.

[0311] Using the Bradford method, the protein-binding ratios of peptide (1) / KLH obtained in this example were measured to be 4.73 and 4.58, respectively.

[0312] Example 21a: Preparation of CHI3L1 antigens (1) and (2)

[0313] Referring to Example 21, the only difference is that the borate buffer used is replaced by an equal volume of carbonate buffer to obtain peptide (1)-KLH coupled protein and peptide (2)-KLH coupled protein, which are CHI3L1 antigen (1) and (2), respectively; the carbonate buffer used is prepared in the following manner: 2.94g of NaHCO3, 1.58g of Na2CO3, 0.25g of ammonium pyruvate, and 0.12g of sodium nitrite are weighed, dissolved in water, and diluted to 1000ml with water to obtain a carbonate buffer with a concentration of 50mM and pH 9.6. Using the [Bradford method], the coupled protein binding ratio of peptide (1) / KLH obtained in this example is measured to be 8.37, and the coupled protein binding ratio of peptide (2) / KLH is 8.17.

[0314] Example 21b: Preparation of CHI3L1 antigens (1) and (2)

[0315] Referring to Example 21, the only difference is that the borate buffer used is replaced by an equal volume of carbonate buffer to prepare peptide (1)-KLH coupled protein and peptide (2)-KLH coupled protein, which are CHI3L1 antigen (1) and (2), respectively; the carbonate buffer used is prepared in the following manner: 2.94 g of NaHCO3, 1.58 g of Na2CO3, and 0.25 g of ammonium pyruvate are weighed, dissolved in water, and diluted to 1000 ml with water to obtain a carbonate buffer with a concentration of 50 mM and a pH of 9.6. Using the Bradford method, the coupled protein binding ratio of peptide (1) / KLH obtained in this example is measured to be 4.93, and the coupled protein binding ratio of peptide (2) / KLH is measured to be 4.74.

[0316] Example 21c: Preparation of CHI3L1 antigens (1) and (2)

[0317] Referring to Example 21, the only difference is that the borate buffer used is replaced by an equal volume of carbonate buffer to obtain peptide (1)-KLH coupled protein and peptide (2)-KLH coupled protein, which are CHI3L1 antigen (1) and (2), respectively; the carbonate buffer used is prepared in the following manner: 2.94g of NaHCO3, 1.58g of Na2CO3, and 0.12g of sodium nitrite are weighed, dissolved in water, and diluted to 1000ml with water to obtain a carbonate buffer with a concentration of 50mM and pH 9.6. Using the Bradford method, the coupled protein binding ratio of peptide (1) / KLH obtained in this example is measured to be 4.82, and the coupled protein binding ratio of peptide (2) / KLH is measured to be 4.66.

[0318] According to the results of Example 21 and Example 21a, Example 21b, and Example 21c, it was unexpectedly found that the use of a 50 mM, pH 9.6 carbonate buffer containing ammonium pyruvate and sodium nitrite can significantly increase the peptide / KLH coupling protein binding ratio.

[0319] Example 22: Preparation of monoclonal antibodies and polyclonal antibodies

[0320] In this example, the antigens (1) and (2) obtained in Example 21 are used to immunize animals respectively, thereby using antigen (1) to prepare specific monoclonal antibodies and polyclonal antibodies, and using antigen (2) to prepare specific monoclonal antibodies and polyclonal antibodies;

[0321] 1. Immunize animals to prepare monoclonal antibodies:

[0322] 1.1. The CHI3L1 antigens (1) and (2) (immunogens) prepared in Example 21 were mixed with Freund's complete adjuvant (purchased from Shanghai Yuanju Biotechnology Co., Ltd.) and immunized Balb / c mice (Experimental Animal Center of Fujian Medical University) at 50 μg antigen / mouse, injected subcutaneously at multiple points; serum titers were measured 4 weeks later, and mice with good immune responsiveness were selected for booster immunization: the antigens were mixed with an equal volume of Freund's incomplete adjuvant, the antigen dose was 25 μg / mouse, injected subcutaneously at multiple points, and the number of booster immunizations was 6 times, with two consecutive booster immunizations before fusion, and then the mice were taken out for 6 times. Spleen cells and Sp2 / 0 myeloma cells were fused by conventional methods using 50% PEG (MW4000) (purchased from Zhongyuan Chemical Company), and selected and cultured using HAT conditioned medium (purchased from Sigma-Aldrich Company); after fusion, the cells were placed in a CO2 incubator and cultured at 37°C for 9 to 11 days, and larger cell clones appeared in the wells; screening was started by indirect ELISA on the 11th day; the wells that were initially positive were cloned and cultured 4 times using the limiting dilution method (even if the screened cells divided and multiplied in large numbers), and then the cells were expanded, frozen, and ascites was prepared.

[0323] 1.2. Balb / c mice were treated with 0.5 ml of pristane (purchased from Sigma-Aldrich) and 2×10 hybridoma cells were inoculated intraperitoneally one week later. 6 The ascites was collected after 10 days.

[0324] 1.3. Determination of antibody titer: The titer of the monoclonal antibody (1) prepared using the CHI3L1 antigen (1) was determined by the indirect ELISA method. The results showed that the titer of the monoclonal antibody reached 1:33500.

[0325] The titer of the monoclonal antibody (2) prepared using the CHI3L1 antigen (2) was also measured using the same method, and its titer also reached 1:32400.

[0326] 2. Immunize animals to prepare polyclonal antibodies:

[0327] 2.1. Three-month-old New Zealand white rabbits weighing about 2 kg were selected as immunized animals. In the basic immunization, 1-2 mg of CHI3L1 antigens (1) and (2) (immunogens) prepared in Example 21 above were mixed with Freund's complete adjuvant, fully emulsified, and then subcutaneously injected at multiple points on the back of the rabbits. For booster immunization every 4 weeks, the antigens were fully emulsified with incomplete Freund's adjuvant and then subcutaneously injected at multiple points on the back at a dose of 100 μg / rabbit. On the 10th day after the last booster immunization, the carotid artery was bled and the serum was separated.

[0328] 2.2. Determination of antibody titer:

[0329] The titer of the polyclonal antibody (1) prepared using the CHI3L1 antigen (1) was measured by indirect ELISA, and the result showed that the antibody titer reached 1:33600;

[0330] The titer of the polyclonal antibody (2) prepared using the CHI3L1 antigen (2) was also measured using the same method, and its titer also reached 1:32800.

[0331] 2.3. Blood collection and serum separation: Blood was collected through carotid artery catheterization and serum was separated.

[0332] 3. Isolation and purification of antibodies: Ascites or serum was precipitated with ammonium sulfate and then affinity purified with Protein G (purchased from Sigma-Aldrich).

[0333] 4. After the antibody is divided and packaged, it is freeze-dried and stored at low temperature.

[0334] Example 22a: Preparation of monoclonal antibodies and polyclonal antibodies

[0335] In this example, the antigens (1) and (2) obtained in Example 21a are used to immunize animals respectively, thereby using antigen (1) to prepare specific monoclonal antibodies and polyclonal antibodies, and using antigen (2) to prepare specific monoclonal antibodies and polyclonal antibodies;

[0336] 1. Immunize animals to prepare monoclonal antibodies:

[0337] 1.1. The CHI3L1 antigens (1) and (2) (immunogens) prepared in Example 21a above were mixed with Freund's complete adjuvant (purchased from Shanghai Yuanju Biotechnology Co., Ltd.) and then immunized Balb / c mice (Experimental Animal Center of Fujian Medical University) with 30 μg antigen / mouse, injected subcutaneously at multiple points; serum titers were measured 4 weeks later, and mice with good immune responsiveness were selected for booster immunization: the antigens were mixed with an equal volume of Freund's incomplete adjuvant, and the antigen dose was 15 μg / mouse, injected subcutaneously at multiple points, and the number of booster immunizations was 6 times, with two consecutive booster immunizations before fusion, and then Spleen cells were taken and fused with Sp2 / 0 myeloma cells by conventional methods using 50% PEG (MW4000) (purchased from Zhongyuan Chemical Company), and selected and cultured using HAT conditioned medium (purchased from Sigma-Aldrich Company); after fusion, the cells were placed in a CO2 incubator and cultured at 37°C for 9 to 11 days, and larger cell clones appeared in the wells; screening was started by indirect ELISA on the 11th day; the wells with positive initial screening were cloned and cultured 4 times using the limiting dilution method (even if the screened cells divided and multiplied in large numbers), and then the cells were expanded, frozen, and ascites was prepared.

[0338] 1.2. Balb / c mice were treated with 0.5 ml of pristane (purchased from Sigma-Aldrich) and 2×10 hybridoma cells were inoculated intraperitoneally one week later. 6 The ascites was collected after 10 days.

[0339] 1.3. Determination of antibody titer: The titer of the monoclonal antibody (1) prepared using the CHI3L1 antigen (1) was determined by an indirect ELISA method. The result showed that the titer of the monoclonal antibody reached 1:33,800.

[0340] The titer of the monoclonal antibody (2) prepared using the CHI3L1 antigen (2) was also measured using the same method, and its titer also reached 1:32100.

[0341] 2. Immunize animals to prepare polyclonal antibodies:

[0342] 2.1. Three-month-old New Zealand white rabbits weighing about 2 kg were selected as immunized animals. In the primary immunization, 1-2 mg of the CHI3L1 antigens (1) and (2) (immunogens) prepared in Example 21a above were mixed with Freund's complete adjuvant, fully emulsified, and then subcutaneously injected at multiple points on the back of the rabbits. For booster immunization every 4 weeks, the antigens were fully emulsified with incomplete Freund's adjuvant and then subcutaneously injected at multiple points on the back at a dose of 60 μg / rabbit. On the 10th day after the last booster immunization, the carotid artery was bled and the serum was separated.

[0343] 2.2. Determination of antibody titer:

[0344] The titer of the polyclonal antibody (1) prepared using the CHI3L1 antigen (1) was determined by the indirect ELISA method, and the result showed that the antibody titer reached 1:33300; the titer of the polyclonal antibody (2) prepared using the CHI3L1 antigen (2) was also determined by the same method, and its titer also reached 1:33100.

[0345] 2.3. Blood collection and serum separation: Blood was collected through carotid artery catheterization and serum was separated.

[0346] 3. Isolation and purification of antibodies: Ascites or serum was precipitated with ammonium sulfate and then affinity purified with Protein G (purchased from Sigma-Aldrich).

[0347] 4. After the antibody is divided and packaged, it is freeze-dried and stored at low temperature.

[0348] Example 3: Specificity Identification of Human CHI3L1 Monoclonal Antibodies (1) and (2)

[0349] ELISA was used for detection. Laminin LN, chitotriosidase Chit1, hyaluronic acid HA, type III procollagen PCⅢ, and type IV collagen CⅣ (all purchased from Shanghai Lianshuo Company) were used as detection antigens to coat ELISA plates. The specific reactions of the CHI3L1 monoclonal antibodies (1) and (2) prepared in Example 22 with the human CHI3L1 protein were detected by ELISA. Normal BALB / c mouse serum was used as a negative control, and PBS solution was used as a blank control. The results showed that the CHI3L1 monoclonal antibodies (1) and (2) only reacted positively with CHI3L1 (P / N>2.1), while they reacted negatively with LN, enzyme Chit1, HA, PCⅢ, and CⅣ, indicating that the CHI3L1 monoclonal antibodies (1) and (2) of the present invention are both specific.

[0350] Example 3a: Referring to the method of Example 3, the specific reactions of the CHI3L1 monoclonal antibodies (1) and (2) prepared in Example 22a with the human CHI3L1 protein were detected by ELISA, with normal BALB / c mouse serum as a negative control and PBS solution as a blank control. The results showed that the CHI3L1 monoclonal antibodies (1) and (2) only reacted positively with CHI3L1 (P / N>2.1), while the reactions with LN, enzyme Chit1, HA, PCⅢ and CⅣ were all negative, indicating that the CHI3L1 monoclonal antibodies (1) and (2) of the present invention are both specific.

[0351] Example 4: Specificity Identification of Human CHI3L1 Polyclonal Antibodies (1) and (2)

[0352] The CHI3L1 polyclonal antibodies (1) and (2) prepared in Example 22 were identified using the same method as the above-mentioned method for identifying the specificity of the monoclonal antibody; the results showed that the CHI3L1 polyclonal antibodies (1) and (2) respectively reacted positively with CHI3L1 (P / N>2.1), while reacting negatively with LN, enzyme Chit1, HA, PCⅢ, and CⅣ, indicating that the CHI3L1 polyclonal antibodies (1) and (2) of the present invention are respectively specific.

[0353] Embodiment 4a: Referring to the method of Example 4, the CHI3L1 polyclonal antibodies (1) and (2) prepared in Example 22a were identified; the results showed that the CHI3L1 polyclonal antibodies (1) and (2) respectively reacted positively with CHI3L1 (P / N>2.1), while reacting negatively with LN, enzyme Chit1, HA, PCⅢ, and CⅣ, indicating that the CHI3L1 polyclonal antibodies (1) and (2) of the present invention are respectively specific.

[0354] Example 5: Preparation of CHI3L1 in vitro diagnostic reagent using CHI3L1 monoclonal antibody and CHI3L1 polyclonal antibody box

[0355] In this example, the monoclonal antibody (1) prepared using the CHI3L1 antigen epitope peptide (1) in Example 22 is used as the coating antibody in this kit, and the polyclonal antibody (2) prepared using the CHI3L1 antigen epitope peptide (2) in Example 22 is used as the binding antibody in this kit.

[0356] 1. The composition of the CHI3L1 in vitro diagnostic kit is as follows:

[0357] (A) a pre-coated plate, each well of which is pre-coated with 0.1 μg of CHI3L1 monoclonal antibody (1) [48 / 96-well plate, the preparation method of which is: dissolving CHI3L1 monoclonal antibody (1) in 0.05 M carbonate buffer at pH 9.6 to prepare a pre-coating solution; adding 100 μl of the pre-coating solution to each well of the ELISA plate at 0.1 μg / well, placing the plate at 4°C for 18-24 hours, removing the plate, discarding the coating solution, washing the plate, blocking the plate with BSA for 16 hours, drying the plate, placing the plate in an aluminum foil bag and vacuum sealing the plate to obtain a pre-coated plate; the plate is usually stored at 4°C; the 0.05 M carbonate buffer at pH 9.6 is also called coating buffer, and the preparation method / formula of the pre-coating solution is: dissolving 16.0 g of Na2CO3, 29.0 g of NaHCO3, and distilled water to 1000 ml];

[0358] (B) CHI3L1 calibrator, which is a recombinant human YKL-40 / CHI3L1 protein solution with a series of concentrations [purchased from abcam, 7 series of concentrations, each 1.0 ml of calibrator solution, the concentrations are 25 ng / ml, 10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1 ng / ml, 0.5 ng / ml, 0.25 ng / ml];

[0359] (C) binding antibody, which is a solution of CHI3L1 polyclonal antibody (2) [which has a concentration of 0.5 μg / 100 μl, and is dissolved and / or diluted with an enzyme marker diluent as a solvent, and 10 ml of the binding antibody solution is provided in each kit; the preparation method / formula of the enzyme marker diluent is: 10 ml of 10×PBS-Tween 20 solution, 20 ml of FCS (calf serum), 1 gram of enzyme stabilizer (DCE0061A, Shanghai Xibao), 1 ml of biological preservative (Proclin 300, Shanghai Xibao), and distilled water dissolved to 1000 ml];

[0360] (D) Enzyme conjugate, which is a horseradish enzyme-labeled goat anti-rabbit IgG antibody solution [ZB-2301 (80 μg / 200 μl, Zhongshan Jinqiao) horseradish enzyme-labeled goat anti-rabbit IgG antibody diluted 1:5000 with enzyme labeling diluent, 10 ml];

[0361] (E) 10× washing buffer, which is 10× PBS-Tween 20 solution [its preparation method / formulation is: 58 g Na2HPO4·12H2O, 4 g KH2PO4, 100 g NaCl, 4 g KCl, 20 ml Tween 20, dissolved in distilled water to 1000 ml, its pH is 7.2; it is diluted 10 times with distilled water when used, and 20 ml of 10× washing buffer is provided in each kit];

[0362] (F) Color developer A [its preparation method / formulation is: 35.5 g citric acid, 10 g urea peroxide, 10 ml Tween 20, distilled water dissolved to 1000 ml, 6 ml of color developer A is provided in each kit];

[0363] (G) Color developer B [its preparation method / formulation is: 120 g citric acid, 1 g EDTA-2Na, 2 g TMB·2HCl, dissolved in distilled water to 1000 ml, 6 ml of color developer B is provided in each kit];

[0364] (H) Stop solution is 2M sulfuric acid solution [its preparation method / formula is: 22.2 ml concentrated sulfuric acid (96%), 177.3 ml distilled water, slowly drip the concentrated sulfuric acid into the distilled water while adding, shaking, each kit provides 6 ml of stop solution].

[0365] TMB·2HCl in the above color developing agent B is 3,3'5,5'-tetramethylbenzidine dihydrochloride, which is a commonly used sensitive color developing substrate of peroxidase.

[0366] 2. Kit operation steps:

[0367] (a) Add 100 μl / well of the blood sample to be tested and CHI3L1 calibrator to each well of the pre-coated plate, in duplicate, incubate at 37°C for 60 min, wash five times with 1× wash buffer, and pat dry;

[0368] (b) Add 100 μl / well of CHI3L1-binding antibody to each well, incubate at 37°C for 30 min, wash five times with 1× wash buffer, and pat dry;

[0369] (c) Add 100 μl / well of enzyme conjugate to each well, incubate at 37°C for 30 min, wash five times with 1× wash buffer, and pat dry;

[0370] (d) Add 50 μl of color developing reagent A and B to each well, mix well, and incubate at 37°C for 15 min;

[0371] (e) Add 50 μl / well of stop solution to terminate the reaction and measure the absorbance at dual wavelengths of 450 nm and 620 nm using an enzyme-linked detector.

[0372] 3. Results:

[0373] The average absorbance of the calibrators of various concentrations was determined (n=6), and a standard curve was drawn using the logarithmic values ​​of the calibrator concentrations and the corresponding absorbances. The CHI3L1 concentrations in the tested samples were calculated based on the standard curve.

[0374] Serum CHI3L1 was detected in sera from 33 healthy volunteers and 46 liver disease patients (samples from confirmed / treated patients provided by a hospital). The CHI3L1 level in the serum of liver disease patients was significantly higher than that in the healthy control group, and the difference was statistically significant (P<0.01). There were also significant differences in CHI3L1 levels between different liver disease groups, as shown in the data of Example 5 in Table 1 below.

[0375] Table 1: CHI3L1 concentrations in serum samples from four groups

[0376]

[0377] The data in Table 1 above are the results of measuring the absorbance of the calibrator and the sample within 2 minutes immediately after the stop solution is added in step (e) and calculated. TMB·2HCl in the color developer B of this example is 3,3'5,5'-tetramethylbenzidine dihydrochloride, which is a commonly used sensitive color substrate for peroxidase; the model of the enzyme-linked detector used in this example is RT-6000 (Raydo Company).

[0378] In view of the fact that in actual detection, there may be a situation where the amount of sample to be detected is large, in step (e) of "2. Operation steps of the kit" of this Example 5, the absorbance is measured within 2 minutes immediately after the stop solution is added. When the amount of sample to be detected is large, the time will be extremely tight, and there will be congestion in the absorbance measurement link of the enzyme-linked detector. Therefore, it is completely necessary to understand the stability of the test plate after adding the stop solution after being placed for a slightly longer period of time, and if the stability is insufficient, it should be improved. To this end, the three groups of patient samples involved in Table 1 were each measured within 2 minutes (the results in Table 1 were taken as the results at 0 min), and then allowed to stand at room temperature for 5 min, 10 min, 20 min, and 30 min, and the absorbance was measured respectively. The CHI3L1 concentration was calculated using the standard curve used in Table 1. The CHI3L1 concentration at 0 min was taken as 100%, and the relative percentage concentration of CHI3L1 at 5 to 30 min was calculated. The results showed that the relative percentage concentrations of CHI3L1 in the three groups of patients were all within the range of 95 to 97% at 5 min, for example, the relative percentage concentrations of CHI3L1 in liver cancer patients were 96.3% at 10 min, and the relative percentage concentrations of CHI3L1 in the three groups of patients were 96.3% at 10 min. The percentage concentrations were all within the range of 88-91%, for example, 90.1% for liver cancer patients; at 20 minutes, the relative percentage concentrations of CHI3L1 in the three groups of patients were all within the range of 78-82%, for example, 79.8% for liver cancer patients; at 30 minutes, the relative percentage concentrations of CHI3L1 in the three groups of patients were all within the range of 61-66%, for example, 63.5% for liver cancer patients. These results show that the test results of the test plate decreased slightly after 5 minutes of placement, and the test results decreased significantly at 10 minutes. The results at 20 and 30 minutes were unacceptable, indicating that the absorbance of the test plate needs to be read on the enzyme-linked detector as soon as possible.

[0379] Example 5a: Preparation of CHI3L1 in vitro diagnostic reagent using CHI3L1 monoclonal antibody and CHI3L1 polyclonal antibody box

[0380] This example was carried out with reference to Example 5, except that the preparation method / formula of the color developer B used was: 120 g citric acid, 1 g EDTA-2Na, 2 g TMB·2HCl, 1.5 g magnesium chloride, 2.5 g serine, dissolved in distilled water to 1000 ml. The absorbance was measured within 2 minutes immediately after the addition of the stop solution in step (e) and the results were calculated using the standard curve obtained in this example. The CHI3L1 concentration (ng / ml) is shown in the data of Example 5a in Table 1. The results showed no difference compared with Example 5. Next, the method of Example 5 is continued. The three groups of patient samples involved in Example 5a of Table 1 are measured within 2 minutes, and then left to stand at room temperature for 5 minutes, 10 minutes, 20 minutes, and 30 minutes. The absorbance is measured respectively, and the CHI3L1 concentration is calculated using the standard curve obtained in this example. The relative percentage concentration of CHI3L1 at 5 to 30 minutes is calculated in the same way. The results of Example 5a are as follows: the relative percentage concentration of CHI3L1 of the three groups of patients at 5 minutes is all within the range of 98 to 101%. For example, the relative percentage concentration of CHI3L1 of the liver cancer patients is 99.4%, and the relative percentage concentration of CHI3L1 of the three groups of patients at 10 minutes is 99.4%. The relative concentrations of CHI3L1 in the three groups of patients were all within the range of 97-99%, for example, 98.6% in liver cancer patients; at 20 minutes, the relative percentage concentrations of CHI3L1 in the three groups of patients were all within the range of 95-98%, for example, 97.3% in liver cancer patients; at 30 minutes, the relative percentage concentrations of CHI3L1 in the three groups of patients were all within the range of 93-95%, for example, 93.9% in liver cancer patients. These results show that the test results of the test plate remained basically unchanged after 20 minutes of placement, and the test results were also greater than 95% at 30 minutes, indicating that the color of the test plate is quite stable, allowing the absorbance to be read on the enzyme-linked detector within a more relaxed time range.

[0381] Example 5b: Preparation of CHI3L1 in vitro diagnostic reagent using CHI3L1 monoclonal antibody and CHI3L1 polyclonal antibody box

[0382] This example is carried out with reference to Example 5, except that the preparation method / formula of the color developer B used is: 120 g of citric acid, 1 g of EDTA-2Na, 2 g of TMB·2HCl, 1.5 g of magnesium chloride, dissolved in distilled water to 1000 ml, and the absorbance is measured within 2 minutes immediately after the stop solution is added in step (e) and the results are calculated using the standard curve obtained in this example. The CHI3L1 concentration (ng / ml) is shown in the data of Example 5b in Table 1, and the results show no difference compared with Example 5. Then, the method of Example 5 is continued, and the three groups of patient samples involved in Example 5b in Table 1 are measured within 2 minutes, and then left to stand at room temperature for 5 min, 10 min, 20 min, and 30 min. The absorbance is measured respectively, and the CHI3L1 concentration is calculated using the standard curve obtained in this example. The relative percentage concentration of CHI3L1 at 5 to 30 min is calculated in the same way. The results of Example 5b are: the relative percentage concentration of CHI3L1 of the three groups of patients at 5 min is all within the range of 96 to 97%. For example, The relative percentage concentration of CHI3L1 in the three groups of patients was 96.7% for liver cancer patients, at 10 minutes, the relative percentage concentration of CHI3L1 in the three groups of patients was all within the range of 88-92%, for example, 89.3% for liver cancer patients, at 20 minutes, the relative percentage concentration of CHI3L1 in the three groups of patients was all within the range of 77-80%, for example, 78.4% for liver cancer patients, and at 30 minutes, the relative percentage concentration of CHI3L1 in the three groups of patients was all within the range of 62-65%, for example, 64.1% for liver cancer patients. These results indicate that the color stability of the detection plate is basically consistent with that in Example 5.

[0383] Example 5c: Preparation of CHI3L1 in vitro diagnostic reagent using CHI3L1 monoclonal antibody and CHI3L1 polyclonal antibody box

[0384] This example is carried out with reference to Example 5, except that the preparation method / formula of the color developer B used is: 120 g citric acid, 1 g EDTA-2Na, 2 g TMB·2HCl, 2.5 g serine, dissolved in distilled water to 1000 ml. The absorbance is measured within 2 minutes immediately after the stop solution is added in step (e) and the result is calculated using the standard curve obtained in this example. The CHI3L1 concentration (ng / ml) is shown in the data of Example 5c in Table 1. The result shows no difference compared with Example 5. Then, the method of Example 5 is continued, and the three groups of patient samples involved in Example 5c in Table 1 are measured within 2 minutes, and then left to stand at room temperature for 5 min, 10 min, 20 min, and 30 min. The absorbance is measured respectively and the CHI3L1 concentration is calculated using the standard curve obtained in this example. The relative percentage concentration of CHI3L1 at 5 to 30 min is calculated in the same way. The result of Example 5c is: the relative percentage concentration of CHI3L1 of the three groups of patients at 5 min is all within the range of 96 to 98%. For example, The relative percentage concentration of CHI3L1 in the three groups of patients was 96.2% at 10 minutes, all of them were in the range of 89-92%, for example, 91.2% in the liver cancer patients, all of them were in the range of 79-83% at 20 minutes, for example, 82.3% in the liver cancer patients, and all of them were in the range of 64-68% at 30 minutes, for example, 65.8% in the liver cancer patients. These results show that the color stability of the detection plate is basically consistent with that in Example 5.

[0385] According to the results of the above Example 5 and Examples 5a to 5c, it was unexpectedly found that when two reagents were added to the developer B at the same time, the color stability of the detection plate could be significantly improved, which provided ample time for large-scale detection.

[0386] Example 6: Preparation of CHI3L1 in vitro diagnostic reagent using CHI3L1 monoclonal antibody and CHI3L1 polyclonal antibody box

[0387] In this example, the monoclonal antibody (1) prepared using the CHI3L1 antigen epitope peptide (1) in Example 22a is used as the coating antibody in this kit, and the polyclonal antibody (2) prepared using the CHI3L1 antigen epitope peptide (2) in Example 22a is used as the binding antibody in this kit. Referring to and using the method and materials of Example 5a (including the use of a color developing agent B containing magnesium chloride and serine), the CHI3L1 concentrations of the sera of 33 healthy volunteers and 46 liver disease patients mentioned in Table 1 were detected, and the results are shown in the data of Example 6 in Table 1. Next, referring to the method of Example 5a, the three groups of patient samples involved in Example 6 in Table 1 were measured within 2 minutes, and then left to stand at room temperature for 5 minutes, 10 minutes, 20 minutes, and 30 minutes. The absorbance was measured respectively, and the CHI3L1 concentration was calculated using the standard curve obtained in this example. The relative percentage concentration of CHI3L1 at 5 to 30 minutes was calculated in the same way. The results of Example 6 were as follows: the relative percentage concentration of CHI3L1 of the three groups of patients at 5 minutes was all within the range of 98 to 102%, for example, the relative percentage concentration of CHI3L1 of the liver cancer patients was 100.3%, and the relative percentage concentration of CHI3L1 of the three groups of patients at 10 minutes was 100.3%. The relative concentrations of CHI3L1 in the three groups of patients were all within the range of 97-98%, for example, 97.8% in liver cancer patients; at 20 minutes, the relative percentage concentrations of CHI3L1 in the three groups of patients were all within the range of 95-97%, for example, 96.7% in liver cancer patients; at 30 minutes, the relative percentage concentrations of CHI3L1 in the three groups of patients were all within the range of 94-96%, for example, 95.6% in liver cancer patients. These results show that the test results of the test plate remained basically unchanged after 20 minutes of placement, and the test results were also greater than 94% at 30 minutes, indicating that the color of the test plate is quite stable, allowing the absorbance to be read on the enzyme-linked detector within a more relaxed time range.

[0388] Example 7: Preparation of CHI3L1 in vitro diagnostic reagent using CHI3L1 monoclonal antibody and CHI3L1 polyclonal antibody box

[0389] In this example, the monoclonal antibody (2) prepared using the CHI3L1 antigen epitope peptide (2) in Example 22a is used as the coating antibody in this kit, and the polyclonal antibody (1) prepared using the CHI3L1 antigen epitope peptide (1) in Example 22a is used as the binding antibody in this kit. Referring to and using the method and materials of Example 5a (including the use of a color developing agent B containing magnesium chloride and serine), the CHI3L1 concentrations of the sera of 33 healthy volunteers and 46 liver disease patients mentioned in Table 1 were detected, and the results are shown in the data of Example 7 in Table 1. Next, referring to the method of Example 5a, the three groups of patient samples involved in Example 7 in Table 1 were measured within 2 minutes, and then left to stand at room temperature for 5 minutes, 10 minutes, 20 minutes, and 30 minutes. The absorbance was measured respectively, and the CHI3L1 concentration was calculated using the standard curve obtained in this example. The relative percentage concentration of CHI3L1 at 5 to 30 minutes was calculated in the same way. The results of Example 7 were as follows: the relative percentage concentration of CHI3L1 of the three groups of patients at 5 minutes was all within the range of 99 to 102%, for example, the relative percentage concentration of CHI3L1 of the liver cancer patients was 101.2%, and the relative percentage concentration of CHI3L1 of the three groups of patients at 10 minutes was 101.2%. The relative concentrations of CHI3L1 in the three groups of patients were all within the range of 97-99%, for example, 98.6% in liver cancer patients; at 20 minutes, the relative percentage concentrations of CHI3L1 in the three groups of patients were all within the range of 96-98%, for example, 97.3% in liver cancer patients; at 30 minutes, the relative percentage concentrations of CHI3L1 in the three groups of patients were all within the range of 95-97%, for example, 95.7% in liver cancer patients. These results show that the test results of the test plate remained basically unchanged after 20 minutes of placement, and the test results were also greater than 95% at 30 minutes, indicating that the color of the test plate is quite stable, allowing the absorbance to be read on the enzyme-linked detector within a more relaxed time range.

[0390] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. CHI3L1 in vitro diagnostic kit, comprising the following parts: (A) A pre-coated plate, each well of which is pre-coated with a monoclonal antibody made from one of the following CHI3L1 antigen epitope peptides (1) or peptides (2): (1)Tyr-Arg-Lys-Ser-Val-Pro-Pro-Phe-Leu-Arg-Thr, (2)Tyr-Arg-Ser-Ala-Ala-Leu-Ser-Ala-Gly-Lys-Val-Thr-Ile-Asp; (B) CHI3L1 calibrator, which is a recombinant human YKL-40 / CHI3L1 protein solution with a series of concentrations; (C) a binding antibody, which is a solution of a polyclonal antibody prepared from one of the CHI3L1 antigen epitope peptide (1) or peptide (2) described in (A); (D) Enzyme conjugate, which is a solution of goat anti-rabbit IgG antibody labeled with horseradish enzyme; (E) 10× washing buffer, which is 10× PBS-Tween 20 solution; (F) Color developer A, prepared by: 35.5 g citric acid, 10 g urea peroxide, 10 ml Tween 20, dissolved in distilled water to 1000 ml; (G) Color developer B, prepared by dissolving 120 g citric acid, 1 g EDTA-2Na, 2 g TMB·2HCl, 1.5 g magnesium chloride, 2.5 g serine in distilled water to 1000 ml; (H) stop solution, which is 2 M sulfuric acid solution; in, The antigen epitope peptides used for both the pre-coated plate and the bound antibody are different; The antigen epitope peptide (1) and the antigen epitope peptide (2) are connected to Tyr in the following manner: To the prepared Fmoc-Arg(Tos)-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin, add 20% hexahydropyridine / dimethylformamide solution, shake at room temperature for decapping reaction to remove the Fmoc protecting group at the nitrogen end, filter out the solvent, then wash the resin twice with dimethylformamide, methanol, and dichloromethane alternately, filter out the solvent, add Fmoc-Tyr(tBu)-OH, HBTU, and HOBT to dimethylformamide to make Dissolve, add DIEA and tromethamine, stir well, transfer into the reactor containing the treated resin, react at room temperature, filter out the reaction solution, then wash the resin twice with dimethylformamide, methanol and dichloromethane alternately, filter out the solvent, and after the peptide connection reaction is completed, filter out the resin and dry it in a vacuum dryer to obtain Fmoc-Tyr(tBu)-Arg(Tos)-Lys(Boc)-Ser(tBu)-Val-Pro-Pro-Phe-Leu-Arg(Tos)-Thr(tBu)-resin, i.e., protected 11-peptide resin; and To the prepared Fmoc-Arg(Tos)-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin, add 20% hexahydropyridine / dimethylformamide solution, shake at room temperature for decapping reaction to remove the nitrogen-terminal Fmoc protecting group, filter out the solvent, then wash the resin twice with dimethylformamide, methanol, and dichloromethane, respectively, and filter out the solvent, add Fmoc-Tyr(tBu)-OH, HBTU, and HOBT to dimethylformamide. Dissolve the mixture, add DIEA and tromethamine, stir well, transfer into a reactor containing the treated resin, react at room temperature, filter out the reaction solution, wash the resin twice with dimethylformamide, methanol and dichloromethane alternately, filter out the solvent, and after the peptide-linking reaction is completed, filter out the resin and dry it in a vacuum dryer to obtain Fmoc-Tyr(tBu)-Arg(Tos)-Ser(tBu)-Ala-Ala-Leu-Ser(tBu)-Ala-Gly-Lys(Boc)-Val-Thr(tBu)-Ile-Asp(OtBu)-resin, i.e., protected tetradecapeptide resin; In the process of preparing peptide (1) or peptide (2) with Tyr insertion, the molar ratio of Fmoc-protected amino acid: HBTU: HOBT: DIEA used is 1:1:1:4, and the amount of tromethamine is 6% of DIEA, calculated as a molar percentage.

2. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein: Each well of the pre-coated plate is pre-coated with 0.1 μg of CHI3L1 monoclonal antibody.

3. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein: The pre-coated plates are 48 / 96 well plates.

4. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein: The method for preparing the pre-coated plate is as follows: dissolving the CHI3L1 monoclonal antibody in a 0.05M carbonate buffer at pH 9.6 to prepare a pre-coating solution; adding 100 μl of the pre-coating solution to each well of the ELISA plate at 0.1 μg / well, placing it at 4°C for 18-24 hours, taking it out, discarding the coating solution, washing, blocking it with BSA for 16 hours, drying it, placing it in an aluminum foil bag and vacuum sealing it to obtain the pre-coated plate.

5. The CHI3L1 in vitro diagnostic kit according to claim 4, wherein: The preparation method / formula of the 0.05M carbonate buffer solution at pH 9.6 is: 16.0 grams of Na2CO3, 29.0 grams of NaHCO3, and distilled water dissolved to 1000 ml.

6. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein: The CHI3L1 calibrator consists of 7 series of 1.0 ml calibrator solutions, with concentrations of 25 ng / ml, 10 ng / ml, 5 ng / ml, 2.5 ng / ml, 1 ng / ml, 0.5 ng / ml, and 0.25 ng / ml, respectively.

7. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein: The CHI3L1 polyclonal antibody solution bound to the antibody has a concentration of 0.1-1 μg / 100 μl or is adjusted to a wider concentration or range as needed or to a concentration or range determined by a square array titration experiment.

8. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein: The antibody-bound CHI3L1 polyclonal antibody solution is dissolved and / or diluted using an enzyme labeling substance diluent as a solvent.

9. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein: Each kit provides 10 ml of antibody-binding solution.

10. The CHI3L1 in vitro diagnostic kit according to claim 8, wherein: The preparation method / formula of the enzyme marker diluent is: 10 ml of 10×PBS-Tween 20 solution, 20 ml of calf serum, 1 gram of enzyme stabilizer DCE0061A, 1 ml of biological preservative Proclin 300, and distilled water dissolved to 1000 ml.

11. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein: The enzyme conjugate was prepared by diluting ZB-2301 horseradish enzyme-labeled goat anti-rabbit IgG antibody at 1:5000 with enzyme labeling diluent.

12. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein: Each kit provides 10 ml of enzyme conjugate solution.

13. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein: The preparation method / formula of 10× washing buffer is: 58 grams of Na2HPO4·12H2O, 4 grams of KH2PO4, 100 grams of NaCl, 4 grams of KCl, 20 ml of Tween 20, and distilled water dissolved to 1000 ml, with a pH of 7.

2.

14. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein each kit is provided with 20 ml of 10× washing buffer.

15. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein each kit contains 6 ml of the color developing reagent A.

16. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein each kit contains 6 ml of the color developing agent B.

17. The CHI3L1 in vitro diagnostic kit according to claim 1, wherein each kit contains 6 ml of the stop solution.

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