An antibody against REG3A protein or its antigen-binding fragment and its use

Abstract

The present application relates to the technical field related to REG3A protein detection, and specifically discloses an antibody or antigen-binding fragment thereof against REG3A protein and its use. The antibody or antigen-binding fragment thereof comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3 and heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3. The antibody or antigen-binding fragment thereof against REG3A protein based on the present application exhibits high affinity and high specificity to REG3A, and can achieve quantitative detection of REG3A protein.

Description

Technical Field

[0001] The present application relates to the technical field related to REG3A protein detection, and specifically to an anti-REG3A protein antibody or an antigen-binding fragment thereof and uses thereof. Background Art

[0002] Regenerative islet-derived protein III-α (REG3A) is a protein with multiple biological activities, belonging to the REG protein family, also known as heptocarcinoma-intestine-pancreas (HIP) or pancreatic associated protein (PAP). As a secreted calcium-dependent lectin protein, REG3A has multiple effects in anti-inflammation, antibacterial, cell proliferation, apoptosis and tissue repair. More and more studies have also demonstrated its potential in tumorigenesis and neural development.

[0003] REG3A is located on human chromosome 2p12 and consists of 6 exons and 5 introns. REG3A encodes a 19kDa protein with a total length of 175 amino acids. The entire domain of REG3A contains a signal peptide (1-26aa), a propeptide (27-37aa) and a C-type lectin-like domain (CLTD, 47-173aa). REG3A is mainly secreted by Paneth cells, some enteroendocrine cells and goblet cells in intestinal microcrypts. It is expressed at low levels in healthy tissues such as the pancreas, liver, and skin, and is highly expressed when the tissue is damaged, but is not found in immune cells.

[0004] REG3A is secreted into the extracellular space after maturation and participates in multiple signaling pathways. Studies have shown that in keratinocytes, REG3A binds to EXTL3 to activate PI3K and AKT, and participates in the PI3K-AKT signaling pathway; in different types of tumors, REG3A protein also participates in the PI3K / Akt-GSK3β signaling pathway, activates the JAK2 / STAT3 signaling pathway or downregulates its key factors, activates the AKT and ERK1 / 2 pathways, and the REG3A / p42 / 44 pathway.

[0005] The REG3A factor in graft-versus-host disease (GVHD) is an important biological marker, especially in intestinal acute graft-versus-host disease (GI-aGVHD), showing significant diagnostic and prognostic value. As an antibacterial protein, REG3A protein can inhibit the activity of G+ bacteria, protect the intestinal crypt stem cell microenvironment from bacteria, and protect intestinal stem cells. At the same time, it can also regulate the intestinal flora to protect the intestinal mucosal epithelial barrier. When GVHD damages the gastrointestinal crypts and destroys the integrity of the intestinal mucosa, REG3A is released into the blood. As the severity of GVHD increases, the serum level of REG3A protein increases. In recent years, literature has reported that REG3A protein can be used as a specific marker for intestinal acute graft-versus-host disease (GI-aGVHD), and a prognostic score (Ann Arbor GVHD score) system for aGVHD has been proposed based on the three biological markers of REG3A, ST2, and TNFRl. REG3A protein can not only be used as a diagnostic indicator, but also predict the therapeutic efficacy and prognosis of GI-aGVHD. Changes in its levels are closely related to the treatment effect and survival status of patients.

[0006] Therefore, detecting the content of REG3A protein in serum can provide strong support for the clinical diagnosis and treatment of GI-aGVHD. Summary of the invention

[0007] The present application provides an antibody or antigen-binding fragment thereof against REG3A protein and its use. The antibody or antigen-binding fragment thereof against REG3A protein of the present application exhibits high affinity and high specificity to REG3A, and can achieve quantitative detection of REG3A.

[0008] In the first aspect of the present application, the present application provides an antibody or an antigen-binding fragment thereof against REG3A protein, wherein the antibody or the antigen-binding fragment thereof comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3 and heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, wherein:

[0009] LCDR1 is selected from the amino acid sequence shown in SEQ ID NO.2;

[0010] LCDR2 is selected from the amino acid sequence shown in SEQ ID NO.3;

[0011] LCDR3 is selected from the amino acid sequence shown in SEQ ID NO.4;

[0012] HCDR1 is selected from the amino acid sequence shown in SEQ ID NO.6;

[0013] HCDR2 is selected from the amino acid sequence shown in SEQ ID NO.7;

[0014] HCDR3 is selected from the amino acid sequence shown in SEQ ID NO.8.

[0015] In some optional embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region; wherein:

[0016] The light chain variable region is selected from the amino acid sequence shown in SEQ ID NO.1;

[0017] And / or, the heavy chain variable region is selected from the amino acid sequence shown in SEQ ID NO.5.

[0018] The antibodies or antigen-binding fragments thereof described in the present application at least comprise a heavy chain variable region and a light chain variable region, both of which comprise the above-mentioned complementarity determining region (CDR) and an intervening framework region (FR), and the arrangement of each domain is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; wherein one or more amino acid substitutions, deletions or insertions or any combination thereof occur in the framework regions FR1, FR2, FR3, and FR4.

[0019] In some specific embodiments, the light chain variable region is selected from the amino acid sequence shown in SEQ ID NO.1, and the heavy chain variable region is selected from the amino acid sequence shown in SEQ ID NO.5.

[0020] In some optional embodiments, the antibody is selected from any form of a monoclonal antibody, a single-chain antibody, a bifunctional antibody, a single-domain antibody, a nanobody, a fully or partially humanized antibody, or a chimeric antibody;

[0021] And / or, the antigen binding fragment is selected from Fab, Fab', Fab'-SH, (Fab')2, Fv, scFv, BsFv, dsFv or (dsFv)2 fragment.

[0022] In some optional embodiments, the antibody or its antigen-binding fragment further comprises a constant region of human or mouse origin. In other optional embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region of human or mouse origin. In other optional embodiments, the antibody or its antigen-binding fragment comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

[0023] In some alternative embodiments, the antibody is a murine, chimeric or humanized monoclonal antibody.

[0024] In some optional embodiments, the heavy chain constant region of the monoclonal antibody is of IgG1, IgG2, IgG3 or IgG4 subtype.

[0025] In some embodiments, the antibody is a monoclonal antibody.

[0026] In some embodiments, the antibody is an IgG antibody

[0027] In a second aspect of the present application, the present application provides a biomaterial, the biomaterial comprising:

[0028] (I), a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the first aspect; and / or,

[0029] (II), a vector comprising the nucleic acid molecule described in (I); and / or,

[0030] (III) A host cell comprising the nucleic acid molecule described in (I) and / or the vector described in (II), or the host cell is transformed or transfected with the nucleic acid molecule described in (I) and / or the vector described in (II).

[0031] Among them, according to the amino acid sequence involved in the antibody or antigen-binding fragment thereof described in the first aspect, a person skilled in the art can obtain a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described in the first aspect. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule is not unique, and all nucleic acid molecules that can encode the antibody or antigen-binding fragment thereof described in the first aspect are within the scope of protection of this application.

[0032] In some optional embodiments, the vector includes but is not limited to a plasmid vector, a phage vector, a virus vector, an artificial chromosome vector, and the like.

[0033] In some alternative embodiments, the host cell includes but is not limited to microbial cells, insect cells or other animal cells.

[0034] In some alternative embodiments, the host cell is selected from a prokaryotic cell or a eukaryotic cell.

[0035] In some optional embodiments, the host cell is selected from yeast cells, CHO cells, 293 cells or plant cells.

[0036] In the third aspect of the present application, the present application provides an antibody conjugate, which comprises the antibody or antigen-binding fragment thereof described in the first aspect, and a label conjugated to the antibody or antigen-binding fragment thereof described in the first aspect.

[0037] In some optional embodiments, the label is selected from at least one of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

[0038] In the fourth aspect of the present application, the present application provides the use of the antibody or antigen-binding fragment thereof described in the first aspect, and / or the biomaterial described in the second aspect, and / or the antibody conjugate described in the third aspect in the preparation of a product for detecting REG3A protein.

[0039] In the fifth aspect of the present application, the present application provides a detection reagent for detecting REG3A protein, wherein the detection reagent comprises the antibody or antigen-binding fragment thereof described in the first aspect, and / or the biomaterial described in the second aspect, and / or the antibody conjugate described in the third aspect.

[0040] In the sixth aspect of the present application, the present application provides a detection kit for detecting REG3A protein, wherein the detection kit comprises the antibody or antigen-binding fragment thereof described in the first aspect, and / or the biological material described in the second aspect, and / or the antibody conjugate described in the third aspect.

[0041] In some optional embodiments, the detection kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

[0042] In some embodiments, the detection kit is selected from an enzyme-linked immunosorbent assay kit.

[0043] In some specific embodiments, the detection kit is selected from a fluorescent immunoassay kit.

[0044] This application has the following beneficial effects:

[0045] First, the monoclonal antibody (anti-REG3A protein antibody) produced by the present application with REG3A protein as the target shows high affinity and high specificity to REG3A protein, and can realize the quantitative detection of REG3A, providing strong support for the clinical diagnosis and treatment of GVHD and even GI-aGVHD.

[0046] Second, the anti-REG3A protein antibody of the present application has specific binding to REG3A protein, is suitable for blood sample detection or sample detection from other tissue sources, and has clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the affinity curve of the anti-REG3A monoclonal antibody and REG3A protein in Example 2.

[0048] Figure 2This is the standard curve for detecting REG3A protein in Example 3. DETAILED DESCRIPTION

[0049] The present application discloses an antibody or antigen-binding fragment thereof against REG3A protein and its use. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

[0050] Definitions or general explanations of terms:

[0051] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0052] The term “and / or” used in this application should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0053] As used herein, “comprising” or “including” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.

[0054] The "antibody (Antibody, Ab)" used in this application is an immunoglobulin with immune function (i.e., able to specifically bind to an antigen) secreted by B lymphocytes after antigen stimulation. Antibodies have a tetrameric structure, and the antibody contains two identical heavy chains (Heavy chain, H chain) and two identical light chains (Light chain, L chain). Among them, the region where the amino acid sequence of the antibody heavy chain and light chain near the N-terminal varies greatly is called the variable region (Variable region, V region), among which the amino acid sequence of the heavy chain and light chain variable region mainly has great variability in three regions, which are called hypervariable regions (Hypervariable region, HVR). Since this region is the site where Ig and antigen epitopes are complementary, it is called the complementarity determining region (Complementarity determing region, CDR), which is represented by HVR1 (CDR1), HVR2 (CDR2), and HVR3 (CDR3), respectively, among which CDR3 has the largest change; the region where the amino acid sequence of the antibody heavy chain and light chain near the C-terminal is relatively constant is called the constant region (constant region, C region).

[0055] As used herein, "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" are used interchangeably and are regions in the variable domain of an antibody that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to antigen epitopes.

[0056] The "monoclonal antibody" used in this application is a specific antibody with a completely identical chemical structure and complementary to the antigenic determinant cluster, which is produced by B lymphocytes under the stimulation of specific antigens.

[0057] As used herein, "amino acid difference" refers to amino acid modification (such as amino acid methylation modification, etc.) or amino acid replacement.

[0058] As used herein, “anti-REG3A protein antibody”, “anti-REG3A protein antibody” and “anti-REG3A monoclonal antibody” are used interchangeably.

[0059] “REG3A”, “REG3a” and “Reg3a” are used interchangeably in this application.

[0060] In order to make the purpose, technical solutions and advantages of the present application clearer, the implementation methods of the present application will be further described in detail below in conjunction with examples.

[0061] Unless otherwise specified, the implementation of this application will adopt the methods of conventional chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology in the art. The experimental methods described, unless otherwise specified, are conventional methods in the art using default parameters, steps, etc.; the experimental materials used, unless otherwise specified, are all commercially available products. If the specific technology or conditions are not specified in the examples, they are carried out according to the technology or conditions described in the literature in the art, or according to the corresponding product instructions. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels.

[0062] Example 1 Preparation of anti-REG3A protein antibodies:

[0063] In this embodiment, the anti-REG3A protein antibody is a monoclonal antibody, which is obtained by using recombinant human REG3A protein as an immunogen and using conventional mouse immunization and ELISA screening methods. The specific method includes the following steps:

[0064] Step 101, animal immunization: Select female Balb / c mice aged 6 to 8 weeks, dilute the recombinant human REG3A protein to 1 mg / mL, mix with an equal volume of Freund's adjuvant and emulsify for immunization, the first immunization is 50 μg / mouse, and the subsequent immunizations are 25 μg / mouse, and the immunization interval is two weeks. After three immunizations, the submandibular vein of the mice is bled, and the antibody titer of the mouse's immune response to the recombinant human REG3A protein is detected by the Elisa method; after five immunizations, the mice are killed by cervical dislocation, the mouse spleen is removed by sterile operation, and a single cell suspension of mouse spleen cells is prepared for standby use.

[0065] Step 102, cell fusion: Mouse spleen cells and SP2 / 0 cells were mixed in a centrifuge tube at a ratio of 5:1, and the supernatant was discarded after centrifugation. The centrifuge tube was placed in a 37°C water bath, and gently shaken. Within 1 minute, the fusion agent PEG1500 solution (50%, sterile) was slowly added. The tube was allowed to stand for 1 minute, and then 2 mL of IMEM cell culture medium was added while shaking within 2 minutes, and then 10 mL of IMEM cell culture medium was slowly added. The supernatant was discarded after centrifugation at 800 rpm. IMEM cell culture medium was added to the centrifuge tube, and the 96-well plate was plated. The 96-well plate was placed in a 37°C cell culture incubator for culture, and the growth of the monoclonal clone was observed after 10 days.

[0066] Step 103, Elisa screening of positive supernatants and clones: The REG3A protein was prepared with coating solution at a concentration of 0.5 μg / mL, and 50 μL was plated on a 96-well plate at 2-8°C overnight. The next day, the unbound antigen in the 96-well plate was shaken off, the plate was washed with 200 μL of PBST buffer, patted dry on paper, and 200 mL of BSA blocking solution (PBS buffer containing 1% (w / v) BSA) was added to each well, and incubated at room temperature for 60 min.

[0067] Step 104, dilute and add the primary antibody according to the dilution requirements and methods of the specific experiment, and incubate at room temperature for 60 minutes;

[0068] Step 105, shake off the unbound primary antibody liquid in the plate, wash the plate three times with PBST (washing with a plate washer), and pat dry on paper;

[0069] Step 106, add 50 μL of correspondingly diluted HRP secondary antibody to each well and incubate at 37° C. for 30 min;

[0070] Step 107, shake off the unbound secondary antibody liquid in the plate, wash the plate three times with PBST buffer, pat dry, add 50 μL of TMB colorimetric reagent to each well, react at room temperature for 5 min; add 50 μL of stop solution to each well to stop the reaction; detect the 450 nm OD value with an enzyme reader in the absorption mode, and process the data.

[0071] Step 108: The PCR product is subjected to first-generation DNA sequencing, and the sequencing results are subjected to homology comparison to obtain the sequence information of the target antibody. The target antibody is an anti-REG3A protein antibody having a light chain variable region with an amino acid sequence as shown in SEQ ID NO.1 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.5.

[0072] The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 in the light chain variable region are shown in SEQ ID NOs. 2, 3 and 4, respectively. The amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 in the heavy chain variable region are shown in SEQ ID NOs. 6, 7 and 8, respectively.

[0073] Table 1 Sequence information of amino acid sequences SEQ ID NO.1~8:

[0074]

[0075] Example 2 Detection of affinity between anti-REG3A protein antibody and REG3A protein based on ELISA method:

[0076] In this example, direct ELISA was used to detect the affinity of the anti-REG3A protein antibody and REG3A protein obtained in Example 1 of the present application. The main reagents included: recombinant human REG3A protein (purchased from Beijing Sino Biological Technology Co., Ltd. (China), catalog number 11235-H08H); HRP-labeled goat anti-mouse IgG antibody (purchased from Jackson ImmunoResearch Laboratories, Inc., catalog number 115-035-062).

[0077] The detection process of anti-REG3A protein antibodies and REG3A protein affinity based on ELISA method specifically includes the following steps:

[0078] Step 201, dilute the recombinant human REG3A protein to 0.5 μg / mL with coating solution, add to the wells of the ELISA plate, 50 μL per well, and incubate at (2-8)°C overnight.

[0079] Step 202, plate washing: shake off the excess coating liquid in the plate, add washing solution, 300uL per well, let stand for 1min, remove the washing solution, and wash the plate 3 times. The washing solution is PBST buffer (0.05% by volume Tween-20, pH=7.4).

[0080] Step 203, blocking: add 1% (w / v) BSA blocking solution to each well, 300uL per well, and block at 37°C for 2 hours. The BSA blocking solution is prepared from bovine serum albumin (BSA) and PBS buffer (pH=7.4).

[0081] Step 204, primary antibody incubation: After washing the plate 3 times, add the anti-REG3A protein antibody solution to the wells of the ELISA plate, 50 μL per well, and incubate at 37°C for 1 hour. The preparation process of the anti-REG3A protein antibody solution is as follows: dilute the anti-REG3A protein antibody to a concentration of 1000 ng / mL anti-REG3A protein antibody solution (numbered S1) in an EP tube with a diluent, and prepare 8 EP tubes containing 100 μL of diluent (numbered S2 to S8 in sequence) for gradient dilution, take 50 μL of the anti-REG3A protein antibody solution from the upper EP tube each time and add it to the lower EP tube, and mix gently. The gradient dilution concentrations of the anti-REG3A protein antibody are: 1000, 333.3, 111.11, 37.04, 12.35, 4.12, 1.23, 0.41, 0.137 ng / mL.

[0082] Step 205, secondary antibody incubation: After washing the plate 3 times, add HRP-labeled goat anti-mouse IgG antibody solution to the wells of the ELISA plate, 50 μL per well, and incubate at 37°C for 1 hour. The HRP-labeled goat anti-mouse IgG antibody solution is prepared by diluting the HRP-labeled goat anti-mouse IgG antibody with a diluent to obtain an HRP-labeled goat anti-mouse IgG antibody solution at a dilution ratio of 1:10000 to 1:20000 (mass volume ratio w / v).

[0083] Step 206, color development: After washing the plate three times, add TMB color developer to the wells of the ELISA plate, 50 μL per well, and react at room temperature in the dark for 15 min.

[0084] Step 207, stop: add stop solution (1M HCl aqueous solution) to the wells of the ELISA plate, 50 μL per well, and gently shake the ELISA plate until the color is evenly developed.

[0085] Step 208: Use an ELISA reader in the absorption mode to detect the OD value of each well in the ELISA plate at a wavelength of 450 nm. The concentration of the anti-REG3A protein antibody is used as the horizontal axis and the OD is used as the horizontal axis. 450nm As the vertical axis, we can draw Figure 1 Affinity curves of anti-REG3A monoclonal antibodies to REG3A protein are shown.

[0086] Depend on Figure 1It can be seen that the binding between the anti-REG3A monoclonal antibody of the present application and the REG3A protein has high specificity and high affinity, and can achieve quantitative detection of REG3A.

[0087] Example 3 Detection kit for detecting REG3A protein based on immunofluorescence method:

[0088] In this example, a detection kit for detecting human REG3A protein was established based on the immunofluorescence method. The kit mainly includes (except for the sources specifically indicated, all reagents are self-produced): microspheres coupled with capture antibodies (Cat. No.: MC10053-1, Luminex), coupled capture antibodies (Cat. No.: 11235-R019, Sino Biological), human REG3A detection antibodies, PE-labeled secondary antibodies (Cat. No.: 115-115-164, Jackson ImmunoResearch), sample diluent, washing solution, and REG3A protein standard (Cat. No. 11235-H08H, Beijing Sino Biological Technology Co., Ltd.).

[0089] The working principle of this kit for detecting human REG3A protein is based on the double antibody sandwich method: first, add the microspheres coupled with the capture antibody to a black 96-well plate; then, add the human REG3A standard or sample to the microwells of the black 96-well plate, and the human REG3A protein in the standard or sample binds to the capture antibody on the microsphere; then, add the REG3A detection antibody to the microwells of the black 96-well plate, so that the antibody binds to the REG3A protein, and wash away the unbound detection antibody; then, add the PE-labeled secondary antibody to the microwells of the black 96-well plate, and wash away the unbound PE-labeled secondary antibody; finally, use a multifunctional flow cytometer (Luminex 200) to measure the fluorescence value, and calculate the concentration of human REG3A protein in the sample by drawing a standard curve.

[0090] Among them, the working process of this detection kit for detecting human REG3A protein based on the double antibody sandwich method specifically includes the following steps:

[0091] Step 301, preparation of standard: prepare the human REG3A calibrator with sample diluent to prepare a standard curve S1 with a concentration of 12000 pg / mL, prepare 6 EP tubes containing 100 μL of sample diluent and number them S2 to S7 respectively, perform gradient dilution, take 50 μL from the previous level calibrator each time and add it to the next level EP tube, and mix gently. (The concentrations of the standard curves used are: 12000, 6000, 2000, 666.67, 222.22, 74.07, 24.69 pg / mL).

[0092] Step 302, sample solution to be tested: dilute the blood sample 10 times with a sample diluent; specifically: 10 μL of blood sample, 90 μL of sample diluent.

[0093] Step 303: First add 50 μL of standard / sample to each well, then add 50 μL of microbead working solution (about 2000 microbeads per well), tap to mix, seal the plate with a sealing film, and incubate at room temperature at 400 rpm for 1 h.

[0094] Step 304: After the incubation is completed, place the 96-well plate in a magnetic stand for separation for 1-2 minutes. While the 96-well plate is still in the magnetic stand, remove the supernatant and add 150 μL of washing solution. Take out the 96-well plate from the magnetic stand and gently shake for 1 minute. Place the 96-well plate in a magnetic stand again for separation for 1-2 minutes, remove the supernatant and add 150 μL of washing solution, and repeat the washing 3 to 4 times.

[0095] Step 305: Add 50 μL of detection antibody to each well and incubate at 400 rpm for 1 h at room temperature in the dark.

[0096] Step 306: After the incubation is completed, place the 96-well plate in a magnetic stand for separation for 1-2 minutes. While the 96-well plate is still in the magnetic stand, remove the supernatant and add 100 μL of washing solution. Remove the 96-well plate from the magnetic stand and gently shake for 1 minute. Repeat the washing 3-4 times.

[0097] Step 307, add 50 μL of PE-labeled secondary antibody to each well, and incubate at room temperature at 400 rpm for 1 h in the dark.

[0098] Step 308: After the incubation is completed, place the 96-well plate in the magnetic stand for separation for 1-2 minutes. While the 96-well plate is still in the magnetic stand, remove the supernatant and add 100 μL of washing solution. Remove the 96-well plate from the magnetic stand and gently shake for 1 minute. Repeat the washing 3 times. Add 100 μL of washing solution to each well and test on the machine.

[0099] Among them, the standard curve for detecting REG3A protein is as follows Figure 2 shown.

[0100] The kit of this example detects the concentration of human REG3A in blood samples numbered 1# to 8#, and the detection results are shown in Table 2.

[0101] Table 2 Detection results of REG3A concentration in blood samples numbered 1# to 8#:

[0102]

[0103] It can be seen from Table 2 that, using the anti-REG3A monoclonal antibody of the present application, the detection kit of the present application is suitable for blood sample detection or sample detection from other tissue sources, and has clinical application prospects.

[0104] The above is a detailed introduction to an anti-REG3A protein antibody or an antigen-binding fragment thereof and its use provided by the present application. This article uses specific examples to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. An antibody or antigen-binding fragment thereof against REG3A protein, characterized in that: The antibody or antigen-binding fragment thereof comprises light chain complementary determining regions LCDR1, LCDR2 and LCDR3 and heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3, wherein: LCDR1 is selected from the amino acid sequence shown in SEQ ID NO.2; LCDR2 is selected from the amino acid sequence shown in SEQ ID NO.3; LCDR3 is selected from the amino acid sequence shown in SEQ ID NO.4; HCDR1 is selected from the amino acid sequence shown in SEQ ID NO.6; HCDR2 is selected from the amino acid sequence shown in SEQ ID NO.7; HCDR3 is selected from the amino acid sequence shown in SEQ ID NO.

8.

2. The anti-REG3A protein antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region; wherein: The light chain variable region comprises the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions or any combination thereof in an amino acid sequence fragment relative to SEQ ID NO.1 and excluding SEQ ID NOs.2, 3 and 4, or consists of the amino acid sequence shown in SEQ ID NO.1; And / or, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.5, or an amino acid sequence containing one or more amino acid substitutions, deletions or insertions or any combination thereof in an amino acid sequence fragment relative to SEQ ID NO.5 but excluding SEQ ID NOs.6, 7 and 8, or consists of the amino acid sequence shown in SEQ ID NO.

5.

3. The anti-REG3A protein antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody is selected from any form of a monoclonal antibody, a single-chain antibody, a bifunctional antibody, a fully or partially humanized antibody, or a chimeric antibody; And / or, the antigen binding fragment is selected from Fab, Fab', Fab'-SH, (Fab')2, Fv, scFv, BsFv, dsFv or (dsFv)2 fragment.

4. The anti-REG3A protein antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region of human or murine origin.

5. The anti-REG3A protein antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region of human or murine origin.

6. The anti-REG3A protein antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a kappa or lambda type light chain constant region.

7. The anti-REG3A protein antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody is a monoclonal antibody.

8. The anti-REG3A protein antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The antibodies are murine, chimeric or humanized monoclonal antibodies.

9. The anti-REG3A protein antibody or antigen-binding fragment thereof according to claim 7, characterized in that: The heavy chain constant region of the monoclonal antibody is of IgG1, IgG2, IgG3 or IgG4 subtype.

10. A biomaterial, characterized in that: The biological material comprises: (I) a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; and / or (II), a vector comprising the nucleic acid molecule described in (I); and / or, (III) A host cell comprising the nucleic acid molecule described in (I) and / or the vector described in (II), or the host cell is transformed or transfected with the nucleic acid molecule described in (I) and / or the vector described in (II).

11. The biomaterial according to claim 10, characterized in that The host cell is selected from a prokaryotic cell or a eukaryotic cell.

12. The biomaterial according to claim 10 or 11, characterized in that: The host cell is selected from yeast cells, CHO cells or 293 cells.

13. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and a label conjugated to the antibody or antigen-binding fragment thereof.

14. The antibody conjugate according to claim 13, characterized in that: The label is selected from at least one of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and / or the biomaterial according to any one of claims 10 to 12, and / or the antibody conjugate according to any one of claims 13 to 14 in the preparation of a product for detecting REG3A protein.

16. A detection reagent for detecting REG3A protein, characterized in that: The detection reagent comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and / or the biomaterial according to any one of claims 10 to 12, and / or the antibody conjugate according to any one of claims 13 to 14.

17. A detection kit for detecting REG3A protein, characterized in that: The detection kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and / or the biomaterial according to any one of claims 10 to 12, and / or the antibody conjugate according to any one of claims 13 to 14.

18. The detection kit according to claim 17, characterized in that The detection kit is selected from an enzyme-linked immunosorbent assay kit, a fluorescent immunoassay kit or a chemiluminescent immunoassay kit.

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