Anti-HbA1c antibody or functional fragment thereof and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-HbA1c or a functional fragment thereof and application of the anti-HbA1c or the functional fragment thereof. The invention not only provides a high-affinity monoclonal antibody (wild type) combined with HbA1c, but also performs site-directed mutagenesis on the wild type, verifies the affinity of each mutation and related application thereof, and provides reference for research in the field of immunodetection of HbA1c.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an anti-HbA1c antibody or a functional fragment thereof and applications thereof. Background Art

[0002] Glycated hemoglobin A1c (HbA1c) is an important biomarker for the diagnosis, monitoring and efficacy observation of diabetes, especially for the evaluation of progressive retinopathy[1]. Normal people have three types of hemoglobin: HbA, HbF, and HbA2, while adult red blood cells mainly contain HbA. When hemoglobin is separated by chromatography, three sugar-containing components can be eluted: HbA1a, HbA1b, and HbA1c, collectively known as glycated hemoglobin. HbA1c is glucose-containing hemoglobin, and the other two are glycated hemoglobin formed by other sugars[2]. Glycated hemoglobin is formed by the combination of HbA and glucose during metabolism, so it can accurately reflect the glucose level in the blood. Glycated hemoglobin is the product of the combination of hemoglobin in red blood cells in human blood and blood sugar. The combination of blood sugar and hemoglobin to form glycated hemoglobin is an irreversible reaction, which is proportional to the blood sugar concentration and remains for about 120 days, so the blood sugar concentration before 120 days can be observed. The glycated hemoglobin test can usually reflect the patient's blood sugar control in the past 8 to 12 weeks. The immunoassay of glycated hemoglobin is one of the commonly used clinical methods. Its principle is to use specific antibodies that can recognize several glycosylated amino acids at the amino terminal of the β-chain of glycated hemoglobin to react with HbA1c to form an antigen-antibody complex for detection. This type of method is fast and simple and can be detected on automated instruments.

[0003] [1] Li Jingjing, Zhao Haibo, Liang Liang, et al. Research and prospects of human glycosylated hemoglobin HbA1c standard materials[J]. Acta Metrologica Sinica, 2023, 44(3): 479-487.

[0004] [2] Nanjing Liding Medical Technology Co., Ltd. A polysaccharide hemoglobin monoclonal antibody and preparation method. 2023, CN 116284370A. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-HbA1c antibody or a functional fragment thereof, to achieve specific binding with the HbA1c protein, and ultimately to be applied to an HbA1c detection kit.

[0006] The details are as follows:

[0007] The anti-HbA1c antibody or its functional fragment has the following complementarity determining region:

[0008] CDR-VH1: S-X1-G-X2-H, where X1 is F or D, and X2 is M;

[0009] CDR-VH2: Y-X1-TGGN-X2-NIFYAD-X3-VKG, wherein X1 is I or N, X2 is V or T, and X3 is T or S;

[0010] CDR-VH3: G-X1-DYRFDVGK-X2-VVY, where X1 is E or F, and X2 is G;

[0011] CDR-VL1: K-X1-SQSLVHNNGN-X2-YLE, where X1 is S or T, and X2 is T or S;

[0012] CDR-VL2: K-X1-SNR-X2-S, where X1 is V or H, and X2 is F or E;

[0013] and CDR-VL3: F-X1-GSHVP-X2-T, wherein X1 is Q and X2 is L or W.

[0014] Furthermore, X2 of CDR-VH1 of the complementary determining region is M, X1 of CDR-VH2 is I, X2 of CDR-VH3 is G, X1 of CDR-VL1 is S, X2 of CDR-VL2 is F, and X1 of CDR-VL3 is Q.

[0015] The CDR segment division described in the present invention adopts the Kabat algorithm.

[0016] "CDR" is used herein to refer to a "complementarity determining region" within an antibody variable sequence. The variable regions of the heavy and light chains each have three CDRs, starting from the N-terminus of the heavy or light chain.

[0017] The antigen binding site may include six CDRs (CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 and CDR-VL3 in the present invention). A polypeptide comprising a single CDR (e.g., CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 or CDR-VL3) may be referred to as a "molecular recognition unit". Crystallographic analysis of antigen-antibody complexes has shown that the amino acid residues of CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Therefore, the molecular recognition unit may be primarily responsible for the specificity of the antigen binding site. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0018] The inventors of the present invention have found that when the mutation sites in each complementary determining region are the amino acid residues, the antibody has a better affinity for the HbA1c protein.

[0019] In the CDR-VH1, X1 is F;

[0020] Preferably, X1 in the CDR-VH1 is D;

[0021] Preferably, X2 in the CDR-VH1 is M;

[0022] Preferably, in the CDR-VH2, X1 is I;

[0023] Preferably, in the CDR-VH2, X1 is N;

[0024] Preferably, X2 in the CDR-VH2 is V;

[0025] Preferably, X2 in the CDR-VH2 is T;

[0026] Preferably, X3 in the CDR-VH2 is T;

[0027] Preferably, X3 in the CDR-VH2 is S;

[0028] Preferably, in the CDR-VH3, X1 is E;

[0029] Preferably, in the CDR-VH3, X1 is F;

[0030] Preferably, X2 in the CDR-VH3 is G;

[0031] Preferably, in the CDR-VL1, X1 is S;

[0032] Preferably, X1 in the CDR-VL1 is T;

[0033] Preferably, X2 in the CDR-VL1 is T;

[0034] Preferably, X2 in the CDR-VL1 is S;

[0035] Preferably, in the CDR-VL2, X1 is V;

[0036] Preferably, in the CDR-VL2, X1 is H;

[0037] Preferably, X2 in the CDR-VL2 is F;

[0038] Preferably, X2 in the CDR-VL2 is E;

[0039] Preferably, in the CDR-VL3, X1 is Q;

[0040] Preferably, in the CDR-VL3, X2 is L;

[0041] Preferably, X2 in the CDR-VL3 is W.

[0042] Preferably, each complementary determining region is selected from any one of the following mutation combinations:

[0043]

[0044]

[0045] Further, the antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H are selected from SEQ ID NOs: 1-4 in sequence; the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NOs: 5-8 in sequence.

[0046] Furthermore, the antibody also comprises a constant region.

[0047] Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. Preferably, the species origin of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans.

[0048] Preferably, the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0049] Further, the antibody is a labeled antibody;

[0050] Furthermore, the labeling substances for labeling antibodies include but are not limited to latex microspheres, biotin, fluorescent dyes, enzymes that catalyze the color development of substrates, radioactive isotopes, chemiluminescent reagents and nanoparticle labels.

[0051] The "labeled antibody" of the present invention is labeled with a detectable marker.

[0052] The term "antibody" includes various forms of antibody structures, including but not limited to complete antibodies and antibody fragments. The antibody according to the present invention is preferably a goat, sheep, mouse, rabbit or rat antibody, a chimeric antibody or a further genetically engineered antibody, as long as the characteristic properties according to the present invention are retained. "Antibody fragment" comprises a portion of a full-length antibody, preferably its variable domain, or at least its antigen binding site. Examples of antibody fragments include diabodies, single-chain antibody molecules and multispecific antibodies formed by antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)2; diabodies; and multispecific antibodies formed by antibody fragments.

[0053] On the other hand, the present invention discloses a detection reagent or a kit, wherein the reagent or the kit comprises the antibody or a functional fragment thereof or the labeled antibody.

[0054] Furthermore, the sample detected by the kit is selected from blood cells in whole blood.

[0055] On the other hand, the present invention discloses the use of the antibody or its functional fragment, the labeled antibody, or the reagent or kit in the preparation of a glycated hemoglobin detection product.

[0056] In another aspect, the present invention discloses a method for detecting progesterone protein in a sample, the method comprising: a) contacting the glycated hemoglobin antigen in the test sample with the antibody or its functional fragment, the labeled antibody, or the reagent or kit to form an immune complex under conditions sufficient for an antibody / antigen binding reaction to occur; and

[0057] b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample;

[0058] Optionally, in step a), the immune complex further comprises a second antibody, which binds to the antibody or antigen-binding fragment;

[0059] Optionally, in step a), the immune complex further comprises a second antibody, and the second antibody binds to the glycated hemoglobin antigen.

[0060] Beneficial effects:

[0061] The present invention not only provides a monoclonal antibody (wild type) with high affinity for binding to HbA1c, but also makes a variety of mutations with excellent performance to the wild type, so that it can be applied to the detection of HbA1c protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1: Comparison of the test results of the reagent of the present invention and the clinical results. DETAILED DESCRIPTION

[0063] Specifically, in order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein. Unless otherwise indicated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0065] As used herein, the terms "including," "comprising," "having," "may," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0066] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (comprising recombinant technology), microbiology, biochemistry and immunology, which are within the capabilities of those skilled in the art. This technology is fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, 1984); "Animal Cell Culture" (RI Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (DM Weir and CC Black, 1989). ell, ed.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0067] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0068] Example 1 Preparation of anti-glycosylated hemoglobin (HbA1c) monoclonal antibody

[0069] A. Mouse immunization and tail blood titer detection

[0070] Select 5 6-8 week old SPF female BALB / c mice, mix and emulsify Freund's complete adjuvant and HbA1c protein at a concentration of 2 mg / ml in equal volumes. Use the emulsified antigen to immunize 6-8 week old SPF female BALB / c mice, and inject 40 μg of antigen protein into each mouse by subcutaneous injection in the foot or back. Two weeks after the first immunization, mix and emulsify the antigen protein with Freund's incomplete adjuvant, and inject 40 μg of antigen protein into each mouse by subcutaneous injection in the foot or back. Two weeks later, collect blood through the tail vein, collect the supernatant by centrifugation, and test the serum titer by ELISA. Immunize and test the serum titer every two weeks. After two immunizations, the serum titer is as high as 2.0 or above after a million-fold dilution. Screening serum titer 10 6 Lymph was taken from the above mice to separate lymphocytes for cell fusion.

[0071] B. Cell fusion, positive hybridoma cell screening and subcloning

[0072] Lymphocytes from isolated immunized mice were fused with cultured SP2 / 0 cells by PEG1500 or electrofusion. The fused cells were cultured in HAT-1640 medium containing 20% ​​FBS serum for screening. The culture medium was replaced after one week, and the culture supernatant was taken for positive clone screening after another 4 days of culture. HbA1c was used for positive well screening. Wells with a higher ratio of ELISA positive value to cell number were selected for multiple subcloning. ELISA plates were coated with HbA1c protein. The culture supernatant of the subclones was taken to screen monoclones that could show affinity under antigen coating conditions, and the monoclonal hybridoma cells with the highest affinity were selected from them. Finally, a hybridoma cell line with a high antibody titer that could secrete HbA1c monoclonal antibodies was obtained, named anti-HbA1c, which had good stability.

[0073] D. Production and purification of monoclonal antibodies

[0074] Two groups of 6-8 week old BALB / c mice were selected and intraperitoneally injected with 500 μL paraffin oil to suppress the immune response of mice. One week after the injection, 0.5 ml of anti-HbA1c cells were injected into the peritoneal cavity of the mice. The number of cells was about 1×10 6 After two weeks, ascites collection was started. The collected ascites was precipitated with ammonium sulfate and affinity purified with protein A to obtain the target antibody, which was named anti-HbA1c antibody.

[0075] E. Identification of monoclonal antibody subtypes and gene sequence cloning

[0076] The SBA Clonotyping System-HRP kit from Southern Biothech was used to identify the subtypes of the heavy and light chains of the monoclonal antibody according to the instructions. The specific operations were as follows:

[0077] 1. Dilute the capture antibody to 1 μg / mL with coating solution (0.05M carbonate and bicarbonate buffer at pH 9.5), add 100 μL / well to the ELISA plate, and coat overnight at 4°C. Wash the plate three times with PBS buffer (washing solution) containing 0.05% Tween-20.

[0078] 2. Dilute the culture supernatant of the hybridoma cells to be tested with diluent (1% BSA, 0.1% PBST) at a ratio of 1:1, add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 minutes. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) at a ratio of 1:3000 with diluent.

[0079] 3. After washing the plate 3 times with the plate washing solution, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate 3 times again, add the color developing solution. After about 5 minutes (depending on the strength of the reaction), add 2M sulfuric acid to terminate the reaction and read the OD450 absorbance value. After identification, the heavy chain subtype of the anti-HbA1c antibody is IgG1, and the light chain is Kappa.

[0080] According to the antibody subtype results, the antibody gene sequence was cloned using a RACE-based method. Hybridoma cells with good growth were collected, and total RNA of hybridoma cells was obtained using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the operating method of Takara's SMARTer RACE manual, and the full-length sequence of the target antibody was amplified.

[0081] Example 2 Verification of Antibody Performance

[0082] 1. ELISA affinity test

[0083] Dilute the goat anti-mouse to 1ug / mL using coating solution (0.05M carbonate and bicarbonate buffer at pH = 9.5), add 100μL per well to a 96-well ELISA plate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20. Dilute the antibody to 1ug / mL using diluent (1% BSA, 0.1% PBST), add 100μL per well to a 96-well ELISA plate, and incubate at 37°C for 30 minutes. Dilute biotin-labeled HbA1c to 100ng / mL with diluent, take out the ELISA plate incubated at 37℃, wash the plate three times, add 150μL of the prepared antibody canine C-reactive protein diluent to the 96-well ELISA plate in row A, add 100μL of the diluent to each well of the 96-well ELISA plate in rows B to H, take 50uL of the antibody diluent in the wells of row A, and dilute it 3 times from row B, and incubate at 37℃ for 30 minutes. Dilute the avidin labeled with horseradish peroxidase at 1:5000 with diluent, take out the ELISA plate and wash the plate three times, add 100μL to each well of the 96-well ELISA plate, and incubate at 37℃ for 30 minutes. Take out the ELISA plate and wash it three times, add the color developing solution, color it at room temperature for 3 minutes, add 0.5M sulfuric acid to terminate the reaction, and read the OD450 value on the plate reader.

[0084] The heavy chain variable region of the anti-HbA1c antibody in Example 1 is shown in SEQ ID NO: 9, wherein the amino acid sequences of the complementary determining regions on the heavy chain variable region are as follows:

[0085] CDR-VH1: SD(X1)-GM(X2)-H;

[0086] CDR-VH2: YN(X1)-TGGNT(X2)-NIFYADS(X3)-VKG;

[0087] CDR-VH3: GF(X1)-DYRFDVGKG(X2)-VVY;

[0088] The light chain variable region is shown in SEQ ID NO: 10, wherein the amino acid sequences of the complementary determining regions on the light chain variable region are as follows:

[0089] CDR-VL1:KT(X1)-SQSLVHNNNGNS(X2)-YLE;

[0090] CDR-VL2: KH(X1)-SNRE(X2)-S;

[0091] and CDR-VL3: FQ(X1)-GSHVPW(X2)-T.

[0092] Based on the antibody anti-HbA1c, mutations were performed on sites related to antibody activity in the complementary determining region, among which X1, X2, and X3 were all mutation sites.

[0093] Table 1 Mutation sites related to antibody activity

[0094]

[0095] Table 2 Antibody activity analysis data

[0096]

[0097] As can be seen from the table above, mutation 1 has the best activity effect. Therefore, mutation 1 is used as the backbone sequence to screen other mutation sites with better affinity. Some results are as follows:

[0098] Table 3 Mutation sites related to antibody affinity

[0099]

[0100]

[0101] HbA1c protein 100ng / ml, measure the absorbance value of each mutant OD450, the results are as follows:

[0102] Table 4 Affinity testing of antibody mutations

[0103]

[0104]

[0105] It can be seen that the affinity of each mutation is higher.

[0106] 2. Stability determination

[0107] The concentration of the antibody of the present invention is 1 ug / mL, and the concentration of the HbA1c protein is 100 ng / mL.

[0108] The antibodies were diluted in a defined buffer (PBS, 0.05% ProClin TM 300) was heat accelerated at 37℃ for 7 days, and the accelerated antibodies were evaluated by SDS-PAGE and ELISA indirect method, and compared with 4℃ to identify the long-term stability of the antibodies. In addition, the test results were displayed as the deviation between the 4℃ value and the accelerated value by repeated freezing and thawing at -20℃ for 5 times. The measurement results are as follows:

[0109] Table 5 Stability study

[0110]

[0111]

[0112] It can be seen from the above that mutations 1 to 1-30 can be stably stored at 4°C and remain stable at 37°C for 0.5 months (the slight decrease in reactivity does not affect the performance of the reagent).

[0113] Example 3 Antibody Application Research

[0114] From the above examples, it can be seen that the performance of mutations 1-30 is comparable. Due to the limitations of the examples, it is impossible to exhaust the effects of all mutations. This example randomly selects mutations 1-20 for verification. In order to detect the effect of the obtained antibodies in biochemical immunoturbidimetric experiments to detect clinical samples, this example applies 1-20 to a biochemical kit. The biochemical detection kit can be purchased from the market.

[0115] The assay reagents include working solution 1 and working solution 2:

[0116] The working solution 1 includes: reagent R1, which can be used directly after opening and can be stable for 30 days when stored at low temperature (4° C.).

[0117] The working solution 2 includes: reagent R2 and reagent R3 are mixed in a ratio of 19:1 to form working solution 2 for use. (Note: Add R3 to R2 and turn it upside down to mix it thoroughly to prevent foaming.) The working solution can be stored at low temperature and away from light (4°C) and is stable for 15 days.

[0118] Clinical samples: 20 random clinical samples can be collected from various hospitals.

[0119] Each component was placed into the fully automatic biochemical analyzer EXC800 (from Zhongyuan Huiji Biotechnology Co., Ltd.), and the HbA1c project was selected for the experiment.

[0120] Table 6 Clinical sample test results

[0121] Sample No. Clinical standard results The test results of this reagent 1 8.7 8.8 2 4.3 4.6 3 4.7 4.9 4 4.9 5.1 5 5 5.2 6 5 5.3 7 5.1 5.1 8 5.1 5.3 9 5.2 5.4 10 5.3 5.5 11 5.3 5.4 12 5.3 5.4 13 5.7 5.8 14 6 6.1 15 6.1 6.3 16 6.3 6.5 17 7.2 7.3 18 7.6 7.7 19 7.8 7.9 20 9.9 9.8

[0122] The results are shown in Table 6 and Figure 1 As shown, the detection results of the reagent of the present invention are highly consistent with the clinical results, indicating that the antibody of the present invention has excellent clinical detection performance.

[0123] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. An anti-HbA1c antibody or a functional fragment thereof, characterized in that: The antibody or its functional fragment comprises the following complementarity determining region: CDR-VH1: S-X1-G-X2-H, where X1 is F or D, and X2 is M; CDR-VH2: Y-X1-TGGN-X2-NIFYAD-X3-VKG, wherein X1 is I or N, X2 is V or T, and X3 is T or S; CDR-VH3: G-X1-DYRFDVGK-X2-VVY, wherein X1 is E or F, and X2 is G; CDR-VL1: K-X1-SQSLVHNNGN-X2-YLE, wherein X1 is S or T, and X2 is T or S; CDR-VL2: K-X1-SNR-X2-S, where X1 is V or H, and X2 is F or E; and CDR-VL3: F-X1-GSHVP-X2-T, wherein X1 is Q and X2 is L or W.

2. The anti-HbA1c antibody or a functional fragment thereof according to claim 1, wherein: X2 of CDR-VH1 of the complementary determining region is M, X1 of CDR-VH2 is I, X2 of CDR-VH3 is G, X1 of CDR-VL1 is S, X2 of CDR-VL2 is F, and X1 of CDR-VL3 is Q.

3. The anti-HbA1c antibody or a functional fragment thereof according to claim 1, wherein: In the CDR-VH1, X1 is F; Preferably, X1 in the CDR-VH1 is D; Preferably, X2 in the CDR-VH1 is M; Preferably, in the CDR-VH2, X1 is I; Preferably, in the CDR-VH2, X1 is N; Preferably, X2 in the CDR-VH2 is V; Preferably, X2 in the CDR-VH2 is T; Preferably, X3 in the CDR-VH2 is T; Preferably, X3 in the CDR-VH2 is S; Preferably, in the CDR-VH3, X1 is E; Preferably, in the CDR-VH3, X1 is F; Preferably, X2 in the CDR-VH3 is G; Preferably, in the CDR-VL1, X1 is S; Preferably, X1 in the CDR-VL1 is T; Preferably, X2 in the CDR-VL1 is T; Preferably, X2 in the CDR-VL1 is S; Preferably, in the CDR-VL2, X1 is V; Preferably, in the CDR-VL2, X1 is H; Preferably, X2 in the CDR-VL2 is F; Preferably, X2 in the CDR-VL2 is E; Preferably, in the CDR-VL3, X1 is Q; Preferably, in the CDR-VL3, X2 is L; Preferably, X2 in the CDR-VL3 is W.

4. The anti-HbA1c antibody or a functional fragment thereof according to any one of claims 1 to 3, characterized in that: Each complementary determining region is selected from any one of the following mutation combinations:

5. The anti-HbA1c antibody or a functional fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody includes light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H are selected from SEQ ID NO: 1-4 in sequence; the light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NO: 5-8 in sequence.

6. The anti-HbA1c antibody or a functional fragment thereof according to claim 5, wherein: The antibody further comprises a constant region; Preferably, the constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; preferably, the species of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans; preferably, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

7. The anti-HbA1c antibody or a functional fragment thereof according to any one of claims 1 to 6, characterized in that: The antibody is a labeled antibody; Preferably, the labeling substances for labeling the antibody include, but are not limited to, latex microspheres, biotin, fluorescent dyes, enzymes that catalyze the color development of substrates, radioactive isotopes, chemiluminescent reagents, and nanoparticle labels.

8. A detection reagent or kit, characterized in that: The reagent or kit comprises the antibody or functional fragment thereof according to any one of claims 1 to 6 or the labeled antibody according to claim 7.

9. Use of the antibody or functional fragment thereof according to any one of claims 1 to 6, the labeled antibody according to claim 7, or the reagent or kit according to claim 8 in the preparation of a glycated hemoglobin detection product.

10. A method for detecting progesterone protein in a sample, characterized in that: The method comprises: a) contacting the glycated hemoglobin antigen in the test sample with the antibody or functional fragment thereof according to any one of claims 1 to 6, the labeled antibody according to claim 7, or the reagent or kit according to claim 8 under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample; Optionally, in step a), the immune complex further comprises a second antibody, which binds to the antibody or antigen-binding fragment; Optionally, in step a), the immune complex further comprises a second antibody, and the second antibody binds to the glycated hemoglobin antigen.

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