Anti-glycated hemoglobin hba1c antibody and use thereof

Abstract

The present disclosure relates to the field of antibodies. Disclosed are an anti-glycated hemoglobin HbA1c antibody and a use thereof. The anti-glycated hemoglobin HbA1c antibody disclosed in the present disclosure comprises a heavy chain complementary determining region and a light chain complementary determining region. The antibody provides an important raw material source for detection of glycated hemoglobin HbA1c, and has good affinity or activity.

Description

Antibody against glycated hemoglobin HbA1c and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese patent application number 202311701674.X filed with the Chinese Patent Office on December 11, 2023, entitled “An Anti-Glycated Hemoglobin HbA1c Antibody and Its Application,” the entire contents of which are incorporated by reference into this disclosure. Technical Field

[0003] The present disclosure relates to the field of antibody technology, and in particular to an antibody against glycated hemoglobin HbA1c and its application. Background Art

[0004] The global prevalence of diabetes is very high, and it is a global public health problem that seriously threatens human health. Its prevention and treatment has always been a hot topic in medical research. Traditional diagnostic and treatment tests use fasting blood sugar, postprandial blood sugar, and oral glucose tolerance tests, but blood sugar parameters only represent the instantaneous blood sugar level when blood is drawn. Glycated hemoglobin (GHb) is the gold standard for reflecting long-term blood sugar levels and is also an important indicator for monitoring diabetes treatment. Due to the different components it binds to, GHb is divided into HbA1a (bound to phospho-glucose), HbA1b (bound to fructose), and HbA1c (bound to glucose). HbA1c is the recognized gold standard for blood sugar control in diabetes management and is also an effective indicator for evaluating diabetes treatment plans. Currently, the World Health Organization and diabetes associations in many countries have used HbA1c as an independent diagnostic indicator for diabetes.

[0005] HbA1c is the product of the slow, continuous, and irreversible non-enzymatic glycation reaction between hemoglobin (Hb) and glucose within red blood cells in human blood, and is directly proportional to blood glucose concentration. Since the life cycle of red blood cells in the human body is generally 120 days, the HbA1c content in the blood remains relatively unchanged before the red blood cells die. Therefore, the HbA1c level can stably and reliably reflect the average blood glucose level within 120 days prior to the test. It is not significantly affected by factors such as the time of blood draw, fasting status, and insulin use, and is considered the "gold standard" for diabetes treatment monitoring. Combined with the characteristics of hemoglobin, we can see that the detection of glycated hemoglobin HbA1c is of great significance for the screening, monitoring, and treatment of diabetes.

[0006] Currently, the main methods for detecting glycated hemoglobin (HbA1c) are colloidal gold immunochromatography and fluorescence immunochromatography. Both are immunological assays based on the specific reaction between antibodies and antigens, and utilize labeling substances (such as colloidal gold or fluorescence) to amplify and display the detected signal. Similar immunological assays include biochemical immunoturbidimetry, radioimmunoassay, and chemiluminescence.

[0007] The above immunological detection methods all require antibodies against glycated hemoglobin HbA1c. Therefore, there is a strong demand in the art for anti-glycated hemoglobin HbA1c antibodies with good performance. Summary of the Invention

[0008] The present application provides an anti-glycated hemoglobin HbA1c antibody, which provides an important source of raw materials for the detection of glycated hemoglobin HbA1c and has good activity or affinity.

[0009] To achieve the above objectives, according to one aspect of the present disclosure, an anti-glycated hemoglobin HbA1c antibody is provided, wherein the antibody comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19.

[0010] To achieve the above object, according to a second aspect of the present disclosure, an anti-glycated hemoglobin HbA1c antibody is provided, wherein the antibody comprises the following complementary determining regions:

[0011] HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0012] HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0013] HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3;

[0014] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0015] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 5;

[0016] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0017] To achieve the above objectives, according to the third aspect of the present disclosure, an anti-glycated hemoglobin HbA1c antibody is provided, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19.

[0018] To achieve the above objectives, according to the fourth aspect of the present disclosure, an anti-glycated hemoglobin HbA1c antibody is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 18; the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0019] In order to achieve the above object, according to a fifth aspect of the present disclosure, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody.

[0020] In order to achieve the above-mentioned object, according to the sixth aspect of the present disclosure, a reagent or a kit is provided, wherein the reagent or the kit comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0021] In order to achieve the above objectives, according to the seventh aspect of the present disclosure, there is provided a use of the above-mentioned antibody or antibody conjugate in detecting glycated hemoglobin HbA1c, diagnosing whether a subject has diabetes, or preparing a product for detecting glycated hemoglobin HbA1c or preparing a product for diagnosing whether a subject has diabetes.

[0022] To achieve the above-mentioned objectives, according to the eighth aspect of the present disclosure, a method for diagnosing whether a subject has diabetes is provided, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with an in vitro sample to be detected under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; and b) detecting the presence of the immune complex.

[0023] To achieve the above objectives, according to the ninth aspect of the present disclosure, a method for detecting glycated hemoglobin HbA1c is provided, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with an in vitro sample to be detected under conditions sufficient for an antibody / antigen binding reaction to occur to form an immune complex; and b) detecting the presence of the immune complex.

[0024] To achieve the above objectives, the present disclosure also provides a nucleic acid, a vector, a cell and a method for preparing the above antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0026] FIG1 shows the results of reducing SDS-PAGE of Anti-HBA1C 30G9 Rmb1.

[0027] Figure 2 is a standard curve diagram of the biochemical platform calibrator detection. DETAILED DESCRIPTION

[0028] In a first aspect, the embodiments of the present disclosure provide an anti-glycated hemoglobin HbA1c antibody, which comprises three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19.

[0029] In the present disclosure, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies or antigen-binding fragments of antibodies, as long as they exhibit the desired binding activity. Antigen-binding fragments of antibodies include any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv, and generally have the same binding specificity as the antibody from which they are derived. It will be readily understood by those skilled in the art based on the contents of the present disclosure that antigen-binding fragments of antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present disclosure, it is easy for those skilled in the art to obtain the antigen-binding fragments of the above-mentioned antibodies.

[0030] Antigen-binding fragments of antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0031] In the present disclosure, the terms "complementarity determining region," "CDR," or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more, or even all, of the primary amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present disclosure, CDRs refer to the hypervariable regions of the heavy and light chains of the antibodies.

[0032] In the present disclosure, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0033] Methods for defining CDRs are well known in the art, and include the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Patent No. 200,255,854,554, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" refers to the definition system described by Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes, but may still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literature vary slightly. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues comprise a specific CDR. It should be noted that CDRs defined by other methods other than those in Table 1 also fall within the scope of protection of the present disclosure.

[0034] Table 1: CDR Definition 1

[0035] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H+number" and amino acid numbers on the light chain represented by "L+number." One of ordinary skill in the art can unambiguously assign this Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0036] 2 "AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0037] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0038] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0039] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0040] According to an embodiment of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

[0041] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0042] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0043] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by an IMGT system.

[0044] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0045] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0046] In some optional embodiments of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0047] In a second aspect, the present disclosure provides an anti-glycated hemoglobin HbA1c antibody, wherein the antibody comprises the following complementary determining regions:

[0048] HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0049] HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0050] HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3.

[0051] LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4.

[0052] LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 5.

[0053] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

[0054] According to an embodiment of the present disclosure, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0055] In the present disclosure, "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region of the antibody heavy chain variable region and the light chain variable region excluding CDR; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0056] In the present disclosure, the heavy chain variable region is obtained by arranging and connecting the following CDRs and FRs with the following numbers in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following CDRs and FRs with the following numbers in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0057] In an alternative embodiment, the antibody of the first aspect or the second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0058] In an alternative embodiment, the HFR1 comprises / is SEQ ID NO: 7 or an amino acid sequence having at least 80% identity thereto;

[0059] The HFR2 comprises / is as SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0060] The HFR3 comprises / is SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0061] The HFR4 comprises / is as SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0062] The LFR1 comprises / is SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

[0063] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0064] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and

[0065] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

[0066] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the anti-glycated hemoglobin HbA1c antibodies provided herein may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0067] In an alternative embodiment, the antibody has a KD < 7.99 × 10 -8 M binds to glycosylated hemoglobin HbA1c with high affinity.

[0068] In an alternative embodiment, the antibody has a KD ≤ 10 -8 M, KD≤10 -9 M, KD≤10 -10M, KD≤10 -11 M or KD≤10 -12 M binds to glycosylated hemoglobin HbA1c with high affinity.

[0069] In an alternative embodiment, the antibody has a KD ≤ 1.17×10 -9 M binds to glycosylated hemoglobin HbA1c with high affinity.

[0070] There are many methods for determining antibody affinity (KD). Based on the detection principle, they can be divided into thermodynamic detection methods, kinetic detection methods, and dynamic equilibrium detection methods. Among them, thermodynamic detection methods such as isothermal titration calorimetry (ITC) are common; kinetic detection methods such as surface plasmon resonance (SPR) and biofilm interferometry (BLI) are common; and dynamic equilibrium detection methods such as enzyme-linked immunosorbent assay (ELISA) are common.

[0071] In alternative embodiments, KD is determined using a kinetic assay; optionally, surface plasmon resonance, for example, by using a kinetic assay such as System of biosensor systems.

[0072] In a third aspect, the embodiments of the present disclosure provide an anti-glycated hemoglobin HbA1c antibody, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19.

[0073] In an optional embodiment, the antibody described in the first, second, and third aspects above further comprises a constant region.

[0074] In alternative embodiments, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0075] In an optional embodiment, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0076] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0077] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0078] In an alternative embodiment, the light chain constant region is selected from a kappa-type or lambda-type light chain constant region.

[0079] In an alternative embodiment, the species origin of the constant region is cow, horse, dairy cow, pig, sheep, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0080] In an alternative embodiment, the species origin of the constant region is mouse.

[0081] In this article, the division of variable and constant region sequences refers to the IMGT division method, see Lefranc, M. and Martinez-Jean C. and Bosc N. or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGHC,IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org.Created:16 / 03 / 2011.Version:17 / 01 / 2020.or Ehrenmann,Patrice Duroux,Chantal Ginestoux,Gene table:house mouse(Mus musculus)IGLC,IMGT Repertoire. the international ImMunoGenetics information http: / / www.imgt.org. Created: 16 / 03 / 2011. Version: 17 / 01 / 2020. Variable regions divided by different methods may differ from the variable region C-terminus or constant region N-terminus divided by IMGT. Variable regions or constant regions divided by other methods known in the art are also within the scope of protection of this disclosure.

[0082] In an optional embodiment, the heavy chain constant region sequence (CH) is shown as SEQ ID NO:15, and the light chain constant region (CL) sequence is shown as SEQ ID NO:16.

[0083] It should be noted that, in other embodiments, the constant region sequence may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned constant region (SEQ ID NO: 15 or 16).

[0084] In an alternative embodiment, the antibody comprises any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv.

[0085] In a fourth aspect, the present disclosure provides an anti-glycated hemoglobin HbA1c antibody, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0086] In a fifth aspect, the present disclosure provides an antibody conjugate comprising the above-mentioned antibody.

[0087] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

[0088] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody.

[0089] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0090] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

[0091] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it falls within the scope of protection of the present disclosure.

[0092] In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0093] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxidase.

[0094] In an optional embodiment, the radioactive isotopes include but are not limited to 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F.

[0095] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0096] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0097] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0098] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0099] In an optional embodiment, the colloidal metal is colloidal gold.

[0100] In an optional embodiment, the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody.

[0101] In an alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0102] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0103] In a sixth aspect, the present disclosure provides a reagent or a kit, which comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0104] As previously mentioned, the antibodies in some embodiments or examples of the present disclosure can effectively bind to glycated hemoglobin HbA1c. Therefore, the reagents or kits containing the glycated hemoglobin HbA1c antibodies can effectively detect glycated hemoglobin HbA1c qualitatively or quantitatively. The reagents or kits provided by the present disclosure can be used, for example, for immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of glycated hemoglobin HbA1c and its antibodies. As previously mentioned, the antibodies in some embodiments or examples of the present disclosure have higher binding activity or affinity for glycated hemoglobin HbA1c. Therefore, the reagents or kits containing the antibodies have higher detection sensitivity or specificity.

[0105] It should be noted that the products disclosed herein include but are not limited to reagents, test kits, test strips or test plates.

[0106] In a seventh aspect, the present disclosure provides a method for detecting glycated hemoglobin HbA1c, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with glycated hemoglobin HbA1c in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur, 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;

[0107] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody.

[0108] In an optional embodiment, the immune complex further comprises a second antibody, which binds to glycated hemoglobin HbA1c.

[0109] In an eighth aspect, the present disclosure provides a method for diagnosing whether a subject has diabetes, comprising: a) contacting the above-mentioned antibody, antibody conjugate, reagent or kit with glycated hemoglobin HbA1c in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to occur, 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;

[0110] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody.

[0111] In an optional embodiment, the immune complex further comprises a second antibody, which binds to glycated hemoglobin HbA1c.

[0112] In a ninth aspect, the present disclosure provides uses of the above-mentioned anti-glycated hemoglobin HbA1c antibodies and antibody conjugates in detecting glycated hemoglobin HbA1c, diagnosing whether a subject has diabetes, preparing glycated hemoglobin HbA1c, or preparing products for diagnosing whether a subject has diabetes.

[0113] In a tenth aspect, the present disclosure provides a nucleic acid molecule encoding the above-mentioned antibody.

[0114] In an eleventh aspect, the present disclosure provides a vector containing the above-mentioned nucleic acid molecule.

[0115] In a twelfth aspect, the present disclosure provides cells containing the above-mentioned vector.

[0116] In a thirteenth aspect, the present disclosure provides a method for preparing an anti-glycated hemoglobin HbA1c antibody, comprising: culturing the cells as described above.

[0117] Based on the amino acid sequence of the anti-glycated hemoglobin HbA1c antibody disclosed in the present disclosure, those skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the anti-glycated hemoglobin HbA1c antibody. For example, the antibody can be isolated and purified from the culture product of recombinant cells that can recombinantly express the antibody as described in any of the above items. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the anti-glycated hemoglobin HbA1c antibody of the present disclosure, it falls within the scope of protection of the present disclosure.

[0118] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, the techniques employed or contemplated herein are standard methods. Materials, methods, and examples are illustrative and non-limiting only.

[0120] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. 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.

[0121] The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

[0122] Example 1 Preparation of Anti-HBA1C 30G9 Monoclonal Antibody

[0123] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was also purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting the anti-HBA1C 30G9 monoclonal antibody was generated in our laboratory and revived for future use.

[0124] (1) Antibody gene preparation

[0125] mRNA was extracted from a hybridoma cell line secreting the Anti-HBA1C 30G9 monoclonal antibody. DNA products were obtained by RT-PCR. This product was PCR-amplified with rTaq DNA polymerase and inserted into the pMD-18T vector. The cells were transformed into DH5α competent cells, and colonies were grown. Heavy chain and light chain gene clones were obtained, and four clones each were sent to a gene sequencing company for sequencing.

[0126] (2) Sequence analysis of the variable region gene of the Anti-HBA1C 30G9 antibody

[0127] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among them, in the gene fragment amplified by the Light Chain, the VL gene sequence was 336 bp, preceded by a 57 bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence was 366 bp, belonging to the VH1 gene family, and preceded by a 57 bp leader peptide sequence.

[0128] (3) Construction of recombinant antibody expression plasmid

[0129] pcDNA TM 3.4 The vector is a recombinant antibody eukaryotic expression vector constructed by using the pMD-18T vector. Multiple cloning restriction sites such as HindIII, BamHI, and EcoRI have been introduced into the vector, and the vector is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector. Based on the sequencing results of the antibody variable region genes in the pMD-18T vector, specific primers for the VL and VH genes of the antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. PCR amplification was used to amplify a 0.71kb Light Chain gene fragment and a 1.40kb Heavy Chain gene fragment.

[0130] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0131] 2. Recombinant Antibody Production

[0132] Resuscitate HEK293 cells in advance and subculture them into 200 ml system to make the cell density reach 3-5×10 6 cells / ml cell density reaches the selected antibody concentration and cells, cell viability>95%; centrifuge and wash the cells, re-dissolve with culture medium, and adjust the cell density to 2.9×10 6 Cells were washed with 100 μg of culture medium and re-dissolved. This was also used as a cell diluent. Plasmid DNA and transfection reagent diluents were prepared separately using culture medium. The transfection reagent diluent was added to the plasmid DNA diluent, mixed, and allowed to stand at room temperature for 15 minutes. The mixture was slowly added to the cell diluent over 1 minute, mixed, and sampled. The viability of the cells after transfection was recorded and observed. The cells were cultured in a 35°C constant temperature incubator at 120 rpm and 8% CO2. After 13 days, the samples were collected by centrifugation. The supernatant was affinity purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE. The electrophoresis pattern is shown in the figure. Two bands were shown after reducing SDS-PAGE, one with an Mr of 50 KD (heavy chain) and the other with an Mr of 28 KD (light chain).

[0133] The obtained antibody was named Anti-HBA1C 30G9Rmb1. The heavy chain amino acid sequence of the antibody Anti-HBA1C 30G9Rmb1 is shown in SEQ ID NO: 18, and the light chain amino acid sequence is shown in SEQ ID NO: 20.

[0134] Example 2 Performance testing of antibodies

[0135] 1. Affinity Analysis

[0136] Purified antibodies were diluted in advance, and HbA1c (from Feipeng Bio) was serially diluted. The antigen-antibody binding and dissociation curves were measured on a Biacore 8K+ instrument using a CM5 chip pre-coupled with goat anti-mouse IgG. The instrument automatically fitted the affinity constant, association rate, and dissociation rate. (KD represents the equilibrium dissociation constant, also known as the affinity constant; ka represents the association rate; and kd represents the dissociation rate.)

[0137] Table 2: Affinity data

[0138] 2. Activity Identification

[0139] The coating solution (main component NaHCO3) was used to dilute glycosylated hemoglobin HbA1c (from Feipeng Bio) to 3ug / ml, 100uL per well, and incubated at 4°C overnight; the next day, the cells were washed twice with washing solution (main component Na2HPO4+NaCl) and patted dry; blocking solution (20% BSA+80% PBS) was added at 120uL per well and incubated at 37°C for 1h and patted dry; diluted purified antibody and control antibody were added at 100uL / well and incubated at 37°C for 30min; the cells were washed 5 times with washing solution and patted dry; goat anti-mouse IgG-HRP was added at 100uL per well and incubated at 37°C for 30min; the cells were washed 5 times with washing solution and patted dry; colorimetric solution A (50uL / well) and colorimetric solution B (50uL / well) were added for 10min; stop solution was added at 50uL / well; the OD value was read at 450nm (reference 630nm) on a microplate reader.

[0140] Note: Solution A (main ingredients: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main ingredients: citric acid + EDTA·2Na + TMB + concentrated HCl); Stop solution (EDTA·2Na + concentrated H2SO4)

[0141] Table 3: Activity data

[0142] 3. Biochemical platform performance testing

[0143] 3.1 Antibodies Anti-HBA1C 30G9Rmb1 and Anti-HBA1C-A (from Feipeng Bio) in the above examples were diluted to 35 μg / ml and 5 μg / ml respectively with diluent, and mixed well to prepare the HbA1c R2 reagent.

[0144] 3.2 Detection method:

[0145] The antibody activity was verified on a Mindray 450 fully automatic biochemical analyzer in the following reaction mode: wavelength 570 nm, 200 μL R1 reagent (Tris buffer + latex microspheres) was added, followed by 1.5 μL of glycated hemoglobin calibrator containing different concentrations, incubated at 37°C for 5 min, 50 μL R2 latex reagent was added, the absorbance A1 was read, and after 5 min of reaction, the absorbance A2 was read, and the reactivity ΔA = A2-A1 was calculated.

[0146] Note: The concentration of the calibrator represents the percentage of HbA1c in total Hb.

[0147] The test results are shown in Table 4, and the standard curve is shown in Figure 2. The results show that the reagent composed of the antibody Anti-HBA1C 30G9Rmb1 has good sensitivity when tested on the biochemical platform, and R 2 Greater than 0.98.

[0148] Table 4: Test results of biochemical platform calibrators

[0149] 4. Stability assessment

[0150] The above-mentioned antibody was placed at 4°C (refrigerator), -80°C (freezer), and 37°C (incubator) for 21 days. Samples were collected at 7, 14, and 21 days for status observation, and the 21-day sample was tested for activity. The results showed that no significant changes in protein status were observed under the three test conditions for 21 days, and the activity did not show a downward trend with increasing test temperature, indicating that the above-mentioned antibody is stable. Table 4 below shows the OD results of the enzyme immunoassay activity assay of the antibody Anti-HBA1C30G9Rmb1 after 21 days of testing.

[0151] Table 5: Stability data

[0152] Some of the amino acid sequences involved in this application are shown in Table 6:

[0153] Table 6: Amino acid sequence listing

[0154] The foregoing description is merely a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection of the present disclosure.

Claims

1. An anti-glycated hemoglobin HbA1c antibody, comprising three complementarity determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17 and three complementarity determining regions of a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

19.

2. The antibody according to claim 1, characterized in that The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

3. An antibody against glycated hemoglobin HbA1c, characterized in that: The antibody comprises the following complementarity determining regions: HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1; HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2; HCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 3; LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4; LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:6; Optionally, the HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto; Optionally, the antibody has a KD of less than 7.99×10 -8 M binds to glycosylated hemoglobin HbA1c with great affinity.

4. An anti-glycosylated hemoglobin HbA1c antibody comprising a heavy chain variable region and / or a light chain variable region, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 19; Optionally, the antibody further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is mouse; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto.

5. An anti-glycosylated hemoglobin HbA1c antibody, comprising a heavy chain and / or a light chain, characterized in that: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 18; the amino acid sequence of the light chain is shown in SEQ ID NO:

20.

6. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a label coupled to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

7. A reagent or a kit, characterized in that: The reagent or kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6 in detecting glycated hemoglobin HbA1c, diagnosing whether a subject has diabetes, preparing glycated hemoglobin HbA1c or preparing a product for diagnosing whether a subject has diabetes.

9. A method for diagnosing whether a subject has diabetes, characterized in that The method comprises: a) contacting the antibody according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with glycated hemoglobin HbA1c in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample; Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to glycated hemoglobin HbA1c.

10. A method for detecting glycated hemoglobin HbA1c, characterized in that: The method comprises: a) contacting the antibody according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with glycated hemoglobin HbA1c in a sample to be tested under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample Optionally, the immune complex further comprises a second antibody, which binds to the antibody; Optionally, the immune complex further comprises a second antibody, which binds to glycated hemoglobin HbA1c.

11. A nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment according to any one of claims 1 to 5.

12. A carrier, wherein The vector comprises the nucleic acid of claim 11.

13. A cell, characterized in that The cell comprises the nucleic acid of claim 11 or the vector of claim 12.

14. A method for preparing the antibody or antigen-binding fragment according to any one of claims 1 to 5, characterized in that: The method comprises culturing the cell of claim 13.

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