Anti-glycosylated hemoglobin HbA1c antibody and application thereof

Abstract

The invention discloses an antibody for resisting glycosylated hemoglobin HbA1c and application of the antibody, and relates to the field of antibodies. The anti-glycosylated hemoglobin HbA1c antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for detection of glycosylated hemoglobin HbA1c, and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular, to an antibody against glycated hemoglobin HbA1c and its application. Background Art

[0002] The global prevalence of diabetes is very high, which is a world-wide public health problem seriously threatening human health, and its prevention and treatment have always been a hot topic in medical research. Traditional diagnostic and treatment tests include fasting blood glucose, postprandial blood glucose, and oral glucose tolerance test, etc. However, blood glucose parameters only represent the instantaneous blood glucose level at the time of blood sampling, while glycated hemoglobin (GHb), as the gold standard for reflecting long-term blood glucose levels, is also an important indicator for detecting diabetes treatment. Due to the different components it binds to, GHb is further divided into HbA1a (binding to phosphorylated glucose), HbA1b (binding to fructose), and HbA1c (binding to glucose). HbA1c is the recognized gold standard for blood glucose control in diabetes management and is also an effective indicator for evaluating diabetes treatment regimens. Currently, the World Health Organization and diabetes societies in many countries have adopted HbA1c as an independent diabetes diagnosis indicator.

[0003] HbA1c is the product of the slow, continuous, and irreversible non-enzymatic protein glycation reaction between hemoglobin (Hb) in human red blood cells and glucose in the blood, and is proportional to the blood glucose concentration. Since the survival period of red blood cells in the human body is generally 120 days, the content of HbA1c in the blood will remain relatively unchanged before the death of red blood cells. Therefore, the HbA1c level can stably and reliably reflect the average blood glucose level within 120 days before the test, and is less affected by factors such as blood sampling time, fasting status, and insulin use. It is considered the "gold standard" for diabetes treatment monitoring. From 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.

[0004] Currently, the main detection methods for glycated hemoglobin HbA1c are colloidal gold immunochromatography and fluorescence immunochromatography, both of which are immunological detection methods based on the specific reaction between antibodies and antigens, and at the same time use labeling substances (such as colloidal gold, fluorescence) to amplify and display the detected signal. Similar immunological detection methods also include biochemical immunoturbidimetry, radioimmunoassay, chemiluminescence assay, etc.

[0005] The above immunological detection methods all require antibodies against glycated hemoglobin HbA1c. Therefore, there is a strong demand in the art for antibodies against glycated hemoglobin HbA1c with good performance. Summary of the Invention

[0006] The present application provides an antibody against glycated hemoglobin HbA1c, which provides an important source of raw materials for the detection of glycated hemoglobin HbA1c and has good activity or affinity.

[0007] To achieve the above object, according to one aspect of the present invention, there is provided an antibody against glycated hemoglobin HbA1c, the antibody comprising three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17 and three complementary determining regions of a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19.

[0008] To achieve the above object, according to a second aspect of the present invention, there is provided an antibody against glycated hemoglobin HbA1c, the antibody comprising the following complementary determining regions:

[0009] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the same;

[0010] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the same;

[0011] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of the same;

[0012] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of the same;

[0013] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of the same;

[0014] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of the same.

[0015] To achieve the above object, according to a third aspect of the present invention, there is provided an antibody against glycated hemoglobin HbA1c, comprising a heavy chain variable region and / or a light chain variable region, the amino acid sequence of the heavy chain variable region being as shown in SEQ ID NO: 17; the amino acid sequence of the light chain variable region being as shown in SEQ ID NO: 19.

[0016] To achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody against glycated hemoglobin HbA1c, comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being as shown in SEQ ID NO: 18; the amino acid sequence of the light chain being as shown in SEQ ID NO: 20.

[0017] To achieve the above object, according to a fifth aspect of the present invention, there is provided an antibody conjugate, the antibody conjugate comprising the above antibody.

[0018] To achieve the above object, according to the sixth aspect of the present invention, a reagent or a kit is provided, and the reagent or the kit includes the above-mentioned antibody or the above-mentioned antibody conjugate.

[0019] To achieve the above object, according to the eighth aspect of the present invention, there is provided a use of the above-mentioned antibody or antibody conjugate in the preparation of a product for detecting glycated hemoglobin HbA1c.

[0020] To achieve the above object, the present invention also provides a nucleic acid, a vector, a cell and a method for preparing the above-mentioned antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 Result of reducing SDS-PAGE for Anti-HBA1C 30G9 Rmb1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] In a first aspect, an embodiment of the present invention provides an antibody against glycated hemoglobin HbA1c, and the antibody includes three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17 and three complementary determining regions of a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 19.

[0024] In the present invention, the term "antibody" is used in the broadest sense, and it may include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies or antigen-binding fragments, as long as they exhibit the required biological activity.

[0025] The above-mentioned antigen-binding fragments generally have the same binding specificity as the antibodies from which they are derived. Those skilled in the art can easily understand from the content described in the present invention that the above-mentioned antigen-binding fragments can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemically reducing and cleaving disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, those skilled in the art can easily obtain the above-mentioned antigen-binding fragments.

[0026] The above antigen-binding fragments can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0027] In the present invention, the terms "complementary determining region", "CDR" or "CDRs" refer to the highly variable regions of the heavy and light chains of an immunoglobulin, and refer to regions containing one or more or even all of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In the specific embodiments of the present invention, the CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0028] In the present invention, the heavy-chain complementary determining regions are denoted as HCDR and include HCDR1, HCDR2, and HCDR3; the light-chain complementary determining regions are denoted as LCDR and include LCDR1, LCDR2, and LCDR3.

[0029] The methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As used herein, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The "Chothia definition" is described in Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR regions defined by Kabat, although they may be shortened or lengthened depending on the prediction or experimental results of specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different literatures are slightly different. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods not limited to Table 1 also fall within the scope of protection of the present disclosure.

[0030] Table 1: CDR Definitions 1

[0031] CDR Kabat AbM2 IMGT Chothia HCDR1 <![CDATA[H31~H35 3 > <![CDATA[H26~H35 3 > <![CDATA[H26~H33..5 5 > <![CDATA[H26~H32..34 4 > HCDR2 H50 - H65 H50 - H58 H51 - H57 H52 - H56 HCDR3 H95 - H102 H95 - H102 H93 - H102 H95 - H102 LCDR1 L24 - L34 L24 - L34 L27 - L32 L24 - L34 LCDR2 L50 - L56 L50 - L56 L50 - L51 L50 - L56 LCDR3 L89 - L97 L89 - L97 L89 - L97 L89 - L97

[0032] 1The numbers of all CDR definitions in Table 1 are based on the Kabat numbering system (see below). The amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number". A person of ordinary skill in the art can clearly correspond this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0033] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDR defined by the "AbM" antibody modeling software of Oxford Molecular.

[0034] 3 If neither H35A nor H35B is present, then CDR-H1 ends at position 35; if only H35A is present, then CDR-H1 ends at position 35A; if both H35A and H35B are present, then CDR-H1 ends at position 35B.

[0035] 4 If neither H35A nor H35B is present, then CDR-H1 ends at position 32; if only H35A is present, then CDR-H1 ends at position 33; if both H35A and H35B are present, then CDR-H1 ends at position 34.

[0036] 5 If neither H35A nor H35B is present, then CDR-H1 ends at position 33; if only H35A is present, then CDR-H1 ends at position 34; if both H35A and H35B are present, then CDR-H1 ends at position 35.

[0037] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

[0038] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0039] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0040] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0041] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the AbM system.

[0042] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0043] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM, or Contact systems.

[0044] In a second aspect, an embodiment of the present invention provides an antibody against glycated hemoglobin HbA1c, the antibody comprising the following complementarity-determining regions:

[0045] HCDR1, which comprises the amino acid sequence shown in SEQ ID NO:1, or consists of the same.

[0046] HCDR2, which comprises the amino acid sequence shown in SEQ ID NO:2, or consists of the same.

[0047] HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:3, or consists of the same.

[0048] LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:4, or consists of the same.

[0049] LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:5, or consists of the same.

[0050] LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:6, or consists of the same.

[0051] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0052] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the regions in the heavy chain variable region and the light chain variable region of the antibody other than the CDRs; wherein, the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3, and LFR4 framework regions.

[0053] In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0054] In an alternative embodiment, the antibody according to the first or second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0055] In an alternative embodiment, the HFR1 comprises / is as shown in SEQ ID NO:7 or an amino acid sequence having at least 80% identity therewith;

[0056] The HFR2 comprises / is as shown in SEQ ID NO:8 or an amino acid sequence having at least 80% identity therewith;

[0057] The HFR3 comprises / is as shown in SEQ ID NO:9 or an amino acid sequence having at least 80% identity therewith;

[0058] The HFR4 comprises / is as shown in SEQ ID NO:10 or an amino acid sequence having at least 80% identity therewith;

[0059] The LFR1 comprises / is as shown in SEQ ID NO:11 or an amino acid sequence having at least 80% identity therewith;

[0060] The LFR2 comprises / is as shown in SEQ ID NO:12 or an amino acid sequence having at least 80% identity therewith;

[0061] The LFR3 comprises / is as shown in SEQ ID NO:13 or an amino acid sequence having at least 80% identity therewith; and

[0062] The LFR4 comprises / is as shown in SEQ ID NO:14 or an amino acid sequence having at least 80% identity therewith.

[0063] It should be noted that in other embodiments, the amino acid sequences of the framework regions of the antibody against glycated hemoglobin HbA1c provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0064] In an alternative embodiment, the antibody binds to glycated hemoglobin HbA1c with an affinity of KD < 7.99×10 -8 M.

[0065] In an alternative embodiment, the antibody binds to glycated hemoglobin HbA1c with an affinity of KD ≤ 10 -8 M, KD ≤ 10 -9 M, KD ≤ 10 -10 M, KD ≤ 10 -11 M or KD ≤ 10 -12 M.

[0066] In an alternative embodiment, the antibody binds to glycated hemoglobin HbA1c with an affinity of KD ≤ 1.17×10 -9 M.

[0067] There are many methods for measuring antibody affinity (KD). According to the detection principle, they can be divided into thermodynamic detection methods, kinetic detection methods and dynamic equilibrium detection methods. Among them, common thermodynamic detection methods are such as isothermal titration calorimetry (ITC); common kinetic detection methods are such as surface plasmon resonance (SPR) and biolayer interferometry (BLI); common dynamic equilibrium detection methods are such as enzyme-linked immunosorbent assay (ELISA), etc.

[0068] In an alternative embodiment, the determination of KD adopts a kinetic detection method; optionally, surface plasmon resonance, for example, by using a biosensor system such as the system.

[0069] In a third aspect, an embodiment of the present invention provides an antibody against glycated hemoglobin HbA1c, 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 as shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 19.

[0070] In an alternative embodiment, the antibodies described in the first aspect, second aspect, and third aspect above further comprise a constant region.

[0071] In an alternative embodiment, the constant region comprises a heavy chain constant region and / or a light chain constant region.

[0072] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments.

[0073] In an alternative embodiment, the heavy chain constant region comprises CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0074] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0075] In an alternative embodiment, the light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0076] In an alternative embodiment, the species origin of the constant region is bovine, equine, dairy cow, porcine, ovine, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0077] In an alternative embodiment, the species origin of the constant region is mouse.

[0078] In this article, the division of variable region and constant region sequences refers to the IMGT division method, see Lefranc, the international ImMunoGeneTics database. Nucl. Acids Res., 29(1):207 - 209(2001). DOI:10.1093 / nar / 29.1.207. PMID:11125093. 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. There will be differences in some amino acids between the variable regions divided by different methods and the C-terminus of the variable region or the N-terminus of the constant region divided by IMGT. The variable regions or constant regions divided by other methods well-known in the art are also within the protection scope of the present invention.

[0079] In an alternative embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO: 15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO: 16.

[0080] It should be noted that in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above constant regions (SEQ ID NO: 15 or 16).

[0081] In an alternative embodiment, the antibody includes any one of F(ab)2, F(ab’)2, Fab’, Fab, Fv and scFv.

[0082] Fourthly, the present invention provides an antibody against glycated hemoglobin HbA1c, including a heavy chain and / or a light chain. The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 18, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 20.

[0083] Fifthly, the present invention provides an antibody conjugate, and the antibody conjugate includes the above antibody.

[0084] In an alternative embodiment, the above antibody conjugate further includes biotin or a biotin derivative conjugated to the antibody.

[0085] In an alternative embodiment, the antibody conjugate further includes a label conjugated to the antibody.

[0086] In an alternative embodiment, the above-mentioned marker refers to a class of substances with characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through these characteristics.

[0087] In an alternative embodiment, the marker includes, but is not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based markers.

[0088] In the actual use process, 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 protection scope of the present invention.

[0089] In an alternative embodiment, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, red rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (preCP), etc.).

[0090] In an alternative embodiment, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0091] In an alternative embodiment, the radioisotopes 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.

[0092] In alternative embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rhodanine and its derivatives, and peroxyoxalate and its derivatives.

[0093] In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0094] In alternative embodiments, the colloids include, but are not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0095] In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0096] In alternative embodiments, the colloidal metal is colloidal gold.

[0097] In alternative embodiments, the above-mentioned antibody conjugate further includes a solid-phase carrier conjugated to the antibody.

[0098] In alternative embodiments, the solid-phase carrier is selected from microspheres, plates, and membranes.

[0099] In alternative embodiments, the solid-phase carrier includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

[0100] In a sixth aspect, the present invention provides a reagent or kit, which includes the above-mentioned antibody or the above-mentioned antibody conjugate.

[0101] As described above, the antibodies in some embodiments or examples of the present invention can effectively bind to glycated hemoglobin HbA1c. Therefore, the reagent or kit containing the glycated hemoglobin HbA1c antibody can effectively perform qualitative or quantitative detection of glycated hemoglobin HbA1c. Applying the reagent or kit provided by the present invention, for example, it can be used in immunoblotting, immunoprecipitation, etc., which involve detecting the specific binding performance of glycated hemoglobin HbA1c and its antibody. As described above, the antibodies in some embodiments or examples of the present invention have higher binding activity or affinity with glycated hemoglobin HbA1c. Therefore, the reagent or kit containing the antibody has higher detection sensitivity or specificity.

[0102] In a seventh aspect, the present invention 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 test sample 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 which indicates the presence of the antigen in the test sample;

[0103] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody.

[0104] In an alternative embodiment, the immune complex further comprises a second antibody that binds to glycated hemoglobin HbA1c.

[0105] In an eighth aspect, the present invention provides the use of the above-mentioned anti-glycated hemoglobin HbA1c antibody and antibody conjugate in the preparation of a product for detecting glycated hemoglobin HbA1c.

[0106] It should be noted that the products of the present invention include but are not limited to reagents, kits, test strips or detection plates.

[0107] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0108] In a tenth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0109] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

[0110] In a twelfth aspect, the present invention provides a method for preparing an anti-glycated hemoglobin HbA1c antibody, which comprises: culturing the cells as described above.

[0111] Based on the disclosure of the amino acid sequence of the anti-glycated hemoglobin HbA1c antibody in the present invention, those skilled in the art can easily conceive of using genetic engineering techniques or other techniques (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 capable of recombinantly expressing the antibody described in any one of the above. This is easily achievable for those skilled in the art. Based on this, regardless of the technique used to prepare the anti-glycated hemoglobin HbA1c antibody of the present invention, it falls within the protection scope of the present invention.

[0112] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 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 formulations or unit doses herein, some methods and materials are now described. Unless otherwise noted, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0114] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those of ordinary skill in the art. Such techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (eds. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (eds. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (eds. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (eds. Mullis et al., 1994); and "Current Protocols in Immunology" (eds. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0115] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0116] Preparation of Anti-HBA1C 30G9 Monoclonal Antibody in Example 1

[0117] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor-RNA extraction kit was purchased from TOYOBO. The BD SMART TM RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting Anti-HBA1C 30G9 monoclonal antibody was the hybridoma cell line prepared in this laboratory and was revived for standby.

[0118] (1) Preparation of Antibody Gene

[0119] mRNA was extracted from the hybridoma cell line secreting Anti-HBA1C 30G9 monoclonal antibody, and a DNA product was obtained by RT-PCR. After the addition of A reaction to this product with rTaq DNA polymerase, it was inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew, 4 clones each of the Heavy Chain and Light Chain genes were cloned and sent to a gene sequencing company for sequencing.

[0120] (2) Sequence Analysis of Anti-HBA1C 30G9 Antibody Variable Region Gene

[0121] The gene sequences obtained from the above sequencing were analyzed in the kabat antibody database, and the 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 the gene fragments amplified by the Light Chain, the VL gene sequence was 336bp, and there was a 57bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 366bp, belonging to the VH1 gene family, and there was a 57bp leader peptide sequence in front of it.

[0122] (3) Construction of Recombinant Antibody Expression Plasmid

[0123] pcDNA TM 3.4 The vector is the eukaryotic expression vector of the constructed recombinant antibody. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI have been introduced into this expression vector, which is named the pcDNA3.4A expression vector, hereinafter simply referred to as the 3.4A expression vector. According to the antibody variable region gene sequencing results in the above pMD-18T, specific primers for the VL and VH genes of this antibody were designed, with HindIII and EcoRI enzyme digestion sites and protective bases at both ends. A 0.71 kb Light Chain gene fragment and a 1.40 kb Heavy Chain gene fragment were amplified by PCR amplification method.

[0124] The Heavy Chain and Light Chain gene fragments were respectively digested with HindIII / EcoRI double enzymes, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After purifying and recovering the fragments and the vector, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector, and the recombinant expression plasmids of the Heavy Chain and the Light Chain were obtained respectively.

[0125] 2. Recombinant antibody production

[0126] Resuscitate HEK293 cells in advance, subculture them to a 200 ml system until the cell density reaches 3 - 5×10 6 cells / ml. Select the antibody concentration and cells with a cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml to wash the cells, resuspend them with the medium, and at the same time, use it as the cell diluent. Prepare plasmid DNA and transfection reagent diluents with the medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell diluent within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in a 35°C constant temperature incubator for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, centrifuge to collect the samples. Affinity purify the centrifuged supernatant with a protein A affinity chromatography column. Take 6 μg of the purified antibody for reducing SDS-PAGE, and the electrophoresis pattern is shown in the figure. Two bands are shown after reducing SDS-PAGE, one with Mr of 50 KD (heavy chain) and the other with Mr of 28 KD (light chain).

[0127] The obtained antibody is named Anti-HBA1C 30G9Rmb1. The amino acid sequence of the heavy chain of the antibody Anti-HBA1C 30G9Rmb1 is as shown in SEQ ID NO:18, and the amino acid sequence of the light chain is as shown in SEQ ID NO:20.

[0128] Performance Detection of the Antibody in Example 2

[0129] 1. Affinity Analysis

[0130] Dilute the purified antibody in advance, and at the same time perform gradient dilution on glycated hemoglobin HbA1c (from Phoenix Biotech); use the CM5 chip that has been pre-coupled with goat anti-mouse IgG to test the binding and dissociation curves of the antigen and antibody on the Biacore 8K+ device, and the instrument automatically fits to obtain the affinity constant, binding rate, and dissociation rate. (KD represents the equilibrium dissociation constant, i.e., the affinity constant; ka represents the binding rate; kd represents the dissociation rate)

[0131] Table 2 Affinity Data

[0132] Sample Name KD ka kd Control 7.99E-08 2.34E+05 1.87E-02 Anti - HBA1C 30G9Rmb1 1.17E-09 1.50E+06 1.76E-03

[0133] 2. Activity Identification

[0134] Dilute glycated hemoglobin HbA1c (from Phoenix Biotech) to 3 μg / ml with the coating solution (main component NaHCO3), 100 μL per well, and incubate overnight at 4°C; the next day, wash 2 times with the washing solution (main components Na2HPO4 + NaCl), and pat dry; add the blocking solution (20% BSA + 80% PBS), 120 μL per well, at 37°C for 1 h, and pat dry; add the diluted purified antibody and control antibody, 100 μL / well, at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, at 37°C for 30 min; wash 5 times with the washing solution, and pat dry; add chromogenic solution A (50 μL / well), add chromogenic solution B (50 μL / well), for 10 min; add the termination solution, 50 μL / well; read the OD value at 450 nm (reference 630 nm) on the microplate reader.

[0135] Note: Solution A (main components citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main components citric acid + EDTA·2Na + TMB + concentrated HCl); Termination solution (EDTA·2Na + concentrated H2SO4)

[0136] Table 3 Activity Data

[0137] Concentration (ng / ml) 125.00 62.50 31.25 15.63 7.81 0.00 Control 1.273 0.977 0.624 0.301 0.112 0.014 Anti - HBA1C 30G9Rmb1 1.829 1.553 1.303 0.776 0.497 0.029

[0138] 3. Stability Assessment

[0139] The above-mentioned antibody was placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days. Samples were taken at 7 days, 14 days, and 21 days for status observation, and the 21-day samples were subjected to activity detection. The results showed that there were no obvious protein status changes after the antibody was placed for 21 days under the three test conditions, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above-mentioned antibody was stable. Table 4 below shows the OD results of the enzyme immunoassay activity detection of the antibody Anti-HBA1C 30G9Rmb1 after 21 days of assessment.

[0140] Table 4 Stability data

[0141] Sample Concentration (ng / ml) 31.25 15.63 0.00 Sample at 4°C for 21 days 1.381 0.719 0.019 Sample at - 80°C for 21 days 1.322 0.772 0.018 Sample at 37°C for 21 days 1.379 0.736 0.018

[0142] The foregoing are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0143] Some of the amino acid sequences involved in this application are shown in Table 5 as follows:

[0144]

[0145]

Claims

1. An anti-glycated hemoglobin HbA1c antibody, wherein the antibody comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 17 and three complementary 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, wherein, the complementary determining regions of the variable regions are defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact.

3. An anti-glycated hemoglobin HbA1c antibody, wherein, the antibody comprises the following complementary determining regions: HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the same; HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the same; HCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of the same; LCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4, or consists of the same; LCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of the same; LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of the same; 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 binds to glycated hemoglobin HbA1c with an affinity of KD < 7.99×10 -8 M.

4. 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 as shown in SEQ ID NO: 17; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 19; Optionally, the antibody further comprises a constant region; Optionally, the constant region comprises a heavy chain constant region and / or a light chain constant region; Optionally, the heavy chain constant region is selected from the heavy chain constant region of any one of IgG, IgA, IgM, IgE, IgD or a combination of multiple constant region segments; Optionally, the heavy chain constant region comprises CH1 of IgG, the hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, canine, camel, feline, 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 therewith; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity therewith; Optionally, the antibody comprises any one of F(ab’)2, Fab’, Fab, Fv and scFv.

5. An anti-glycated hemoglobin HbA1c antibody, comprising a heavy chain and / or a light chain, characterized in that the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 18; the amino acid sequence of the light chain is as 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 conjugated to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle-based labels; Optionally, the antibody conjugate further comprises a solid phase carrier conjugated to the antibody.

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

9. A nucleic acid, vector, cell or method for preparing the antibody according to any one of claims 1-5, the nucleic acid encoding the antibody according to any one of claims 1 to 5; the vector contains the nucleic acid encoding the antibody according to any one of claims 1 to 5; the cell contains the above nucleic acid or vector; the method comprises the above cell.

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