CN117402239B - Anti-glycosylated hemoglobin antibody, reagent for detecting glycosylated hemoglobin and kit - Google Patents

Anti-glycosylated hemoglobin antibody, reagent for detecting glycosylated hemoglobin and kit Download PDF

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CN117402239B
CN117402239B CN202210794266.2A CN202210794266A CN117402239B CN 117402239 B CN117402239 B CN 117402239B CN 202210794266 A CN202210794266 A CN 202210794266A CN 117402239 B CN117402239 B CN 117402239B
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antibody
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antigen
binding fragment
amino acid
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CN117402239A (en
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孟媛
钟冬梅
唐丽娜
李艳
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Dongguan Pengzhi Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G01N33/723—Glycosylated haemoglobin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
    • G01N2333/805—Haemoglobins; Myoglobins

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Abstract

The invention discloses an anti-glycosylated hemoglobin antibody, a reagent for detecting glycosylated hemoglobin and a kit, and relates to the technical field of antibodies. The anti-glycosylated hemoglobin antibodies disclosed herein comprise a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody provides an important raw material source for the detection of glycosylated hemoglobin and has improved affinity and activity.

Description

Anti-glycosylated hemoglobin antibody, reagent for detecting glycosylated hemoglobin and kit
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-glycosylated hemoglobin antibody, a reagent for detecting glycosylated hemoglobin and a kit.
Background
The prevalence of diabetes mellitus worldwide is high, and the disease control is always a hot spot of medical research, and is a worldwide public health problem seriously threatening human health. Traditional diagnosis and treatment tests adopt fasting blood glucose, postprandial blood glucose, oral glucose tolerance experiments and the like, but blood glucose parameters only represent the instant blood glucose level when blood is drawn, and glycosylated hemoglobin (glcated hemoglobin, GHb) is used as a gold standard reflecting the long-term blood glucose level and is also an important index for detecting diabetes treatment. GHb is classified into HbA1a (bound to phosphoryl glucose), hbA1b (bound to fructose), and HbA1c (bound to glucose) depending on the component bound thereto. HbA1c is a recognized gold standard for glycemic control in diabetes management and is also an effective indicator for evaluating a diabetes treatment regimen. Currently, hbA1c has been used as an independent diagnostic index for diabetes by the world health organization and diabetes society in many countries.
HbA1c is the product of a slow, sustained and irreversible non-enzymatic glycation reaction of hemoglobin (Hb) in erythrocytes in human blood with glucose and is proportional to blood glucose concentration. Since the life cycle of red blood cells in a human body is generally 120 days, the HbA1c content in blood can be kept relatively unchanged before the red blood cells die, so that the HbA1c level can reflect the average blood glucose level in 120 days before detection, and is not greatly interfered by factors such as blood drawing time, fasting, insulin usage and the like, and is considered as a gold standard for monitoring diabetes treatment. By combining the characteristics of hemoglobin, the detection of glycosylated hemoglobin has important significance for screening, monitoring and treating diabetes.
Currently, the detection methods of HbA1c mainly include a colloidal gold immunochromatography method and a fluorescence immunochromatography method, which are immunological detection methods based on specific reactions of antibodies and antigens, and amplifying and displaying detected signals by using a labeling substance (such as colloidal gold and fluorescence). Similar immunological detection methods include biochemical immunoturbidimetry, radioimmunoassay, chemiluminescence, and the like. The immunological detection methods described above all require antibodies against HbA1 c.
Thus, there is a strong need in the art for antibodies that efficiently bind and detect glycosylated hemoglobin.
In view of this, the present invention has been made.
Disclosure of Invention
The application provides a glycosylated hemoglobin antibody with improved affinity and activity, which is used for improving the detection of glycosylated hemoglobin and providing an important raw material source for the detection of glycosylated hemoglobin.
In order to achieve the above object, according to one aspect of the present invention, there is provided an antibody or a functional fragment thereof comprising HCDR1 having an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 15, HCDR2 having an amino acid sequence shown as SEQ ID NO. 2, HCDR3 having an amino acid sequence shown as SEQ ID NO. 3, and LCDR1, LCDR2, LCDR3 having amino acid sequences shown as SEQ ID NO. 4 to 6.
In order to achieve the above object, according to a second aspect of the present invention, there is provided an anti-glycosylated hemoglobin antibody or a functional fragment thereof, the antibody or the functional fragment thereof comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above.
In order to achieve the above object, according to a third aspect of the present invention, there is provided an anti-glycosylated hemoglobin antibody comprising a heavy chain comprising the heavy chain variable region described above and/or a light chain comprising the light chain variable region described above.
In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody conjugate comprising the above antibody or a functional fragment thereof.
In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a reagent or kit for detecting glycosylated hemoglobin, the reagent or kit comprising the above-described antibody or a functional fragment thereof or the above-described antibody conjugate.
In order to achieve the above object, according to a sixth aspect of the present invention, there is also provided a vector, a cell and a method for producing the above antibody or a functional fragment thereof.
In order to achieve the above object, according to a seventh aspect of the present invention, there is provided a method for detecting glycosylated hemoglobin, the method comprising contacting the above-mentioned antibody or functional fragment thereof with glycosylated hemoglobin in a sample to be detected to form an immune complex.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of reducing SDS-PAGE of Anti-HBA1C-10C7mut1 to Anti-HBA1C-10C7mut 6.
Detailed Description
The invention provides an antibody or a functional fragment thereof, which comprises HCDR1 with an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO.15, HCDR2 with an amino acid sequence shown as SEQ ID NO.2, HCDR3 with an amino acid sequence shown as SEQ ID NO. 3, and LCDR1, LCDR2 and LCDR3 with amino acid sequences shown as SEQ ID NO. 4-6. The antibodies have improved affinity and activity.
In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity.
In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues contributing to the binding affinity of an antibody or antigen binding fragment to the antigen or epitope it recognizes. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.
In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining regions are denoted by LCDR and include LCDR1, LCDR2 and LCDR3. CDR labeling methods commonly used in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering schemes, 1997 Lefranc et al, all protein sequences of the immunoglobulin superfamily. Kabat et al were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of KABATMAN databases, and the Kabat numbering scheme is generally considered as a widely adopted standard for numbering antibody residues. The invention adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the invention.
In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to regions other than CDRs in an antibody heavy chain variable region and a light chain variable region; wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions may be further subdivided into contiguous regions separated by CDRs comprising LFR1, LFR2, LFR3 and LFR4 framework regions.
In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs with FRs in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
In alternative embodiments, the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having amino acid sequences shown in SEQ ID NO. 7 through SEQ ID NO. 10, and LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NO. 11 through SEQ ID NO. 14.
In other embodiments, each of the framework region amino acid sequences of the antibodies or functional fragments thereof provided herein 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 to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.
In an alternative embodiment, the antibody or functional fragment thereof binds glycosylated hemoglobin with an affinity of KD.ltoreq.2.52X10 -7 M.
In an alternative embodiment, the antibody or functional fragment thereof binds glycosylated hemoglobin with an affinity of KD.ltoreq.2.39X10 -9 M.
In an alternative embodiment, the antibody or functional fragment thereof binds glycosylated hemoglobin with an affinity of KD.ltoreq.2.38X10 -9M、KD≤2.32×10-9M、KD≤1.98×10-9M、KD≤1.85×10-9 M.
In an alternative embodiment, the antibody or functional fragment thereof binds glycosylated hemoglobin with an affinity of KD.ltoreq.1.71×10 -9 M.
In an alternative embodiment, the amino acid sequence of HFR1 of the antibody or functional fragment thereof is set forth in SEQ ID NO. 16.
In an alternative embodiment, the amino acid sequence of HFR3 of the antibody or functional fragment thereof is set forth in SEQ ID NO. 17.
In an alternative embodiment, the amino acid sequence of HFR4 of the antibody or functional fragment thereof is set forth in SEQ ID NO. 18.
In an alternative embodiment, the amino acid sequence of LFR3 of said antibody or functional fragment thereof is shown in SEQ ID NO. 19.
In another aspect, embodiments of the present invention provide an anti-glycosylated hemoglobin antibody or a functional fragment thereof, the antibody or the functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprises a sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, and the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR3, HFR1, HFR2, HFR4, LFR1, HFR2, LFR3, LFR4 is the amino acid sequence of HFR1, HFR2, HFR3, LFR4, LFR2, LFR 4.
In an alternative embodiment, the heavy chain variable region amino acid sequence is set forth in any one of SEQ ID NOs 22 to 37.
In an alternative embodiment, the light chain variable region amino acid sequence is as set forth in any one of SEQ ID NOs 38 to 39.
In alternative embodiments, the antibody further comprises a constant region.
In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.
In alternative embodiments, the heavy chain constant region is selected from the group consisting of an IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD heavy chain constant region, and the light chain constant region is selected from the group consisting of kappa-type or lambda-type light chain constant regions.
In alternative embodiments, the constant region is of a species derived from a cow, horse, cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.
In an alternative embodiment, the constant region is of murine species origin.
In an alternative embodiment, the constant region is of rat species origin.
In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 20 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 21.
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 to the constant region (SEQ ID NO:19 or 20) described above.
In alternative embodiments, the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.
The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.
Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.
In another aspect, the invention provides an anti-glycosylated hemoglobin antibody comprising a heavy chain and/or a light chain, said heavy chain comprising a heavy chain variable region as described above and a heavy chain constant region as described above; the light chain comprises the light chain variable region described above and the light chain constant region described above.
In an alternative embodiment, the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NOs 40 to 55.
In an alternative embodiment, the amino acid sequence of the light chain is as shown in any one of SEQ ID NOs 56 to 57.
In an alternative embodiment, the monoclonal antibody Anti-HBA1C-10C7mut1 heavy chain has the amino acid sequence shown in SEQ ID NO. 51 and the light chain has the amino acid sequence shown in SEQ ID NO. 56.
In an alternative embodiment, the monoclonal antibody Anti-HBA1C-10C7mut2 heavy chain has the amino acid sequence shown in SEQ ID NO. 53 and the light chain has the amino acid sequence shown in SEQ ID NO. 56.
In an alternative embodiment, the monoclonal antibody Anti-HBA1C-10C7mut3 heavy chain has the amino acid sequence shown in SEQ ID NO. 48 and the light chain has the amino acid sequence shown in SEQ ID NO. 56.
In an alternative embodiment, the monoclonal antibody Anti-HBA1C-10C7mut4 heavy chain has the amino acid sequence shown in SEQ ID NO. 43 and the light chain has the amino acid sequence shown in SEQ ID NO. 56.
In an alternative embodiment, the monoclonal antibody Anti-HBA1C-10C7mut5 heavy chain has the amino acid sequence shown in SEQ ID NO. 46 and the light chain has the amino acid sequence shown in SEQ ID NO. 56.
In an alternative embodiment, the monoclonal antibody Anti-HBA1C-10C7mut6 heavy chain has the amino acid sequence shown in SEQ ID NO. 40 and the light chain has the amino acid sequence shown in SEQ ID NO. 56.
In an alternative embodiment, the monoclonal antibody Anti-HBA1C-10C7mut7 heavy chain has the amino acid sequence shown in SEQ ID NO. 51 and the light chain has the amino acid sequence shown in SEQ ID NO. 57.
The monoclonal antibodies Anti-HBA1C-10C7mut1-Anti-HBA1C-10C7mut7 have improved affinity and activity, and the Anti-HBA1C-10C7mut1 and Anti-HBA1C-10C7mut7 have comparable affinity and activity.
In another aspect, the invention provides an antibody conjugate comprising an antibody or functional fragment thereof as described above.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is labeled with a label.
In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), and the like).
In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.
In alternative embodiments, the radioisotope includes, but is 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 18 F.
In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.
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.
In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is coated onto a solid phase.
In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.
In alternative embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.
In an alternative embodiment, the solid phase is a nitrocellulose membrane.
In another aspect, the present invention provides a reagent or kit for detecting glycosylated hemoglobin, the reagent or kit comprising the antibody or functional fragment thereof or the antibody conjugate.
In another aspect, the present invention provides the use of the above antibody or functional fragment thereof, antibody conjugate or the above reagent or kit for glycosylated hemoglobin detection.
In another aspect, the invention provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.
In another aspect, the invention provides a vector comprising the nucleic acid molecule described above.
In another aspect, the invention provides a cell comprising the vector described above.
In another aspect, the invention provides a method of making an antibody or functional fragment thereof comprising: the cells as described above were cultured.
On the basis of the present invention, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present invention to prepare the antibody or the functional fragment thereof by any technique.
In another aspect, the present invention also provides a method for detecting glycosylated hemoglobin, comprising: contacting the antibody or functional fragment thereof with glycosylated hemoglobin in a sample to be tested to form an immune complex;
In an alternative embodiment, the immune complex described above further comprises a second antibody, said second antibody being bound to said antibody or a functional fragment thereof;
In an alternative embodiment, the immunocomplex described above further comprises a second antibody, which binds to glycosylated hemoglobin.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Unless defined otherwise, 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 formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
Unless otherwise indicated, 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 ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait, eds., 1984); animal cell Culture (ANIMAL CELL Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.)), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C. Blackwell, inc.), gene transfer Vectors for mammalian cells (GENE TRANSFER vector for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, 1987), polymerase chain reaction (PCR: the Polymerase Chain Reaction) (Mullis et al, 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which are expressly incorporated herein by reference.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
EXAMPLE 1 preparation of Anti-HBA1C-10C7 monoclonal antibodies
Restriction enzymes, PRIME STAR DNA polymerase in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation.
1 Construction of recombinant plasmid
(1) Antibody Gene production
MRNA is extracted from hybridoma cell strain secreting monoclonal antibody against glycosylated hemoglobin, DNA product is obtained through RT-PCR method, the product is inserted into pMD-18T vector after adding A reaction by rTaq DNA polymerase, and is transformed into DH5 alpha competent cells, HEAVY CHAIN and LIGHT CHAIN gene clones are respectively taken after colony growth, and each 4 clones are sent to gene sequencing company for sequencing.
(2) Sequence analysis of Anti-HBA1C-10C7 antibody variable region Gene
The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI 11.5.5 software is utilized for analysis to determine that the amplified genes of the heavy chain primer pair and the light chain primer pair are correct, wherein in the LIGHT CHAIN amplified gene fragment, the VL gene sequence is 327bp, and the front of the VL gene fragment is 57bp leader peptide sequence; the gene fragment amplified by HEAVY CHAIN primer pair has 357bp VH gene sequence, belonging to VH1 gene family, and 57bp leader peptide sequence in front of it.
(3) Construction of recombinant antibody expression plasmids
pcDNATM 3.4Vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is introduced with HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and named pcDNA3.4A expression vector, and is hereinafter abbreviated as 3.4A expression vector; according to the result of the gene sequencing of the antibody variable region in pMD-18T, VL and VH gene specific primers of the antibody are designed, and the two ends of the VL and VH gene specific primers are respectively provided with HindIII, ecoRI digestion sites and protective bases, and a LIGHT CHAIN gene fragment of 0.72kb and a HEAVY CHAIN gene fragment of 1.40kb are amplified by a PCR amplification method.
The HEAVY CHAIN and LIGHT CHAIN gene fragments are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the HEAVY CHAIN gene and LIGHT CHAIN gene after the fragments and the vector are purified and recovered are respectively connected with the 3.4A expression vector, and recombinant expression plasmids of HEAVY CHAIN and LIGHT CHAIN are respectively obtained.
2 Stable cell line selection
(1) Recombinant antibody expression plasmid transient transfection CHO cells, determination of expression plasmid activity
The plasmid was diluted to 40ug/100ul with ultrapure water, CHO cells were adjusted to 1.43X10 7 cells/ml in centrifuge tubes, 100. Mu.L of plasmid was mixed with 700. Mu.L of cells, transferred to an electrotransfer cup, electrotransferred, sampled and counted on days 3,5, 7, and collected on day 7.
The coating solution (NaHCO 3 as the main ingredient) diluted human glycosylated hemoglobin (available from Yinmet under the trade designation KAY 0025) to 3ug/ml, 100 uL per well, overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.L per well, 37℃for 1h, and the mixture was dried by shaking; adding diluted cell supernatant at 100. Mu.L/well, 37℃for 30min (1 h for part of supernatant); washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 mu L of each hole, and 30min at 37 ℃; washing with washing liquid for 5 times, and drying; adding a color development solution A (50 mu L/hole, containing citric acid, sodium acetate, acetanilide and carbamide peroxide), and adding a color development solution B (50 mu L/hole, containing citric acid, EDTA, 2Na+TMB and concentrated HCL) for 10min; adding stop solution (50 mu L/hole, EDTA.2Na+ concentrated H 2SO4); OD was read on the microplate reader at 450nm (reference 630 nm). The results showed that the reaction OD after 1000-fold dilution of the cell supernatant was still greater than 1.0, and that the reaction OD without cell supernatant was less than 0.1, indicating that antibodies produced after transient transformation of the plasmid were active on human glycosylated hemoglobin (purchased from Yimmett, cat. KAY 0025).
(2) Linearization of recombinant antibody expression plasmids
The following reagents were prepared: buffer 50 mu L, DNA mu g/tube, puv I enzyme 10 mu L, sterile water to 500 mu L, water bath at 37 ℃ for enzyme digestion overnight; firstly, extracting with equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25:24:1, and then sequentially extracting with chloroform (water phase); precipitating 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol on ice, rinsing the precipitate with 70% ethanol, removing organic solvent, completely volatilizing ethanol, re-thawing with appropriate amount of sterilized water, and measuring concentration.
(3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell strain
Diluting the plasmid to 40ug/100ul with ultrapure water, regulating CHO cells to 1.43X10 7 cells/ml in a centrifuge tube, mixing 100 mu L of plasmid with 700 mu L of cells, transferring into an electrorotating cup, electrorotating, and counting the next day; 25umol/L MSX 96-well pressure culture for about 25 days.
Observing the clone holes with the cells under a microscope, and recording the confluency; taking culture supernatant, and carrying out sample feeding detection; selecting cell strains with high antibody concentration and relative concentration, turning 24 holes, and turning 6 holes about 3 days; seed preservation batch culture is carried out after 3 days, cell density is adjusted to be 0.5X10 6 cells/ml, batch culture is carried out by 2.2ml, and seed preservation is carried out by 2ml, wherein the cell density is 0.3X10 6 cells/ml; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.
3 Recombinant antibody production
(1) Cell expansion culture
After cell recovery, the cells were first cultured in 125ml shake flasks with an inoculation volume of 30ml and a medium of 100% dynamis and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/ml inoculating density, and calculating the expanding culture volume according to production requirements, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/ml for production.
(2) Shake flask production and purification
Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8%. Feeding: feeding is started every day when the culture is carried out in a shake flask for 72 hours, hyCloneTM Cell BoostTM Feed a is fed with 3% of the initial culture volume every day, and the feeding amount of Feed 7b is one thousandth of the initial culture volume every day until the 12 th day (feeding on 12 th day). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 6 μg of purified antibody was subjected to reducing SDS-PAGE, the electrophoretogram is shown in FIG. 1, and two bands were shown after reducing SDS-PAGE, 1 Mr was 50KD and the other Mr was 28KD.
Example 2 affinity and Activity optimization
The Anti-HBA1C-10C7 monoclonal antibody obtained in example 1 has an ability to bind glycosylated hemoglobin, but has insufficient affinity and antibody activity, and thus the applicant has performed directed mutation on the light chain CDR and the heavy chain CDR of the antibody. The method comprises the steps of performing structural simulation of an antibody variable region, structural simulation of an antigen-antibody variable region acting complex, analysis of key amino acids of an antibody and mutation design by using a computer, designing and synthesizing a two-way primer covering a mutation site according to a mutation scheme, synthesizing primers at two ends of target DNA, performing high-fidelity PCR reaction, cloning a PCR product to a vector, and preparing the mutant antibody according to the method described in the example 1. Monoclonal antibodies with remarkably improved affinity and antibody activity are obtained through screening and are named as: anti-HBA1C-10C7mut1 to Anti-HBA1C-10C7mut7. The amino acid sequences of the respective monoclonal antibodies are shown in the following table:
TABLE 1 antibody sequences
Sample name Heavy chain sequence number Light chain sequence number
Anti-HBA1C-10C7mut1 SEQ ID NO:51 SEQ ID NO:56
Anti-HBA1C-10C7mut2 SEQ ID NO:53 SEQ ID NO:56
Anti-HBA1C-10C7mut3 SEQ ID NO:48 SEQ ID NO:56
Anti-HBA1C-10C7mut4 SEQ ID NO:43 SEQ ID NO:56
Anti-HBA1C-10C7mut5 SEQ ID NO:46 SEQ ID NO:56
Anti-HBA1C-10C7mut6 SEQ ID NO:40 SEQ ID NO:56
Anti-HBA1C-10C7mut7 SEQ ID NO:51 SEQ ID NO:57
Example 3 detection of Performance of antibodies
1 Affinity assay
Purified antibodies were diluted to 10ug/ml with PBST using AMC sensor, and human glycosylated hemoglobin (purchased from inflett, cat No. KAY 0025) was gradient diluted with PBST:
The operation flow is as follows: equilibration for 60s in buffer 1 (PBST), antibody 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM pH 1.69GLY solution and buffer 3 (PBST), output data. (KD represents equilibrium dissociation constant, i.e., affinity; kon represents binding rate; kdis represents dissociation rate. PBST major component Na2 HPO4+NaCl+TW-20).
Table 2 affinity data
Sample name KD(M) kon(1/Ms) kdis(1/s)
Control 2.52E-07 3.68E+04 9.28E-03
Anti-HBA1C-10C7mut1 2.38E-09 3.60E+04 8.57E-05
Anti-HBA1C-10C7mut2 1.98E-09 3.23E+04 6.40E-05
Anti-HBA1C-10C7mut3 1.71E-09 3.27E+04 5.60E-05
Anti-HBA1C-10C7mut4 1.85E-09 3.03E+04 6.20E-05
Anti-HBA1C-10C7mut5 2.39E-09 3.54E+04 8.46E-05
Anti-HBA1C-10C7mut6 2.32E-09 3.71E+04 8.62E-05
2 Activity assay
The coating solution (NaHCO 3 as the main ingredient) diluted human glycosylated hemoglobin (available from Yinmet under the trade designation KAY 0025) to 3ug/ml, 100 uL per well, overnight at 4 ℃; the next day, washing with washing liquid for 2 times, and drying; blocking solution (20% BSA+80% PBS) was added, 120. Mu.l per well, 37℃for 1h, and the mixture was dried by pipetting; adding the diluted monoclonal antibody into the mixture at the temperature of 37 ℃ for 30-60 min at the concentration of 100 mu l/hole; washing with washing liquid for 5 times, and drying; adding goat anti-mouse IgG-HRP, 100 μl per well, 37deg.C, 30min; washing with washing solution (PBS) for 5 times, and drying; adding a color development solution A (50. Mu.l/hole, containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), and adding a color development solution B (50. Mu.l/hole, containing 1.05g/L citric acid, 0.186 g/LEDTA.2Na, 0.45g/L TMB and 0.2ml/L concentrated HCl) for 10min; stop solution (50. Mu.l/well, 0.75 g/EDTA.2Na and 10.2ml/L concentrated H 2SO4) was added; OD was read on the microplate reader at 450nm (reference 630 nm). The results are shown in Table 3 below.
TABLE 3 Anti-HBA1C-10C7mut1-mut6 Activity data
3 Stability assessment
Placing the antibody at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days samples for state observation, and detecting the activity of the 21 days samples, wherein the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. Table 4 below shows the results of the detection of OD after 21 days of antibody examination.
TABLE 4 Anti-HBA1C-10C7mut1-mut6 stability data
Sample concentration (ng/ml) 62.5 3.91 0
4 ℃,21 Days sample 0.999 0.203 0.045
Sample at-80℃for 21 days 0.959 0.225 0.064
37 ℃ And 21 days of sample 0.965 0.218 0.021
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
The partial amino acid sequence related to the application is as follows:
Numbering device Sequence fragments
SEQ ID NO:1 SYDIN
SEQ ID NO:2 WIFPGDGSFQYNEKFKG
SEQ ID NO:3 GPWDFYG
SEQ ID NO:4 RSSTGAVTTSNYAN
SEQ ID NO:5 GTHNRAP
SEQ ID NO:6 VLWYSNHW
SEQ ID NO:15 SYDLN

Claims (40)

1. An anti-glycosylated hemoglobin antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof comprises HCDR1 with an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 15, HCDR2 with an amino acid sequence shown as SEQ ID NO. 2, HCDR3 with an amino acid sequence shown as SEQ ID NO. 3, and LCDR1, LCDR2, LCDR3 with amino acid sequences shown as SEQ ID NO. 4 to 6 in sequence.
2. The antibody or antigen-binding fragment thereof according to claim 1, further comprising an HFR1, an HFR2, an HFR3, an HFR4 having the amino acid sequences shown in SEQ ID No. 7 to SEQ ID No. 10, and an LFR1, an LFR2, an LFR3 and an LFR4 having the amino acid sequences shown in SEQ ID No. 11 to SEQ ID No. 14, or an amino acid sequence having at least 90% identity to each of said sequences.
3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of HFR1 of the antibody or antigen-binding fragment thereof is replaced with the amino acid sequence shown in SEQ ID No. 16.
4. The antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of HFR3 of the antibody or antigen-binding fragment thereof is replaced with the amino acid sequence shown in SEQ ID No. 17.
5. The antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of HFR4 of the antibody or antigen-binding fragment thereof is replaced with the amino acid sequence shown in SEQ ID No. 18.
6. The antibody or antigen-binding fragment thereof of claim 2, wherein the amino acid sequence of LFR3 of the antibody or antigen-binding fragment thereof is replaced with the amino acid sequence shown in SEQ ID No. 19.
7. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds glycosylated hemoglobin with an affinity of KD ∈2.52×10 -7 M.
8. An anti-glycosylated hemoglobin antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, HFR4, LFR1, HFR2, LFR3, LFR4 is the amino acid sequence of HFR1, HFR2, HFR3, LFR4, LFR3, LFR4 of claim 1, HFR2, HFR3, LFR 4.
9. An anti-glycosylated hemoglobin antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region having amino acid sequences as shown in SEQ ID No. 33 and SEQ ID No. 38, respectively; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 35 and SEQ ID NO. 38; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 30 and SEQ ID NO. 38; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 25 and SEQ ID NO. 38; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 28 or SEQ ID NO. 38; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 22 and SEQ ID NO. 38; or the amino acid sequences of the heavy chain variable region and the light chain variable region are respectively shown as SEQ ID NO. 33 or SEQ ID NO. 39.
10. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof further comprises a constant region.
11. The antibody or antigen-binding fragment thereof of claim 10, wherein the constant regions comprise a heavy chain constant region and a light chain constant region.
12. The antibody or antigen-binding fragment thereof of claim 11, wherein the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.
13. The antibody or antigen-binding fragment thereof of claim 10, wherein the constant region is of a species origin of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.
14. The antibody or antigen-binding fragment thereof of claim 10, wherein the constant region is of murine origin.
15. The antibody or antigen-binding fragment thereof of claim 11, wherein the heavy chain constant region sequence is as shown in SEQ ID No. 20 or has at least 80% identity thereto and the light chain constant region sequence is as shown in SEQ ID No. 21 or has at least 80% identity thereto.
16. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the antigen-binding fragment is selected from any one of F (ab ') 2, fab', fab, fv of the antibody.
17. The antibody or antigen-binding fragment thereof of claim 16, wherein the antigen-binding fragment is selected from the group consisting of scFv of the antibody.
18. An anti-glycosylated hemoglobin antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the heavy chain variable region of any one of claims 8 to 9 and the heavy chain constant region of any one of claims 11, 12, 15; the light chain comprises the light chain variable region of any one of claims 8 to 9 and the light chain constant region of any one of claims 11, 12, 15.
19. An anti-glycosylated hemoglobin antibody comprises a heavy chain and a light chain, and is characterized in that the heavy chain and the light chain have amino acid sequences shown as SEQ ID NO. 51 and SEQ ID NO. 56 respectively; or the heavy chain and the light chain are respectively shown as SEQ ID NO. 53 and SEQ ID NO. 56; or the heavy chain and the light chain are respectively shown as SEQ ID NO. 48 and SEQ ID NO. 56; or the heavy chain and the light chain are respectively shown as SEQ ID NO. 43 and SEQ ID NO. 56; or the heavy chain and the light chain are respectively shown as SEQ ID NO. 46 and SEQ ID NO. 56; or the heavy chain and the light chain are respectively shown as SEQ ID NO. 40 and SEQ ID NO. 56; or the heavy chain and the light chain have the amino acid sequences shown as SEQ ID NO. 51 and SEQ ID NO. 57 respectively.
20. An antibody conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 17 or the antibody of any one of claims 18 to 19; the antibody or antigen binding fragment thereof is labeled with a label.
21. The antibody conjugate of claim 20, wherein the label is selected from the group consisting of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.
22. The antibody conjugate of claim 21, wherein the fluorescent dye is selected from the group consisting of fluorescein-based dyes, rhodamine-based dyes, cy-based dyes, alexa-based dyes, and protein-based dyes.
23. The antibody conjugate of claim 21, wherein the enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
24. The antibody conjugate of claim 21, wherein the radioisotope is selected from the group consisting of 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 18 F.
25. The antibody conjugate of claim 21, wherein the chemiluminescent reagent is selected from the group consisting of luminol, luciferin, crustacean fluorescein, ruthenium bipyridine, acridinium esters, dioxane, lomustine and peroxyoxalate.
26. The antibody conjugate of claim 21, wherein the nanoparticle-based label is selected from the group consisting of a nanoparticle or a colloid.
27. The antibody conjugate of claim 26, wherein the nanoparticle comprises an organic nanoparticle, a magnetic nanoparticle, a quantum dot nanoparticle, and a rare earth complex nanoparticle.
28. The antibody conjugate of claim 26, wherein the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
29. The antibody conjugate of claim 28, wherein the colloidal metal is selected from the group consisting of colloidal gold, colloidal silver, and colloidal selenium.
30. The antibody conjugate of claim 20, wherein the antibody or antigen binding fragment thereof is coated onto a solid phase.
31. The antibody conjugate of claim 30, wherein the solid phase is selected from the group consisting of a microsphere, a plate, and a membrane.
32. The antibody conjugate of claim 30, wherein the solid phase is selected from the group consisting of magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.
33. A reagent or kit for detecting glycosylated hemoglobin, characterized in that the reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 or the antibody according to any one of claims 18 to 19 or the antibody conjugate according to any one of claims 20 to 32.
34. A nucleic acid encoding the antibody or antigen binding fragment thereof of any one of claims 1 to 17 or the antibody of any one of claims 18 to 19.
35. A vector comprising the nucleic acid of claim 34.
36. A cell comprising the nucleic acid of claim 34 or the vector of claim 35.
37. A method of preparing the antibody or antigen-binding fragment thereof of any one of claims 1 to 17 or the antibody of any one of claims 18 to 19, comprising: culturing the cell of claim 36.
38. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 or an antibody according to any one of claims 18 to 19 for the preparation of a reagent or kit for detecting glycosylated hemoglobin, comprising:
Contacting the antibody of any one of claims 1 to 17 or an antigen-binding fragment thereof or the antibody of any one of claims 18 to 19 with glycosylated hemoglobin in a sample to be tested to form an immune complex.
39. The use of claim 38, wherein the immune complex further comprises a second antibody that binds to the antibody or antigen binding fragment thereof.
40. The use of claim 38, wherein the immune complex further comprises a second antibody that binds to glycosylated hemoglobin.
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