CN117327177A - Anti-adiponectin antibody, reagent for detecting adiponectin and kit - Google Patents

Anti-adiponectin antibody, reagent for detecting adiponectin and kit Download PDF

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CN117327177A
CN117327177A CN202210724959.4A CN202210724959A CN117327177A CN 117327177 A CN117327177 A CN 117327177A CN 202210724959 A CN202210724959 A CN 202210724959A CN 117327177 A CN117327177 A CN 117327177A
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antibody
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孟媛
钟冬梅
覃文新
游辉
曹慧方
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Dongguan Pengzhi Biotechnology Co Ltd
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Dongguan Pengzhi Biotechnology Co Ltd
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    • 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
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • G—PHYSICS
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    • 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
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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    • C07K2317/515—Complete light chain, i.e. VL + CL
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • 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
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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    • G01N2333/575—Hormones
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/321—Arterial hypertension
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

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Abstract

The invention discloses an anti-adiponectin antibody, a reagent for detecting adiponectin and a kit, and relates to the technical field of antibodies. The anti-adiponectin 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 adiponectin and has improved affinity and activity.

Description

Anti-adiponectin antibody, reagent for detecting adiponectin and kit
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-adiponectin antibody, a reagent for detecting adiponectin and a kit.
Background
Adiponectin (APN), also known as Acrp30, GBP28, adipoQ, or Adiponectin, is a protein hormone secreted primarily by adipocytes. Human adiponectin monomer consists of 244 amino acids, including 3 regions: amino terminal signal sequence, collagen domain and carboxy terminal globular domain (globular domain of APN, gAd). Adiponectin exists in blood as three subtypes, trimer (65 kDa), hexamer (150 kDa) and high molecular weight (HMW, 18-36 monomers, > 280 kDa) polymer. The concentration range of the serum is 5-30 mg/L, and the serum accounts for about 0.01% of the total amount of human serum albumin.
The main biological function of adiponectin is to regulate energy metabolism, promote catabolism of glucose and fat, and improve insulin sensitivity. In recent years, more and more researches show that the adiponectin level is closely related to cardiovascular risk factors such as obesity, diabetes, hypertension, hyperlipidemia, atherosclerosis and insulin resistance, and the serum adiponectin level of patients is obviously reduced compared with healthy people. Adiponectin has the effects of enhancing insulin sensitivity, resisting Atherosclerosis (AS) formation, resisting inflammation and resisting intimal hyperplasia after vascular injury. Therefore, the detection of the content of adiponectin in blood is of great significance for understanding the physiological functions, regulation and control mechanisms and the relation between the adiponectin content in blood and the occurrence and development of diseases such as type II diabetes, coronary heart disease, hypertension and the like.
Currently, the detection method of adiponectin mainly includes a fluorescent immunochromatography method, which is an immunological detection method based on the specific reaction of an antibody and an antigen, and amplifying and displaying a detected signal by using fluorescence. Similar immunological detection methods include radioimmunoassay, enzyme-linked immunoassay, chemiluminescent method, etc. The immunological detection methods described above all require antibodies directed against adiponectin. Thus, there is a strong need in the art for antibodies that are effective and bind to and detect adiponectin.
In view of this, the present invention has been made.
Disclosure of Invention
The present application provides an anti-adiponectin antibody with increased affinity and/or activity to improve adiponectin detection, providing an important source of raw material for adiponectin detection.
In order to achieve the above object, according to one aspect of the present invention, there is provided an anti-adiponectin antibody or a functional fragment thereof, comprising:
a) HCDR1, HCDR2, HCDR3 with amino acid sequence shown in SEQ ID NO 1-3, and LCDR1 with amino acid sequence shown in SEQ ID NO 4, LCDR2 with amino acid sequence shown in SEQ ID NO 5, and LCDR3 with amino acid sequence shown in SEQ ID NO 6 or 7; or (b)
b) A heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO 10 to 11, and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO 12 to 15; or (b)
c) A heavy chain variable region and a light chain variable region having an amino acid sequence having 80% or more identity to the sequence set forth in b), and comprising HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence set forth in a); or (b)
d) A heavy chain with an amino acid sequence shown in any one of SEQ ID NO 16 to 17, and a light chain with an amino acid sequence shown in any one of SEQ ID NO 18 to 21.
In order to achieve the above object, according to a second 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 third aspect of the present invention, there is provided a reagent or kit for detecting adiponectin, comprising the antibody or functional fragment thereof or the antibody conjugate described above.
In order to achieve the above object, according to a fourth aspect of the present invention, there is provided a method for detecting adiponectin, comprising: contacting the antibody or functional fragment, conjugate or reagent or kit thereof with adiponectin in the sample to be detected to form an immune complex.
In order to achieve the above object, the present invention also provides a vector, a cell and a method for preparing the above antibody or a functional fragment thereof.
Drawings
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-APN 6C2mut1 to mut7.
Detailed Description
The present invention provides an anti-adiponectin antibody or functional fragment thereof comprising:
a) HCDR1, HCDR2, HCDR3 with amino acid sequences shown in SEQ ID NO 1-3, and LCDR1 with amino acid sequence shown in SEQ ID NO 4, LCDR2 with amino acid sequence shown in SEQ ID NO 5, and LCDR3 with amino acid sequence shown in SEQ ID NO 6 or 7. The antibodies have improved affinity and/or 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 responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. 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, and the 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 Kabat numbering schemes, which are generally considered as widely adopted criteria 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 another aspect, the invention provides an anti-adiponectin antibody or functional fragment thereof comprising:
b) A heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO 10 to 11, and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO 12 to 15. The antibodies have improved affinity and/or activity.
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 another aspect, the invention provides an anti-adiponectin antibody or functional fragment thereof comprising:
c) Heavy and light chain variable regions having an amino acid sequence having 80% or more identity to the sequence set forth in b) above, and comprising HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence set forth in a) above. The antibodies have improved affinity and/or activity.
In alternative embodiments, the antibody or functional fragment thereof comprises a heavy chain framework region in a heavy chain variable region as set forth in any one of SEQ ID NOS 10 to 11, and a light chain framework region in a light chain variable region as set forth in any one of SEQ ID NOS 12 to 15.
In alternative embodiments, the framework region amino acid sequence of the antibody or functional fragment thereof 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 framework region described above.
In an alternative embodiment, the antibody or functional fragment thereof has a KD of < 8.71×10 -08 The affinity of M binds adiponectin.
In an alternative embodiment, the antibody or functional fragment thereof has a KD of 10 or less -08 M、KD≤10 -09 M、KD≤10 -10 M、KD≤10 -11 M or KD is less than or equal to 10 -12 The affinity of M binds adiponectin.
In an alternative embodiment, the antibody or functional fragment thereof has a KD of 4.7X10 ∈ -09 The affinity of M binds adiponectin.
In an alternative embodiment, the antibody or functional fragment thereof has a KD of 3.7X10 ∈ -10 The affinity of M binds adiponectin.
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 heavy chain constant region sequence (CH) is shown in SEQ ID NO. 8 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 9.
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:8 or 9) 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-adiponectin antibody comprising:
d) A heavy chain with an amino acid sequence shown in any one of SEQ ID NO 16 to 17, and a light chain with an amino acid sequence shown in any one of SEQ ID NO 18 to 21. The antibodies have improved affinity and/or activity.
In another aspect, the invention provides an antibody conjugate comprising an antibody as described above. Wherein the antibody is directly or indirectly covalently coupled to the conjugate. Alternatively, the antibody is coupled to the conjugate to be conjugated in a non-covalent adsorption manner.
In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to biotin or a biotin derivative.
In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to 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 (chlorophyll), 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, 110 msin, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.
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, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metal includes, but is not limited to, colloidal gold or colloidal silver.
In an alternative embodiment, the colloidal metal is colloidal gold.
In an alternative embodiment, the antibody in the above antibody conjugate is conjugated to 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 invention provides a reagent or kit for detecting adiponectin, the reagent or kit comprising an antibody or functional fragment thereof as described above or an antibody conjugate as described above.
In another aspect, the invention provides the use of an antibody as described above or a functional fragment thereof, an antibody conjugate or a reagent or kit as described above in the detection of adiponectin.
In another aspect, the invention provides the use of an antibody or functional fragment thereof or antibody conjugate as described above for the preparation of a diagnostic reagent or diagnostic kit for a disease associated with adiponectin metabolism.
In alternative embodiments, the disease includes, but is not limited to, diabetes, coronary heart disease, hypertension, or atherosclerosis.
In another aspect, the invention provides a method of detecting adiponectin comprising: contacting the antibody or functional fragment thereof, antibody conjugate or reagent or kit with adiponectin in the sample to be tested to form an immune complex.
In an alternative embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.
In an alternative embodiment, the immune complex further comprises a second antibody, which binds to adiponectin.
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 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 et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (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 Vectors 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, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is 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-APN 6C2 monoclonal antibody
Restriction enzymes, prime Star DNA polymerase in this example were purchased from Takara Corp. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMARTTMRACE 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 strains secreting anti-adiponectin monoclonal antibodies, DNA products are obtained through an RT-PCR method, rTaq DNA polymerase is used for carrying out an A adding reaction on the products, the products are inserted into a pMD-18T vector and are transformed into DH5 alpha competent cells, after colonies grow out, the Heavy Chain gene and the Light Chain gene are respectively taken for cloning, and 4 clones are sent to a gene sequencing company for sequencing.
(2) Sequence analysis of Anti-APN 6C2 antibody variable region Gene
The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI11.5 software is utilized for analysis to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 321bp, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 360bp, belongs to the VH1 gene family, and a 57bp leader peptide sequence is arranged in front of the VH gene family.
(3) Construction of recombinant antibody expression plasmids
pcDNA TM 3.4vector is a constructed eukaryotic expression vector of the recombinant antibody, and the expression vector is introduced into a HindIII, bamHI, ecoRI polyclonal enzyme cutting site, 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 the pMD-18T, VL and VH gene specific primers of the antibody are designed, and the two ends of the primers are respectively provided with HindIII, ecoRI enzyme cutting sitesAnd a protecting base, and amplifying a Light Chain gene fragment of 0.73kb and a Heavy Chain gene fragment of 1.40kb by a PCR amplification method.
The Heavy Chain gene fragment and the Light Chain gene fragment are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, and the Heavy Chain gene fragment and the Light Chain gene fragment after the fragment and the vector are purified and recovered are respectively connected into the 3.4A expression vector to respectively obtain recombinant expression plasmids of the Heavy Chain gene fragment and the Light Chain gene fragment.
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 and CHO cells were conditioned to 1.43X 10 7 100. Mu.L of plasmid was mixed with 700. Mu.L of cells in a centrifuge tube, transferred to an electrocuvette, electroblotted, sample counted on days 3, 5, 7, and harvested on day 7.
Coating (main ingredient NaHCO 3) diluted adiponectin antigen (purchased from HYTEST,8AN 7) to 0.5ug/ml, 100 μl 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/well, EDTA. 2Na+ concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the 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 wells was less than 0.1, indicating that antibodies produced after transient plasmid transformation were active against adiponectin antigen.
(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
The plasmid was diluted to 40ug/100ul with ultrapure water and CHO cells were conditioned to 1.43X 10 7 Placing cells/ml in a centrifuge tube, mixing 100 μl of plasmid with 700 μl of cells, transferring into an electrorotor, 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 and batch culture are carried out after 3 days, and cell density is regulated to be 0.5x10 6 Batch culture was performed with cells/ml,2.2ml, and cell density was 0.3X10 6 Performing seed preservation by using cells/ml and 2 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 was started every day until 72h of culture in shake flasks, hyCloneTM Cell BoostTM Feed a fed-batch was 3% of the initial culture volume every day, feed 7b fed-batch was one thousandth of the initial culture volume every day, and fed-batch was continued until day 12 (day 12 Feed). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 3 μg of purified antibody was subjected to reducing SDS-PAGE, and the electrophoretogram shows two bands after reducing SDS-PAGE, 1 Mr at 50KD and the other Mr at 28KD.
Example 2 affinity and Activity optimization
The Anti-APN 6C2 monoclonal antibody obtained in example 1 had an ability to bind to an adiponectin antigen, but was not satisfactory in affinity and antibody activity, and thus, the applicant had 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-APN 6C2mut1 to Anti-APN 6C2mut7. The heavy and light chain amino acid sequences of each monoclonal antibody are shown in the following table, respectively.
TABLE 1 antibody sequences
Example 3 detection of Performance of antibodies
1 affinity assay
Using the AMC sensor, the purified antibodies were diluted to 10ug/ml with PBST and adiponectin antigen (from HYTEST,8AN 7) was diluted gradient 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 8.71E-08 1.06E+05 9.23E-03
Anti-APN 6C2mut1 3.74E-10 5.58E+06 2.09E-03
Anti-APN 6C2mut2 3.57E-09 3.42E+05 1.22E-03
Anti-APN 6C2mut3 4.72E-09 2.35E+05 1.11E-03
Anti-APN 6C2mut4 4.19E-10 3.22E+06 1.35E-03
Anti-APN 6C2mut5 9.81E-10 3.21E+06 3.15E-03
Anti-APN 6C2mut6 3.78E-10 5.61E+06 2.12E-03
Anti-APN 6C2mut7 3.75E-10 5.47E+06 2.05E-03
2 Activity assay
Coating (main ingredient NaHCO 3) diluted adiponectin antigen (purchased from HYTEST,8AN 7) to 0.5ug/ml, 100 μl 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;adding stop solution (50. Mu.l/well, 0.75 g/EDTA.2Na and 10.2ml/L of concentrated H) 2 SO 4 ) The method comprises the steps of carrying out a first treatment on the surface of the OD was read on the microplate reader at 450nm (reference 630 nm). The results are shown in the following table.
TABLE 3 Activity data
Sample concentration (ng/ml) 6.25 3.13 1.56 0.78 0.39 0
Control 1.802 1.425 0.802 0.451 0.261 0.055
Anti-APN 6C2mut1 2.332 2.264 2.063 1.792 1.019 0.074
Anti-APN 6C2mut2 2.281 2.196 2.035 1.792 1.082 0.021
Anti-APN 6C2mut3 2.225 2.141 1.784 1.315 0.786 0.042
Anti-APN 6C2mut4 2.412 2.336 2.241 2.275 1.676 0.078
Anti-APN 6C2mut5 2.269 2.220 2.062 2.004 1.595 0.083
Anti-APN 6C2mut6 2.232 2.065 1.967 1.691 0.932 0.043
Anti-APN 6C2mut7 2.301 2.115 1.956 1.702 1.019 0.074
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. The following table shows the OD results of the enzyme-free activity assay for 21 days of antibody assessment.
Table 4 stability data
Sample concentration (ng/ml) 0.78 0.39 0
4 ℃,21 days sample 1.532 0.712 0.027
Sample at-80℃for 21 days 1.550 0.730 0.031
37 ℃ and 21 days of sample 1.519 0.689 0.025
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 sequences referred to in this application are as follows:
sequence numbering Sequence fragments
SEQ ID NO:1 GYFIN
SEQ ID NO:2 RINPYNGDTFYNEKFKG
SEQ ID NO:3 GGGLQRPY
SEQ ID NO:4 KASDHVNNWLA
SEQ ID NO:5 GATNLET
SEQ ID NO:6 QQYWIS
SEQ ID NO:7 QQYWLS
SEQUENCE LISTING
<110> Dongguan City, pengzhi biotechnology Co., ltd
<120> anti-adiponectin antibody, and reagent and kit for detecting adiponectin
<130> P2022069CN01
<160> 21
<170> PatentIn version 3.5
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Gly Tyr Phe Ile Asn
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Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Glu Lys Phe Lys
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Gly
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Gly Gly Gly Leu Gln Arg Pro Tyr
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Lys Ala Ser Asp His Val Asn Asn Trp Leu Ala
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Gly Ala Thr Asn Leu Glu Thr
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Gln Gln Tyr Trp Ile Ser
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Gln Gln Tyr Trp Leu Ser
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Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
1 5 10 15
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
100 105 110
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
115 120 125
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
130 135 140
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
145 150 155 160
Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
165 170 175
Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
180 185 190
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
195 200 205
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
210 215 220
Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
225 230 235 240
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
260 265 270
Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
275 280 285
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
290 295 300
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
305 310 315 320
Ser Pro Gly Lys
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Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
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Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
20 25 30
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
65 70 75 80
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
85 90 95
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Phe Ile Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Ile Ser Ser Ser Ala Ala His
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Gly Arg Gly Gly Gly Leu Gln Arg Pro Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Pro
115 120
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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Phe Ile Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Leu Ser Ser Ser Ala Ala His
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Gly Arg Gly Gly Gly Leu Gln Arg Pro Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Pro
115 120
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Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly
1 5 10 15
Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Val Asn Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Thr Ser Val Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ile Ser Ala Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Asn
100 105
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Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly
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Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Val Asn Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Thr Ser Val Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ile Ser Ala Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Asn
100 105
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Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly
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Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Val Asn Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Thr Ser Val Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Leu Ser Ala Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Asn
100 105
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Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly
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Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Val Asn Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Thr Ser Val Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Leu Ser Ala Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Asn
100 105
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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Phe Ile Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Ile Ser Ser Ser Ala Ala His
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Gly Arg Gly Gly Gly Leu Gln Arg Pro Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Pro Ala Lys Thr Thr Pro Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser
180 185 190
Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys
210 215 220
Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val
245 250 255
Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe
260 265 270
Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro
290 295 300
Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val
305 310 315 320
Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335
Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys
340 345 350
Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp
355 360 365
Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro
370 375 380
Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser
385 390 395 400
Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala
405 410 415
Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His
420 425 430
His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440
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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Phe Ile Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Gly Asp Thr Phe Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Leu Ser Ser Ser Ala Ala His
65 70 75 80
Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Gly Arg Gly Gly Gly Leu Gln Arg Pro Tyr Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Pro Ala Lys Thr Thr Pro Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser
180 185 190
Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys
210 215 220
Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val
245 250 255
Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe
260 265 270
Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro
275 280 285
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro
290 295 300
Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val
305 310 315 320
Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
325 330 335
Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys
340 345 350
Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp
355 360 365
Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro
370 375 380
Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser
385 390 395 400
Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala
405 410 415
Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His
420 425 430
His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440
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Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly
1 5 10 15
Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Val Asn Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Thr Ser Val Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ile Ser Ala Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Asn Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
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Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly
1 5 10 15
Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Val Asn Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Thr Ser Val Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ile Ser Ala Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Asn Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 20
<211> 214
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 20
Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly
1 5 10 15
Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Val Asn Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Thr Ser Val Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Leu Ser Ala Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Asn Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 21
<211> 214
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 21
Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu Ser Val Ser Leu Gly
1 5 10 15
Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Val Asn Asn Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile
35 40 45
Ser Gly Ala Thr Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Thr Ser Val Gln Thr
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Leu Ser Ala Leu
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Asn Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210

Claims (11)

1. An anti-adiponectin antibody or functional fragment thereof, comprising:
a) HCDR1, HCDR2, HCDR3 with amino acid sequence shown in SEQ ID NO 1-3, and LCDR1 with amino acid sequence shown in SEQ ID NO 4, LCDR2 with amino acid sequence shown in SEQ ID NO 5, and LCDR3 with amino acid sequence shown in SEQ ID NO 6 or 7; or (b)
b) A heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NO 10 to 11, and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NO 12 to 15; or (b)
c) A heavy chain variable region and a light chain variable region having an amino acid sequence having 80% or more identity to the sequence set forth in b), and comprising HCDR1 to HCDR3 and LCDR1 to LCDR3 of the sequence set forth in a); or (b)
d) A heavy chain with an amino acid sequence shown in any one of SEQ ID NO 16 to 17, and a light chain with an amino acid sequence shown in any one of SEQ ID NO 18 to 21.
2. An antibody or functional fragment thereof according to claim 1, wherein it is capable ofAlternatively, the antibody or functional fragment thereof is produced with a KD < 8.71×10 -08 The affinity of M binds adiponectin.
3. The antibody or functional fragment thereof according to any one of claims 1 to 2, wherein the antibody or functional fragment thereof further comprises a constant region;
optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region;
alternatively, 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 a kappa-type or lambda-type light chain constant region;
alternatively, the constant region is of bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin;
alternatively, the constant region is of mouse species origin;
alternatively, the heavy chain constant region sequence is as shown in SEQ ID NO. 8 or at least 80% identical thereto;
alternatively, the light chain constant region sequence is as shown in SEQ ID NO. 9 or at least 80% identical thereto.
4. An antibody or functional fragment thereof according to any one of claims 1 to 3, wherein the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv and scFv of the antibody.
5. An antibody conjugate comprising the antibody or functional fragment thereof of any one of claims 1 to 4;
optionally, the antibody is conjugated to biotin or a biotin derivative;
optionally, the antibody is coupled to a solid phase;
optionally, the antibody is conjugated to a label;
optionally, the label is selected from the group consisting of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels; preferably colloidal gold.
6. A reagent or kit for detecting adiponectin, comprising the antibody or functional fragment thereof according to any one of claims 1 to 4 or the antibody conjugate according to claim 5.
7. A method for detecting adiponectin, comprising:
contacting the antibody of any one of claims 1 to 4, a functional fragment thereof, the antibody conjugate of claim 5 or the reagent or kit of claim 6 with adiponectin in a sample to be detected to form an immune complex;
preferably, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof;
preferably, the immune complex further comprises a second antibody, which binds to adiponectin.
8. A nucleic acid encoding the antibody or functional fragment thereof of any one of claims 1 to 4.
9. A vector comprising the nucleic acid of claim 8.
10. A cell comprising the nucleic acid of claim 8 or the vector of claim 9.
11. A method of preparing an antibody or functional fragment thereof according to any one of claims 1 to 4, comprising: culturing the cell of claim 10.
CN202210724959.4A 2022-06-24 2022-06-24 Anti-adiponectin antibody, reagent for detecting adiponectin and kit Pending CN117327177A (en)

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