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CN119320450B - Antibodies against calcitonin gene-related peptide - Google Patents

Antibodies against calcitonin gene-related peptide

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Publication number
CN119320450B
CN119320450B CN202411835080.2A CN202411835080A CN119320450B CN 119320450 B CN119320450 B CN 119320450B CN 202411835080 A CN202411835080 A CN 202411835080A CN 119320450 B CN119320450 B CN 119320450B
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antibody
seq
antigen
binding fragment
amino acid
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CN119320450A (en
Inventor
乔春霞
邢聪
冯健男
罗龙龙
陈国江
李新颖
肖鹤
于吉军
刘成华
沈倍奋
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Academy of Military Medical Sciences AMMS of PLA
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Academy of Military Medical Sciences AMMS of PLA
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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
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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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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    • 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
    • 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
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    • 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
    • C07K2317/567—Framework region [FR]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/5753—Calcitonin gene related peptide

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Abstract

本发明公开了针对降钙素基因相关肽的抗体，属于生物医药领域，本发明提供了一种抗体或其抗原结合片段，包含其的核酸分子，包含核酸分子的载体，包含核酸分子或载体的细胞，本发明还提供了与前述抗体或其抗原结合片段相关的产品和药物组合物以及相关的应用与方法。

The present invention discloses an antibody against calcitonin gene-related peptide, belonging to the field of biomedicine. The present invention provides an antibody or an antigen-binding fragment thereof, a nucleic acid molecule comprising the antibody, a vector comprising the nucleic acid molecule, and a cell comprising the nucleic acid molecule or the vector. The present invention also provides products and pharmaceutical compositions related to the above-mentioned antibody or the antigen-binding fragment thereof, as well as related applications and methods.

Description

Antibodies against calcitonin gene-related peptide
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an antibody aiming at calcitonin gene related peptide.
Background
Calcitonin gene-RELATED PEPTIDE (CGRP), a 37 amino acid peptide widely expressed in the central and peripheral nervous systems, belongs to the calcitonin polypeptide family and plays an important role in regulating metabolism, inflammatory response, vascular tone, pain, etc. in sensory neurotransmission. The main advantages of CGRP receptor antagonists are more obvious efficacy, less adverse reactions and no addiction, so that the use of most analgesics is not contraindicated.
Compared with chemical small molecules, the antibody antagonizing CGRP and the receptor thereof has higher specificity and affinity, longer half-life and smaller hepatotoxicity, can prolong the medication interval, improve the compliance of patients and improve the drug effect. Therefore, developing antibodies against CGRP has good application prospects.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides the following technical scheme.
The present invention provides an antibody or antigen-binding fragment thereof comprising:
heavy chain variable region complementarity determining region:
VH CDR1 comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO. 1,
VH CDR2 comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID No.2,
VH CDR3 comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID No. 3,
Light chain variable region complementarity determining region:
VL CDR1 comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO. 9,
VL CDR2 comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO. 10,
VL CDR3 comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID No. 11;
The antibodies or antigen binding fragments thereof specifically bind to and/or neutralize calcitonin gene-related peptide.
Further, the amino acid sequence of the VH CDR1 is shown as SEQ ID NO. 1,
The amino acid sequence of the VH CDR2 is shown as SEQ ID NO. 2,
The amino acid sequence of the VH CDR3 is shown as SEQ ID NO. 3,
The amino acid sequence of VL CDR1 is shown as SEQ ID NO 9,
The amino acid sequence of VL CDR2 is shown as SEQ ID NO. 10,
The amino acid sequence of VL CDR3 is shown in SEQ ID NO. 11.
Further, the antibody or antigen-binding fragment thereof also includes,
Heavy chain variable region framework regions:
VH FR1 comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID No. 4,
VH FR2 comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID No. 5,
VH FR3 comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID No. 6,
VH FR4 comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID No. 7,
Light chain variable region framework regions:
VL FR1 comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO. 12,
VL FR2 comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO. 13,
VL FR3 comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO. 14,
VL FR4 comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence shown in SEQ ID NO. 15.
Further, the amino acid sequence of the VH FR1 is shown as SEQ ID NO. 4,
The amino acid sequence of the VH FR2 is shown as SEQ ID NO. 5,
The amino acid sequence of the VH FR3 is shown as SEQ ID NO. 6,
The amino acid sequence of the VH FR4 is shown as SEQ ID NO. 7,
The amino acid sequence of VL FR1 is shown as SEQ ID NO. 12,
The amino acid sequence of VL FR2 is shown as SEQ ID NO. 13,
The amino acid sequence of VL FR3 is shown as SEQ ID NO. 14,
The amino acid sequence of VL FR4 is shown as SEQ ID NO. 15.
Further, the heavy chain variable region VH of the antibody or antigen binding fragment thereof has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence shown in SEQ ID NO. 8,
The light chain variable region VL has an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence shown in SEQ ID NO. 16.
Further, the amino acid sequence of the heavy chain variable region VH is shown as SEQ ID NO. 8,
The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO. 16.
Further, the antibody or antigen binding fragment thereof further comprises a heavy chain variable region signal peptide having an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 17.
Further, the antibody or antigen binding fragment thereof also includes a light chain variable region signal peptide having an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 18.
Further, the amino acid sequence of the heavy chain variable region signal peptide in the antibody or the antigen binding fragment thereof is shown as SEQ ID NO. 17.
Further, the amino acid sequence of the light chain variable region signal peptide in the antibody or the antigen binding fragment thereof is shown as SEQ ID NO. 18.
Further, the antibody or antigen binding fragment thereof also comprises a heavy chain constant region, and the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO. 19.
Further, the antibody or antigen binding fragment thereof also comprises a light chain constant region, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 20.
Further, the antibody or antigen-binding fragment thereof also includes a defucosylated antibody or antigen-binding fragment thereof.
In the present invention, the term "identity" is synonymous with "sequence identity" and refers to the degree of identity between two sequences, and specific calculation rules are that two optimally aligned sequences in a window are aligned, the number of positions at which the same nucleobase (e.g., A, T, C, G, I) or the same amino acid residue (e.g., ala, pro, ser, thr, gly, val, leu, ile, phe, tyr, trp, lys, arg, his, asp, glu, asn, gln, cys and Met) occurs in the two sequences is determined to yield the number of matched positions, the number of matched positions is divided by the total number of positions in the window of comparison (i.e., window size), and the result is multiplied by 100 to yield the percent sequence identity.
Further, the antibodies include diabodies, fab ', F (ab') 2, fd, fv fragments, disulfide stabilized Fv fragments, (dsFv) 2, dual specificity dsFv, disulfide stabilized diabodies, single chain antibody molecules, scFv dimers, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.
The present invention provides nucleic acid molecules encoding the antibodies or antigen binding fragments thereof described previously.
Further, the nucleic acid molecule encodes a nucleotide sequence of VH CDR1, VH CDR2, VH CDR3 in an antibody or antigen binding fragment thereof, which has a nucleotide sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequences shown in SEQ ID NO. 21, 22, 23, respectively,
Nucleotide sequences encoding VL CDR1, VL CDR2, VL CDR3 in an antibody or antigen binding fragment thereof, which have nucleotide sequences at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequences shown in SEQ ID NOs 29, 30, 31, respectively.
Further, the nucleotide sequences of the VH CDR1, the VH CDR2 and the VH CDR3 in the nucleic acid molecule encoding antibody or antigen binding fragment thereof are respectively shown as SEQ ID NO. 21, 22 and 23,
Nucleotide sequences encoding VL CDR1, VL CDR2, VL CDR3 of an antibody or antigen binding fragment thereof are shown in SEQ ID NOS 29, 30, 31, respectively.
Further, the nucleic acid molecule encodes a nucleotide sequence of VH FR1, VH FR2, VH FR3, VH FR4 in an antibody or antigen binding fragment thereof, which has a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence shown in SEQ ID NO. 24, 25, 26, 27, respectively,
Nucleotide sequences encoding VL FR1, VL FR2, VL FR3, VL FR4 in the antibody or antigen binding fragment thereof, which have nucleotide sequences at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequences shown in SEQ ID NOs 32, 33, 34, 35, respectively.
Further, the nucleotide sequences of the VH FR1, the VH FR2, the VH FR3 and the VH FR4 in the nucleic acid molecule encoding antibody or antigen binding fragment thereof are respectively shown as SEQ ID NO. 24, 25, 26 and 27,
Nucleotide sequences encoding VL FR1, VL FR2, VL FR3 and VL FR4 of the antibody or antigen-binding fragment thereof are shown in SEQ ID NOS 32, 33, 34 and 35, respectively.
Further, the nucleic acid molecule encodes a heavy chain variable region of an antibody or antigen binding fragment thereof having a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence set forth in SEQ ID NO. 28,
A light chain variable region encoding an antibody or antigen binding fragment thereof having a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence set forth in SEQ ID No. 36.
Further, the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule encoding the antibody or antigen binding fragment thereof is shown as SEQ ID NO. 28,
The nucleotide sequence of the light chain variable region of the nucleic acid molecule encoding the antibody or antigen binding fragment thereof is shown in SEQ ID NO. 36.
Further, the nucleic acid molecule encodes a heavy chain variable region signal peptide in an antibody or antigen binding fragment thereof having a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence set forth in SEQ ID NO. 37,
A light chain variable region signal peptide encoding an antibody or antigen binding fragment thereof, having a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence set forth in SEQ ID No. 38.
Further, the nucleotide sequence of the heavy chain variable region signal peptide in the nucleic acid molecule encoding antibody or antigen binding fragment thereof is shown as SEQ ID NO. 37,
The nucleotide sequence of the light chain variable region signal peptide in the coded antibody or antigen binding fragment thereof is shown as SEQ ID NO. 38.
Further, the nucleotide sequence of the heavy chain constant region in the nucleic acid molecule encoding antibody or antigen binding fragment thereof is shown as SEQ ID NO. 39;
the nucleotide sequence encoding the light chain constant region of the antibody or antigen binding fragment thereof is shown in SEQ ID NO. 40.
Further, the nucleic acid molecule is codon optimized.
In some embodiments, the nucleotide sequence in the nucleic acid molecule is substituted with a conservative nucleic acid. Conserved nucleic acids refer to those nucleic acids that encode amino acid sequences that are identical or related in basic structure and sequence, if the nucleic acid molecule does not encode an amino acid sequence. In the substitution of conserved nucleic acids, the degeneracy of the genetic code applies.
The present invention provides a vector comprising a nucleic acid molecule as described above.
Further, the vector also includes elements for controlling expression.
Further, the carrier also includes a material that aids in the entry of the carrier into the cell.
In some embodiments, the most commonly used type of vector is a plasmid. In other embodiments, the vector is a viral vector, specifically including common retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses, poxviruses, baculoviruses, papillomaviruses, and the like.
The present invention provides a cell comprising a nucleic acid molecule as described above or a vector as described above.
Further, the cells include prokaryotic cells and eukaryotic cells.
Further, the prokaryotic cells include gram-negative or gram-positive cells.
Further, the eukaryotic cells include protozoan cells, animal cells, fungal cells.
Further, the animal cells include mammalian cells, avian cells, insect cells.
Further, the mammalian cells include 293F cells, 293T cells, CHO cells, AT1080 cells, a549 cells.
In some embodiments, the mammalian cells include human and non-human mammalian cells, which refer to fetal animal cells other than humans, including murine cells, sheep cells, rabbit cells, horse cells, bovine cells, porcine cells, dog cells, monkey cells, and the like.
The present invention provides a product for detecting a calcitonin gene-related peptide, said product comprising an antibody or antigen-binding fragment thereof as described hereinbefore.
Further, the product may further comprise reagents for performing antigen-antibody reactions or reagents for detecting reactions.
Further, the reagent for performing an antigen-antibody reaction includes a buffer, a salt.
Further, the product includes a conjugate of an antibody linked to a detectable label.
Further, the product comprises a kit and a test strip.
The present invention provides a pharmaceutical composition for preventing or treating inflammatory pain, comprising an antibody or antigen-binding fragment thereof as described above, or a nucleic acid molecule as described above, or a vector as described above, or a cell as described above.
Further, the inflammatory pain includes migraine, visceral inflammatory pain in the peritoneum.
Further, the migraine includes chronic migraine, migraine without aura, tension headache, and cluster pain.
Further, the pharmaceutical composition further comprises pharmaceutically acceptable adjuvants.
Further, the pharmaceutical composition also includes other antibodies.
The invention provides any one of the following applications.
The invention provides the use of an antibody or antigen binding fragment thereof as described hereinbefore, a nucleic acid molecule as described hereinbefore, a vector as described hereinbefore, or a cell as described hereinbefore in the manufacture of a product for detecting a calcitonin gene-related peptide.
The present invention provides the use of an antibody or antigen binding fragment thereof as described above, a nucleic acid molecule as described above, a vector as described above, or a cell as described above for the preparation of a pharmaceutical composition for the prevention and/or treatment of inflammatory pain.
Further, the inflammatory pain includes migraine, visceral inflammatory pain in the peritoneum.
Further, the migraine includes chronic migraine, migraine without aura, tension headache, and cluster pain.
Further, the pharmaceutical composition is used for Balb/c mice inflammatory pain.
The present invention provides any one of the following methods.
The present invention provides a method of producing an antibody or antigen-binding fragment thereof as described above, comprising culturing the cells as described above, and recovering the antibody or antigen-binding fragment thereof.
The present invention provides a method for detecting a calcitonin gene-related peptide in a sample for non-diagnostic purposes, which comprises contacting the sample to be tested with the aforementioned antibody or antigen-binding fragment thereof, thereby detecting the level of the calcitonin gene-related peptide in the sample to be tested.
Drawings
FIG. 1 is a diagram showing the result of SDS-PAGE detection of antibodies.
FIG. 2 is a graph of the results of antibody SEC-HPLC detection.
FIG. 3 is a graph of the results of an antibody binding assay, wherein A is 5-fold dilution from 4ug/ml for ELISA assay, and B is 2-fold dilution from 100ug/ml for increasing the initial concentration of antibody.
Fig. 4 is a graph of the results of a torsion body experiment in mice with visceral inflammatory pain in the peritoneum treated with antibodies.
Detailed Description
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 invention belongs.
The invention is further illustrated below in connection with specific embodiments. It should be understood that the particular embodiments described herein are presented by way of example and not limitation. The principal features of the invention may be used in various embodiments without departing from the scope of the invention.
EXAMPLE 1 ZRQ1Ab antibody preparation and purification method
1. Experimental method
Expression method CHO-S cell expression.
The heavy and light chain genes of the antibodies were synthesized, the specific sequences were as shown in Table 1, and optimized to accommodate the expression of CHO-S cells, the CHO-S cells were transfected, cultured for 8-10 days, the expression levels of the antibodies were detected by ELISA, and the antibodies were harvested at the appropriate time. Then purifying by a protein purifier, and concentrating. Finally, the purity of the antibodies was verified by SDS and HPLC.
Wherein transfection is performed when CHO-S cells are cultured to a suitable confluence and the viability is above 95%. High quality plasmid DNA, OD260/OD280 ratio between 1.8-2.0, concentration of hundreds of nanograms/microliter to microgram/microliter. Under aseptic conditions, an appropriate amount of plasmid DNA was diluted into serum-free medium and gently mixed. Appropriate amounts of transfection reagent were also diluted into serum-free medium and gently mixed. The diluted plasmid DNA solution and transfection reagent solution are slowly mixed, gently mixed upside down, and left to stand at room temperature for a certain period of time (usually 1-5 minutes) to form a plasmid-transfection reagent complex. Added to CHO-S cell shake flasks and slowly added with shaking. The cell shake flask was placed back on the cell shake flask and the culture was continued with 37 ℃ 125r,8% co 2.
ELISA to detect antibody expression levels ELISA plates with good adsorption, low blank values, high transparency at the bottom of wells were selected and CGRP protein was diluted to the appropriate concentration (e.g., 2. Mu.g/ml) using coating buffer (e.g., pH7.2-7.4 phosphate buffer) and added to a volume (e.g., 0.1 ml) per well and coated overnight at 4℃or 37℃for 2 hours. After coating, the non-specific binding sites are blocked with a blocking solution (e.g., 5% BSA), background noise is reduced, a volume (e.g., 0.20 ml) is added per well, and blocking is performed at 37℃for 1-2 hours. The diluted antibody to be tested is added and incubated for 1 hour at 37℃in a given volume (e.g., 0.1 ml) per well. Unbound sample is removed by washing 3 times for 1 minute with wash buffer (e.g., PBST). Enzyme-labeled secondary antibody was added to a volume (e.g., 0.1 ml) per well and incubated at 37℃for 30-60 minutes. The unbound enzyme-labeled antibody was removed by washing again 3 times with washing buffer for 1 minute each. Enzyme substrate (e.g., TMB) was added to a volume (e.g., 0.1 ml) per well and incubated at 37℃for 1-15 minutes in the dark until a significant color gradient change was observed. Stop solution (e.g., 2M sulfuric acid) is added to stop the enzyme reaction at a certain volume (e.g., 0.1 ml) per well. The absorbance (OD value) of each well was measured at a wavelength of 450nm using an enzyme-labeled instrument, and the concentration of the antibody in the sample was calculated from a standard curve.
SDS-PAGE, in which an antibody sample is mixed with a loading buffer containing SDS (sodium dodecyl sulfate) and a reducing agent (e.g., beta-mercaptoethanol) to disrupt the spatial structure and disulfide bonds of the protein and depolymerize the protein into polypeptide chains. The mixing ratio of sample and loading buffer is typically 4:1 or 5:1. The mixed sample was heated in boiling water for 5-10 minutes to completely denature the protein. Polyacrylamide gels are typically prepared comprising two layers, a concentrated gel (5% strength) and a split gel (the strength is chosen based on the molecular weight of the protein, e.g., 10%, 12%, etc.). After the gel preparation is completed, the gel is placed in an electrophoresis tank, and an electrophoresis buffer solution is added. The treated sample and a protein molecular weight standard (Marker) were added to the sample wells of the gel, respectively. And (5) switching on a power supply to start electrophoresis. During electrophoresis, protein molecules separate in gels of different concentrations according to their molecular weight and charge. The electrophoresis conditions are usually that the voltage of the gel concentration stage is 80-100V, the voltage of the gel separation stage is 120-150V, and the electrophoresis time is determined according to the migration condition of the bromophenol blue indicator. After electrophoresis is completed, the gel is removed and stained with coomassie blue staining solution, typically for 1-2 hours. After staining, the protein bands were evident by decolorizing with a decolorizing solution until the background was clear. The protein bands on the gel were observed, indicating higher antibody purity if the antibody sample had only one distinct band and the molecular weight was consistent with the expectations.
HPLC, preparing a sample of the antibody to be tested, ensuring that it is dissolved in the appropriate solvent and brought to the appropriate concentration. The appropriate chromatographic column and mobile phase are chosen according to the nature of the antibody. Common chromatographic columns include reverse phase columns, ion exchange columns, and the like. The HPLC system was equilibrated with the same buffer as the sample dilution until baseline stabilized. The prepared sample is loaded into the chromatographic system by an autosampler or manually. The antibody will elute sequentially according to its size and nature at a constant flow rate through the column. The elution profile is recorded by a detector, such as an ultraviolet detector. The elution profile is analyzed to identify protein peaks and possible impurity peaks. The purity of the antibodies was calculated by quantitative analysis of the peak area integration.
TABLE 1
2. Experimental results
After antibody culture using CHO-S cells (using the Human IgG1-Kappa tag), its expression level was 283.60mg/L. Molecular analysis was then used to obtain a predicted MW of 49/23kDa under antibody reduction conditions and 144kDa under non-reduction conditions.
The purity of the antibody under reducing conditions was then detected to be greater than 95% using SDS-PAGE, and the results are shown in FIG. 1. Antibody purity was measured using SEC-HPLC and found to be 98.463% pure, as shown in figure 2. Endotoxin levels were measured at 0-1EU/mg.
The result of binding of antibody ZRQ1Ab is shown in FIG. 3, and the IC50 value thereof was 15.45. Mu.g/ml. ZR8Ab was Galcanezuma, a Gift's commercial antibody, as a positive control.
Example 2 animal torsion assay for determining therapeutic Functions of ZRQ1Ab antibodies
1. Experimental method
The acetic acid induced abdominal contractions (writhing) test is a model of visceral inflammatory pain in the peritoneum. Acetic acid activates visceral and somatic nociceptors of the peritoneum, causing inflammation and tissue damage of the abdominal wall muscles and internal organs, thereby causing central as well as peripheral sensitization and hyperalgesia. In mice, the nociceptive cascade following acetic acid administration results in repeated abdominal contractions or twists.
The Balb/c mice were used for 20+ -2 g each (n=4 per group), the ICR mice were 40 g+ -2 g (n=4 per group), acetic acid as material was injected, and the antibodies (5 mg/Kg) were injected into the control group by subcutaneous injection of the antibodies (5 mg/Kg) each day before the experiment according to the method of using the marketed drug. The following day, mice were acclimatized in plexiglas cages for 15 minutes and injected intraperitoneally with 100 μl/10g of 0.6% acetic acid solution (v/v).
Typical torsion responses were recorded within 30 minutes after acetic acid injection, characterized by longitudinal extension of the trunk, concave extension of the back, and extension of the hind limbs, ending with a torsion at the time of recovery from normal posture.
2. Experimental results
The torsion test results are shown in fig. 4, and the results show that compared with the control group, the torsion strength of the Balb/c mouse can be remarkably reduced by using the ZRQ1Ab antibody, and the specific results are shown in table 2.
TABLE 2
Table 3 shows the effect of using different antibodies on the torque strength in ICR mice, and the use of ZRQ1Ab antibody according to the present invention did not act to reduce the torque strength in ICR mice, and the specific results are shown in FIG. 4.
TABLE 3 Table 3
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (38)

1.一种抗体或其抗原结合片段，所述抗体或其抗原结合片段包括：1. An antibody or an antigen-binding fragment thereof, comprising: 重链可变区互补决定区：Heavy chain variable region complementarity determining region: VH CDR1的氨基酸序列如SEQ ID NO:1所示，The amino acid sequence of VH CDR1 is shown in SEQ ID NO: 1, VH CDR2的氨基酸序列如SEQ ID NO:2所示，The amino acid sequence of VH CDR2 is shown in SEQ ID NO: 2, VH CDR3的氨基酸序列如SEQ ID NO:3所示，The amino acid sequence of VH CDR3 is shown in SEQ ID NO: 3, 轻链可变区互补决定区：Light chain variable region complementarity determining region: VL CDR1的氨基酸序列如SEQ ID NO:9所示，The amino acid sequence of VL CDR1 is shown in SEQ ID NO:9, VL CDR2的氨基酸序列如SEQ ID NO:10所示，The amino acid sequence of VL CDR2 is shown in SEQ ID NO: 10, VL CDR3的氨基酸序列如SEQ ID NO:11所示；The amino acid sequence of VL CDR3 is shown in SEQ ID NO: 11; 所述抗体或其抗原结合片段特异性的结合降钙素基因相关肽和/或中和降钙素基因相关肽。The antibody or antigen-binding fragment thereof specifically binds to the CGRP and/or neutralizes the CGRP. 2.如权利要求1所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段还包括，2. The antibody or antigen-binding fragment thereof according to claim 1, further comprising: 重链可变区框架区：Heavy chain variable region framework region: VH FR1的氨基酸序列如SEQ ID NO:4所示，The amino acid sequence of VH FR1 is shown in SEQ ID NO:4, VH FR2的氨基酸序列如SEQ ID NO:5所示，The amino acid sequence of VH FR2 is shown in SEQ ID NO:5, VH FR3的氨基酸序列如SEQ ID NO:6所示，The amino acid sequence of VH FR3 is shown in SEQ ID NO:6, VH FR4的氨基酸序列如SEQ ID NO:7所示，The amino acid sequence of VH FR4 is shown in SEQ ID NO:7, 轻链可变区框架区：Light chain variable region framework region: VL FR1的氨基酸序列如SEQ ID NO:12所示，The amino acid sequence of VL FR1 is shown in SEQ ID NO: 12, VL FR2的氨基酸序列如SEQ ID NO:13所示，The amino acid sequence of VL FR2 is shown in SEQ ID NO: 13, VL FR3的氨基酸序列如SEQ ID NO:14所示，The amino acid sequence of VL FR3 is shown in SEQ ID NO: 14, VL FR4的氨基酸序列如SEQ ID NO:15所示。The amino acid sequence of VL FR4 is shown in SEQ ID NO:15. 3.如权利要求1所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段的重链可变区VH的氨基酸序列如SEQ ID NO:8所示，3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the amino acid sequence of the heavy chain variable region VH of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 8, 轻链可变区VL的氨基酸序列如SEQ ID NO:16所示。The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:16. 4.如权利要求1所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段还包括重链可变区信号肽，所述重链可变区信号肽的氨基酸序列如SEQ ID NO:17所示。4. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a heavy chain variable region signal peptide, the amino acid sequence of the heavy chain variable region signal peptide being as shown in SEQ ID NO: 17. 5.如权利要求1所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段还包括轻链可变区信号肽，所述轻链可变区信号肽的氨基酸序列如SEQ ID NO:18所示。5. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a light chain variable region signal peptide, the amino acid sequence of the light chain variable region signal peptide being as shown in SEQ ID NO: 18. 6.如权利要求1所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段还包括重链恒定区，所述重链恒定区的氨基酸序列如SEQ ID NO:19所示。6. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a heavy chain constant region, the amino acid sequence of the heavy chain constant region being as shown in SEQ ID NO: 19. 7.如权利要求1所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段还包括轻链恒定区，所述轻链恒定区的氨基酸序列如SEQ ID NO:20所示。7. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a light chain constant region, the amino acid sequence of the light chain constant region being as shown in SEQ ID NO: 20. 8.如权利要求1所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段还包括去岩藻糖基化的抗体或其抗原结合片段。8. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a defucosylated antibody or antigen-binding fragment thereof. 9.核酸分子，所述核酸分子编码权利要求1-8任一项所述的抗体或其抗原结合片段。9. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. 10.如权利要求9所述的核酸分子，所述核酸分子编码抗体或其抗原结合片段中的VHCDR1、VH CDR2、VH CDR3的核苷酸序列，其分别具有与SEQ ID NO:21、22、23所示的核苷酸序列至少95%、至少96%、至少97%、至少98%、至少99%相同性的核苷酸序列，10. The nucleic acid molecule of claim 9, wherein the nucleic acid molecule encodes the nucleotide sequences of VH CDR1, VH CDR2, and VH CDR3 in the antibody or antigen-binding fragment thereof, and has a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 21, 22, and 23, respectively. 编码抗体或其抗原结合片段中的VL CDR1、VL CDR2、VL CDR3的核苷酸序列，其分别具有与SEQ ID NO:29、30、31所示的核苷酸序列至少95%、至少96%、至少97%、至少98%、至少99%相同性的核苷酸序列。The nucleotide sequences encoding VL CDR1, VL CDR2, and VL CDR3 in the antibody or antigen-binding fragment thereof have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequences shown in SEQ ID NOs: 29, 30, and 31, respectively. 11.如权利要求9所述的核酸分子，所述核酸分子编码抗体或其抗原结合片段中的VHCDR1、VH CDR2、VH CDR3的核苷酸序列分别如SEQ ID NO:21、22、23所示，11. The nucleic acid molecule according to claim 9, wherein the nucleotide sequences of VH CDR1, VH CDR2, and VH CDR3 in the antibody or antigen-binding fragment thereof are as shown in SEQ ID NOs: 21, 22, and 23, respectively, 编码抗体或其抗原结合片段中的VL CDR1、VL CDR2、VL CDR3的核苷酸序列分别如SEQID NO:29、30、31所示。The nucleotide sequences encoding VL CDR1, VL CDR2, and VL CDR3 in the antibody or its antigen-binding fragment are shown in SEQ ID NOs: 29, 30, and 31, respectively. 12.如权利要求9所述的核酸分子，所述核酸分子编码抗体或其抗原结合片段中的VHFR1、VH FR2、VH FR3、VH FR4的核苷酸序列，其分别具有与SEQ ID NO:24、25、26、27所示的核苷酸序列至少95%、至少96%、至少97%、至少98%、至少99%相同性的核苷酸序列，12. The nucleic acid molecule of claim 9, wherein the nucleic acid molecule encodes the nucleotide sequence of VHFR1, VH FR2, VH FR3, and VH FR4 in an antibody or an antigen-binding fragment thereof, and has a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 24, 25, 26, and 27, respectively. 编码抗体或其抗原结合片段中的VL FR1、VL FR2、VL FR3、VL FR4的核苷酸序列，其分别具有与SEQ ID NO:32、33、34、35所示的氨基酸序列至少95%、至少96%、至少97%、至少98%、至少99%相同性的核苷酸序列。The nucleotide sequences encoding VL FR1, VL FR2, VL FR3, and VL FR4 in the antibody or its antigen-binding fragment have nucleotide sequences that are at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the amino acid sequences shown in SEQ ID NOs: 32, 33, 34, and 35, respectively. 13.如权利要求9所述的核酸分子，所述核酸分子编码抗体或其抗原结合片段中的VHFR1、VH FR2、VH FR3、VH FR4的核苷酸序列分别如SEQ ID NO:24、25、26、27所示，13. The nucleic acid molecule of claim 9, wherein the nucleotide sequences of VHFR1, VH FR2, VH FR3, and VH FR4 in the antibody or antigen-binding fragment thereof are as shown in SEQ ID NOs: 24, 25, 26, and 27, respectively, 编码抗体或其抗原结合片段中的VL FR1、VL FR2、VL FR3、VL FR4的核苷酸序列分别如SEQ ID NO:32、33、34、35所示。The nucleotide sequences encoding VL FR1, VL FR2, VL FR3, and VL FR4 in the antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 32, 33, 34, and 35, respectively. 14.如权利要求9所述的核酸分子，所述核酸分子编码抗体或其抗原结合片段的重链可变区，其具有与SEQ ID NO:28所示的核苷酸序列至少70%、至少75%、至少80%、至少85%、至少90%、至少92%、至少93%、95%、至少96%、至少97%、至少98%、至少99%相同性的核苷酸序列，14. The nucleic acid molecule of claim 9, encoding a heavy chain variable region of an antibody or antigen-binding fragment thereof, having a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 28, 编码抗体或其抗原结合片段的轻链可变区，其具有与SEQ ID NO:36所示的核苷酸序列至少70%、至少75%、至少80%、至少85%、至少90%、至少92%、至少93%、95%、至少96%、至少97%、至少98%、至少99%相同性的核苷酸序列。A light chain variable region encoding an antibody or antigen-binding fragment thereof having a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence shown in SEQ ID NO:36. 15.如权利要求9所述的核酸分子，所述核酸分子编码抗体或其抗原结合片段的重链可变区的核苷酸序列如SEQ ID NO:28所示，15. The nucleic acid molecule according to claim 9, wherein the nucleotide sequence encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 28, 核酸分子编码抗体或其抗原结合片段的轻链可变区的核苷酸序列如SEQ ID NO:36所示。The nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:36. 16.如权利要求9所述的核酸分子，所述核酸分子编码抗体或其抗原结合片段中的重链可变区信号肽，其具有与SEQ ID NO:37所示的核苷酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%相同性的核苷酸序列，16. The nucleic acid molecule of claim 9, wherein the nucleic acid molecule encodes a heavy chain variable region signal peptide in an antibody or antigen-binding fragment thereof, and has a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the nucleotide sequence shown in SEQ ID NO: 37, 编码抗体或其抗原结合片段中的轻链可变区信号肽，其具有与SEQ ID NO:38所示的核苷酸序列至少90%、至少95%、至少96%、至少97%、至少98%、至少99%相同性的核苷酸序列。A nucleotide sequence encoding a light chain variable region signal peptide in an antibody or an antigen-binding fragment thereof having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence shown in SEQ ID NO:38. 17.如权利要求9所述的核酸分子，所述核酸分子编码抗体或其抗原结合片段中的重链可变区信号肽的核苷酸序列如SEQ ID NO:37所示，17. The nucleic acid molecule according to claim 9, wherein the nucleotide sequence encoding the heavy chain variable region signal peptide in the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 37, 编码抗体或其抗原结合片段中的轻链可变区信号肽的核苷酸序列如SEQ ID NO:38所示。The nucleotide sequence encoding the light chain variable region signal peptide in the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:38. 18.如权利要求9所述的核酸分子，所述核酸分子编码抗体或其抗原结合片段中的重链恒定区的核苷酸序列如SEQ ID NO:39所示；编码抗体或其抗原结合片段中的轻链恒定区的核苷酸序列如SEQ ID NO:40所示。18. The nucleic acid molecule of claim 9, wherein the nucleotide sequence encoding the heavy chain constant region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:39; the nucleotide sequence encoding the light chain constant region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:40. 19.载体，所述载体包括权利要求9-18任一项所述的核酸分子。19. A vector comprising the nucleic acid molecule according to any one of claims 9 to 18. 20.如权利要求19所述的载体，所述载体还包括用于控制表达的元件。20. The vector of claim 19, further comprising an element for controlling expression. 21.如权利要求19所述的载体，所述载体还包括有助于载体进入细胞的材料。21. The vector of claim 19, further comprising a material that facilitates entry of the vector into cells. 22.细胞，所述细胞包括权利要求9-18任一项所述的核酸分子或权利要求19-21任一项所述的载体。22. A cell comprising the nucleic acid molecule of any one of claims 9 to 18 or the vector of any one of claims 19 to 21. 23.如权利要求22所述的细胞，所述细胞包括原核细胞、真核细胞。23. The cell of claim 22, wherein the cell comprises a prokaryotic cell or a eukaryotic cell. 24.如权利要求23所述的细胞，所述原核细胞包括革兰氏阴性或革兰氏阳性细胞。24. The cell of claim 23, wherein the prokaryotic cell comprises a Gram-negative or Gram-positive cell. 25.如权利要求23所述的细胞，所述真核细胞包括原生生物细胞、动物细胞、真菌细胞。25. The cell of claim 23, wherein the eukaryotic cell comprises a protist cell, an animal cell, or a fungal cell. 26.如权利要求25所述的细胞，所述动物细胞包括哺乳动物细胞、鸟类细胞、昆虫细胞。26. The cell of claim 25, wherein the animal cell comprises a mammalian cell, an avian cell, or an insect cell. 27.如权利要求26所述的细胞，所述哺乳动物细胞包括293F细胞、293T细胞、CHO细胞、AT1080细胞、A549细胞。27. The cell of claim 26, wherein the mammalian cell comprises 293F cell, 293T cell, CHO cell, AT1080 cell, A549 cell. 28.一种检测降钙素基因相关肽的产品，所述产品包括权利要求1-8任一项所述的抗体或其抗原结合片段。28. A product for detecting calcitonin gene-related peptide, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. 29.如权利要求28所述的产品，所述产品还包括用于执行抗原-抗体反应的试剂或用于检测反应的试剂。29. The product of claim 28, further comprising a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction. 30.如权利要求29所述的产品，所述用于执行抗原-抗体反应的试剂包括缓冲剂、盐。30. The product of claim 29, wherein the reagents for performing an antigen-antibody reaction include a buffer, a salt. 31.如权利要求28所述的产品，所述产品包括连接有可检测标记物的抗体的缀合物。31. The product of claim 28, comprising a conjugate of an antibody linked to a detectable label. 32.一种预防或治疗腹膜内脏炎性疼痛的药物组合物，所述药物组合物包括权利要求1-8任一项所述的抗体或其抗原结合片段，或权利要求9-18任一项所述的核酸分子，或权利要求19-21任一项所述的载体，或权利要求22-27任一项所述的细胞。32. A pharmaceutical composition for preventing or treating peritoneal visceral inflammatory pain, the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the nucleic acid molecule according to any one of claims 9-18, or the vector according to any one of claims 19-21, or the cell according to any one of claims 22-27. 33.如权利要求32所述的药物组合物，所述药物组合物还包括药学上可接受的辅剂。33. The pharmaceutical composition of claim 32, further comprising a pharmaceutically acceptable adjuvant. 34.如权利要求32所述的药物组合物，所述药物组合物还包括其他的抗体。34. The pharmaceutical composition of claim 32, further comprising other antibodies. 35.权利要求1-8任一项所述的抗体或其抗原结合片段，权利要求9-18任一项所述的核酸分子，权利要求19-21任一项所述的载体，或权利要求22-27任一项所述的细胞在制备检测降钙素基因相关肽的产品中的应用。35. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the nucleic acid molecule according to any one of claims 9 to 18, the vector according to any one of claims 19 to 21, or the cell according to any one of claims 22 to 27 in the preparation of a product for detecting calcitonin gene-related peptide. 36.权利要求1-8任一项所述的抗体或其抗原结合片段，权利要求9-18任一项所述的核酸分子，权利要求19-21任一项所述的载体，或权利要求22-27任一项所述的细胞在制备预防和/或治疗腹膜内脏炎性疼痛的药物组合物中的应用。36. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the nucleic acid molecule according to any one of claims 9 to 18, the vector according to any one of claims 19 to 21, or the cell according to any one of claims 22 to 27 in the preparation of a pharmaceutical composition for preventing and/or treating peritoneal visceral inflammatory pain. 37.一种制备权利要求1-8任一项所述的抗体或其抗原结合片段的方法，所述方法包括：培养权利要求22-27任一项所述的细胞，回收抗体或其抗原结合片段。37. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the method comprising: culturing the cell according to any one of claims 22 to 27, and recovering the antibody or antigen-binding fragment thereof. 38.一种非诊断目的的检测样本中降钙素基因相关肽的方法，所述方法包括：使用权利要求1-8任一项所述的抗体或其抗原结合片段与待测样本接触，从而检测待测样本中降钙素基因相关肽的水平。38. A method for detecting calcitonin gene-related peptide in a sample for non-diagnostic purposes, the method comprising: contacting the sample to be tested with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, thereby detecting the level of calcitonin gene-related peptide in the sample to be tested.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN104292332A (en) * 2010-06-10 2015-01-21 伊莱利利公司 CGRP antibodies

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CN104292332A (en) * 2010-06-10 2015-01-21 伊莱利利公司 CGRP antibodies

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降钙素基因相关肽受体单克隆抗体的制备与初步鉴定;王佳星;许永亮;付楚溪;张丹雨;薛冲;张惟材;王升厚;熊向华;;生物技术通讯;20191130(第06期);49-53 *

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