CN116836273B - Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit - Google Patents

Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit Download PDF

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CN116836273B
CN116836273B CN202210289973.6A CN202210289973A CN116836273B CN 116836273 B CN116836273 B CN 116836273B CN 202210289973 A CN202210289973 A CN 202210289973A CN 116836273 B CN116836273 B CN 116836273B
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antibody
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CN116836273A (en
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孟媛
钟冬梅
唐丽娜
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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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • 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
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Abstract

The invention discloses an anti-serum amyloid A antibody, a reagent and a kit for detecting serum amyloid A, and relates to the technical field of antibodies. The antiserum amyloid A 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 serum amyloid A.

Description

Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-serum amyloid A antibody, a reagent for detecting serum amyloid A and a kit.
Background
Serum Amyloid A (SAA) is a nonspecific acute phase response protein consisting of 104 amino acids, belonging to a heterogeneous class of proteins in the apolipoprotein family. In the acute phase response, SAA is synthesized in the liver by activated macrophages and fibroblasts stimulated by IL-1, IL-6 and TNF, and can be raised to 100-1000 times the initial concentration, but has a very short half-life of only about 50 minutes.
SAA is elevated in both viral and bacterial infections, whereas CRP is hardly elevated or not significantly elevated in viral infections. For weak inflammatory stimuli, SAA is more sensitive than CRP, and exceeds the upper limit of the reference range for a period of time earlier than CRP, rising earlier, falling faster and amplitude greater during recovery. Thus, SAA is a more sensitive indicator in patients with normal CRP viral infections, non-invasive or early invasive bacterial infections. In the early stages of pediatric infectious diseases, it is difficult to identify whether a pediatric patient is a bacterial infection or a viral infection due to ambiguous signs and symptoms of the pediatric patient. Therefore, the method for rapidly identifying the bacterial infection and the viral infection of the pediatric patient in early stage has important significance for effectively treating and preventing various complications in time. Since CRP lacks sensitivity to viral infection, SAA can be elevated at both the early stages of bacterial and viral infection. The combined detection of the two can improve the diagnosis efficiency of early virus infection and provide valuable reference information for the identification of virus and bacterial infection and the selection of treatment schemes. In addition, the ratio of SAA to CRP has correlation with the severity of the pediatric infectious diseases, and the monitoring of SAA/CRP has greater application value than the independent detection of SAA or CRP. Meanwhile, research shows that SAA in patients with various tumors such as liver cancer, lung cancer, breast cancer, prostate cancer, endometrial cancer and the like is increased to different degrees, SAA levels are obviously related to the activity stage, malignancy degree and metastasis of the tumors, and SAA increase in the metastasis stage of the malignant tumors usually shows higher values than those in the stationary phase of the tumors.
Currently, the detection method of SAA mainly includes a fluorescent immunochromatography method, which is an immunological detection method based on the specific reaction of an antibody and an antigen, and simultaneously 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 SAA. Thus, there is a strong need in the art for antibodies that efficiently bind and detect SAA.
In view of this, the present invention has been made.
Disclosure of Invention
Aiming at the problem of low sources of the existing anti-serum amyloid A antibodies, the application provides an anti-serum amyloid A antibody with improved affinity and activity so as to improve the detection of serum amyloid A and provide an important raw material source for the detection of serum amyloid A.
In order to achieve the above object, according to one aspect of the present invention, there is provided an anti-serum amyloid a antibody or a functional fragment thereof comprising the following complementarity determining regions:
a) The amino acid sequences are shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 1 to SEQ ID NO 3, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4 to SEQ ID NO 6; or (b)
b) The amino acid sequences are shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO. 21 to SEQ ID NO. 23 and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 24 to SEQ ID NO. 26.
In order to achieve the above object, according to a second aspect of the present invention, there is provided an antibody against serum amyloid a or a functional fragment thereof, which comprises a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above.
In order to achieve the above object, according to a third aspect of the present invention, there is provided an antibody against serum amyloid a, comprising a heavy chain comprising the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region and/or a light chain; the light chain comprises the light chain variable region described above and the light chain constant region described above.
In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody conjugate comprising the above antibody or a functional fragment thereof.
In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a reagent or kit for detecting serum amyloid a, the reagent or kit comprising the above antibody or a functional fragment thereof or the above antibody conjugate.
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 result of reducing SDS-PAGE of Anti-SAA 4B3mut 1.
FIG. 2 shows the results of reducing SDS-PAGE of Anti-SAA 9G5mut1 and Anti-SAA 9G5mut2.
Detailed Description
The present invention provides an anti-serum amyloid a antibody or functional fragment thereof having improved affinity and activity, comprising the complementarity determining regions:
a) The amino acid sequences are shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 1 to SEQ ID NO 3, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4 to SEQ ID NO 6; or (b)
b) The amino acid sequences are shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO. 21 to SEQ ID NO. 23 and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO. 24 to SEQ ID NO. 26.
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 the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, and refers to regions other than CDRs in an antibody heavy chain variable region and a light chain variable region; wherein the heavy chain framework regions can be further subdivided into contiguous regions separated by CDRs comprising HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework regions may be further subdivided into contiguous regions separated by CDRs comprising LFR1, LFR2, LFR3 and LFR4 framework regions.
In the present invention, the heavy chain variable region is obtained by connecting the following numbered CDRs with FRs in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
In alternative embodiments, the antibody or functional fragment thereof further comprises the following framework regions:
c) The amino acid sequences are shown as HFR1, HFR2, HFR3 and HFR4 shown in SEQ ID NO 7 to SEQ ID NO 10 and LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NO 11 to SEQ ID NO 14; or (b)
d) The amino acid sequences are shown as HFR1, HFR2, HFR3 and HFR4 shown as SEQ ID NO 27 to SEQ ID NO 30 and LFR1, LFR2, LFR3 and LFR4 shown as SEQ ID NO 31 to SEQ ID NO 34; or (b)
e) A framework region having at least 80% homology to the amino acid sequence set forth in c) or d);
in other embodiments, each framework region amino acid sequence of an antibody or functional fragment thereof provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the corresponding framework region described above.
In an alternative embodiment, the amino acid sequence of LFR1 in said e) framework region is shown as SEQ ID NO. 35.
In an alternative embodiment, the antibody or functional fragment thereof is in K D ≤10 -8 M、K D ≤10 -9 M、K D ≤10 - 10 M or K D ≤10 -11 The affinity of M binds serum amyloid a.
In an alternative embodiment, the antibody or functional fragment thereof is in K D ≤7.98×10 -9 The affinity of M binds serum amyloid a.
In an alternative embodiment, the antibody or functional fragment thereof is in K D ≤9.06×10 -10 The affinity of M binds serum amyloid a.
K D Reference is made to the method in the embodiment of the invention.
In another aspect, embodiments of the present invention provide an antibody or a functional fragment thereof against serum amyloid a, the antibody or the functional fragment thereof comprises a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFR1, HFR2, HFR3, HFR4, HFR3, LFR4 is the amino acid sequence of HFR1, HFR2, HCDR3, LCDR 4, LFR2, LFR4.
In alternative embodiments, the heavy chain variable region amino acid sequence is set forth in any one of SEQ ID NOs 17, 38, 39.
In an alternative embodiment, the light chain variable region amino acid sequence is set forth in any one of SEQ ID NOs 18, 40, 41.
In an alternative embodiment, the heavy chain variable region amino acid sequence is shown in SEQ ID NO. 17 and the light chain variable region amino acid sequence is shown in SEQ ID NO. 18; or (b)
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 38, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 40; or (b)
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 39, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 41.
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 species origin from cattle, horses, cows, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, camels, geese, turkeys, cocks or humans.
In an alternative embodiment, the constant region is of murine species origin.
In alternative embodiments, the heavy chain constant region sequence is as shown in SEQ ID NO. 15 or SEQ ID NO. 36 or has at least 80% homology thereto, and the light chain constant region sequence is as shown in SEQ ID NO. 16 or SEQ ID NO. 37 or has at least 80% homology thereto.
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% homology to the above-described constant region (SEQ ID NOS: 15, 36, 16, 37).
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 antibody against serum amyloid a comprising a heavy chain variable region as described above and a heavy chain constant region as described above and/or a light chain; the light chain comprises the light chain variable region described above and the light chain constant region described above.
In an alternative embodiment, the heavy chain has an amino acid sequence as set forth in any one of SEQ ID NOs 19, 42, 43; the amino acid sequence of the light chain is shown in any one of SEQ ID NO. 20, 44 and 45.
In an alternative embodiment, the heavy chain amino acid sequence is shown in SEQ ID NO. 19 and the light chain amino acid sequence is shown in SEQ ID NO. 20.
In an alternative embodiment, the heavy chain amino acid sequence is shown as SEQ ID NO. 42 and the light chain amino acid sequence is shown as SEQ ID NO. 44.
In an alternative embodiment, the heavy chain amino acid sequence is shown in SEQ ID NO. 43 and the light chain amino acid sequence is shown in SEQ ID NO. 45.
In another aspect, the invention provides an antibody conjugate comprising an antibody or functional fragment thereof as described above.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is labeled with a label.
In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.
In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.
In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.
In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (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 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
in alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.
In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.
In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.
In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.
In an alternative embodiment, the antibody or functional fragment thereof in the above antibody conjugate is coated onto a solid phase.
In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.
In alternative embodiments, the solid phase includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.
In an alternative embodiment, the solid phase is a magnetic microsphere.
In another aspect, the invention provides a reagent or kit for detecting serum amyloid a, comprising the antibody or functional fragment thereof or the antibody conjugate.
In another aspect, the invention provides the use of the above-described reagent or kit in serum amyloid a detection.
In another aspect, the invention provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.
In another aspect, the invention provides a vector comprising the nucleic acid molecule described above.
In another aspect, the invention provides a cell comprising the vector described above.
In another aspect, the invention provides a method of making an antibody or functional fragment thereof comprising: the cells as described above were cultured.
On the basis of the present invention, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present invention to prepare the antibody or the functional fragment thereof by any technique.
In 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-SAA 4B3 monoclonal antibodies
Restriction enzymes, prime Star DNA polymerase in this example were purchased from Takara Corp. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer setThe adult gene sequencing was performed by Invitrogen corporation.
1 construction of recombinant plasmid
(1) Antibody Gene production
mRNA is extracted from hybridoma cell strains secreting Anti-SAA 4B3 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-SAA 4B3 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 339bp, and the front of the VL gene sequence is 57bp leader peptide sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 336bp, 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 antibody variable region gene sequencing in pMD-18T, VL and VH gene specific primers of the antibody are designed, hindIII, ecoRI restriction sites and protective bases are respectively arranged at two ends, and a 0.73kb Light Chain gene fragment and a 1.42kb Heavy Chain gene fragment are amplified 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 solution (main ingredient NaHCO 3) diluted SAA (available from Yashraj biotechnology limited, FRSAA-61) to 2ug/ml, 100 μl per well, overnight at 4deg.C; 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 was less than 0.1, indicating that the antibodies produced after transient plasmid transformation were active on SAA.
(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; serum amyloid A is detected by sending samples from the culture supernatants of the batch culture with 6 holes for 7 days, and cell strains with smaller antibody concentration and cell diameter are selected to be transferred to 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 protein A affinity chromatography columns. 3 μg of purified antibody was subjected to reducing SDS-PAGE, and after reducing SDS-PAGE, two bands were shown, 1 Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).
Example 2 preparation of Anti-SAA 9G5 monoclonal antibody the same as in example 1 above.
Example 3 affinity and Activity optimization
The two wild-type monoclonal antibodies (Anti-SAA 4B3 and Anti-SAA 9G 5) obtained in examples 1 and 2 were not ideal in affinity and antibody activity, although they had the ability to bind SAA, and thus the applicant had performed directed mutations on the light chain CDRs and heavy chain CDRs of the antibodies. 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. Screening to obtain monoclonal antibodies with obviously improved affinity and antibody activity compared with the respective wild type, and naming the monoclonal antibodies as follows: anti-SAA 4B3mut1, anti-SAA 9G5mut2. The heavy and light chain amino acid sequences of the mutants are shown in the following table, respectively.
TABLE 1 antibody sequences
Sample name Heavy chain sequence number Light chain sequence number
Anti-SAA 4B3mut1 SEQ ID NO:19 SEQ ID NO:20
Anti-SAA 9G5mut1 SEQ ID NO:42 SEQ ID NO:44
Anti-SAA 9G5mut2 SEQ ID NO:43 SEQ ID NO:45
Example 4 detection of Performance of antibodies
1 affinity assay
Using the AMC sensor, purified antibodies were diluted to 10ug/ml with PBST and SAA (available from Yashraj biotechnology limited, cat. FRSAA-61) 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 7.98E-09 1.23E+04 9.82E-05
Anti-SAA 4B3mut1 8.97E-10 7.52E+04 6.75E-05
Anti-SAA 9G5mut1 9.06E-10 7.36E+04 6.67E-05
Anti-SAA 9G5mut2 8.75E-10 7.45E+04 6.52E-05
2 Activity assay
Coating solution (main ingredient NaHCO 3) diluted SAA (available from Yashraj biotechnology limited, FRSAA-61) to 2ug/ml, 100 μl per well, overnight at 4deg.C; 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
Concentration (ng/ml) 60 30 15 7.5 3.75 0
Control 1.793 1.630 0.912 0.562 0.316 0.009
Anti-SAA 4B3mut1 2.215 1.506 1.133 0.635 0.386 0.007
Anti-SAA 9G5mut1 2.261 1.954 1.234 0.767 0.474 0.011
Anti-SAA 9G5mut2 2.305 2.011 1.298 0.801 0.501 0.011
3 stability assessment
Placing the mutant 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 the mutant antibody has no obvious protein state change and has no activity decreasing trend along with the increase of the examination temperature after being placed for 21 days under three examination conditions, thus indicating that the mutant antibody is stable. The following table shows the OD results of the enzyme-free activity assay for 21 days.
Table 4 stability data
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 NKYIY
SEQ ID NO:2 EIDPSNGATKFNEKFTN
SEQ ID NO:3 GAYWGHGT
SEQ ID NO:4 KSSQSLLDRDGKTYLN
SEQ ID NO:5 LVSKLDS
SEQ ID NO:6 WQGTHFP
SEQ ID NO:21 DTYMH
SEQ ID NO:22 RIDPANGNTKYDPKFQG
SEQ ID NO:23 GGN
SEQ ID NO:24 KASQSVDYDGYSYLN
SEQ ID NO:25 AASNLES
SEQ ID NO:26 QQSNEDPY
SEQUENCE LISTING
<110> Dongguan City, pengzhi biotechnology Co., ltd
<120> anti-serum amyloid A antibody, reagent and kit for detecting serum amyloid A
<130> P2022038CN01
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<170> PatentIn version 3.5
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Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
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Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
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Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
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Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
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Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
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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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Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
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Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
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Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
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Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
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His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
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Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
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Tyr Ile Tyr Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
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Gly Glu Ile Asp Pro Ser Asn Gly Ala Thr Lys Phe Asn Glu Lys Phe
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Thr Asn Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Thr Thr Ala Tyr
65 70 75 80
Val Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
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Ala Arg Gly Ala Tyr Trp Gly His Gly Thr Leu Val Thr Val Ser Ala
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Asp Val Val Leu Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly
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Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Arg
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Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Phe Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly
85 90 95
Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys
100 105 110
Arg
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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
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Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asn Lys
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Tyr Ile Tyr Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
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Gly Glu Ile Asp Pro Ser Asn Gly Ala Thr Lys Phe Asn Glu Lys Phe
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Thr Asn Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Thr Thr Ala Tyr
65 70 75 80
Val Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Ala Tyr Trp Gly His Gly Thr Leu Val Thr Val Ser Ala
100 105 110
Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
115 120 125
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
130 135 140
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
145 150 155 160
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
165 170 175
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
180 185 190
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
195 200 205
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
210 215 220
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
225 230 235 240
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
245 250 255
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
260 265 270
Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
275 280 285
Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
290 295 300
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
325 330 335
Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
340 345 350
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
355 360 365
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
370 375 380
Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
385 390 395 400
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
405 410 415
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
420 425 430
Ser Pro Gly Lys
435
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Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Phe Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly
85 90 95
Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys
100 105 110
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
115 120 125
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
130 135 140
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
145 150 155 160
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
165 170 175
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
180 185 190
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
195 200 205
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
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<223> Artificial sequence
<400> 29
Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln
1 5 10 15
Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg
20 25 30
<210> 30
<211> 13
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 30
Tyr Asp Trp Gly Gln Gly Thr Thr Leu Ser Val Ser Ser
1 5 10
<210> 31
<211> 23
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 31
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys
20
<210> 32
<211> 15
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 32
Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Ser
1 5 10 15
<210> 33
<211> 32
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 33
Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys
20 25 30
<210> 34
<211> 12
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 34
Thr Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg
1 5 10
<210> 35
<211> 23
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 35
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Leu Ser Cys
20
<210> 36
<211> 330
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 36
Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly
1 5 10 15
Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Leu 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 Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys
100 105 110
Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
115 120 125
Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys
130 135 140
Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp
145 150 155 160
Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
165 170 175
Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln
180 185 190
His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn
195 200 205
Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly
210 215 220
Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu
225 230 235 240
Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met
245 250 255
Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu
260 265 270
Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe
275 280 285
Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
290 295 300
Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr
305 310 315 320
Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
325 330
<210> 37
<211> 106
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 37
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
1 5 10 15
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
20 25 30
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
35 40 45
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
65 70 75 80
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
85 90 95
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 38
<211> 114
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 38
Glu Val His Leu Gln Gln Ser Gly Ala Glu Leu Val Thr Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Asp Trp Gly Gln Gly Thr Thr Leu Ser Val
100 105 110
Ser Ser
<210> 39
<211> 114
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 39
Glu Val His Leu Gln Gln Ser Gly Ala Glu Leu Val Thr Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Asp Trp Gly Gln Gly Thr Thr Leu Ser Val
100 105 110
Ser Ser
<210> 40
<211> 112
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 40
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Tyr Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Ser Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg
100 105 110
<210> 41
<211> 112
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 41
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Tyr Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Ser Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg
100 105 110
<210> 42
<211> 444
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 42
Glu Val His Leu Gln Gln Ser Gly Ala Glu Leu Val Thr Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Asp Trp Gly Gln Gly Thr Thr Leu Ser Val
100 105 110
Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val
115 120 125
Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys
130 135 140
Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu
145 150 155 160
Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr
165 170 175
Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln
180 185 190
Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp
195 200 205
Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys
210 215 220
Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe
225 230 235 240
Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile
260 265 270
Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
275 280 285
His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro
290 295 300
Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val
305 310 315 320
Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro
325 330 335
Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu
340 345 350
Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp
355 360 365
Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr
370 375 380
Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu
405 410 415
Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His
420 425 430
His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
435 440
<210> 43
<211> 444
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 43
Glu Val His Leu Gln Gln Ser Gly Ala Glu Leu Val Thr Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Gly Asn Tyr Asp Trp Gly Gln Gly Thr Thr Leu Ser Val
100 105 110
Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val
115 120 125
Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys
130 135 140
Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu
145 150 155 160
Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr
165 170 175
Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln
180 185 190
Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp
195 200 205
Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys
210 215 220
Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe
225 230 235 240
Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile
260 265 270
Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
275 280 285
His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro
290 295 300
Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val
305 310 315 320
Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro
325 330 335
Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu
340 345 350
Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp
355 360 365
Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr
370 375 380
Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu
405 410 415
Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His
420 425 430
His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
435 440
<210> 44
<211> 218
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 44
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Tyr Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Ser Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg
100 105 110
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
115 120 125
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
130 135 140
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
145 150 155 160
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
180 185 190
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
195 200 205
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215
<210> 45
<211> 218
<212> PRT
<213> Artificial
<220>
<223> Artificial sequence
<400> 45
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Tyr Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Ser Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg
100 105 110
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
115 120 125
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
130 135 140
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
145 150 155 160
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
180 185 190
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
195 200 205
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215

Claims (34)

1. An antibody or functional fragment thereof against serum amyloid a, wherein the antibody or functional fragment thereof comprises a heavy chain variable region and a light chain variable region comprising complementarity determining regions:
a) The amino acid sequences are shown as HCDR1, HCDR2 and HCDR3 shown as SEQ ID NO 1 to SEQ ID NO 3, and LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 4 to SEQ ID NO 6; or (b)
b) The amino acid sequences are shown as HCDR1, HCDR2, HCDR3 and SEQ ID NO. 21-23, and LCDR1, LCDR2, LCDR3 shown as SEQ ID NO. 24-26.
2. The antibody or functional fragment thereof according to claim 1, further comprising the following framework regions:
c) The amino acid sequences are shown as HFR1, HFR2, HFR3 and HFR4 shown in SEQ ID NO 7 to SEQ ID NO 10 and LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NO 11 to SEQ ID NO 14; or (b)
d) The amino acid sequences are shown as HFR1, HFR2, HFR3 and HFR4 shown as SEQ ID NO 27 to SEQ ID NO 30 and LFR1, LFR2, LFR3 and LFR4 shown as SEQ ID NO 31 to SEQ ID NO 34; or (b)
e) A framework region having at least 80% homology with the amino acid sequence depicted in c) or d).
3. The antibody or functional fragment thereof according to claim 2, wherein the amino acid sequence of LFR1 in the e) framework region is shown in SEQ ID No. 35.
4. The antibody or functional fragment thereof according to claim 2, wherein the antibody or functional fragment thereof has a KD.ltoreq.10 -8 The affinity of M binds serum amyloid a.
5. An antibody against serum amyloid a or a functional fragment thereof, characterized in that the antibody or the functional fragment thereof comprises a heavy chain variable region comprising the sequence structure of HFR1-HCDR 2-HFR3-HCDR3-HFR4 and a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 as defined in claim 1, wherein the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, HFR2, LFR3, LFR4 is the amino acid sequence of any one of claims 2-4 HFR1, HCDR2, LCDR3, LFR4, LFR2, LFR4.
6. An antibody or functional fragment thereof against serum amyloid a, wherein the antibody or functional fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID No. 17 and a light chain variable region having an amino acid sequence as shown in SEQ ID No. 18; or (b)
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 38, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 40; or (b)
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 39, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 41.
7. The antibody or functional fragment thereof of any one of claims 1 to 6, wherein the antibody or functional fragment thereof further comprises a constant region.
8. The antibody or functional fragment thereof of claim 7, wherein the constant regions comprise a heavy chain constant region and a light chain constant region.
9. The antibody or functional fragment thereof of claim 8, wherein the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD; the light chain constant region is selected from kappa-type or lambda-type light chain constant regions.
10. The antibody or functional fragment thereof according to claim 8, wherein the constant region is of bovine, equine, porcine, ovine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human origin.
11. The antibody or functional fragment thereof according to claim 8, wherein the constant region is of murine origin.
12. The antibody or functional fragment thereof according to claim 8, wherein the heavy chain constant region sequence is shown in SEQ ID No. 15 or SEQ ID No. 36 or has at least 80% homology thereto and the light chain constant region sequence is shown in SEQ ID No. 16 or SEQ ID No. 37 or has at least 80% homology thereto.
13. The antibody or functional fragment thereof according to any one of claims 1 to 6, wherein the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv and scFv of the antibody.
14. An antibody against serum amyloid a comprising a heavy chain and a light chain, characterized in that the heavy chain comprises a heavy chain variable region as defined in claim 5 or 6 and a heavy chain constant region as defined in any one of claims 7 to 12; the light chain comprises the light chain variable region of claim 5 or 6 and the light chain constant region of any one of claims 7 to 12.
15. An antibody against serum amyloid a, wherein the antibody comprises a heavy chain and a light chain, the heavy chain amino acid sequence is shown as SEQ ID No. 19, and the light chain amino acid sequence is shown as SEQ ID No. 20; or (b)
The heavy chain amino acid sequence is shown as SEQ ID NO. 42, and the light chain amino acid sequence is shown as SEQ ID NO. 44; or (b)
The heavy chain amino acid sequence is shown as SEQ ID NO. 43, and the light chain amino acid sequence is shown as SEQ ID NO. 45.
16. An antibody conjugate comprising the antibody or functional fragment thereof of any one of claims 1-15.
17. The antibody conjugate of claim 16, wherein the antibody or functional fragment thereof is labeled with a label.
18. The antibody conjugate of claim 17, wherein the label is selected from the group consisting of a fluorescent dye, an enzyme, a radioisotope, a chemiluminescent reagent, and a nanoparticle-based label.
19. The antibody conjugate of claim 18, wherein the fluorescent dye is selected from the group consisting of fluorescein-based dyes and derivatives thereof, rhodamine-based dyes and derivatives thereof, cy-based dyes and derivatives thereof, alexa-based dyes and derivatives thereof, and protein-based dyes and derivatives thereof.
20. The antibody conjugate of claim 18, wherein the enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.
21. The antibody conjugate of claim 18, wherein the radioisotope is selected from the group consisting of 212 Bi、 131 I、 111 In、 90 Y、 186 Re、 211 At、 125 I、 188 Re、 153 Sm、 213 Bi、 32 P、 94 mTc、 99 mTc、 203 Pb、 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn、 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb、 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F。
22. the antibody conjugate of claim 18, wherein the chemiluminescent reagent is selected from the group consisting of luminol and derivatives thereof, lucigenin, crustacean fluorescein and derivatives thereof, ruthenium bipyridine and derivatives thereof, acridinium esters and derivatives thereof, dioxane and derivatives thereof, lotensine and derivatives thereof, and peroxyoxalate and derivatives thereof.
23. The antibody conjugate of claim 18, wherein the nanoparticle-based label is selected from the group consisting of a nanoparticle and a colloid.
24. The antibody conjugate of claim 23, wherein the nanoparticle is selected from the group consisting of an organic nanoparticle, a magnetic nanoparticle, a quantum dot nanoparticle, and a rare earth complex nanoparticle.
25. The antibody conjugate of claim 23, wherein the colloid is selected from the group consisting of latex, colloidal selenium, colloidal metal, disperse dye, and dye-labeled microsphere.
26. The antibody conjugate of claim 25, wherein the colloidal metal is selected from the group consisting of colloidal gold and colloidal silver.
27. The antibody conjugate of claim 16, wherein the antibody or functional fragment thereof is coated onto a solid phase.
28. The antibody conjugate of claim 27, wherein the solid phase is selected from the group consisting of a microsphere, a plate, and a membrane.
29. The antibody conjugate of claim 28, wherein the solid phase is selected from the group consisting of magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.
30. A reagent or kit for detecting serum amyloid a, comprising the antibody or functional fragment thereof of any one of claims 1-15 or the antibody conjugate of any one of claims 16-29.
31. A nucleic acid encoding the antibody or functional fragment thereof of any one of claims 1-15.
32. A vector comprising a nucleic acid fragment encoding the antibody or functional fragment thereof of any one of claims 1-15.
33. A cell comprising the nucleic acid of claim 31 or the vector of claim 32.
34. A method of preparing the antibody or functional fragment thereof of any one of claims 1-15, comprising: culturing the cell of claim 33.
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CN110484512A (en) * 2019-08-12 2019-11-22 杭州伊佰新生物技术有限公司 Secrete hybridoma cell strain and its application of human serum amyloid A monoclonal antibody
CN114044822A (en) * 2021-10-28 2022-02-15 杭州博茵生物技术有限公司 Heavy chain and light chain variable regions of serum amyloid protein A antibody, antibody and application

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