CN116925216A - 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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CN116925216A
CN116925216A CN202210372088.4A CN202210372088A CN116925216A CN 116925216 A CN116925216 A CN 116925216A CN 202210372088 A CN202210372088 A CN 202210372088A CN 116925216 A CN116925216 A CN 116925216A
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thr
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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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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

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Abstract

The application 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, and has improved affinity and activity.

Description

Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit
Technical Field
The application 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 application has been made.
Disclosure of Invention
Aiming at the problem of less sources of the existing anti-serum amyloid A antibodies, the application provides the anti-serum amyloid A antibody with improved affinity and activity, and provides an important raw material source for detecting serum amyloid A.
In order to achieve the above object, according to one aspect of the present application, there is provided an antibody or a functional fragment thereof comprising HCDR1, HCDR2, HCDR3 having amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3, and LCDR1, LCDR2, LCDR3 having amino acid sequences shown in SEQ ID NO. 4 to SEQ ID NO. 6.
In order to achieve the above object, according to a second aspect of the present application, there is provided an antisera amyloid a antibody or a functional fragment thereof comprising a heavy chain variable region comprising the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 and/or a light chain variable region comprising the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described above.
In order to achieve the above object, according to a third aspect of the present application, there is provided an anti-serum amyloid a antibody comprising a heavy chain comprising the heavy chain variable region described above and/or a light chain comprising the light chain variable region described above.
In order to achieve the above object, according to a fourth aspect of the present application, 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 application, 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, according to a sixth aspect of the present application there is provided the use of an antibody as defined above or a functional fragment thereof, an antibody conjugate as defined above or a reagent or kit as defined above in serum amyloid a detection.
In order to achieve the above object, the present application 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 application, 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 application and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of reducing SDS-PAGE of Anti-SAA-7F5mut1 and Anti-SAA-7F5mut2.
Detailed Description
The application provides an antibody or a functional fragment thereof, which comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3, and LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID NO. 4 to SEQ ID NO. 6. The antibodies have improved affinity and activity.
In the present application, 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 application, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues contributing to the binding affinity of an antibody or antigen binding fragment to the antigen or epitope it recognizes. In a specific embodiment of the application, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.
In the present application, 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 application adopts Kabat annotation standard to mark CDR regions, but other methods to mark CDR regions also belong to the protection scope of the application.
In the present application, 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 application, the heavy chain variable region is obtained by connecting the following numbered CDRs with FRs in the following combination arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by ligating the following numbered CDRs with the FR in the following combination arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.
In alternative embodiments, the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having amino acid sequences shown in SEQ ID NO. 7 through SEQ ID NO. 10, and LFR1, LFR2, LFR3 and LFR4 having amino acid sequences shown in SEQ ID NO. 11 through SEQ ID NO. 14.
In other embodiments, each of the framework amino acid sequences of the antibodies or functional fragments thereof provided herein may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.
In an alternative embodiment, HFR2 of the antibody or functional fragment thereof is set forth in SEQ ID NO. 15.
In an alternative embodiment, the antibody or functional fragment thereof has a KD of 10 or less -8 M、KD≤10 -9 M、KD≤10 -10 M、KD≤10 -11 M or KD is less than or equal to 10 -12 The affinity of M binds serum amyloid a.
In an alternative embodiment, the antibody or functional fragment thereof has a KD of 9.00×10 or less -9 The affinity of M binds serum amyloid a.
In an alternative embodiment, the antibody or functional fragment thereof has a KD of 8.59X10 ∈ -11 The affinity of M binds serum amyloid a.
In another aspect, embodiments of the present application 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 SEQ ID NO. 18 or 19.
In an alternative embodiment, the light chain variable region amino acid sequence is set forth in SEQ ID NO. 20.
In alternative embodiments, the antibody further comprises a constant region.
In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.
In alternative embodiments, the heavy chain constant region is selected from the group consisting of an IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD heavy chain constant region, and the light chain constant region is selected from the group consisting of kappa-type or lambda-type light chain constant regions.
In alternative embodiments, the constant region is of a species derived from a cow, horse, cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.
In an alternative embodiment, the constant region is of murine species origin.
In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 16 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 17.
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 NO:16 or 17).
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 application 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 alternative embodiments, the heavy chain has an amino acid sequence as set forth in SEQ ID NO. 21 or 22.
In an alternative embodiment, the amino acid sequence of the light chain is shown in SEQ ID NO. 23.
In another aspect, the application 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 application.
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 nitrocellulose membrane.
In another aspect, the application 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 application provides the use of an antibody as described above or a functional fragment thereof, an antibody conjugate, or a reagent or kit as described above in serum amyloid a detection.
In another aspect, the application provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.
In another aspect, the application provides a vector comprising the nucleic acid molecule described above.
In another aspect, the application provides a cell comprising the vector described above.
In another aspect, the application 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 application, 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 application 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 application more clear, the technical solutions of the embodiments of the present application 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 application 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 application are described in further detail below in connection with the examples.
EXAMPLE 1 preparation of Anti-SAA-7F5 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 synthesis and gene sequencing were accomplished by Invitrogen corporation.
1 construction of recombinant plasmid
(1) Antibody Gene production
mRNA is extracted from hybridoma cell strains secreting monoclonal antibodies of antiserum amyloid A, a DNA product is obtained through an RT-PCR method, the product is inserted into a pMD-18T vector after an A adding reaction by rTaq DNA polymerase, and is transformed into DH5 alpha competent cells, after colony growth, 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-7F5 antibody variable region Gene
The gene sequence obtained by sequencing is placed in a Kabat antibody database for analysis, and VNTI11.5 software is utilized for analysis to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 321bp, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 345bp, 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 recombinant antibody, which is introduced with HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and named pcDNA3.4A expression vector, and is subsequently expressed for short as 3.4AA carrier; 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 0.72kb Light Chain gene fragment and a 1.40kb 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 3ug/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; and (3) carrying out sample feeding detection on the culture supernatant of the 6-hole batch culture for 7 days, and selecting cell strains with smaller antibody concentration and smaller cell diameter to transfer TPP for seed preservation and passage.
3 recombinant antibody production
(1) Cell expansion culture
After cell recovery, the cells were first cultured in 125ml shake flasks with an inoculation volume of 30ml and a medium of 100% dynamis and placed in a shaker at a speed of 120r/min at 37℃and with 8% carbon dioxide. Culturing for 72h, inoculating and expanding culture at 50 ten thousand cells/ml inoculating density, and calculating the expanding culture volume according to production requirements, wherein the culture medium is 100% Dynamis culture medium. After that, the culture was spread every 72 hours. When the cell quantity meets the production requirement, the inoculation density is strictly controlled to be about 50 ten thousand cells/ml for production.
(2) Shake flask production and purification
Shake flask parameters: the rotating speed is 120r/min, the temperature is 37 ℃, and the carbon dioxide is 8%. Feeding: feeding was started every day until 72h of culture in shake flasks, hyCloneTM Cell BoostTM Feed a fed-batch was 3% of the initial culture volume every day, feed 7b fed-batch was one thousandth of the initial culture volume every day, and fed-batch was continued until day 12 (day 12 Feed). Glucose was fed at 3g/L on day six. Samples were collected on day 13. Affinity purification was performed using a proteona affinity column. 6 μg of purified antibody was subjected to reducing SDS-PAGE, and the electrophoretogram shows two bands after reducing SDS-PAGE, 1 Mr 50KD (heavy chain) and the other Mr 28KD (light chain).
Example 2 affinity and Activity optimization
Although the Anti-SAA-7F5 monoclonal antibody obtained in example 1 has the ability to bind to serum amyloid A, the affinity and antibody activity are not satisfactory, and thus the applicant has performed directed mutation on the light chain CDR and the heavy chain CDR of the antibody. The method comprises the steps of performing structural simulation of an antibody variable region, structural simulation of an antigen-antibody variable region acting complex, analysis of key amino acids of an antibody and mutation design by using a computer, designing and synthesizing a two-way primer covering a mutation site according to a mutation scheme, synthesizing primers at two ends of target DNA, performing high-fidelity PCR reaction, cloning a PCR product to a vector, and preparing the mutant antibody according to the method described in the example 1. Monoclonal antibodies with remarkably improved affinity and antibody activity are obtained through screening and are named as: anti-SAA-7F5mut1 and Anti-SAA-7F5mut2. The amino acid sequences of the respective monoclonal antibodies are shown in the following table:
TABLE 1 antibody sequences
Example 3 detection of Performance of antibodies
1 affinity assay
Using the AMC sensor, purified antibodies were diluted to 20ug/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 9.00E-09 2.41E+04 2.17E-04
Anti-SAA-7F5mut1 8.57E-11 3.58E+05 3.07E-05
Anti-SAA-7F5mut2 8.59E-11 3.62E+05 3.11E-05
2 Activity assay
Coating solution (main ingredient NaHCO 3) diluted SAA (available from Yashraj biotechnology limited, FRSAA-61) to 3ug/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
Sample concentration (ng/ml) 50 25 12.5 6.25 3.125 0
Control 1.752 1.431 0.803 0.387 0.199 0.021
Anti-SAA-7F5mut1 1.958 1.553 0.923 0.452 0.247 0.018
Anti-SAA-7F5mut2 2.010 1.592 1.103 0.492 0.301 0.022
3 stability assessment
Placing the antibody at 4 ℃ (refrigerator), 80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days samples for state observation, and detecting the activity of the 21 days samples, wherein the result shows that no obvious protein state change is seen for the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. Table 4 below shows the results of the detection of OD after 21 days of antibody examination.
Table 4 stability data
Sample concentration (ng/ml) 25 6.25 0
4 ℃,21 days sample 1.552 0.503 0.011
Sample at-80℃for 21 days 1.578 0.498 0.012
37 ℃ and 21 days of sample 1.568 0.475 0.011
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
The partial amino acid sequence related to the application is as follows:
sequence numbering Sequence fragments
SEQ ID NO:1 NYWMH
SEQ ID NO:2 YINPSTGYTEYNQKFKE
SEQ ID NO:3 NYPL
SEQ ID NO:4 SASSSVSYMH
SEQ ID NO:5 DTSKLAS
SEQ ID NO:6 QQWSRNPP
SEQUENCE LISTING
<110> Dongguan City, pengzhi biotechnology Co., ltd
<120> anti-serum amyloid A antibody, reagent and kit for detecting serum amyloid A
<130> P2022040CN01
<160> 23
<170> PatentIn version 3.5
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Asn Tyr Trp Met His
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Ser Ala Ser Ser Ser Val Ser Tyr Met His
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Asp Thr Ser Lys Leu Ala Ser
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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala
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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
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Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly
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Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln
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Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
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Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
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Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
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Glu Lys Val Thr Met Thr Cys
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Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
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Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser
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Leu Thr Ile Ser Thr Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
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Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Leu Gly
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Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
1 5 10 15
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
100 105 110
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
115 120 125
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
130 135 140
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
145 150 155 160
Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
165 170 175
Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
180 185 190
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
195 200 205
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
210 215 220
Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
225 230 235 240
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
260 265 270
Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
275 280 285
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
290 295 300
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
305 310 315 320
Ser Pro Gly Lys
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Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
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Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
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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
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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala
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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Glu Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Tyr Pro Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala
115
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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala
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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Glu Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Tyr Pro Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala
115
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Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
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Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Thr Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Arg Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Glu Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Tyr Pro Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro
115 120 125
Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val
130 135 140
Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser
145 150 155 160
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
165 170 175
Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser
180 185 190
Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
195 200 205
Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys
210 215 220
Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys
225 230 235 240
Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val
245 250 255
Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
260 265 270
Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
275 280 285
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp
290 295 300
Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe
305 310 315 320
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys
325 330 335
Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys
340 345 350
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
355 360 365
Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
370 375 380
Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser
385 390 395 400
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
405 410 415
Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser
420 425 430
Leu Ser His Ser Pro Gly Lys
435
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Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Trp Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Leu
35 40 45
Gly Tyr Ile Asn Pro Ser Thr Gly Tyr Thr Glu Tyr Asn Gln Lys Phe
50 55 60
Lys Glu Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Tyr Pro Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro
115 120 125
Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val
130 135 140
Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser
145 150 155 160
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
165 170 175
Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser
180 185 190
Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
195 200 205
Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys
210 215 220
Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys
225 230 235 240
Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val
245 250 255
Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
260 265 270
Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
275 280 285
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp
290 295 300
Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe
305 310 315 320
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys
325 330 335
Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys
340 345 350
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
355 360 365
Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
370 375 380
Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser
385 390 395 400
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
405 410 415
Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser
420 425 430
Leu Ser His Ser Pro Gly Lys
435
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Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Thr Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Arg Asn Pro Pro Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro
100 105 110
Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly
115 120 125
Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn
130 135 140
Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn
145 150 155 160
Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser
165 170 175
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr
180 185 190
Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe
195 200 205
Asn Arg Asn Glu Cys
210

Claims (12)

1. An anti-serum amyloid a antibody or a functional fragment thereof, characterized in that the antibody or the functional fragment thereof comprises HCDR1, HCDR2, HCDR3 having amino acid sequences as shown in SEQ ID No. 1 to SEQ ID No. 3, and LCDR1, LCDR2, LCDR3 having amino acid sequences as shown in SEQ ID No. 4 to SEQ ID No. 6.
2. The antibody or functional fragment thereof according to claim 1, wherein the antibody or functional fragment thereof further comprises HFR1, HFR2, HFR3, HFR4 having the amino acid sequences shown in SEQ ID No. 7 to SEQ ID No. 10 and LFR1, LFR2, LFR3 and LFR4 having the amino acid sequences shown in SEQ ID No. 11 to SEQ ID No. 14, or an amino acid sequence having at least 80% homology with each of said sequences;
optionally, the amino acid sequence of HFR2 of the antibody or functional fragment thereof is shown in SEQ ID NO. 15;
alternatively, the antibody or functional fragment thereof is produced with a KD of 9.00×10 or less -9 The affinity of M binds serum amyloid a.
3. An anti-serum amyloid a antibody 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/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, HFR4, LFR1, HFR2, LFR3, r4 is the amino acid sequence of HFR1, HFR2, HFR3, LFR4, LFR3, LFR4 of claim 1, HCDR3;
alternatively, the heavy chain variable region amino acid sequence is shown as SEQ ID NO. 18 or 19;
alternatively, the light chain variable region amino acid sequence is shown in SEQ ID NO. 20.
4. The antibody or functional fragment thereof according to any one of claims 1 to 3, wherein the antibody or functional fragment thereof further comprises a constant region;
optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region;
alternatively, the heavy chain constant region is selected from the group consisting of the heavy chain constant region of IgG1, igG2, igG3, igG4, igA, igM, igE or IgD; the light chain constant region is selected from a kappa-type or lambda-type light chain constant region;
optionally, the constant region is derived from bovine, equine, porcine, ovine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human;
alternatively, the constant region is of mouse species origin;
alternatively, the heavy chain constant region sequence is as shown in SEQ ID NO. 16 or has at least 80% homology thereto;
alternatively, the light chain constant region sequence is as shown in SEQ ID NO. 17 or has at least 80% homology thereto.
5. The antibody or functional fragment thereof according to any one of claims 1 to 4, wherein the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv and scFv of the antibody.
6. An anti-serum amyloid a antibody comprising a heavy chain and/or a light chain, characterized in that the heavy chain comprises the heavy chain variable region of claim 3 and the heavy chain constant region of claim 4; the light chain comprises the light chain variable region of claim 3 and the light chain constant region of claim 4;
alternatively, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 21 or 22;
alternatively, the amino acid sequence of the light chain is shown in SEQ ID NO. 23.
7. An antibody conjugate comprising the antibody or functional fragment thereof of any one of claims 1 to 6;
optionally, the antibody or functional fragment thereof is labeled with a label;
optionally, the label is selected from the group consisting of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels;
optionally, the fluorescent dye is selected from fluorescein dye and its derivative, rhodamine dye and its derivative, cy series dye and its derivative, alexa series dye and its derivative, and protein dye and its derivative;
alternatively, the enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase;
optionally, the radioisotope is selected from 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110 msin, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu and 18F;
optionally, the chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, clorine and its derivatives, and peroxyoxalate and its derivatives;
optionally, the nanoparticle-based label is selected from the group consisting of nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;
optionally, the colloid is selected from the group consisting of colloidal metals, disperse dyes, dye-labeled microspheres, and latex;
optionally, the colloidal metal is selected from the group consisting of colloidal gold, colloidal silver, and colloidal selenium;
optionally, the antibody or functional fragment thereof is coated to a solid phase;
alternatively, the solid phase is selected from the group consisting of microspheres, plates, and membranes;
alternatively, the solid phase is selected from the group consisting of magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon and nitrocellulose membranes.
8. A reagent or kit for detecting serum amyloid a, comprising the antibody or functional fragment thereof of any one of claims 1 to 6 or the antibody conjugate of claim 7.
9. A nucleic acid encoding the antibody or functional fragment thereof of any one of claims 1 to 6.
10. A vector comprising the nucleic acid of claim 9.
11. A cell comprising the nucleic acid of claim 9 or the vector of claim 10.
12. A method of preparing an antibody or functional fragment thereof according to any one of claims 1 to 6, comprising: culturing the cell of claim 11.
CN202210372088.4A 2022-04-11 2022-04-11 Anti-serum amyloid A antibody, reagent for detecting serum amyloid A and kit Pending CN116925216A (en)

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CN116925216A true CN116925216A (en) 2023-10-24

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