Anti-serum amyloid A antibodies, reagents and kits for detecting serum amyloid A

Abstract

The present 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 anti-serum amyloid A antibody disclosed in the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region. The antibody provides an important source of raw materials for the detection of serum amyloid A and has improved affinity and activity.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-serum amyloid A antibody, a reagent and a kit for detecting serum amyloid A. Background Art

[0002] Serum amyloid A (SAA) is a non-specific acute phase protein, composed of 104 amino acids, and belongs to the heterogeneous protein class in the apolipoprotein family. In the acute phase reaction, SAA is synthesized by activated macrophages and fibroblasts in the liver after stimulation by IL-1, IL-6 and TNF, and can be increased to 100-1000 times the initial concentration, but the half-life is extremely short, only about 50 minutes.

[0003] SAA increases in both viral and bacterial infections, while CRP hardly increases or increases insignificantly in viral infections. For weak inflammatory stimulation, SAA is more sensitive than CRP, and exceeds the upper limit of the reference range earlier than CRP, increases earlier, and decreases faster and more significantly when recovering. Therefore, SAA is a more sensitive indicator in patients with viral infections and non-invasive or early invasive bacterial infections with normal CRP. In the early stages of pediatric infectious diseases, it is difficult to distinguish whether the pediatric patient has a bacterial infection or a viral infection because the signs and symptoms of pediatric patients are unclear. Therefore, early and rapid identification of bacterial and viral infections in pediatric patients is of great significance for timely and effective treatment and prevention of various complications. Since CRP lacks sensitivity to viral infections, SAA can increase in the early stages of bacterial and viral infections. The combined detection of the two can improve the efficiency of early diagnosis of viral infections and provide valuable reference information for the identification of viral and bacterial infections and the selection of treatment plans. In addition, the ratio of SAA to CRP is correlated with the severity of pediatric infectious diseases, and monitoring SAA / CRP has greater application value than detecting SAA or CRP alone. At the same time, studies have shown that SAA levels in patients with many tumors, such as liver cancer, lung cancer, breast cancer, prostate cancer, and endometrial cancer, are elevated to varying degrees. SAA levels are significantly correlated with the activity, malignancy, and metastasis of the tumor. The increase in SAA in the metastasis stage of malignant tumors usually shows higher values ​​than in the stable stage of the tumor.

[0004] At present, the main detection method of SAA is fluorescent immunochromatography, which is an immunological detection method based on the specific reaction of antibodies and antigens, and uses fluorescence to amplify and display the detected signal. Similar immunological detection methods include radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence, etc. The above immunological detection methods all require antibodies against SAA. Therefore, there is a strong demand in the field for antibodies that are effective and bind to SAA and detect it.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In view of the problem that the existing sources of anti-serum amyloid A antibodies are scarce, the present application provides an anti-serum amyloid A antibody with improved affinity and activity, which provides an important source of raw materials for the detection of serum amyloid A.

[0007] To achieve the above-mentioned object, according to one aspect of the present invention, an antibody or a functional fragment thereof is provided, wherein the antibody or the functional fragment thereof comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:1, HCDR2 with an amino acid sequence as shown in SEQ ID NO:2, HCDR3 with an amino acid sequence as shown in SEQ ID NO:3 or 15, and LCDR1 with an amino acid sequence as shown in SEQ ID NO:4, LCDR2 with an amino acid sequence as shown in SEQ ID NO:5, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:6 or 16.

[0008] In order to achieve the above-mentioned object, according to the second aspect of the present invention, an anti-serum amyloid A antibody or a functional fragment thereof is provided, wherein the antibody or the functional fragment thereof comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region comprises a sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are the amino acid sequences of the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

[0009] In order to achieve the above object, according to the third aspect of the present invention, an anti-serum amyloid A antibody is provided, comprising a heavy chain and / or a light chain, wherein the heavy chain comprises the above-mentioned heavy chain variable region, and the light chain comprises the above-mentioned light chain variable region.

[0010] In order to achieve the above object, according to the fourth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody or a functional fragment thereof.

[0011] In order to achieve the above object, according to the fifth aspect of the present invention, a reagent or kit for detecting serum amyloid A is provided, wherein the reagent or kit comprises the above antibody or its functional fragment or the above antibody conjugate.

[0012] In order to achieve the above object, according to the sixth aspect of the present invention, there is provided use of the above antibody or its functional fragment, the above antibody conjugate or the above reagent or kit in the detection of serum amyloid A.

[0013] 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 its functional fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0015] Figure 1 The figure shows the result of reducing SDS-PAGE of Anti-SAA-8D4mut1. DETAILED DESCRIPTION

[0016] The present invention provides an antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 1, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 2, HCDR3 with an amino acid sequence as shown in SEQ ID NO: 3 or 15, and LCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 6 or 16. The antibody has improved affinity and / or activity.

[0017] 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.

[0018] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0019] In the present invention, the heavy chain complementary determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. 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 the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark the CDR region, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0020] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region other than the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0021] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0022] In an optional embodiment, the antibody or its functional fragment further comprises HFR1, HFR2, HFR3, HFR4 having amino acid sequences as shown in SEQ ID NO:7 to SEQ ID NO:10 and LFR1, LFR2, LFR3 and LFR4 having amino acid sequences as shown in SEQ ID NO:11 to SEQ ID NO:14.

[0023] It should be noted that, in other embodiments, the amino acid sequences of each framework region of the antibodies or functional fragments 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 with the above-mentioned corresponding framework regions (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14).

[0024] In an optional embodiment, the amino acid sequence of HFR1 of the antibody or its functional fragment is as shown in SEQ ID NO:17.

[0025] In an optional embodiment, the amino acid sequence of HFR2 of the antibody or its functional fragment is as shown in SEQ ID NO:18.

[0026] In an optional embodiment, the antibody or its functional fragment has a KD≤10 -8 M, KD≤10 -9 M, KD≤10 -10 M, KD≤10 -11 M or KD≤10 -12 The affinity of M binds to serum amyloid A.

[0027] In an optional embodiment, the antibody or its functional fragment has a KD≤8.19×10 -9 The affinity of M binds to serum amyloid A.

[0028] In an optional embodiment, the antibody or its functional fragment has a KD≤2.14×10 -10 The affinity of M binds to serum amyloid A.

[0029] On the other hand, an embodiment of the present invention provides an anti-serum amyloid A antibody or a functional fragment thereof, wherein the antibody or the functional fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, and the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, and the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR 1, LCDR2, and LCDR3 are the above-mentioned amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and the amino acid sequences of the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are the above-mentioned amino acid sequences of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0030] In an optional embodiment, the heavy chain variable region amino acid sequence is shown in any one of SEQ ID NOs: 21 to 28.

[0031] In an optional embodiment, the light chain variable region amino acid sequence is shown in any one of SEQ ID NOs: 29 to 30.

[0032] In an alternative embodiment, the antibody further comprises a constant region.

[0033] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0034] In an alternative embodiment, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, and the light chain constant region is selected from the κ type or λ type light chain constant region.

[0035] In an alternative embodiment, the constant region is derived from a species of cow, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0036] In an alternative embodiment, the species origin of the constant region is mouse.

[0037] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:19, and the light chain constant region (CL) sequence is as shown in SEQ ID NO:20.

[0038] It should be noted that, 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 with the above-mentioned constant region (SEQ ID NO: 19 or 20).

[0039] In an optional embodiment, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0040] The functional fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, a person skilled in the art can easily obtain the above functional fragments.

[0041] The functional fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0042] On the other hand, the present invention provides an anti-serum amyloid A antibody, comprising a heavy chain and / or a light chain, wherein the heavy chain comprises the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; and the light chain comprises the above-mentioned light chain variable region and the above-mentioned light chain constant region.

[0043] In an alternative embodiment, the amino acid sequence of the heavy chain is as shown in any one of SEQ ID NOs: 31 to 38.

[0044] In an optional embodiment, the amino acid sequence of the light chain is as shown in any one of SEQ ID NOs: 39 to 40.

[0045] In another aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the above-mentioned antibody or a functional fragment thereof.

[0046] In an optional embodiment, the antibody or its functional fragment in the above-mentioned antibody conjugate is labeled with a marker.

[0047] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0048] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.

[0049] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0050] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0051] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0052] In an optional embodiment, the radioactive isotope 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 Such as 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.

[0053] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0054] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0055] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0056] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0057] In an optional embodiment, the antibody or its functional fragment in the above-mentioned antibody conjugate is coated on a solid phase.

[0058] In alternative embodiments, the solid phase is selected from the group consisting of microspheres, plates, and membranes.

[0059] In an optional embodiment, the solid phase includes, but is not limited to, magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

[0060] In an alternative embodiment, the solid phase is a nitrocellulose membrane.

[0061] On the other hand, the present invention provides a reagent or kit for detecting serum amyloid A, wherein the reagent or kit comprises the above-mentioned antibody or its functional fragment or the above-mentioned antibody conjugate.

[0062] On the other hand, the present invention provides use of the above-mentioned antibody or its functional fragment, antibody conjugate or the above-mentioned reagent or kit in the detection of serum amyloid A.

[0063] In another aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or a functional fragment thereof.

[0064] In another aspect, the present invention provides a vector comprising the above nucleic acid molecule.

[0065] In another aspect, the present invention provides a cell containing the above-mentioned vector.

[0066] In another aspect, the present invention provides a method for preparing an antibody or a functional fragment thereof, comprising: culturing the cell as described above.

[0067] Based on the amino acid sequence of the antibody or its functional fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the antibody or its functional fragment, for example, separating and purifying the antibody or its functional fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its functional fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the antibody or its functional fragment of the present invention, it belongs to the protection scope of the present invention.

[0068] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0070] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0071] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0072] Example 1 Preparation of Anti-SAA-8D4 Monoclonal Antibody

[0073] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMRACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen.

[0074] 1 Construction of recombinant plasmid

[0075] (1) Antibody gene preparation

[0076] mRNA was extracted from the hybridoma cell line secreting monoclonal antibodies against serum amyloid A, and the DNA product was obtained by RT-PCR. The product was inserted into the pMD-18T vector after A addition reaction with rTaq DNA polymerase and transformed into DH5α competent cells. After the colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

[0077] (2) Sequence analysis of the variable region gene of Anti-SAA-8D4 antibody

[0078] The gene sequences obtained by the above sequencing were placed in the Kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 336bp, preceded by a 57bp leader peptide sequence; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 360bp, belonging to the VH1 gene family, and preceded by a 57bp leader peptide sequence.

[0079] (3) Construction of recombinant antibody expression plasmid

[0080] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, respectively, and the 0.73kb Light Chain gene fragment and the 1.42kb Heavy Chain gene fragment were amplified by the PCR amplification method.

[0081] The Heavy Chain and Light Chain gene fragments were double-digested with HindIII / EcoRI, and the 3.4A vector was double-digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0082] 2. Screening of stable cell lines

[0083] (1) Transient transfection of recombinant antibody expression plasmid into CHO cells to determine the activity of the expression plasmid

[0084] The plasmid was diluted to 40ug / 100ul with ultrapure water and the CHO cells were adjusted to 1.43×10 7 cells / ml in a centrifuge tube, mix 100 μL of plasmid with 700 μL of cells, transfer to an electroporation cup, electroporate, take samples and count on the 3rd, 5th and 7th days, and collect samples for detection on the 7th day.

[0085] The coating solution (main component NaHCO3) was used to dilute SAA (purchased from Yashraj biotechnology limited, product number FRSAA-61) to 1ug / ml, 100μL per well, and incubated at 4°C overnight; the next day, the cells were washed twice with the washing solution and patted dry; the blocking solution (20% BSA + 80% PBS) was added, 120μL per well, incubated at 37°C for 1h, and patted dry; the diluted cell supernatant was added, 100μL / well, incubated at 37°C for 30min (part of the supernatant was removed for 1h); the cells were washed 5 times with the washing solution and patted dry; goat anti-mouse IgG-HRP was added, 100μL per well, and ...). μL, 37℃, 30min; wash 5 times with washing solution, pat dry; add colorimetric solution A (50μL / well, containing citric acid + sodium acetate + acetanilide + carbamide peroxide), add colorimetric solution B (50μL / well, containing citric acid + EDTA·2Na + TMB + concentrated HCL), 10min; add stop solution (50μL / well, containing EDTA·2Na + concentrated H2SO4); read OD value at 450nm (reference 630nm) on the microplate reader. The results showed that the reaction OD was still greater than 1.0 after the cell supernatant was diluted 1000 times, and the reaction OD of the well without cell supernatant was less than 0.1, indicating that the antibody produced after transient plasmid transfection was active against SAA.

[0086] (2) Linearization of recombinant antibody expression plasmid

[0087] Prepare the following reagents: 50 μL of Buffer, 100 μg of DNA / tube, 10 μL of PuvⅠ enzyme, and make up to 500 μL with sterile water. Perform enzymatic digestion in a 37°C water bath overnight. First, use an equal volume of phenol / chloroform / isoamyl alcohol (lower layer) 25:24:1, and then use chloroform (aqueous phase) for extraction in sequence. Precipitate on ice with 0.1 volume (aqueous phase) of 3M sodium acetate and 2 volumes of ethanol, rinse the precipitate with 70% ethanol, remove the organic solvent, and reconstitute with an appropriate amount of sterile water after the ethanol has completely evaporated. Finally, measure the concentration.

[0088] (3) Stable transfection of recombinant antibody expression plasmid and pressure screening of stable cell lines

[0089] The plasmid was diluted to 40ug / 100ul with ultrapure water and the CHO cells were adjusted to 1.43×10 7 cells / ml in a centrifuge tube, mix 100μL plasmid with 700μL cells, transfer to an electroporation cup, electrotransform, and count the next day; culture under pressure in 25umol / L MSX 96-well for about 25 days.

[0090] Observe the clone wells with cells under a microscope and record the confluence; take the culture supernatant and send it for testing; select cell lines with high antibody concentration and relative concentration and transfer them to 24 wells, and transfer them to 6 wells after about 3 days; after 3 days, keep the seed batch culture and adjust the cell density to 0.5×10 6 cells / ml, 2.2 ml for batch culture, cell density 0.3×10 6 cells / ml, 2ml for seed preservation; 7-day 6-well batch culture supernatant samples were sent for testing, and cell lines with smaller antibody concentrations and cell diameters were selected for TPP seed preservation and subculture.

[0091] 3. Recombinant Antibody Production

[0092] (1) Cell expansion

[0093] After cell recovery, the cells are first cultured in a 125ml shake flask with an inoculation volume of 30ml and a culture medium of 100% Dynamis medium. The cells are placed in a shaker at a speed of 120r / min, a temperature of 37°C, and a carbon dioxide content of 8%. After 72 hours of culture, the cells are inoculated and expanded at an inoculation density of 500,000 cells / ml. The expansion volume is calculated according to production requirements, and the culture medium is 100% Dynamis medium. The cells are expanded every 72 hours. When the cell volume meets the production requirements, the inoculation density is strictly controlled to be around 500,000 cells / ml for production.

[0094] (2) Shake flask production and purification

[0095] Shake flask parameters: speed 120r / min, temperature 37℃, carbon dioxide 8%. Feeding: Start feeding every day when the culture reaches 72h in the shake flask. HyCloneTM Cell BoostTM Feed 7a is fed daily at 3% of the initial culture volume, and Feed 7b is fed daily at one thousandth of the initial culture volume until the 12th day (feeding on the 12th day). Glucose is added at 3g / L on the sixth day. Samples are collected on the 13th day. Affinity purification is performed using a proteinA affinity chromatography column. Take 6μg of the purified antibody for reducing SDS-PAGE. The electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands are shown, one Mr is 50KD (heavy chain) and the other Mr is 28KD (light chain).

[0096] Example 2 Affinity and activity optimization

[0097] Although the Anti-SAA-8D4 monoclonal antibody obtained in Example 1 has the ability to bind to serum amyloid A, its affinity and antibody activity are not ideal. Therefore, the applicant carried out directed mutation of the light chain CDR and heavy chain CDR of the antibody. That is, a computer is used to simulate the structure of the antibody variable region, the structure of the complex of the antigen and the antibody variable region, the key amino acid analysis of the antibody and the mutation design. According to the mutation scheme, bidirectional primers covering the mutation site are designed and synthesized, primers at both ends of the target DNA are synthesized, a high-fidelity PCR reaction is carried out, the PCR product is cloned into a vector, and then the mutant antibody is prepared according to the method described in Example 1. After screening, monoclonal antibodies with significantly improved affinity and antibody activity were obtained, and they were named: Anti-SAA-8D4mut1 to Anti-SAA-8D4mut16. The amino acid sequence of each monoclonal antibody is shown in the following table:

[0098] Table 1 Antibody sequences

[0099] Sample name Heavy chain number Light chain number Anti-SAA-8D4mut1 SEQ ID NO:31 SEQ ID NO:39 Anti-SAA-8D4mut2 SEQ ID NO:32 SEQ ID NO:39 Anti-SAA-8D4mut3 SEQ ID NO:33 SEQ ID NO:39 Anti-SAA-8D4mut4 SEQ ID NO:34 SEQ ID NO:39 Anti-SAA-8D4mut5 SEQ ID NO:35 SEQ ID NO:39 Anti-SAA-8D4mut6 SEQ ID NO:36 SEQ ID NO:39 Anti-SAA-8D4mut7 SEQ ID NO:37 SEQ ID NO:39 Anti-SAA-8D4mut8 SEQ ID NO:38 SEQ ID NO:39 Anti-SAA-8D4mut9 SEQ ID NO:31 SEQ ID NO:40 Anti-SAA-8D4mut10 SEQ ID NO:32 SEQ ID NO:40 Anti-SAA-8D4mut11 SEQ ID NO:33 SEQ ID NO:40 Anti-SAA-8D4mut12 SEQ ID NO:34 SEQ ID NO:40 Anti-SAA-8D4mut13 SEQ ID NO:35 SEQ ID NO:40 Anti-SAA-8D4mut14 SEQ ID NO:36 SEQ ID NO:40 Anti-SAA-8D4mut15 SEQ ID NO:37 SEQ ID NO:40 Anti-SAA-8D4mut16 SEQ ID NO:38 SEQ ID NO:40

[0100] Example 3 Performance Testing of Antibodies

[0101] 1 Affinity analysis

[0102] Using the AMC sensor, the purified antibody was diluted to 20ug / ml with PBST, and SAA (purchased from Yashrajbiotechnology limited, catalog number FRSAA-61) was graded diluted with PBST:

[0103] Operation process: 60s of equilibrium in buffer 1 (PBST), 300s of antibody fixation in antibody solution, 180s of incubation in buffer 2 (PBST), 420s of binding in antigen solution, 1200s of dissociation in buffer 2, sensor regeneration with 10mM pH 1.69 GLY solution and buffer 3 (PBST), and data output. (KD represents equilibrium dissociation constant, i.e. affinity; kon represents binding rate; kdis represents dissociation rate. The main components of PBST are Na2HPO4+NaCl+TW-20).

[0104] Table 2 Affinity data

[0105]

[0106]

[0107] 2. Activity identification

[0108] The coating solution (main component NaHCO3) was used to dilute SAA (purchased from Yashraj biotechnology limited, product number FRSAA-61) to 1ug / ml, 100μL per well, and incubated at 4°C overnight; the next day, the cells were washed twice with the washing solution and patted dry; blocking solution (20% BSA + 80% PBS) was added, 120μl per well, incubated at 37°C for 1h, and patted dry; the diluted monoclonal antibodies were added, 100μl / well, incubated at 37°C for 30min-60min; the cells were washed 5 times with the washing solution and patted dry; goat anti-mouse IgG- HRP, 100 μl per well, 37°C, 30 min; wash 5 times with washing solution (PBS), pat dry; add colorimetric solution A (50 μl / well, containing 2.1 g / L citric acid, 12.25 g / L citric acid, 0.07 g / L acetanilide and 0.5 g / L urea peroxide), add colorimetric solution B (50 μl / well, containing 1.05 g / L citric acid, 0.186 g / EDTA·2Na, 0.45 g / L TMB and 0.2 ml / L concentrated HCl), 10 min; add stop solution (50 μl / well, containing 0.75 g / EDTA·2Na and 10.2 ml / L concentrated H2SO4); read OD value at 450 nm (reference 630 nm) on the microplate reader. The results are shown in the table below.

[0109] Table 3 Activity data

[0110]

[0111]

[0112] 3. Stability Assessment

[0113] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant temperature box) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and the activity of the 21-day samples was tested. The results showed that there was no obvious protein state change in the antibodies under the three test conditions for 21 days, and the activity did not show a downward trend with the increase of the test temperature, indicating that the above antibodies are stable. Table 4 below shows the OD results of the enzyme immunoassay activity test for 21 days of antibody test.

[0114] Table 4 Stability data

[0115] Sample concentration (ng / ml) 100 25 0 4℃, 21-day samples 1.698 0.702 0.013 -80℃, 21 days sample 1.669 0.687 0.014 37℃, 21 days sample 1.657 0.692 0.011

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0117] The partial amino acid sequences involved in this application are as follows:

[0118] Sequence number Sequence fragment SEQ ID NO:1 G JZ SEQ ID NO:2 YINCYNGATSYNQKFKG SEQ ID NO:3 TITTAEAWF SEQ ID NO:4 RASQSVSTSSYSYMH SEQ ID NO:5 YASNLES SEQ ID NO:6 QHSWEIP SEQ ID NO:15 TLTTAEAWF SEQ ID NO:16 QHSWELP

Claims

1. An anti-serum amyloid A antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises an amino acid sequence such as HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3 or 15, and an amino acid sequence such as LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, LCDR3 shown in SEQ ID NO: 6 or 16.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or its antigen-binding fragment also includes HFR1, HFR2, HFR3, HFR4 whose amino acid sequences are shown in SEQ ID NO:7 to SEQ ID NO:10 and LFR1, LFR2, LFR3 and LFR4 whose amino acid sequences are shown in SEQ ID NO:11 to SEQ ID NO:14, or amino acid sequences that have at least 80% homology with each of the sequences.

3. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The amino acid sequence of HFR1 of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:

17.

4. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The amino acid sequence of HFR2 of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:

18.

5. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The antibody or antigen-binding fragment thereof has a KD of ≤8.19×10 -9 The affinity of M binds to serum amyloid A.

6. An anti-serum amyloid A antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, the light chain variable region comprises a sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described in claim 1, and the amino acid sequences of the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 are the amino acid sequences of the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 described in any one of claims 2 to 4.

7. An anti-serum amyloid A antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences of the heavy chain variable region and the light chain variable region are selected from any combination of the following:

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region.

9. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that: The constant region includes a heavy chain constant region and a light chain constant region.

10. The antibody or antigen-binding fragment thereof according to claim 9, characterized in that: The heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

11. The antibody or antigen-binding fragment thereof according to claim 9, characterized in that: The species origin of the constant region is cow, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

12. The antibody or antigen-binding fragment thereof according to claim 9, characterized in that: The species origin of the constant region is mouse.

13. The antibody or antigen-binding fragment thereof according to claim 9, characterized in that: The heavy chain constant region sequence is as shown in SEQ ID NO: 19 or has at least 80% homology thereto; The light chain constant region sequence is as shown in SEQ ID NO: 20 or has at least 80% homology thereto.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that: The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

15. An anti-serum amyloid A antibody comprising a heavy chain and a light chain, characterized in that: The heavy chain comprises the heavy chain variable region described in claim 6 or 7 and the heavy chain constant region described in any one of claims 9, 10, and 13; the light chain comprises the light chain variable region described in claim 6 or 7 and the light chain constant region described in any one of claims 9, 10, and 13.

16. An anti-serum amyloid A antibody comprising a heavy chain and a light chain, characterized in that: The amino acid sequences of the heavy chain and the light chain are selected from any combination of the following:

17. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the anti-serum amyloid A antibody according to claim 15 or 16, wherein the antibody or antigen-binding fragment thereof is labeled with a marker.

18. The antibody conjugate according to claim 17, characterized in that The label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents and nanoparticle labels.

19. The antibody conjugate according to claim 18, characterized in that The fluorescent dye is selected from fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, Alexa series dyes and their derivatives, and protein dyes and their derivatives.

20. The antibody conjugate according to claim 18, characterized in that The enzyme is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and 6-phosphate glucose deoxidase.

21. The antibody conjugate according to claim 18, characterized in that The radioisotope is selected from 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 Such as 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 according to claim 18, characterized in that The chemiluminescent reagent is selected from luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

23. The antibody conjugate according to claim 18, characterized in that The nanoparticle marker is selected from nanoparticles and colloids.

24. The antibody conjugate according to claim 23, characterized in that The nanoparticle marker is selected from organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

25. The antibody conjugate according to claim 23, characterized in that The colloid is selected from latex, colloidal selenium, colloidal metal, disperse dye and dye-labeled microspheres.

26. The antibody conjugate according to claim 25, characterized in that The colloidal metal is selected from colloidal gold and colloidal silver.

27. The antibody conjugate according to claim 17, characterized in that The antibody or antigen-binding fragment thereof is coated onto a solid phase.

28. The antibody conjugate according to claim 27, characterized in that The solid phase is selected from the group consisting of microspheres, plates and membranes.

29. The antibody conjugate according to claim 28, characterized in that The solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

30. A reagent or kit for detecting serum amyloid A, characterized in that: The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, the anti-serum amyloid A antibody according to claim 15 or 16, or the antibody conjugate according to any one of claims 17 to 29.

31. A nucleic acid, characterized in that It encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the anti-serum amyloid A antibody according to claim 15 or 16.

32. A carrier, characterized in that It contains the nucleic acid according to claim 31.

33. A cell, characterized in that It contains the nucleic acid according to claim 31 or the vector according to claim 32.

34. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14 or the anti-serum amyloid A antibody according to claim 15 or 16, characterized in that: It includes: Cultivate the cell of claim 33.

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