Pairs of antibodies, reagents and methods for detecting serum amyloid a

Abstract

The application discloses an antibody pair for detecting serum amyloid A, a reagent and a method, and relates to the field of immunodiagnosis. The antibody pair for detecting serum amyloid A disclosed by the application comprises a first antibody and a second antibody, and the reagent and the detection method based on the antibody combination can accurately detect the presence of serum amyloid A.

Description

Technical Field

[0001] This invention relates to the field of immunodiagnostic technology, and more specifically, to an antibody pair, reagent, and method for detecting serum amyloid A. Background Technology

[0002] SAA, or serum amyloid A, is an acute-phase reactive protein belonging to the heterogeneous protein family of apolipoproteins. During the acute phase response, its expression increases under the regulation of cytokines (IL-1, IL-6, TNFα). SAA is synthesized in the liver by activated macrophages and fibroblasts. Synthesized SAA binds to high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL), especially HDL3. SAA is present in trace amounts in the blood of healthy individuals, but its levels can significantly increase within 8-24 hours when the body experiences infection or injury. It has a short half-life of only 50 minutes, and plasma SAA concentrations can increase by approximately 100-1000 times in the acute phase. Changes in SAA levels have significant clinical value for the early diagnosis, risk assessment, efficacy monitoring, and prognostic evaluation of infectious diseases.

[0003] Currently, immunodiagnostic methods are important for detecting SAA. The main immunodiagnostic methods available on the market for detecting SAA protein include colloidal gold assay, fluorescence assay, chemiluminescence assay, and latex-enhanced immunoturbidimetric assay. Through long-term research, the inventors discovered that the sandwich pairing of SAA antibody raw materials is, besides the antibody raw materials themselves, the key to achieving SAA immunodetection. Summary of the Invention

[0004] This application provides an antibody pair that provides an important source of paired antibody raw materials for the detection of serum amyloid A, and has good detection performance.

[0005] To achieve the above objectives, according to one aspect of the present invention, an antibody pair for detecting serum amyloid A is provided. The antibody pair comprises a first antibody, which includes: three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region shown in SEQ ID NO:9, and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) in the light chain variable region shown in SEQ ID NO:11;

[0006] The antibody pair includes a second antibody, the second antibody comprising: the heavy chain variable region shown in SEQ ID NO:20

[0007] The three heavy chain complementarity determination regions HCDR1, HCDR2 and HCDR3 and the three light chain complementarity determination regions LCDR1, LCDR2 and LCDR3 in the light chain variable region shown in SEQ ID NO:22.

[0008] The complementary decision region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0009] To achieve the above objective, according to a second aspect of the present invention, an antibody pair for detecting serum amyloid A is provided, the antibody pair comprising a first antibody having HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6.

[0010] The antibody pair includes a second antibody having HCDR1 as shown in SEQ ID NO:13, HCDR2 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO:15, LCDR1 as shown in SEQ ID NO:16, and LCDR1 as shown in SEQ ID NO:17.

[0011] LCDR2, LCDR3 as shown in SEQ ID NO:18.

[0012] To achieve the above objectives, according to a third aspect of the present invention, an antibody pair for detecting serum amyloid A is provided;

[0013] The antibody pair comprises a first antibody, which comprises at least one of (1)-(2):

[0014] (1) The heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:11;

[0015] (2) The heavy chain shown in SEQ ID NO:10 and the light chain shown in SEQ ID NO:12;

[0016] The antibody pair comprises a second antibody, the second antibody comprising at least one of (a)-(b):

[0017] (a) The heavy chain variable region shown in SEQ ID NO:20 and the light chain variable region shown in SEQ ID NO:22;

[0018] (b) The heavy chain shown in SEQ ID NO:21 and the light chain shown in SEQ ID NO:23.

[0019] To achieve the above objectives, according to a fourth aspect of the present invention, an antibody pair for detecting serum amyloid A is provided;

[0020] The antibody pair comprises a first antibody, wherein the variable region of the heavy chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:9, and the variable region of the light chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:11; or;

[0021] The antibody pair comprises a second antibody, wherein the variable region of the heavy chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:20, and the variable region of the light chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:22.

[0022] In one alternative implementation, the mutation is the addition, deletion, substitution, or modification of one or more amino acids in the framework region of the first or second antibody.

[0023] In one alternative implementation, the framework region of the conserved variant formed by the mutation of the first antibody has at least 80% identity with the framework region of the first antibody before the mutation; or;

[0024] The framework region of the conserved variant formed by the mutation of the second antibody has at least 80% identity with the framework region of the second antibody before the mutation.

[0025] To achieve the above objectives, according to a fifth aspect of the present invention, an antibody pair for detecting serum amyloid A is provided;

[0026] The antibody pair comprises a first antibody that binds to an epitope, the epitope being the same as the epitope bound by the antibody comprising the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:11.

[0027] The antibody pair comprises a second antibody that binds to an epitope: the epitope is the same as the epitope bound to the antibody comprising the heavy chain variable region shown in SEQ ID NO:20 and the light chain variable region shown in SEQ ID NO:22.

[0028] To achieve the above objectives, according to a sixth aspect of the present invention, an antibody pair for detecting serum amyloid A is provided, the antibody comprising a constant region, wherein the heavy chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, or a combination of multiple constant regions; and the light chain constant region is selected from κ-type or λ-type light chain constant regions.

[0029] In an optional embodiment, the heavy chain constant region of the first antibody is SEQ ID NO:7 or a sequence having at least 80% identity with it, and the light chain constant region of the first antibody is SEQ ID NO:8 or a sequence having at least 80% identity with it.

[0030] In an optional embodiment, the heavy chain constant region of the second antibody is SEQ ID NO:19 or a sequence having at least 80% identity with it, and the light chain constant region of the second antibody is SEQ ID NO:8 or a sequence having at least 80% identity with it.

[0031] To achieve the above objectives, according to a seventh aspect of the present invention, a reagent for detecting serum amyloid A is provided, the reagent comprising a first group of antibodies and a second group of antibodies, wherein the first group of antibodies comprises a first antibody in an antibody pair;

[0032] The second group of antibodies includes the second antibody in the antibody pair;

[0033] Optionally, one of the first group of antibodies and the second group of antibodies is a coating antibody, and the other is a labeled antibody;

[0034] The reagent includes the first antibody and the second antibody in the above antibody pair; the first antibody is a coating antibody and the second antibody is a labeled antibody, or the second antibody is a coating antibody and the first antibody is a labeled antibody;

[0035] In an optional embodiment, the coated antibody or labeled antibody is conjugated with biotin or a biotin derivative;

[0036] In one optional embodiment, the label conjugated to the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels;

[0037] In one optional embodiment, the coated antibody is coupled to a solid-phase support;

[0038] In one alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0039] To achieve the above objectives, according to an eighth aspect of the present invention, a method for detecting serum amyloid A is provided, comprising:

[0040] a) Under conditions sufficient to induce antibody / antigen binding, the aforementioned antibody pair or reagent is brought into contact with the sample to be tested to form an immune complex; and

[0041] b) Detect the presence of the immune complex, the presence of which indicates the presence of serum amyloid A in the test sample.

[0042] To achieve the above objectives, according to a ninth aspect of the present invention, the use of the above-described antibody pair or reagent in detecting serum amyloid A or in preparing a product for detecting serum amyloid A is provided. Attached Figure Description

[0043] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 This is a standard curve based on the test results of the calibrators;

[0045] Figure 2 The linear correlation of clinical samples tested to compare the proposed method with the Siemens method;

[0046] Figure 3 The linear correlation of low values ​​in clinical samples tested in comparison with the Siemens protocol was used to examine the protocol in this application. Detailed Implementation

[0047] In a first aspect, embodiments of the present invention provide an antibody pair for detecting serum amyloid A. The antibody pair includes a first antibody, which comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region shown in SEQ ID NO:9 and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) in the light chain variable region shown in SEQ ID NO:11. The antibody pair also includes a second antibody, which comprises three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region shown in SEQ ID NO:20 and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) in the light chain variable region shown in SEQ ID NO:22.

[0048] The complementary decision region is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM, or Contact.

[0049] In this invention, the term "antibody" is used in the broadest sense, and the antibody in the method of this invention can be a whole antibody, an antigen-binding fragment, or a polymeric antibody capable of binding to serum amyloid A.

[0050] Whole antibodies can be monoclonal. Such whole antibodies are typically prepared by any suitable method known in the art. For example, antibody molecules are isolated from the serum of a mammal, typically a rabbit or mouse, by immunizing it with serum amyloid A under suitable conditions, for example, said mammal. Monoclonal antibodies can be obtained by hybridoma or recombinant methods. The antigen-binding fragment includes an antigen-binding site, such as a Fab or F(ab)2 fragment. In an alternative embodiment, the antigen-binding fragment is selected from any one of the F(ab')2, Fab', Fab, Fv, and scFv of said antibody. The antigen-binding fragment of the above-described antibody typically has the same binding specificity as the antibody from which it originates. As will be readily understood by those skilled in the art according to the description of the present invention, the antigen-binding fragment of the above-described antibody can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction of disulfide bonds. Based on the structure of the complete antibody disclosed in this invention, those skilled in the art can readily obtain the above-described antigen-binding fragment. The antigen-binding fragment of the above-described antibody can also be obtained by recombinant genetic techniques, also known to those skilled in the art, or by, for example, an automated peptide synthesizer, such as those sold by AppliedBioSystems. Polymerized antibodies are polymers formed by the polymerization of whole antibodies and antigen-binding fragments.

[0051] In a second aspect, embodiments of the present invention provide an antibody pair for detecting serum amyloid A, the antibody pair comprising a first antibody and a second antibody; the first antibody having HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6; the second antibody having HCDR1 as shown in SEQ ID NO:13, HCDR2 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO:15, LCDR1 as shown in SEQ ID NO:16, LCDR2 as shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18.

[0052] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0053] Methods for defining CDRs are well-known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat definition" refers to the definition system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" is found in Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow any of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may be shortened or lengthened based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, with slight variations in labeling across different literature. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods, not limited to those in Table 1, are also within the scope of this disclosure.

[0054] Table 1: CDR Definition 1

[0055] HCDR1 <![CDATA[H31~H35 3 ]]> <![CDATA[H26~H35 3 ]]> <![CDATA[H26~H33..5 5 ]]> <![CDATA[H26~H32..34 4 ]]> HCDR2 H50~H65 H50~H58 H51~H57 H52~H56 HCDR3 H95~H102 H95~H102 H93~H102 H95~H102 LCDR1 L24~L34 L24~L34 L27~L32 L24~L34 LCDR2 L50~L56 L50~L56 L50~L51 L50~L56 LCDR3 L89~L97 L89~L97 L89~L97 L89~L97

[0056] 1 The CDRs defined in Table 1 are numbered according to the Kabat numbering system (see below), with amino acid numbers on the heavy chain represented by "H + number" and amino acid numbers on the light chain represented by "L + number". Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., USD ept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0057] 2 As used in Table 1, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

[0058] 3If neither H35A nor H35B exists, then CDR-H1 ends at bit 35; if only H35A exists, then CDR-H1 ends at bit 35A; if both H35A and H35B exist, then CDR-H1 ends at bit 35B.

[0059] 4 If neither H35A nor H35B exists, then CDR-H1 ends at bit 32; if only H35A exists, then CDR-H1 ends at bit 33; if both H35A and H35B exist, then CDR-H1 ends at bit 34.

[0060] 5 If neither H35A nor H35B exists, then CDR-H1 ends at bit 33; if only H35A exists, then CDR-H1 ends at bit 34; if both H35A and H35B exist, then CDR-H1 ends at bit 35.

[0061] According to embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact.

[0062] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Kabat system.

[0063] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Chothia system.

[0064] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT system.

[0065] In some optional embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the AbM system.

[0066] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the Contact system.

[0067] In some alternative embodiments of the present invention, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM, or Contact systems.

[0068] The antibody of the present invention also includes a frame region. In the present invention, the "frame region" or "FR" region includes a heavy chain frame region and a light chain frame region, which refers to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR. The heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including LFR1, LFR2, LFR3 and LFR4 frame regions.

[0069] In this invention, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0070] In some optional embodiments of the present invention, the antibody described herein may also be an antibody having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the first or second antibody in the antibody pair in the FR region.

[0071] Thirdly, embodiments of the present invention provide an antibody pair for detecting serum amyloid A.

[0072] The antibody pair comprises a first antibody having a heavy chain variable region shown in SEQ ID NO:9 and a light chain variable region shown in SEQ ID NO:11; the antibody pair comprises a second antibody having a heavy chain variable region shown in SEQ ID NO:20 and a light chain variable region shown in SEQ ID NO:22.

[0073] In an optional embodiment, the antibody pair comprises a first antibody having a heavy chain as shown in SEQ ID NO:10 and a light chain as shown in SEQ ID NO:12; and the antibody pair comprises a second antibody having a heavy chain as shown in SEQ ID NO:21 and a light chain as shown in SEQ ID NO:23.

[0074] Fourthly, embodiments of the present invention provide an antibody pair for detecting serum amyloid A, the antibody pair comprising a first antibody, wherein the variable region of the heavy chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:9, and the variable region of the light chain of the first antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:11; the antibody pair comprising a second antibody, wherein the variable region of the heavy chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:20, and the variable region of the light chain of the second antibody is a conserved variant formed by the amino acid sequence mutation shown in SEQ ID NO:22.

[0075] The conserved variant refers to a mutant obtained by replacing the amino acid at the mutation site with an amino acid with the same chemical properties as the original amino acid.

[0076] The mutation includes the addition, deletion, substitution, or modification of one or more amino acids.

[0077] Fifthly, embodiments of the present invention provide an antibody pair for detecting serum amyloid A, the antibody pair comprising a first antibody that binds to an epitope, the epitope being identical to the epitope bound by an antibody containing the heavy chain variable region shown in SEQ ID NO:9 and the light chain variable region shown in SEQ ID NO:11; the antibody pair comprising a second antibody that binds to an epitope, the epitope being identical to the epitope bound by an antibody containing the heavy chain variable region shown in SEQ ID NO:20 and the light chain variable region shown in SEQ ID NO:22.

[0078] The epitope, also known as an antigen epitope (AE), determines the ability of an antigen to specifically bind to an antibody. Antibodies bind to the same epitope because the amino acid fragments they bind to the target antigen are identical. Whether an antibody recognizes the same epitope as other antibodies can be confirmed by their competition for the epitope. Competition between antibodies can be evaluated using competitive binding assays, such as ELISA, fluorescence energy transfer assay (FRET), or fluorescence microassay (FMAT). The amount of antibody binding to the antigen is indirectly related to the binding ability of candidate competing antibodies (the antibody being tested) that compete for the same epitope. That is, the greater the binding amount or affinity of the antibody being tested for the same epitope, the lower the amount of antibody binding to the antigen, and the higher the amount of antibody binding to the antigen. Specifically, an appropriately labeled antibody and the antibody to be evaluated are added simultaneously to the antigen, and the binding of the antibody is detected using the labeling. By pre-labeling the antibody, the amount of antibody binding to the antigen can be easily determined. There are no particular limitations on the labeling; a labeling method appropriate to the assay technique is selected. Labeling methods include: fluorescent labeling, radioactive labeling, enzyme labeling, etc.

[0079] In an optional embodiment, the antibody further includes a constant region, wherein the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; the light chain constant region is selected from the κ-type or λ-type light chain constant region. The λ-type light chain constant region may be selected from the λ1, λ2, λ3, and λ4 subtypes.

[0080] It should be noted that, in some embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the aforementioned constant region (SEQ ID NO: 7, 8, or 19).

[0081] In one alternative embodiment, the antibody is a polymer formed by the polymerization of antibody monomers.

[0082] In some alternative implementations, the species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, donkeys, deer, mink, chickens, ducks, geese, or humans.

[0083] In a seventh aspect, embodiments of the present invention provide a reagent for detecting serum amyloid A.

[0084] The reagent includes a first group of antibodies and a second group of antibodies, wherein the first group of antibodies includes the first antibody in the antibody pair described above.

[0085] The second group of antibodies includes the second antibody in the antibody pair described above.

[0086] In one optional implementation, one of the first group of antibodies and the second group of antibodies is a coating antibody, and the other is a labeled antibody.

[0087] It should be noted that the meaning of "reagent" in this application can be considered equivalent to the meaning of "kit".

[0088] In an optional embodiment, the coated antibody or labeled antibody is conjugated with biotin or a biotin derivative;

[0089] In one optional embodiment, the label conjugated to the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle labels;

[0090] In optional embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), and Cy series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, C...). y5, Cy5.5, Cy3 and other similar substances), Alexa series dyes and their derivatives (including but not limited to Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750 and other similar substances) and protein dyes and their derivatives (including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydiophytoxanthin-chlorophyll protein (preCP) and other similar substances).

[0091] In optional embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0092] In optional embodiments, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F.

[0093] In optional embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridine ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxazone and its derivatives.

[0094] In optional embodiments, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0095] In optional embodiments, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latexes.

[0096] In optional embodiments, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0097] In an optional embodiment, the colloidal metal is colloidal gold.

[0098] In one optional embodiment, the coated antibody is coupled to a solid-phase support;

[0099] In one alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0100] Eighthly, embodiments of the present invention provide a method for detecting serum amyloid A, comprising:

[0101] a) Under conditions sufficient to induce antibody / antigen binding, the aforementioned antibody pair or reagent is brought into contact with the sample to be tested to form an immune complex; and

[0102] b) Detect the presence of the immune complex, the presence of which indicates the presence of serum amyloid A in the test sample.

[0103] To achieve the above objectives, according to a ninth aspect of the present invention, the use of the above-described antibody pair or reagent in detecting serum amyloid A or in preparing a product for detecting serum amyloid A is provided.

[0104] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0105] Unless otherwise defined, 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 pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0106] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0107] Based on long-term and creative research on serum amyloid A, the inventors discovered two antibodies that can meet the requirements for serum amyloid A detection.

[0108] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0109] Example 1 Antibody Preparation

[0110] In this embodiment, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. The MagExtractor RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit and pMD-18T vector were purchased from Takara. The plasmid extraction kit was purchased from Tiangen Biotech. Primer synthesis and gene sequencing were performed by Invitrogen. The hybridoma cell line secreting serum amyloid A monoclonal antibody was an existing hybridoma cell line, which was revived and ready for use.

[0111] (1) Antibody gene preparation

[0112] mRNA was extracted from hybridoma cell lines that secrete monoclonal antibodies against serum amyloid A, and DNA products were obtained by RT-PCR. The product was then inserted into the pMD-18T vector after an A-addition reaction with rTaq DNA polymerase. The vector was then transformed into DH5α competent cells. After bacterial growth, four clones of the Heavy Chain and Light Chain genes were collected and sent to a gene sequencing company for sequencing.

[0113] (2) Sequence analysis of the variable region gene of serum amyloid A antibody

[0114] The gene sequences obtained from the sequencing were analyzed in the Kabat antibody database and VNTI11.5 software was used to confirm that the genes amplified by both heavy and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain primer pair, the VL gene sequence was approximately 320 bp, with a 57 bp leader peptide sequence preceding it. Among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was approximately 360 bp, belonging to the VH1 gene family, with a 57 bp leader peptide sequence preceding it.

[0115] (3) Construction of recombinant antibody expression plasmid

[0116] pcDNA TM 3.4 The vector is a constructed recombinant antibody eukaryotic expression vector. This expression vector has been introduced with 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. Based on the sequencing results of the variable region gene of the antibody in pMD-18T, VL and VH gene-specific primers of this antibody were designed, with HindIII and EcoRI restriction sites and protective bases at both ends, respectively. The Light Chain gene fragment and Heavy Chain gene fragment were amplified by PCR.

[0117] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI, and the 3.4A vector was also digested with HindIII / EcoRI. After purification and recovery of the fragments and vector, the Heavy Chain gene and Light Chain gene were ligated into the 3.4A expression vector to obtain recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0118] 2. Recombinant antibody production

[0119] HEK293 cells were revived early and passaged to a 200ml volume to achieve a cell density of 3–5 × 10⁻⁶ cells / mL. 6 Cell density reached the required antibody concentration and cell viability >95%; cells were washed by centrifugation, reconstituted with culture medium, and the cell density was adjusted to 2.9 × 10⁻⁶ cells / ml. 6 Cells were washed at a concentration of cells / ml and reconstituted with culture medium, which served as a cell dilution buffer. Plasmid DNA and transfection reagent dilution buffers were prepared separately using culture medium. The transfection reagent dilution buffer was added to the plasmid DNA dilution buffer, mixed well, and incubated at room temperature for 15 min. This mixture was then slowly added to the cell dilution buffer over 1 min, mixed well, and samples were taken for cell counting. Cell viability after transfection was recorded and observed. The cells were then incubated at 35°C with a rotation speed of 120 rpm and a CO2 concentration of 8%. After 13 days, the samples were centrifuged and collected. The supernatant was purified using a protein A affinity chromatography column. 6 μg of the purified antibody was subjected to reducing SDS-PAGE, as shown in the figure. The reducing SDS-PAGE showed two bands: one with a Mr of 50 kDa (heavy chain) and the other with a Mr of 28 kDa (light chain).

[0120] The resulting antibodies were named Anti-SAA08 and Anti-SAA09. The heavy chain (H) and light chain (L) sequences of the above antibodies are shown in the table below:

[0121] Table 2 Antibody Sequences

[0122] Anti-SAA08 SEQ ID NO:10 SEQ ID NO:12 Anti-SAA09 SEQ ID NO:21 SEQ ID NO:23

[0123] Example 2: Preparation of Detection Reagents

[0124] Using the Anti-SAA08 antibody as the coating antibody and the Anti-SAA09 antibody as the labeling antibody, the experimental procedure is as follows:

[0125] 1. Antibody labeling: Add 100 μL of 1% solid content fluorescent microspheres to 900 μL of activation buffer and mix well. After centrifugation to remove the supernatant, add 1 mL of activation buffer and sonicate to mix well. Then add the activator, and mix well in the dark by shaking for 20 min. After centrifugation to remove the supernatant, add an equal volume of coupling buffer (MES buffer recommended, pH=6.5) to the microspheres and sonicate to mix well. Then add 0.2-0.4 mg of SAA-labeled antibody, and mix well in the dark by shaking for 3 h. Finally, add blocking buffer for blocking, and mix well in the dark by shaking for 45 min to stop labeling. After centrifugation to remove the supernatant, reconstitute the microspheres with microsphere preservation solution, sonicate to mix well, and store at 4℃ for use.

[0126] 2. Preparation of microsphere working solution: After diluting the SAA marker to 10-20% with microsphere diluent, spray the marker onto the glass fiber using a spray pad device.

[0127] 3. Preparation of dried microsphere pads: Place the sprayed fluorescent pads in a 50℃ oven and dry for more than 2 hours.

[0128] 4. Sample pad treatment: Dilute the blocking agent to 0.2-0.4 mg / ml with sample pad diluent, spread it on glass fiber, and dry it in a 50℃ oven overnight.

[0129] 5. NC membrane coating: Dilute the SAA-coated antibody and the anti-labeled antibody secondary antibody to 0.5-1.0 mg / ml with coating diluent and then streak them evenly at the T line and C line; dry in an oven at 50℃ overnight.

[0130] 6. Preparation of fluorescence chromatography strips: Cut the fluorescence chromatography strips to the required width using a strip cutter, assemble them, and then add samples for detection.

[0131] Example 3: Antibody Detection Performance Evaluation

[0132] The cross-reactivity of the evaluation reagents was assessed.

[0133] (1) Dilute the calibrators, quality control materials, and clinical test samples with sample diluent;

[0134] (2) Use a pipette to transfer 75 μL of the diluted sample and add it onto the prepared immunoassay strip for chromatography.

[0135] (3) After 5-10 minutes, the instrument is read to obtain the T / C data, and the sample concentration is calculated based on the T / C value.

[0136] The test results for calibrators with known concentrations are shown in the table below:

[0137] mark Anti-SAA09 Calibrator (mg / L) T / C 0 0.08 4.17 0.18 9.67 0.34 20.47 0.62 48.85 1.02 97.71 1.51 291.90 1.97 755.36 2.30

[0138] Based on the T / C values ​​obtained from the above calibrators, the following calculations were performed: Figure 1 The calibration curve shown.

[0139] To assess the accuracy of the proposed method for testing clinical samples, the applicant compared the results with those of Siemens' SAA testing method, which is widely recognized in the industry. The test results of the two methods for different concentrations of clinical samples are shown in the table below:

[0140]

[0141]

[0142] Based on the above data, the inventors obtained the following respectively: Figure 2 The linear correlation of all clinical samples compared to the test results is shown. Figure 3 The linear correlation of low values ​​in clinical samples is shown. As can be seen from the above data and figures, the results of clinical sample testing using the proposed method are highly consistent with currently industry-recognized SAA testing products, with a coefficient of determination R0 for samples with concentrations ranging from 0 mg / L to 50 mg / L. 2 The coefficient of determination (R²) can reach 0.9281 for the detection results of samples with various concentration ranges. 2 It can reach 0.9724.

[0143] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0144] The partial amino acid sequence involved in this application is shown below:

[0145]

[0146]

Claims

1. An antibody pair for detecting serum amyloid A, characterized in that, The antibody pair includes a first antibody, the complementarity-determining regions of which are: three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:9, and three light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 in the light chain variable region as shown in SEQ ID NO:11; and The antibody pair includes a second antibody, the complementarity-determining regions of which are: three heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3 in the heavy chain variable region as shown in SEQ ID NO:20, and three light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3 in the light chain variable region as shown in SEQ ID NO:

22. The complementary decision region is defined by any one of the systems Kabat, Chothia, IMGT, AbM, or Contact.

2. An antibody pair for detecting serum amyloid A, characterized in that, The antibody pair includes a first antibody, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antibody being as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and The antibody pair includes a second antibody, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the second antibody being shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively.

3. An antibody pair for detecting serum amyloid A, characterized in that, The antibody pair comprises a first antibody, which comprises at least one of (1)-(2): (1) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:9 and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:11; (2) The heavy chain with the amino acid sequence shown in SEQ ID NO:10 and the light chain with the amino acid sequence shown in SEQ ID NO:12; and The antibody pair comprises a second antibody, the second antibody comprising at least one of (a)-(b): (a) The heavy chain variable region with amino acid sequences as shown in SEQ ID NO:20 and the light chain variable region with amino acid sequences as shown in SEQ ID NO:22; (b) The heavy chain with the amino acid sequence shown in SEQ ID NO:21 and the light chain with the amino acid sequence shown in SEQ ID NO:

23.

4. The antibody pair for detecting serum amyloid A according to claim 2, characterized in that, The antibody pair comprises a first antibody, wherein the variable region of the heavy chain of the first antibody has the amino acid sequence shown in SEQ ID NO:9, and the variable region of the light chain of the first antibody has the amino acid sequence shown in SEQ ID NO:11; the framework region of the conserved variant formed by the mutation of the first antibody has at least 80% identity with the framework region of the first antibody before the mutation; the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antibody are shown in SEQ ID NO:1~6 in sequence; and The antibody pair comprises a second antibody, wherein the variable region of the heavy chain of the second antibody is the amino acid sequence shown in SEQ ID NO:20, and the variable region of the light chain of the second antibody is the amino acid sequence shown in SEQ ID NO:22; the frame region of the conserved variant formed by the mutation of the second antibody has at least 80% identity with the frame region of the second antibody before the mutation; the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the second antibody are shown in SEQ ID NO:13-18 in sequence.

5. The antibody pair for detecting serum amyloid A according to any one of claims 1-4, characterized in that, The antibody further includes a constant region; wherein the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, or a combination of multiple constant regions; the light chain constant region is selected from the κ-type or λ-type light chain constant region.

6. The antibody pair for detecting serum amyloid A according to claim 5, characterized in that, The heavy chain constant region of the first antibody is a sequence with at least 80% identity to SEQ ID NO:7, and the light chain constant region of the first antibody is a sequence with at least 80% identity to SEQ ID NO:8; the heavy chain constant region of the second antibody is a sequence with at least 80% identity to SEQ ID NO:19, and the light chain constant region of the second antibody is a sequence with at least 80% identity to SEQ ID NO:

8.

7. The antibody pair for detecting serum amyloid A according to claim 6, characterized in that, The amino acid sequence of the heavy chain constant region of the first antibody is shown in SEQ ID NO:7, and the amino acid sequence of the light chain constant region of the first antibody is shown in SEQ ID NO:8; the amino acid sequence of the heavy chain constant region of the second antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain constant region of the second antibody is shown in SEQ ID NO:

8.

8. A reagent for detecting serum amyloid A, characterized in that, The reagent includes a first group of antibodies and a second group of antibodies, wherein the first group of antibodies includes the first antibody as described in any one of claims 1-7; and the second group of antibodies includes the second antibody as described in any one of claims 1-7.

9. The reagent for detecting serum amyloid A according to claim 8, characterized in that, One of the first and second antibodies is a coating antibody, and the other is a labeling antibody.

10. The reagent for detecting serum amyloid A according to claim 9, characterized in that, The coated antibody or labeled antibody is conjugated with biotin.

11. The reagent for detecting serum amyloid A according to claim 9, characterized in that, The labeled antibody-conjugated markers are selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent markers, electrochemiluminescent markers, latex and nanoparticle markers.

12. The reagent for detecting serum amyloid A according to claim 9, characterized in that, The coated antibody is coupled to a solid-phase carrier.

13. The reagent for detecting serum amyloid A according to claim 12, characterized in that, The solid support is selected from microspheres, plates, and membranes.

14. Use of the antibody pair according to any one of claims 1-7 or the reagent according to any one of claims 8-13 in the preparation of a product for detecting serum amyloid A.

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