Antibody pair, reagent and method for detecting CA15-3

Abstract

The invention discloses an antibody pair, a reagent and a method for detecting CA15-3, and relates to the field of immunodiagnosis. The antibody pair for detecting the CA15-3 disclosed by the invention comprises the first antibody and the second antibody, and the reagent and the detection method based on the antibody combination can accurately detect the existence of the CA15-3.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular, to an antibody pair, a reagent and a method for detecting CA15-3. Background Art

[0002] Carbohydrate antigen CA15-3 is an antigenic determinant on a large mucin glycoprotein. This glycoprotein is encoded by the MUC1 gene and is the first carbohydrate antigen substance discovered on the cell membrane of breast cancer. Its structure includes a membrane region, an intracellular region, and a glycosyl-rich extracellular region. CA15-3 on breast cancer cells detaches from the cell membrane and is released into the blood, and the antigenic determinant in its extracellular region can be specifically bound by a monoclonal antibody in the detection reagent for determination. In addition, abnormal levels of CA15-3 are often found in many other adenocarcinomas such as lung cancer, ovarian cancer, and endometrial adenocarcinoma.

[0003] CA15-3 is the most commonly used tumor marker for breast cancer. In patients with breast cancer, the level of CA15-3 will increase significantly, so it is an accepted effective indicator for the auxiliary diagnosis and dynamic monitoring of breast cancer. Usually, an increase in the level of CA15-3 in the blood may indicate the progression of breast cancer, and a decrease in its level indicates the improvement of the condition and the positivity of treatment. Summary of the Invention

[0004] The present application provides an antibody pair, which provides an important raw material source for the detection of CA15-3 and has good detection performance.

[0005] To achieve the above object, according to one aspect of the present invention, there is provided an antibody pair for detecting CA15-3. The antibody pair includes a first antibody, and the first antibody includes: HCDR1, HCDR2, and

[0006] HCDR3, three heavy-chain complementarity-determining regions in the heavy-chain variable region shown in SEQ ID NO: 9, and LCDR1, LCDR2, and LCDR3, three light-chain complementarity-determining regions in the light-chain variable region shown in SEQ ID NO: 11;

[0007] The antibody pair includes a second antibody, and the second antibody includes:

[0008] HCDR1, HCDR2, and HCDR3, three heavy-chain complementarity-determining regions in the heavy-chain variable region shown in SEQ ID NO: 21, and LCDR1, LCDR2, and LCDR3, three light-chain complementarity-determining regions in the light-chain variable region shown in SEQ ID NO: 23.

[0009] The complementarity-determining regions are defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM, or Contact.

[0010] To achieve the above object, according to the second aspect of the present invention, there is provided an antibody pair for detecting CA15-3, the antibody pair comprising a first antibody, the first antibody having HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6;

[0011] The antibody pair comprises a second antibody, the second antibody having HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16,

[0012] LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:18.

[0013] To achieve the above object, according to the third aspect of the present invention, there is provided an antibody pair for detecting CA15-3;

[0014] The antibody pair comprises a first antibody, the first antibody comprising at least one of (1)-(2):

[0015] (1) A heavy chain variable region shown in SEQ ID NO:9 and a light chain variable region shown in SEQ ID NO:11;

[0016] (2) A heavy chain shown in SEQ ID NO:10 and a light chain shown in SEQ ID NO:12;

[0017] The antibody pair comprises a second antibody, the second antibody comprising at least one of (a)-(b):

[0018] (a) A heavy chain variable region shown in SEQ ID NO:21 and a light chain variable region shown in SEQ ID NO:23;

[0019] (b) A heavy chain shown in SEQ ID NO:22 and a light chain shown in SEQ ID NO:24.

[0020] To achieve the above object, according to the fourth aspect of the present invention, there is provided an antibody pair for detecting CA15-3;

[0021] The antibody pair comprises a first antibody, wherein the heavy chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 11; or;

[0022] The antibody pair comprises a second antibody, wherein the heavy chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 23;

[0023] In an alternative embodiment, the mutation is an addition, deletion, substitution or modification of one or more amino acids in the framework region of the first antibody or the second antibody.

[0024] In an alternative embodiment, the framework region of the conservative variant formed by mutation of the first antibody has at least 80% identity with the framework region of the first antibody before mutation; or;

[0025] The framework region of the conservative variant formed by mutation of the second antibody has at least 80% identity with the framework region of the second antibody before mutation.

[0026] To achieve the above object, according to the fifth aspect of the present invention, there is provided an antibody pair for detecting CA15-3;

[0027] The antibody pair comprises a first antibody, and the first antibody binds to an epitope, and the epitope is the same as the epitope bound by an 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;

[0028] The antibody pair comprises a second antibody, and the second antibody binds to an epitope: the epitope is the same as the epitope bound by an antibody comprising the heavy chain variable region shown in SEQ ID NO: 21 and the light chain variable region shown in SEQ ID NO: 23.

[0029] To achieve the above object, according to the sixth aspect of the present invention, there is provided an antibody pair for detecting CA15-3, wherein the heavy chain constant region of the antibody is selected from the heavy chain constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, 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;

[0030] In an alternative embodiment, the heavy chain constant region of the first antibody is SEQ ID NO:7 or a sequence having at least 80% identity therewith, and the light chain constant region of the first antibody is SEQ ID NO:8 or a sequence having at least 80% identity therewith;

[0031] In an alternative embodiment, the heavy chain constant region of the second antibody is SEQ ID NO:19 or a sequence having at least 80% identity therewith, and the light chain constant region of the second antibody is SEQ ID NO:20 or a sequence having at least 80% identity therewith;

[0032] In an alternative embodiment, the heavy chain constant region comprises CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM and / or CH4 of IgM;

[0033] In an alternative embodiment, the antibody is a multimer formed by polymerization of antibody monomers.

[0034] To achieve the above object, according to the seventh aspect of the present invention, there is provided a reagent for detecting CA15-3, the reagent comprising a first group of antibodies and a second group of antibodies, the first group of antibodies comprising the first antibody in the antibody pair;

[0035] The second group of antibodies comprises the second antibody in the antibody pair;

[0036] 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;

[0037] The reagent comprises 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;

[0038] In an alternative embodiment, the coating antibody or the labeled antibody is conjugated with biotin or a biotin derivative;

[0039] In an alternative embodiment, the label conjugated to the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, latexes and nanoparticle-based labels;

[0040] In an alternative embodiment, the coating antibody is conjugated to a solid phase carrier;

[0041] In an alternative embodiment, the solid phase carrier is selected from microspheres, plates and membranes.

[0042] To achieve the above object, according to the eighth aspect of the present invention, a method for detecting CA15-3 is provided, including:

[0043] a) contacting the above antibody pair or reagent with a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; and

[0044] b) detecting the presence of the immune complex, the presence of which indicates the presence of CA15-3 in the test sample.

[0045] To achieve the above object, according to the ninth aspect of the present invention, the use of the above antibody pair or reagent in detecting CA15-3 or in preparing a product for detecting CA15-3 is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 . Clinical correlation between commercially available antibody pairing reagents and Roche's CA15-3 reagent

[0047] Figure 2 . Clinical correlation between the antibody pairing reagent of this patent and Roche's CA15-3 reagent DETAILED DESCRIPTION OF THE INVENTION

[0048] In a first aspect, an embodiment of the present invention provides an antibody pair for detecting CA15-3. The antibody pair includes a first antibody, and the first antibody 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; the antibody pair includes a second antibody, and the second antibody includes: three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 in the heavy chain variable region shown in SEQ ID NO:21 and three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 in the light chain variable region shown in SEQ ID NO:23.

[0049] The complementarity determining regions are defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM, or Contact.

[0050] In the present invention, the term "antibody" is used in the broadest sense, and the antibody in the method of the present invention can be a whole antibody, an antigen-binding fragment, or a polymeric antibody capable of binding to CA15-3.

[0051] A whole antibody can be monoclonal. Such whole antibodies are generally antibodies prepared by any suitable method known in the art. For example, by immunizing a mammal, usually a rabbit or a mouse, with CA15-3 under suitable conditions and isolating antibody molecules from, for example, the serum of said mammal. Monoclonal antibodies can be obtained by hybridoma or recombinant methods. Antigen-binding fragments include antigen-binding sites, such as Fab or F(ab)2 fragments. In an alternative embodiment, the antigen-binding fragment is selected from any one of F(ab’)2, Fab’, Fab, Fv, and scFv of the antibody. The antigen-binding fragments of the above antibodies generally have the same binding specificity as the antibodies from which they are derived. Those skilled in the art can easily understand from the content described in the present invention that the antigen-binding fragments of the above antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds. Based on the disclosure of the structure of the complete antibody in the present invention, those skilled in the art can easily obtain the above antigen-binding fragments. The antigen-binding fragments of the above antibodies can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as the automated peptide synthesizer sold by Applied BioSystems, etc. A polymeric antibody refers to a multimer formed by polymerization of whole antibodies and antigen-binding fragments.

[0052] In a second aspect, an embodiment of the present invention provides an antibody pair for detecting CA15-3, the antibody pair including a first antibody and a second antibody; the first antibody has HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; the second antibody has HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:18.

[0053] In the present invention, the terms "complementary determining region", "CDR", or "CDRs" refer to the highly variable regions of the heavy and light chains of an immunoglobulin, and refer to regions containing one or more or even all of the main amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the specific embodiments of the present invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0054] The 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, the "Kabat definition" refers to the definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For the "Chothia definition", see Chothia et al., J Mol Biol 196: 901-917 (1987). There are other CDR definition methods that may not strictly follow one 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 for specific residues or groups of residues. Exemplary defined CDRs are listed in Table 1 below, with slightly different annotations in different literatures. 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 also fall within the scope of protection of the present disclosure.

[0055] Table 1: CDR Definitions 1

[0056] CDR Kabat <![CDATA[AbM 2 > IMGT Chothia 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

[0057] 1 The numbers for all CDR definitions in Table 1 are based on the Kabat numbering system (see below), and the amino acid numbers on the heavy chain are represented by "H + number", and the amino acid numbers on the light chain are represented by "L + number". Those of ordinary skill in the art can clearly map the Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, the "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0058] 2 As used in Table 1, "AbM" with a lowercase "b" refers to the CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

[0059] 3If neither H35A nor H35B exists, then CDR-H1 ends at position 35; if only H35A exists, then CDR-H1 ends at position 35A; if both H35A and H35B exist, then CDR-H1 ends at position 35B.

[0060] 4 If neither H35A nor H35B exists, then CDR-H1 ends at position 32; if only H35A exists, then CDR-H1 ends at position 33; if both H35A and H35B exist, then CDR-H1 ends at position 34.

[0061] 5 If neither H35A nor H35B exists, then CDR-H1 ends at position 33; if only H35A exists, then CDR-H1 ends at position 34; if both H35A and H35B exist, then CDR-H1 ends at position 35.

[0062] According to an embodiment of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 is defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM or Contact.

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

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

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

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

[0067] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0068] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of the Kabat, Chothia, IMGT, AbM or Contact systems.

[0069] The antibody of the present invention further includes a framework region. In the present invention, the "framework region" or "FR" region includes a heavy-chain framework region and a light-chain framework region, which refers to the regions other than the CDRs in the variable regions of the heavy and light chains of the antibody. Among them, the heavy-chain framework region can be further subdivided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3 and HFR4 framework regions; the light-chain framework region can be further subdivided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3 and LFR4 framework regions.

[0070] In the present invention, the variable region of the heavy chain is obtained by connecting the CDRs numbered as follows with the FRs in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the variable region of the light chain is obtained by connecting the CDRs numbered as follows with the FRs in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0071] In some alternative embodiments of the present invention, the antibody of the present invention 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 antibody and the second antibody in the antibody pair in the FR region.

[0072] In a third aspect, an embodiment of the present invention provides an antibody pair for detecting CA15-3.

[0073] The antibody pair includes 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 includes a second antibody having a heavy-chain variable region shown in SEQ ID NO: 21 and a light-chain variable region shown in SEQ ID NO: 23.

[0074] In an alternative embodiment, the antibody pair includes a first antibody having a heavy chain shown in SEQ ID NO: 10 and a light chain shown in SEQ ID NO: 12; the antibody pair includes a second antibody having a heavy chain shown in SEQ ID NO: 22 and a light chain shown in SEQ ID NO: 24.

[0075] Fourth aspect, an embodiment of the present invention provides an antibody pair for detecting CA15-3. The antibody pair includes a first antibody. The heavy chain variable region of the first antibody is a conservative variant formed by mutating the amino acid sequence shown in SEQ ID NO:9, and the light chain variable region of the first antibody is a conservative variant formed by mutating the amino acid sequence shown in SEQ ID NO:11. The antibody pair includes a second antibody. The heavy chain variable region of the second antibody is a conservative variant formed by mutating the amino acid sequence shown in SEQ ID NO:21, and the light chain variable region of the second antibody is a conservative variant formed by mutating the amino acid sequence shown in SEQ ID NO:23.

[0076] The conservative variant refers to a mutant obtained by substituting the amino acid at the mutation site with an amino acid having the same chemical property as the original amino acid.

[0077] The mutation is an addition, deletion, substitution or modification of one or more amino acids in the framework region of the first antibody or the second antibody.

[0078] Fifth aspect, an embodiment of the present invention provides an antibody pair for detecting CA15-3. The antibody pair includes a first antibody. The first antibody binds to an epitope, and the epitope is the same as the epitope bound by an 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. The antibody pair includes a second antibody. The second antibody binds to an epitope, and the epitope is the same as the epitope bound by an antibody comprising the heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:23.

[0079] The epitope is also called an antigenic epitope (Antigen epitope, AE). The epitope determines the ability of the antigen to specifically bind to the antibody. Antibodies binding to the same epitope means that the amino acid fragments to which the antibodies bind to the target antigen are identical. Whether an antibody recognizes the same epitope as other antibodies can be confirmed by the competition between them for the epitope. The competition between antibodies can be evaluated by a competitive binding assay. The methods include: ELISA, fluorescence energy transfer assay (FRET) or fluorescence microassay technology (FMAT), etc. The amount of the antibody binding to the antigen is indirectly related to the binding ability of the candidate competing antibody (test antibody) competing for binding to the same epitope. That is, the greater the binding amount or affinity of the test antibody to the same epitope, the lower the binding amount of the antibody to the antigen, and the binding amount of the test antibody to the antigen increases. Specifically, the antibody and the antibody to be evaluated that have been appropriately labeled are added to the antigen at the same time, and the bound antibody is detected using the label. By pre-labeling the antibody, the amount of the antibody binding to the antigen can be easily measured. There is no particular limitation on the label, and a labeling method corresponding to the assay technique is selected. The labeling methods specifically include: fluorescence labeling, radioisotope labeling, enzyme labeling, etc.

[0080] In an alternative embodiment, the antibody further comprises a constant region, wherein the heavy chain constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, 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.

[0081] 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 above constant regions (SEQ ID NO: 7, 8, 19 or 20).

[0082] In an alternative embodiment, the heavy chain constant region comprises CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM and / or CH4 of IgM;

[0083] In an alternative embodiment, the antibody is a multimer formed by polymerization of antibody monomers.

[0084] In some alternative embodiments, the species source of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

[0085] In a seventh aspect, an embodiment of the present invention provides a reagent for detecting CA15-3,

[0086] The reagent comprises a first group of antibodies and a second group of antibodies, and the first group of antibodies comprises the first antibody in the above antibody pair;

[0087] The second group of antibodies comprises the second antibody in the above antibody pair.

[0088] In an alternative embodiment, one of the first group of antibodies and the second group of antibodies is a coating antibody, and the other is a labeled antibody.

[0089] It should be noted that in this application, the meaning of the reagent and the meaning of the kit can be mutually recognized as equivalent.

[0090] In an alternative embodiment, the coating antibody or the labeled antibody is conjugated with biotin or a biotin derivative;

[0091] In an alternative embodiment, the label conjugated to the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, latex, and nanoparticle-based labels;

[0092] In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0093] In alternative 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.

[0094] In alternative embodiments, the radioisotopes 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.

[0095] In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetane and its derivatives, rosamide and its derivatives, and peroxyoxalate and its derivatives.

[0096] In an alternative embodiment, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0097] In an alternative embodiment, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0098] In an alternative embodiment, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0099] In an alternative embodiment, the colloidal metal is colloidal gold.

[0100] In an alternative embodiment, the coating antibody is conjugated to a solid-phase support;

[0101] In an alternative embodiment, the solid-phase support is selected from microspheres, plates, and membranes.

[0102] In an eighth aspect, an embodiment of the present invention provides a method for detecting CA15-3, comprising:

[0103] a) contacting the above antibody pair or reagent with a sample to be detected under conditions sufficient to allow an antibody / antigen binding reaction to form an immune complex; and

[0104] b) detecting the presence of the immune complex, the presence of which indicates the presence of CA15-3 in the test sample.

[0105] To achieve the above object, according to a ninth aspect of the present invention, there is provided the use of the above antibody pair or reagent in detecting CA15-3 or in preparing a product for detecting CA15-3.

[0106] To make the objects, 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. Those not specified in the embodiments are carried out under conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer are all conventional products that can be obtained by commercial purchase.

[0107] 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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the formulations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0108] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", 2nd Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (eds. D.M. Weir and C.C. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (eds. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (eds. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (eds. Mullis et al., 1994); and "Current Protocols in Immunology" (eds. J.E. Coligan et al., 1991), each of which is hereby expressly incorporated by reference.

[0109] Based on long-term and creative research on CA15-3, the applicant has discovered two antibodies that can meet the requirements for CA15-3 detection.

[0110] The features and properties of the present invention will be further described in detail below in conjunction with the examples.

[0111] Example 1 Antibody Preparation

[0112] In this example, the restriction endonuclease 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 was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting the CA15-3 monoclonal antibody was an existing hybridoma cell line and was revived for standby.

[0113] (1) Preparation of antibody genes

[0114] mRNA was extracted from the hybridoma cell line secreting the CA15-3 monoclonal antibody. DNA products were obtained by RT-PCR. After adding A to the products with rTaq DNA polymerase, they were inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones of each were sent to a gene sequencing company for sequencing.

[0115] (2) Sequence analysis of the variable region genes of the CA15-3 antibody

[0116] The gene sequences obtained from the above sequencing were analyzed in the kabat antibody database, and the VNTI 11.5 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 about 320 bp, and there was a 57-bp leader peptide sequence in front of it; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was about 360 bp, belonging to the VH1 gene family, and there was a 57-bp leader peptide sequence in front of it.

[0117] (3) Construction of recombinant antibody expression plasmids

[0118] pcDNA TM 3.4 The vector pcDNA3.4 was the recombinant antibody eukaryotic expression vector constructed. Multiple cloning enzyme digestion sites such as HindIII, BamHI, and EcoRI had been introduced into this expression vector, and it was named the pcDNA3.4A expression vector, hereinafter referred to as the 3.4A expression vector for short; according to the antibody variable region gene sequencing results in the above pMD-18T, specific primers for the VL and VH genes of this antibody were designed, with HindIII and EcoRI enzyme digestion sites and protective bases at both ends, and the Light Chain gene fragment and Heavy Chain gene fragment were amplified by PCR.

[0119] The Heavy Chain and Light Chain gene fragments were digested with HindIII / EcoRI double enzymes respectively, and the 3.4A vector was digested with HindIII / EcoRI double enzymes. After purifying and recovering the fragments and the vector, the Heavy Chain gene and the Light Chain gene were respectively ligated into the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain respectively.

[0120] 2. Recombinant antibody production

[0121] Resuscitate HEK293 cells in advance, passage and culture them to a 200 ml system to make the cell density reach 3 - 5×10 6 cells / ml. Select antibodies with the appropriate concentration and cells with a cell viability > 95%; centrifuge and wash the cells, resuspend them with the culture medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml. Wash the cells, resuspend them with the culture medium, and at the same time, use it as the cell diluent. Prepare the plasmid DNA and transfection reagent diluents with the culture medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let it stand at room temperature for 15 min; slowly add this mixture to the cell diluent within 1 min, mix well, sample and count, record and observe the viability of the cells after transfection, and place them in an incubator at 35°C for culture, with a rotation speed of 120 rmp and a CO2 content of 8%. After 13 days, centrifuge to collect the samples. Purify the centrifuged supernatant with a protein A affinity chromatography column.

[0122] The obtained antibodies were named 5D11 and 3F10. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table:

[0123] Table 2 Antibody sequences

[0124] Antibody Name Heavy Chain Light Chain 5D11 SEQ ID NO:10 SEQ ID NO:12 3F10 SEQ ID NO:22 SEQ ID NO:24

[0125] Example 2 Preparation of detection reagents

[0126] (1) Preparation method of acridinium ester antibody conjugate

[0127] Antibody 3F10 (5 mg / mL) was replaced into PBS (10 mM PB, 50 mM sodium chloride, pH 7.4, 5 mM EDTA) buffer with a Zeba TM desalting column (10K MWCO). 10 mM NHS - AE (10 eq, dissolved in DMSO) was added to the 30 μM antibody solution, and the reaction was carried out at 25°C for 1 hour. After the reaction, desalt to remove the excess reagents and store them at 2 - 8°C for later use.

[0128] (2) Preparation method of biotin antibody conjugate

[0129] The antibody 5D11 (5 mg / mL) was exchanged into PBS (10 mM PB, 50 mM sodium chloride, pH 7.4) buffer using a Zeba TM desalting column (10K MWCO). 10 mM LC-biotin (10 eq, dissolved in DMSO) was added to the 30 μM antibody solution, and the reaction was carried out at 25 °C for 1 hour. After the reaction, excess reagents were removed by desalting and stored at 2 - 8 °C for later use.

[0130] (3) Preparation method of avidin magnetic bead coating

[0131] a) Incubation: Take the magnetic beads and wash them once with the washing buffer, resuspend the magnetic beads with the activation buffer at a concentration of 10 mg / mL, add avidin (SA) with a coating amount of 30 μg / mg, and incubate at 25 °C for 30 min;

[0132] b) Coating: Add the cross-linking agent EDC with a final concentration of 0.2 mg / mL and react at 25 °C for 24 h;

[0133] c) Blocking: After magnetic separation, remove the supernatant, wash once with the blocking solution (10 mM PBS, 1% BSA, pH 7.4), add the blocking solution, and the reaction concentration is 10 mg / mL;

[0134] d) Storage: Wash once with the blocking solution, add the blocking solution, and store at a concentration of 10 mg / mL. Store at 2 - 8 °C for later use.

[0135] (4) Kit preparation

[0136] a) Dilute the avidin magnetic bead coating with the magnetic bead coating diluent (25 mM HEPES, 1% BSA, pH 7.2) to a working solution concentration of 0.5 mg / mL, which is CA15-3 Ra; dilute the biotin antibody conjugate with the biotin label diluent (25 mM MES, 5% NBS, pH 6.0) to a working solution concentration of 1.0 μg / mL, which is CA15-3 Rc; dilute the acridinium ester antibody conjugate with the acridinium ester label diluent (20 mM PB, 1% BSA, pH 6.8) to a working solution concentration of 1.0 μg / mL, which is CA15-3 Rd. Dilute the CA15-3 antigen with the calibrator diluent (25 mM HEPES, 1% BSA, pH 7.2) to antigen working solutions with concentrations of approximately 0.1, 0.5, 2.5, 7.5, 30 U / mL and store at 2 - 8 °C for later use.

[0137] b) Detection was carried out on the Yingkai Shine i2910 fully automatic chemiluminescent immunoassay analyzer. The double antibody sandwich method was used, and the reaction mode was the one-step delay method. That is, 10 μL of the sample, 50 μL of Rc, and 50 μL of Rd were added to the reaction cup in sequence, mixed well and incubated for 10 min, then 50 μL of Ra was added, mixed well again and incubated for 10 min to form a sandwich structure of SA magnetic bead-(Bio-antibody)-antigen-(antibody-AE). After incubation in the reaction tube was completed, the magnetic beads were adsorbed by the magnetic field, and the unbound substances were washed away. The pre-excitation solution (acidic solution containing H2O2) and the excitation solution (alkaline solution containing NaOH) were added to detect the relative luminescence intensity (RLU).

[0138] (5) Performance detection

[0139] a) Results of activity detection

[0140]

[0141] In terms of activity, when detecting the same sample, the relative luminescence intensity of the antibody pairing of this patent increased by about 23.7% compared with that of the commercially available antibody pairing, and the signal-to-noise ratio increased by about 1.8 times. Compared with the commercially available antibody pairing, the activity and sensitivity of the antibody pairing of this patent have been improved to a certain extent.

[0142] b) Results of correlation detection

[0143]

[0144]

[0145]

[0146]

[0147] In terms of correlation, when detecting 117 serum samples, the R of the linear fitting between the relative luminescence intensity of the commercially available antibody pairing and the Roche measurement value 2 was 0.846, and the R 2 of the antibody pairing of this patent was 0.9648. Compared with the commercially available antibody pairing, the correlation between the antibody pairing of this patent and the Roche reagent has been greatly improved.

[0148] c) Results of accelerated stability detection of the working solution

[0149]

[0150]

[0151] In terms of the accelerated stability of the working fluid, when the commercially available antibody pair is accelerated at 37°C for 7 days, the activity decreases by about 13.2% compared with the group stored at 4°C, while the decrease amplitude of the antibody pair of this patent is about 3.2%. Compared with the commercially available antibody pair, the accelerated stability of the working fluid of the antibody pair of this patent is significantly improved.

[0152] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0153] Some of the amino acid sequences involved in this application are shown as follows:

[0154]

[0155]

Claims

1. An antibody pair for detecting CA15-3, characterized in that, the antibody pair includes a first antibody, and the first antibody comprises: three heavy-chain complementarity-determining regions, namely HCDR1, HCDR2, and HCDR3, in the heavy-chain variable region shown in SEQ ID NO:9 and three light-chain complementarity-determining regions, namely LCDR1, LCDR2, and LCDR3, in the light-chain variable region shown in SEQ ID NO:11; the antibody pair includes a second antibody, and the second antibody comprises: three heavy-chain complementarity-determining regions, namely HCDR1, HCDR2, and HCDR3, in the heavy-chain variable region shown in SEQ ID NO:21 and three light-chain complementarity-determining regions, namely LCDR1, LCDR2, and LCDR3, in the light-chain variable region shown in SEQ ID NO:

23.

2. The antibody pair according to claim 1, wherein the complementarity-determining regions are defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM, or Contact.

3. An antibody pair for detecting CA15-3, characterized in that, the antibody pair includes a first antibody, and the first antibody has HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; the antibody pair includes a second antibody, and the second antibody has HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:

18.

4. An antibody pair for detecting CA15-3, characterized in that, the antibody pair comprises a first antibody, and the first antibody comprises at least one of (1)-(2): (1) the heavy-chain variable region shown in SEQ ID NO:9 and the light-chain variable region shown in SEQ ID NO:11; (2) the heavy chain shown in SEQ ID NO:10 and the light chain shown in SEQ ID NO:12; the antibody pair comprises a second antibody, and the second antibody comprises at least one of (a)-(b): (a) the heavy-chain variable region shown in SEQ ID NO:21 and the light-chain variable region shown in SEQ ID NO:23; (b) the heavy chain shown in SEQ ID NO:22 and the light chain shown in SEQ ID NO:

24.

5. The antibody pair for detecting CA15-3 according to claim 4, characterized in that, the antibody pair comprises a first antibody, the heavy-chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO:9, and the light-chain variable region of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO:11; The antibody pair comprises a second antibody, wherein the heavy chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 23; or; Optionally, the mutation is an addition, deletion, substitution or modification of one or more amino acids in the framework region of the first antibody or the second antibody; The framework region of the conservative variant formed by mutation of the first antibody has at least 80% identity with the framework region of the first antibody before mutation; or; The framework region of the conservative variant formed by mutation of the second antibody has at least 80% identity with the framework region of the second antibody before mutation.

6. An antibody pair for detecting CA15-3, characterized in that The antibody pair comprises a first antibody, and the first antibody binds to an epitope, and the epitope is the same as the epitope bound by an 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; The antibody pair comprises a second antibody, and the second antibody binds to an epitope: the epitope is the same as the epitope bound by an antibody comprising the heavy chain variable region shown in SEQ ID NO: 21 and the light chain variable region shown in SEQ ID NO:

23.

7. The antibody pair for detecting CA15-3 according to any one of claims 1-6, characterized in that, The heavy chain constant region of the antibody is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, 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; Optionally, the heavy chain constant region of the first antibody is SEQ ID NO: 7 or a sequence having at least 80% identity therewith, and the light chain constant region of the first antibody is SEQ ID NO: 8 or a sequence having at least 80% identity therewith; Optionally, the heavy chain constant region of the second antibody is SEQ ID NO: 19 or a sequence having at least 80% identity therewith, and the light chain constant region of the second antibody is SEQ ID NO: 20 or a sequence having at least 80% identity therewith; Optionally, the heavy chain constant region includes CH1 of IgG1, the hinge region of IgG1, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

8. A reagent for detecting CA15-3, characterized in that, The reagent includes a first group of antibodies and a second group of antibodies, and the first group of antibodies includes the first antibody according to any one of claims 1-7; The second group of antibodies includes the second antibody according to any one of claims 1-7; 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; Optionally, the coating antibody is conjugated with biotin or a biotin derivative; Optionally, the label conjugated to the labeled antibody is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioisotopes, chemiluminescent labels, electrochemiluminescent labels, latex and nanoparticle-based labels; Optionally, the coating antibody is conjugated to a solid phase carrier; Optionally, the solid phase carrier is selected from microspheres, plates and membranes.

9. A method for detecting CA15-3, characterized in that, Comprising: a) Under conditions sufficient to cause an antibody / antigen binding reaction, contacting the antibody pair according to any one of claims 1-7 or the reagent according to claim 8 with a sample to be tested to form an immune complex; and b) Detecting the presence of the immune complex, the presence of which indicates the presence of CA15-3 in the test sample.

10. Use of the antibody pair according to any one of claims 1-7 or the reagent according to claim 8 in detecting CA15-3 or in preparing a product for detecting CA15-3.

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