Antibody pair, reagent and method for detecting CA19-9

Abstract

The invention discloses an antibody pair, a reagent and a method for detecting CA19-9, and relates to the field of immunodiagnosis. The antibody pair for detecting the CA19-9 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 CA19-9.

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 CA19-9. Background Art

[0002] Tumor markers refer to substances that are characteristically present in malignant tumor cells, or abnormally produced by malignant tumor cells, or substances produced by the host's response to tumors, and can reflect the occurrence and development of tumors and monitor the response of tumors to treatment.

[0003] CA19-9 is a glycopolymer structure containing sialic acid, secreted by human glandular epithelial cells, with a molecular weight of more than 1000 kDa. Its antigenic epitope is a glycopolymer 1 containing the sialylated Lewis a structural motif, and it belongs to one of the tumor markers. The CA19-9 antigen mainly exists in glycolipids on the surface of tissues or cells, and a part is also contained in serum, mainly in the form of mucin (highly glycosylated glycoprotein) 2. CA19-9 is a related marker for pancreatic cancer, gastric cancer, colorectal cancer, and gallbladder cancer, and it is also the marker with the highest sensitivity to pancreatic cancer reported so far. Steinberg 3 et al. found that for patients suspected of having pancreatic cancer, when the serum level of CA19-9 is 37-40 U / mL, the sensitivity and specificity are 81% and 90% respectively, the positive predictive value (PPV) of the elevated serum CA19-9 level is 72.3%, and the negative predictive value (NPV) is 95.8%; when the diagnostic threshold of serum CA19-9 for pancreatic cancer is increased to 100 U / mL or 1000 U / mL, the specificity is increased to 98% and 99.8% respectively, and the sensitivity is decreased to 68% and 41% respectively. In the in vitro diagnostic industry, the determination of carbohydrate antigen CA19-9 generally uses a two-site sandwich chemiluminescence immunoassay.

[0004] A decrease or normalization of the postoperative CA-19-9 serum level is associated with an improved survival rate 4. Such detection kits are mainly used for the dynamic detection of the condition of patients with confirmed malignant tumors. Tumor markers are not the only basis for tumor diagnosis, and clinical symptoms, imaging examinations, and other means need to be comprehensively considered clinically. Summary of the Invention

[0005] The present application provides an antibody pair, which provides an important raw material source for the detection of CA19-9 and has good detection performance.

[0006] To achieve the above object, according to one aspect of the present invention, there is provided an antibody pair for detecting CA19-9. The antibody pair includes a first antibody, and the first antibody includes: HCDR1, HCDR2, and

[0007] The three heavy-chain complementarity-determining regions of HCDR3 and the three light-chain complementarity-determining regions of LCDR1, LCDR2, and LCDR3 in the light-chain variable region shown in SEQ ID NO: 11;

[0008] The antibody pair includes a second antibody, which comprises: in the heavy-chain variable region shown in SEQ ID NO: 21

[0009] The three heavy-chain complementarity-determining regions of HCDR1, HCDR2, and HCDR3 and the three light-chain complementarity-determining regions of LCDR1, LCDR2, and LCDR3 in the light-chain variable region shown in SEQ ID NO: 23.

[0010] The complementarity-determining regions are defined by any one system or a combination of multiple systems among Kabat, Chothia, IMGT, AbM, or Contact.

[0011] To achieve the above object, according to the second aspect of the present invention, there is provided an antibody pair for detecting CA19-9, the antibody pair includes a first 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;

[0012] The antibody pair includes a second antibody, 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, and

[0013] LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 18.

[0014] To achieve the above object, according to the third aspect of the present invention, there is provided an antibody pair for detecting CA19-9;

[0015] The antibody pair contains a first antibody, and the first antibody includes at least one of (1)-(2):

[0016] (1) The heavy-chain variable region shown in SEQ ID NO: 9 and the light-chain variable region shown in SEQ ID NO: 11;

[0017] (2) The heavy chain shown in SEQ ID NO: 10 and the light chain shown in SEQ ID NO: 12;

[0018] The antibody pair comprises a second antibody, which comprises at least one of (a)-(b):

[0019] (a) A heavy chain variable region shown in SEQ ID NO:21 and a light chain variable region shown in SEQ ID NO:23;

[0020] (b) A heavy chain shown in SEQ ID NO:22 and a light chain shown in SEQ ID NO:24.

[0021] To achieve the above object, according to the fourth aspect of the present invention, there is provided an antibody pair for detecting CA19-9;

[0022] 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; or;

[0023] The antibody pair comprises a second antibody, 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;

[0024] 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.

[0025] 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;

[0026] 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.

[0027] To achieve the above object, according to the fifth aspect of the present invention, there is provided an antibody pair for detecting CA19-9;

[0028] The antibody pair comprises a first antibody, the first antibody binds to an epitope, and the epitope is the same as the epitope bound by an antibody comprising a heavy chain variable region shown in SEQ ID NO:9 and a light chain variable region shown in SEQ ID NO:11;

[0029] The antibody pair comprises a second antibody, the second antibody binds to an epitope: the epitope is the same as the epitope bound by an antibody comprising a heavy chain variable region shown in SEQ ID NO:21 and a light chain variable region shown in SEQ ID NO:23.

[0030] To achieve the above object, according to the sixth aspect of the present invention, there is provided an antibody pair for detecting CA19-9, wherein 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;

[0031] 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;

[0032] 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;

[0033] In an alternative embodiment, 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;

[0034] In an alternative embodiment, the antibody is a multimer formed by polymerization of antibody monomers.

[0035] To achieve the above object, according to the seventh aspect of the present invention, there is provided a reagent for detecting CA19-9, 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;

[0036] The second group of antibodies comprises the second antibody in the antibody pair;

[0037] 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;

[0038] 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;

[0039] In an alternative embodiment, the coating antibody or the labeled antibody is conjugated with biotin or a biotin derivative;

[0040] 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;

[0041] In an alternative embodiment, the coated antibody is conjugated to a solid phase carrier;

[0042] In an alternative embodiment, the solid phase carrier is selected from microspheres, plates, and membranes.

[0043] To achieve the above object, according to the eighth aspect of the present invention, there is provided a method for detecting CA19-9, comprising:

[0044] 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

[0045] b) detecting the presence of the immune complex, the presence of which indicates the presence of CA19-9 in the test sample.

[0046] To achieve the above object, according to the ninth aspect of the present invention, there is provided the use of the above antibody pair or reagent in detecting CA19-9 or in preparing a product for detecting CA19-9. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 . Clinical correlation of commercially available antibody pair chemiluminescent reagent and Roche's CA19-9 reagent

[0048] Figure 2 . Clinical correlation of the chemiluminescent reagent paired in the present invention patent and Roche's CA19-9 reagent DETAILED DESCRIPTION OF THE INVENTION

[0049] In a first aspect, an embodiment of the present invention provides an antibody pair for detecting CA19-9, the antibody pair comprising a first antibody, the first antibody comprising: 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 comprises a second antibody, the second antibody comprising: 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.

[0050] The complementary determining regions are defined by any one or a combination of multiple systems among Kabat, Chothia, IMGT, AbM, or Contact.

[0051] In the present invention, the term "antibody" is used in the broadest sense, and the antibodies in the methods of the present invention can be whole antibodies, antigen-binding fragments, or polymeric antibodies that are capable of binding to CA19-9.

[0052] The whole antibody can be monoclonal. Such whole antibodies are usually antibodies prepared by any suitable method known in the art. For example, by immunizing a mammal, usually a rabbit or a mouse, with CA19-9 under suitable conditions and isolating the antibody molecules from, for example, the serum of the mammal. Monoclonal antibodies can be obtained by hybridoma or recombinant methods. The 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 antibody 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 synthesizers sold by Applied BioSystems, etc. A polymeric antibody refers to a multimer formed by polymerization of whole antibodies and antigen-binding fragments.

[0053] In a second aspect, an embodiment of the present invention provides an antibody pair for detecting CA19-9, 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.

[0054] In the present invention, the terms "complementary determining region", "CDR" or "CDRs" refer to the hypervariable regions of the heavy and light chains of an immunoglobulin, and refer to 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 the specific embodiments of the present invention, the CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0055] Methods for defining CDRs are well known in the art and include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As used 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). The "Chothia definition" can be found in 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 regions defined by Kabat, although they may be shortened or lengthened based on the prediction or experimental results of 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 comprise a particular 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.

[0056] Table 1: CDR Definitions 1

[0057] 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

[0058] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), where 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 unambiguously map this Kabat numbering system to any variable region sequence without relying on any experimental data outside of the sequence itself. As used herein, "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).

[0059] 2 "AbM" as used in Table 1 with a lower case "b" refers to the CDR defined by Oxford Molecular's "AbM" antibody modeling software.

[0060] 3 When neither H35A nor H35B is present, CDR-H1 ends at position 35; when only H35A is present, CDR-H1 ends at position 35A; when both H35A and H35B are present, CDR-H1 ends at position 35B.

[0061] 4 When neither H35A nor H35B is present, CDR-H1 ends at position 32; when only H35A is present, CDR-H1 ends at position 33; when both H35A and H35B are present, CDR-H1 ends at position 34.

[0062] 5 When neither H35A nor H35B is present, CDR-H1 ends at position 33; when only H35A is present, CDR-H1 ends at position 34; when both H35A and H35B are present, CDR-H1 ends at position 35.

[0063] 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 of Kabat, Chothia, IMGT, AbM or Contact.

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

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

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

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

[0068] In some alternative embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Contact system.

[0069] 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.

[0070] The antibody of the present invention further includes framework regions. In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, which refers to the regions other than the CDRs in the variable regions of the heavy chain and light chain of the antibody; wherein, the heavy chain framework region can be further divided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further divided into adjacent regions separated by CDRs, including the LFR1, LFR2, LFR3 and LFR4 framework regions.

[0071] In the present invention, the variable region of the heavy chain is obtained by connecting the following numbered CDRs and 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 following numbered CDRs and FRs in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0072] 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.

[0073] In a third aspect, an embodiment of the present invention provides an antibody pair for detecting CA19-9,

[0074] 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.

[0075] In an alternative 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; the antibody pair comprises a second antibody having a heavy chain as shown in SEQ ID NO: 22 and a light chain as shown in SEQ ID NO: 24.

[0076] In a fourth aspect, an embodiment of the present invention provides an antibody pair for detecting CA19-9, the antibody pair comprising a first antibody, wherein the variable region of the heavy chain of the first antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 9, and the variable region of the light chain 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 variable region of the heavy chain of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 21, and the variable region of the light chain of the second antibody is a conservative variant formed by mutation of the amino acid sequence shown in SEQ ID NO: 23.

[0077] 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.

[0078] 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.

[0079] In a fifth aspect, an embodiment of the present invention provides an antibody pair for detecting CA19-9, the antibody pair comprising a first antibody, the first antibody binding to an epitope that is the same as the epitope bound by an antibody comprising a heavy chain variable region as shown in SEQ ID NO: 9 and a light chain variable region as shown in SEQ ID NO: 11; the antibody pair comprises a second antibody, the second antibody binding to an epitope: the epitope is the same as the epitope bound by an antibody comprising a heavy chain variable region as shown in SEQ ID NO: 21 and a light chain variable region as shown in SEQ ID NO: 23.

[0080] The epitope is also known as the antigenic epitope (AE). The epitope determines the ability of the antigen to specifically bind to the antibody. That is, the antibody binds to the same epitope, which means the amino acid fragments to which the antibody binds to the target antigen are the same. 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 competitive binding assays, and the methods include: ELISA, fluorescence resonance energy transfer assay (FRET), or fluorescence microplate assay technology (FMAT), etc. The amount of the antibody bound to the antigen is indirectly related to the binding ability of the candidate competing antibody (the antibody to be tested) that competes for binding to the same epitope. That is, the greater the binding amount or affinity of the antibody to be tested to the same epitope, the lower the binding amount of the antibody to the antigen, and the binding amount of the antibody to be tested to the antigen increases. Specifically, the antibody appropriately labeled and the antibody to be evaluated are added to the antigen simultaneously, and the labeled antibody bound is detected using the label. By pre-labeling the antibody, the amount of the antibody bound to the antigen can be easily measured. There is no particular limitation on the label, and a labeling method corresponding to the assay technology is selected. Specific labeling methods include: fluorescence labeling, radiolabeling, enzyme labeling, etc.

[0081] In an alternative embodiment, the antibody further includes 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 can be selected from the λ1, λ2, λ3, and λ4 subtypes.

[0082] 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).

[0083] In an alternative embodiment, 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;

[0084] In an alternative embodiment, the antibody is a multimer formed by polymerization of antibody monomers.

[0085] In some alternative embodiments, the species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

[0086] In a seventh aspect, an embodiment of the present invention provides a reagent for detecting CA19-9,

[0087] The reagent includes a first group of antibodies and a second group of antibodies. The first group of antibodies includes the first antibody in the above antibody pair;

[0088] The second group of antibodies includes the second antibody in the above antibody pair.

[0089] In an optional embodiment, one of the first group of antibodies and the second group of antibodies is a coated antibody, and the other is a labeled antibody.

[0090] 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.

[0091] In an optional embodiment, the coated antibody or the labeled antibody is conjugated with biotin or a biotin derivative;

[0092] In an optional 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;

[0093] In an optional embodiment, 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 (preCP), etc.).

[0094] In an optional embodiment, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphogluconate dehydrogenase.

[0095] In an alternative embodiment, 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.

[0096] In an alternative embodiment, 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 ester and its derivatives, dioxetane and its derivatives, rhodamine and its derivatives, and peroxyoxalate and its derivatives.

[0097] In an alternative embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0098] In an alternative embodiment, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0099] In an alternative embodiment, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0100] In an alternative embodiment, the colloidal metal is colloidal gold.

[0101] In an alternative embodiment, the coated antibody is conjugated to a solid support;

[0102] In an alternative embodiment, the solid support is selected from microspheres, plates, and membranes.

[0103] In an eighth aspect, an embodiment of the present invention provides a method for detecting CA19-9, comprising:

[0104] a) contacting the above antibody pair or reagent with a sample to be tested under conditions sufficient to allow an antibody / antigen binding reaction to form an immune complex; and

[0105] b) detecting the presence of the immune complex, the presence of which indicates the presence of CA19-9 in the test sample.

[0106] 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 CA19-9 or in preparing a product for detecting CA19-9.

[0107] 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. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0108] 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 considered herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0109] 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 well 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.

[0110] Based on long-term and creative research on CA19-9, the applicant has discovered two antibodies that can meet the requirements for CA19-9 detection.

[0111] The features and properties of the present invention will be further described in detail below in conjunction with the examples.

[0112] Example 1 Antibody Preparation

[0113] 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 CA19-9 monoclonal antibody was an existing hybridoma cell line and was revived for standby.

[0114] (1) Preparation of antibody gene

[0115] mRNA was extracted from the hybridoma cell line secreting the CA19-9 monoclonal antibody. DNA products were obtained by RT-PCR method. After adding A reaction to the products with rTaq DNA polymerase, they were inserted into the pMD-18T vector and transformed into DH5α competent cells. After colonies grew, Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones of each were sent to a gene sequencing company for sequencing.

[0116] (2) Sequence analysis of the variable region genes of CA19-9 antibody

[0117] The gene sequences obtained from the above sequencing were analyzed in the kabat antibody database, and 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.

[0118] (3) Construction of recombinant antibody expression plasmid

[0119] pcDNA TM 3.4 The pcDNA3.4 vector was the constructed recombinant antibody eukaryotic expression vector. 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 amplification method.

[0120] The Heavy Chain and Light Chain gene fragments were respectively digested with HindIII / EcoRI double enzymes, 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.

[0121] 2. Recombinant antibody production

[0122] 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 the antibody concentration and cells with a cell viability > 95%; centrifuge and wash the cells, resuspend them with the medium, and at the same time adjust the cell density to 2.9×10 6 cells / ml. Wash the cells, resuspend them with the medium, and at the same time, use it as the cell diluent. Prepare the plasmid DNA and transfection reagent diluents with the 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 it in a 35°C constant temperature incubator 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.

[0123] The obtained antibodies were named 8H8 and 12D10. The sequences of the heavy chain (H) and light chain (L) of the above antibodies are shown in the following table:

[0124] Table 2 Antibody sequences

[0125] Antibody Name Heavy Chain Light Chain 8H8 SEQ ID NO:10 SEQ ID NO:12 12D10 SEQ ID NO:22 SEQ ID NO:24

[0126] Example 2 Preparation of detection reagents

[0127] 1. Reagent preparation

[0128] A) SA coating of magnetic particles: Use carboxyl magnetic particles as the solid-phase carrier. Take 100 mg of carboxyl magnetic particle suspension, after magnetic separation, take the supernatant and resuspend it with MES buffer (0.02M MES, pH 6.0), add an EDC aqueous solution (EDC concentration is 15 mg / mL) to activate the surface carboxyl groups of the carboxyl magnetic particles, then add SA (SA4), suspend it at room temperature for 5 h, after magnetic separation to remove the supernatant, resuspend it with a magnetic particle diluent (25 mM HEPES, 1% BSA, pH 7.2) to obtain carboxylated magnetic particles with a SA coating concentration of 10 mg / mL.

[0129] The mass ratio of EDC to carboxyl magnetic particles is 1:50.

[0130] The mass ratio of SA to carboxyl magnetic particles is 1:5.

[0131] The average particle size of the carboxyl magnetic particles is about 0.4 μm.

[0132] B) Acridinium ester labeling of the antibody: Take the labeling buffer solution (0.02 M PBS, pH 7.2) in a centrifuge tube, add the CA19-9 monoclonal antibody (12D10), and mix well; add the acridinium ester solution, mix well, and react with light avoidance and oscillation at room temperature for 2 h, then remove impurities to obtain the CA19-9 monoclonal antibody labeled with the acridinium ester label.

[0133] The operation of removing impurities is desalting with a centrifugal desalting column. First, treat the centrifugal desalting column with pure water and PBS buffer solution (0.02 M PBS, pH 7.2) respectively. Finally, add the CA19-9 monoclonal antibody solution labeled with the acridinium ester label, and collect the liquid in the centrifuge tube.

[0134] The molar ratio of acridinium ester to CA19-9 monoclonal antibody is about 10:1.

[0135] C) Biotin labeling of the antibody: Take the labeling buffer solution (0.02 M PBS, pH 7.4) in a centrifuge tube, add the CA19-9 monoclonal antibody (8H-10), and mix well; add the biotin solution, mix well, and react at room temperature for 1 h to 2 h, then remove impurities to obtain the CA19-9 monoclonal antibody labeled with the biotin label.

[0136] The operation of removing impurities is desalting with a centrifugal desalting column. First, treat the centrifugal desalting column with pure water and PBS buffer solution (0.02 M PBS, pH 7.4) respectively. Finally, add the CA19-9 monoclonal antibody solution labeled with the biotin label, and collect the liquid in the centrifuge tube.

[0137] The molar ratio of biotin to CA19-9 monoclonal antibody is about 5:1.

[0138] D) Dilute the SA-coated carboxylated magnetic particles with a magnetic particle diluent (50 mM Tris, 1% BSA, 9% NaCl, pH 7.2) to a magnetic particle working solution of 0.2 mg / mL. Dilute the acridinium ester-labeled CA19-9 monoclonal antibody with an acridinium ester label diluent (20 mM PBS, 1% BSA, 1% NaCl, pH 7.2) to an acridinium ester working solution of 0.25 μg / mL. Dilute the biotin-labeled CA19-9 monoclonal antibody with a biotin label diluent (20 mM PBS, 1% BSA, 1% NaCl, pH 7.4) to a biotin working solution of 0.25 μg / mL, and store at 2 - 8 °C for later use. Combining the three working solutions can obtain a CA19-9 chemiluminescent immunoassay kit.

[0139] E) Preparation of CA19-9 calibrator: Prepare CA19-9 antigen with a calibrator diluent (20 mM HEPES, 0.5% BSA, 1% NaCl, pH 7.2) at concentrations of 0 U / mL, 0.244 U / mL, 0.977 U / mL, 3.91 U / mL, 15.63 U / mL, 62.50 U / mL, 250.0 U / mL, and 1000 U / mL, and store at 2 - 8 °C for later use.

[0140] F) The pre-excitation solution is a H2O2 solution with a concentration of 0.1 mol / L, and the excitation solution is a NaOH solution with a concentration of 0.25 mol / L.

[0141] 2. Detection process

[0142] Perform the detection on a Yingkai shine i2910 model fully automatic chemiluminescent immunoassay analyzer. Use the double antibody sandwich method, that is, the instrument sequentially adds 20 μL of the sample, 70 μL of the biotin working solution, and 70 μL of the acridinium ester working solution. After mixing and incubating for 10 minutes, incubate with 40 μL of the magnetic particle working solution and then incubate for another 10 minutes to form a sandwich structure of SA magnetic beads - (Bio-antibody) - antigen - (antibody-AE). After the incubation in the reaction tube is completed, adsorb the magnetic beads with a magnetic field and wash away the unbound substances. Add the pre-excitation solution and the excitation solution, and detect the relative luminescence intensity (RLU). Obtain the luminescence value (RLU) of the CA19-9 calibrator and the luminescence values (RLU) of 68 Roche-assigned clinical blood samples, and compare the performance differences between the paired antibodies in this patent and commercially available antibodies.

[0143] 3. Detection results and summary

[0144] A) The detection data of the CA19-9 calibrator are as follows:

[0145]

[0146] The paired activity of this patent is significantly improved compared to the paired activity (signal value) of commercially available antibodies, about 775% higher.

[0147] B) The results of the CA19-9 correlation test are as follows:

[0148]

[0149]

[0150]

[0151] The correlation R between the 68 clinical blood samples paired in this patent and Roche is 2 = 0.962, which is significantly improved compared to the paired antibodies on the market; there is an obvious HOOK phenomenon when the sample concentration of the commercially available antibody pair is higher than 913.7 U / mL, while there is no obvious HOOK phenomenon within the range of 5.72 - 1383 U / mL for the paired antibodies of this patent, and the linear range is wider.

[0152] C) The results of the batch-to-batch difference test of the new CA19-9 antibody are as follows:

[0153]

[0154]

[0155] The difference between the three batches of the paired antibodies of this patent is small (the batch-to-batch difference of the activity of the three large samples ≤ 15%). 43 clinical sera were tested and compared with the Roche sample fixed value: the slope is between 0.75 - 1.2, and R 2 is not less than 0.99.

[0156] The above are only the preferred embodiments of the present invention and are not used 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 in the protection scope of the present invention.

[0157] Some of the amino acid sequences involved in this application are shown as follows:

[0158]

[0159]

Claims

1. An antibody pair for detecting CA19-9, 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 CA19-9, 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 CA19-9, characterized in that the antibody pair contains 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 contains 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 CA19-9 according to claim 4, characterized in that the antibody pair contains 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 CA19-9, 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 CA19-9 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 CA19-9, characterized in that, The reagent comprises a first group of antibodies and a second group of antibodies, and the first group of antibodies comprises the first antibody according to any one of claims 1-7; The second group of antibodies comprises 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 or the labeled 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 CA19-9, 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 CA19-9 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 CA19-9 or in preparing a product for detecting CA19-9.

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