Anti-ApoB antibody or functional fragment thereof and application thereof

Abstract

The invention discloses an antibody of apolipoprotein B (ApoB) related to low density lipoprotein (LDL) and application of the antibody, and relates to the field of antibodies. The anti-ApoB antibody disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for detection of ApoB, and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-ApoB antibody and application thereof. Background Art

[0002] This antibody can be used in the detection of ApoB, and can also be used as a supporting antibody for the detection of Ox-ApoB. This article focuses on its role in the detection of Ox-ApoB from the application end.

[0003] The relationship between ApoB (apolipoprotein B) and Ox-ApoB (oxidized apolipoprotein B) involves the oxidative modification of lipoproteins and its impact on cardiovascular health. ApoB is a key protein that makes up LDL (low-density lipoprotein) and some other lipoproteins such as VLDL and IDL. ApoB-100 is the most common form, which is the main apolipoprotein of LDL and the main marker recognized by the LDL receptor. ApoB-100 is essential for the structural stability and function of LDL, and it helps regulate the circulation time and clearance rate of LDL in the blood. Ox-ApoB refers to the form of ApoB that has undergone oxidative modification in the body. This oxidation usually occurs during the oxidation of LDL by free radicals or other oxidants. After the ApoB-100 in oxidized LDL (ox-LDL) is oxidized, its structure and function will change, which makes ox-LDL more likely to deposit in the blood vessel wall and trigger an inflammatory response, thereby promoting the development of atherosclerosis. Ox-ApoB may lose its normal receptor recognition properties and is no longer easily cleared by the LDL receptor, which leads to its continued presence in the blood. ApoB is the core component of LDL, and Ox-ApoB is the oxidized form of ApoB in LDL. Oxidized ApoB causes changes in the properties of LDL particles, making them more likely to deposit on the blood vessel walls, thereby promoting the progression of atherosclerosis. Due to the accumulation of oxidized LDL (ox-LDL), the risk of cardiovascular disease increases. The presence and level of Ox-ApoB can be used as a marker for cardiovascular disease risk.

[0004] Commonly used detection methods for Ox-LDL include enzyme-linked immunosorbent assay, chemiluminescence assay, etc. The reaction principle is based on the sandwich method of antigen-antibody reaction to detect ApoB and Ox-ApoB. When detecting Ox-LDL, one solution is to fix the anti-Ox-ApoB antibody on the plate, incubate it with the sample to be tested, and then use HRP or other peroxidase-coupled anti-ApoB antibodies to achieve double antibody sandwich detection of Ox-LDL content. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 This is the calibration curve of ApoB-A antibody. Specific embodiments

[0006] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0007] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0008] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. The methods and materials described herein, and any similar or equivalent methods and materials can be used for the practice or testing of the preparations or unit doses herein. Unless otherwise indicated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative only and not limiting.

[0009] As used herein, the terms "includes," "including," "having," "may," and variations thereof are intended to be open-ended, transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0010] In a first aspect, an embodiment of the present invention provides an anti-ApoB antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a complementary determining region of any one of the following groups:

[0011] (1) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively;

[0012] (2) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 16, SEQ ID NO: 23 and SEQ ID NO: 18, respectively;

[0013] (3) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 16, SEQ ID NO: 23 and SEQ ID NO: 24, respectively;

[0014] (4) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 19, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 17 and SEQ ID NO: 18, respectively;

[0015] (5) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 18, respectively;

[0016] (6) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 respectively.

[0017] It should be noted that HCDR1, HCDR2 and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2 and HCDR3 of the same heavy chain variable region defined in the antibody or antigen-binding fragment thereof described in the first aspect, and LCDR1, LCDR2 and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2 and LCDR3 of the same light chain variable region defined in the antibody or antigen-binding fragment thereof described in the first aspect.

[0018] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological activity.

[0019] In the present invention, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that play a role in the binding of an antibody or antigen-binding fragment to its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refers to the highly variable region of the heavy and light chains of the antibody.

[0020] In the present invention, the heavy chain complementarity determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3.

[0021] Methods for defining CDRs are well known in the art, and CDR definition methods include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As used herein, "Kabat definition" refers to the definition system described by Kabat et al., US Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" refers to 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 extended based on predictions or experimental results of specific residues or residue groups.

[0022] 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, preferably defined by the Kabat system.

[0023] In the present invention, the "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region other than the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDR, including HFR1, HFR2, HFR3 and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by CDR, including LFR1, LFR2, LFR3 and LFR4 framework regions.

[0024] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combination:

[0025] LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0026] In some specific embodiments, the antibody or antigen-binding fragment thereof of the present invention further comprises framework regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4;

[0027] The HFR1 comprises SEQ ID NO: 3 or an amino acid sequence having at least 80% identity thereto;

[0028] The HFR2 comprises SEQ ID NO:4 or an amino acid sequence having at least 80% identity thereto;

[0029] The HFR3 comprises SEQ ID NO:5 or an amino acid sequence having at least 80% identity thereto;

[0030] The HFR4 comprises SEQ ID NO:6 or an amino acid sequence having at least 80% identity thereto;

[0031] The LFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

[0032] The LFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

[0033] The LFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto;

[0034] The LFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto.

[0035] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the anti-ApoB antibodies or antigen-binding fragments thereof provided by the present invention may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above-mentioned corresponding framework regions (SEQ ID NO: XX).

[0036] In a second aspect, an embodiment of the present invention provides an anti-ApoB antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and / or a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NO:1, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:33; and the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:34.

[0037] In an optional embodiment, the antibody or antigen-binding fragment thereof described in the first aspect or the second aspect further comprises a constant region.

[0038] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0039] In an optional embodiment, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments.

[0040] In an alternative embodiment, the heavy chain constant region includes CH1 of IgG, a hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM.

[0041] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0042] In an alternative embodiment, the light chain constant region is selected from a kappa-type or a lambda-type light chain constant region.

[0043] In an alternative embodiment, the constant region is derived from a species of cow, horse, dairy cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, fighting cock or human.

[0044] In an alternative embodiment, the species origin of the constant region is mouse.

[0045] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:11, and the light chain constant region (CL) sequence is as shown in SEQ ID NO:12.

[0046] It should be noted that, in other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above-mentioned constant region (SEQ ID NO: 11-12).

[0047] In an optional embodiment, the antigen-binding fragment is selected from any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

[0048] The antigen-binding fragments of the above antibodies usually have the same binding specificity as the antibodies from which they are derived. It is easy for those skilled in the art to understand based on the contents 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 split disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, those skilled in the art can easily obtain the above antigen-binding fragments.

[0049] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0050] In a third aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the above-mentioned antibody or antigen-binding fragment thereof.

[0051] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof.

[0052] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or antigen-binding fragment thereof.

[0053] In an optional embodiment, the above-mentioned marker refers to a class of substances with properties that can be directly observed by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0054] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.

[0055] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0056] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0057] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0058] In an optional 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.

[0059] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0060] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0061] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0062] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0063] In an optional embodiment, the colloidal metal is colloidal gold.

[0064] In an optional embodiment, the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

[0065] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0066] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.

[0067] In a fourth aspect, the present invention provides a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned antibody conjugate.

[0068] As mentioned above, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention can effectively bind to anti-ApoB, and therefore, the reagents or kits containing the anti-ApoB antibodies or antigen-binding fragments thereof can effectively detect anti-ApoB qualitatively or quantitatively. The reagents or kits provided by the present invention can be used, for example, for detections involving the use of anti-ApoB and its antibody-specific binding properties by immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, latex immunoturbidimetry, etc. As mentioned above, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention have higher binding activity or affinity with anti-ApoB, and therefore the reagents or kits containing the antibodies or antigen-binding fragments thereof have higher detection sensitivity or specificity.

[0069] In a fifth aspect, the present invention provides a method for detecting anti-ApoB, comprising: a) contacting the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit with anti-ApoB in a sample to be detected to form an immune complex under conditions sufficient for an antibody / antigen binding reaction to occur; and b) detecting the presence of the immune complex, wherein the presence of the complex indicates the presence of the antigen in the test sample;

[0070] In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody or antigen-binding fragment thereof.

[0071] In an alternative embodiment, the immune complex further comprises a second antibody that binds to anti-ApoB.

[0072] In a sixth aspect, the present invention provides use of the above-mentioned anti-ApoB antibody or antigen-binding fragment thereof, antibody conjugate or the above-mentioned reagent or kit in the preparation of a product for detecting anti-ApoB.

[0073] In a seventh aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0074] In an eighth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0075] In a ninth aspect, the present invention provides a cell containing the above-mentioned vector.

[0076] In a tenth aspect, the present invention provides a method for preparing an anti-ApoB antibody or an antigen-binding fragment thereof, comprising: culturing the cells as described above.

[0077] Based on the amino acid sequence of the anti-ApoB antibody or its antigen-binding fragment disclosed in the present invention, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the anti-ApoB antibody or its antigen-binding fragment, for example, separating and purifying the antibody or its antigen-binding fragment from the culture product of a recombinant cell that can recombinantly express the antibody or its antigen-binding fragment as described in any of the above items, which is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the anti-ApoB antibody or its antigen-binding fragment of the present invention, it belongs to the protection scope of the present invention.

[0078] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0080] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0081] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0082] Example 1 Preparation of anti-ApoB antibodies

[0083] (1) Mouse immunization and antibody detection

[0084] Select 5 6-8 week old SPF female BALB / c mice, mix and emulsify Freund's complete adjuvant and ApoB protein at a concentration of 1 mg / ml in equal volumes. Use the emulsified antigen to immunize 6-8 week old SPF female BALB / c mice, and inject 50 μg of antigen protein into each mouse by subcutaneous injection in the foot or back. Two weeks after the first immunization, mix the antigen protein with Freund's incomplete adjuvant and emulsify it, and inject 50 μg of antigen protein into each mouse by subcutaneous injection in the foot or back. Two weeks later, collect blood through the tail vein, collect the supernatant by centrifugation, and test the serum titer by ELISA. Immunize once every two weeks and test the serum titer. After two immunizations, the serum titer is as high as 2.0 or above after a million-fold dilution. Screening serum titer 10 6 Lymph was taken from the above mice to separate lymphocytes for cell fusion.

[0085] (2) Cell fusion, positive hybridoma cell screening and subcloning

[0086] Lymphocytes of isolated immune mice were fused with cultured SP2 / 0 cells by PEG1500 or electrofusion. The fused cells were cultured in HAT-1640 medium containing 20% ​​FBS serum for screening. The culture medium was replaced after one week, and the culture supernatant was taken for positive clone screening after another 4 days of culture. The antibody screening scheme is the key to the successful development of the antibody of the present invention. In the antibody screening stage, ApoB protein was used to coat the ELISA plate for positive well screening. The wells with the highest ratio of ELISA positive value to cell number were selected for multiple subcloning. The ELISA plate was then coated with ApoB protein. The culture supernatant of the subclone was taken to screen the monoclones that can show affinity under the antigen coating conditions, and the monoclonal hybridoma cells with the highest affinity were selected from them, and finally a hybridoma cell line with a high antibody titer that can secrete ApoB monoclonal antibodies was obtained, named 158#, which has good stability.

[0087] (3) Production and purification of monoclonal antibodies

[0088] Two groups of 6-8 week old BALB / c mice were selected and intraperitoneally injected with 500 μL paraffin oil to suppress the immune response of mice. One week after the injection, 0.5 ml of anti-ApoB hybridoma cell 158# was injected into the peritoneal cavity of the mice. The number of cells was about 1×10 6 After two weeks, ascites was collected. The collected ascites was precipitated with ammonium sulfate and affinity purified with protein A to obtain the target antibody 158#.

[0089] (4) Monoclonal antibody subtype identification and gene sequence cloning

[0090] The SBA Clonotyping System-HRP kit from Southern Biothech was used to identify the subtypes of the heavy and light chains of the monoclonal antibody according to the instructions. The specific procedures were as follows:

[0091] a. Dilute the capture antibody to 1 μg / mL with coating solution (0.05M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the ELISA plate, and coat overnight at 4°C. Wash the plate three times with PBS buffer (washing solution) containing 0.05% Tween-20;

[0092] b. Dilute the culture supernatant of the hybridoma cells to be tested with diluent (1% BSA, 0.1% PBST) at a ratio of 1:1, add 100 μL / well to the ELISA plate, and incubate at 37°C for 30 minutes. Dilute the corresponding enzyme-labeled antibodies (Ig-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, IgM-HRP, kappa-HRP, lamda-HRP) at a ratio of 1:3000 with diluent;

[0093] After washing the plate three times with the plate washing solution, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate three times again, add the color developing solution. After about 5 minutes (depending on the strength of the reaction), add 2M sulfuric acid to terminate the reaction and read the OD450 absorbance value. After identification, the heavy chain subtype of the anti-ApoB-A antibody is IgG1 and the light chain is Kappa. According to the antibody subtype results, the antibody gene sequence was cloned using a method based on the RACE technology route. Hybridoma cells with good growth status were collected, and the total RNA of the hybridoma cells was obtained using a total RNA extraction kit. According to the operating method of Takara's SMARTer RACE manual, the mRNA was reversely transcribed into cDNA, and the full-length sequence of the target antibody was amplified.

[0094] (5) Antibody expression in vitro

[0095] According to the full-length sequence of the antibody, the heavy and light chains were synthesized separately into the pcDNA3.1(+) vector and transfected into HEK293 for expression verification. The fermentation supernatant was purified by protein G to obtain antibody 158#.

[0096] Antibody 158# was named anti-ApoB-A, anti-ApoB-A was mutated, and 5 mutant antibodies with equivalent performance were screened out. The antibody sequences are shown in Table 1 below (CDR region sequences are defined by the Kabat system):

[0097] Table 1 Antibody sequences

[0098]

[0099] Example 2 Performance testing of antibodies

[0100] 1. Reactivity Identification

[0101] ApoB antigen was diluted to 1 μg / mL with 50 mM carbonate buffer coating solution for microplate coating, 100 μL per well, overnight at 4°C; the next day, the washing solution was washed twice with PBST and patted dry; the above antibodies were added respectively, starting from 1000 ng / ml and diluted 3 times, loaded, 100 μL / well, 37°C, 60 min; washed 3 times with PBST washing solution, patted dry; horseradish peroxidase-labeled goat anti-mouse IgG was added, 100 μL per well, 37°C, 30 min; washed 3 times with PBST washing solution, patted dry; carbamide peroxide (50 μL / well) and tetramethylbenzidine (50 μL / well) were added for 10 min; dilute sulfuric acid was added to terminate the reaction, 50 μL / well; OD values ​​were read at 450 nm (reference 630 nm) on the microplate reader; the reactivity of the above antibodies was identified as shown in Table 2 below:

[0102] Table 2 Reactivity data

[0103]

[0104] The experimental results show that the above six anti-ApoB antibodies have good reactivity

[0105] 2. Functional testing

[0106] (1) Application of antibodies in Ox-LDL luminescence detection kits

[0107] ① Reagent preparation

[0108] R1 reagent: Aliquot 7 ml of Ox-LDL specific reagent 1 buffer.

[0109] R2 reagent: dilute the ApoBA-F6 antibody labeled with alkaline phosphatase to 1ug / ml with Ox-LDL special reagent 2 buffer, prepare 7mL each, mark and place on a vortex mixer, mix for no less than 60s, and set aside.

[0110] Magnetic bead reagent: Dilute the magnetic beads labeled with Ox-ApoB antibody (Biorbyt, orb330200) to 7 mL with Ox-LDL special reagent buffer 1, place on a vortex mixer after marking, mix for no less than 60 seconds, and set aside.

[0111] ② Computer test

[0112] Set up the instrument according to the "EXI1800 Fully Automatic Chemiluminescence Immunoassay Instrument Operation and Maintenance Instructions" and Ox-LDL project parameters, and load R1, R2, and magnetic bead reagents.

[0113] Test corporate reference products and clinical samples: Place Ox-ApoB corporate reference products and clinical samples into the EXI1800 (Zhongyuan Huiji Biotechnology Co., Ltd.) instrument sample tray, and apply for testing according to the "EXI1800 Fully Automatic Chemiluminescence Immunoanalyzer Use and Maintenance Operation Guide".

[0114] ③Data results

[0115] a. Calibration experiment

[0116] Table 3 Anti-ApoB AF calibration test data

[0117]

[0118] The calibration was completed by multiple dilution calibration. The results are shown in Table 3. The above 6 anti-ApoB AF antibodies were used as enzyme-labeled antibodies in anti-ApoB chemiluminescence detection, and all had good linear effects. The anti-ApoB-A calibration results met the requirements: the linear regression equation of the standard curve was R 2 ≥0.99 (if attached Figure 1 (shown)

[0119] b. Clinical testing

[0120] Clinical samples were collected for anti-ApoB-A antibody testing, and the clinical results are shown in Table 4 below:

[0121] Table 4. Detection data of positive and negative samples

[0122] sample Sample No. RLU mean concentration Positive dilution 2 times S1 19082275 143.45 Positive dilution 20 times S2 2499781 18.2 Positive dilution 200 times S3 282253 1.7 Total negative blood S4 2861 0

[0123] It can be seen from the test results that this strain of antibody can be effectively used in the quantitative detection of Ox-LDL projects and can be used for quantitative determination of positive samples.

[0124] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. An anti-ApoB antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises a complementarity determining region of any of the following groups: (1) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; (2) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 16, SEQ ID NO: 23 and SEQ ID NO: 18, respectively; (3) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 16, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; (4) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 19, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; (5) the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 18, respectively; (6) The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The complementarity determining regions are defined by any one system or a combination of multiple systems of Kabat, Chothia, IMGT, AbM or Contact, preferably by the Kabat system.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof further comprises framework regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; The HFR1 comprises SEQ ID NO: 3 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO:4 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO:5 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO:6 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 9 or an amino acid sequence having at least 80% identity thereto; The LFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto.

4. An anti-ApoB antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof further includes a heavy chain variable region and / or a light chain variable region, the amino acid sequence of the heavy chain variable region being as shown in any one of SEQ ID NO:1, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:33; the amino acid sequence of the light chain variable region being as shown in any one of SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:

34.

5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region is selected from any one of the heavy chain constant regions of IgG, IgA, IgM, IgE, and IgD, or a combination of multiple constant region segments; Preferably, the species of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans; Preferably, the heavy chain constant region comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto, and the light chain constant region comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto.

6. An antibody conjugate, characterized in that: The antibody conjugate comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; Preferably, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or antigen-binding fragment thereof; Preferably, the antibody conjugate further comprises a label coupled to the antibody or antigen-binding fragment thereof; Preferably, the marker is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle markers; Preferably, the antibody conjugate further comprises a solid phase carrier coupled to the antibody or antigen-binding fragment thereof; Preferably, the solid support is selected from at least one of microspheres, plates or membranes; Preferably, the solid phase carrier is selected from at least one of magnetic microspheres, plastic microspheres, plastic microparticles, microplates, glass, capillaries, nylon or nitrocellulose membranes.

7. A reagent or a kit, characterized in that: The reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or the antibody conjugate according to claim 6; Preferably, the method is selected from at least one of immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, and latex immunoturbidimetry.

8. A method for detecting anti-ApoB, characterized in that: include: a) contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 with anti-ApoB in a sample to be detected to form an immune complex under conditions sufficient for an antibody / antigen binding reaction to occur; and b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample; Preferably, the immune complex further comprises a second antibody, which binds to the antibody or antigen-binding fragment thereof; Preferably, the immune complex further comprises a second antibody, which binds to anti-ApoB.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody conjugate according to claim 6, or the reagent or kit according to claim 7 in the preparation of a product for detecting anti-ApoB.

10. A nucleic acid, a vector, a cell or a method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the nucleic acid encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; the vector contains a nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; the cell contains the above-mentioned nucleic acid or vector; and the method comprises the above-mentioned cell.

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