Anti-Ox-ApoB antibody or functional fragment thereof and application thereof

Abstract

The invention discloses an apolipoprotein B (ApoB) antibody related to oxidized low density lipoprotein (Ox-LDL) or a functional fragment of the apolipoprotein B antibody, and an application of the apolipoprotein B antibody and the functional fragment. The anti-Ox-ApoB antibody or the functional fragment thereof disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, and the antibody or the functional fragment thereof has relatively good specificity and sensitivity to Ox-ApoB, and can be used for detecting Ox-ApoB and / or Ox-LDL and used for auxiliary diagnosis of atherosclerosis by taking Ox-ApoB and / or Ox-LDL as a marker.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to anti-Ox-ApoB antibodies or their functional fragments and their applications. Background Technology

[0002] Oxidized low-density lipoprotein (ox-LDL) is a type of low-density lipoprotein (LDL) that has undergone oxidative modification in vivo. LDL is a lipid in the human body, whose main function is to carry cholesterol and lipids to various cells, supplying energy and building cell membranes. Due to the presence of abundant polyunsaturated fatty acids within LDL, malondialdehyde (MDA) is produced under the influence of excess free radicals and other oxygen-producing factors. MDA binds to the lysine residues of apolipoprotein B (ApoB) in LDL, resulting in a chemical modification product (Ox-ApoB), which is also an important detection target. When LDL is subjected to oxidative damage, the structure and function of apolipoprotein B change, forming oxidized low-density lipoprotein. Oxidized low-density lipoprotein plays a crucial role in the development of atherosclerosis, an arterial intima-media disease caused by lipid deposition and inflammatory responses in blood vessels. Oxidized low-density lipoprotein is considered a key pathological factor in this process. Therefore, the detection of oxidized low-density lipoprotein can serve as a specific diagnostic indicator for atherosclerotic cardiovascular disease and has an early warning effect on cardiovascular disease.

[0003] Common methods for detecting Ox-LDL include enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (chemiluminescence immunoassay). These methods all utilize an antigen-antibody sandwich approach to detect oxidized apolipoprotein B. One solution for Ox-LDL detection involves immobilizing anti-Ox-ApoB antibodies on a plate, incubating them with the sample, and then using HRP or other peroxidase-conjugated anti-ApoB antibodies to achieve a double-antibody sandwich detection of Ox-LDL levels. Currently, the sources of antibodies for detecting Ox-ApoB are limited, and most detection kits are imported. However, my country has a significant demand for early warning detection of cardiovascular and cerebrovascular diseases. Therefore, preparing antibodies against Ox-ApoB can effectively improve domestic detection capabilities for this biomarker. Summary of the Invention

[0004] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0005] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0006] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The methods and materials described herein, and any similar or equivalent methods and materials, may be used in the practice or testing of formulations or unit doses described herein. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0007] As used herein, the terms “comprising,” “including,” “having,” “may,” and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures.

[0008] The purpose of this invention is to provide an antibody against Ox-ApoB or a functional fragment thereof, and a reagent for detecting Ox-LDL and / or Ox-ApoB. The antibody or functional fragment thereof has good affinity for Ox-ApoB, and the detection of Ox-LDL or Ox-ApoB using the antibody or functional fragment thereof has good sensitivity and specificity.

[0009] Specifically as follows:

[0010] On one hand, the present invention provides an anti-Ox-ApoB antibody or a functional fragment thereof, wherein the anti-Ox-ApoB antibody or the functional fragment thereof has the following complementarity-determining region:

[0011] CDR-VH1: GYT-X1-TE-X2-T, where X1 is P or F, and X2 is F or Y;

[0012] CDR-VH2: IN-X1-D-X2-GGI, where X1 is P or F, and X2 is N or Q;

[0013] CDR-VH3: T-X1-KG-X2-A, where X1 is R or A, and X2 is W or D;

[0014] CDR-VL1: ES-X1-Q-X2-YG-X2-SL, where X1 is L or V, X2 is F or Y, and X3 is S or T;

[0015] CDR-VL2: A-X1-S, where X1 is G or A;

[0016] CDR-VL3: QQS-X1-KV-X2-T, where X1 is H or R, and X2 is Y or W.

[0017] Furthermore, in the complementary determination region, X2 of CDR-VH1 is Y, X2 of CDR-VH2 is N, X1 of CDR-VH3 is R, X1 of CDR-VL1 is V, X1 of CDR-VL2 is A, and X2 of CDR-VL3 is W.

[0018] It should be noted that CDR-VH1, CDR-VH2, and CDR-VH3 are amino acid sequences identical to those of CDR-VH1, CDR-VH2, and CDR-VH3 in the same heavy chain variable region defined in the antibody or its antigen-binding fragment described in the first aspect, and CDR-VL1, CDR-VL2, and CDR-VL3 are amino acid sequences identical to those of CDR-VL1, CDR-VL2, and CDR-VL3 in the same light chain variable region defined in the antibody or its antigen-binding fragment described in the first aspect.

[0019] In this invention, the term "antibody" is used in the broadest sense, and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity.

[0020] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0021] In this invention, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of immunoglobulins, specifically regions containing one or more, or even all, of the major amino acid residues that contribute to the binding of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In specific embodiments of this invention, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

[0022] In this article, "CDR" refers to the "complementarity-determining region" within the variable sequence of the antibody. Each of the heavy and light chains has three CDRs, starting from the N-terminus of either the heavy or light chain.

[0023] The antigen-binding site may include six CDRs (CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 in this invention). A polypeptide containing a single CDR (e.g., CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, or CDR-VL3) can be termed a "molecular recognition unit." Crystallographic analysis of antigen-antibody complexes has demonstrated that the amino acid residues of the CDR form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Therefore, the molecular recognition unit may primarily be responsible for the specificity of the antigen-binding site; generally, CDR residues directly and substantially participate in influencing antigen binding.

[0024] The methods for defining CDRs are well-known in the art and include: the Kabat definition, the Chothia definition, the IMGT definition, the Contact definition, and the AbM definition. As described herein, the “Kabat definition” refers to the definition system described by Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). For the “Chothia definition,” see Chothia et al., J Mol Biol 196:901-917 (1987). Other CDR definition methods may not strictly follow one of the above schemes but will still overlap with at least a portion of the CDR region defined by Kabat, although they may shorten or lengthen them based on predictions or experimental results for specific residues or residue groups.

[0025] According to an embodiment of the present invention, the CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2 or CDR-VL3 is defined by any one or a combination of systems such as Kabat, Chothia, IMGT, AbM or Contact, preferably defined by the Kabat system.

[0026] In an optional implementation, X1 in CDR-VH1 is P;

[0027] In an optional implementation, X2 in CDR-VH1 is F;

[0028] In an optional implementation, X2 in CDR-VH1 is Y;

[0029] In an optional implementation, X1 in CDR-VH2 is P;

[0030] In an optional implementation, X1 in CDR-VH2 is F;

[0031] In an optional implementation, X2 in CDR-VH2 is N;

[0032] In an optional implementation, X2 in CDR-VH2 is Q;

[0033] In an optional implementation, X1 in CDR-VH3 is R;

[0034] In an optional implementation, X1 in CDR-VH3 is A;

[0035] In an optional implementation, X2 in CDR-VH3 is W;

[0036] In an optional implementation, X2 in CDR-VH3 is D;

[0037] In an optional implementation, X1 in CDR-VL1 is L;

[0038] In an optional implementation, X1 in CDR-VL1 is V;

[0039] In an optional implementation, X2 in CDR-VL1 is F;

[0040] In an optional implementation, X2 in CDR-VL1 is Y;

[0041] In an optional implementation, X1 in CDR-VL2 is G;

[0042] In an optional implementation, X1 in CDR-VL2 is A;

[0043] In an optional implementation, X1 in CDR-VL3 is H;

[0044] In an optional implementation, X1 in CDR-VL3 is R;

[0045] In an optional implementation, X2 in CDR-VL3 is Y;

[0046] In an optional implementation, X2 in the CDR-VL3 is W.

[0047] In an optional implementation, each complementary determination region is selected from any of the following combinations of mutations:

[0048]

[0049]

[0050] In this invention, the "frame region" or "FR" region includes the heavy chain frame region and the light chain frame region, referring to the regions in the antibody heavy chain variable region and light chain variable region other than the CDR; wherein, the heavy chain frame region can be further subdivided into adjacent regions separated by the CDR, including the HFR1, HFR2, HFR3 and HFR4 frame regions; the light chain frame region can be further subdivided into adjacent regions separated by the CDR, including the LFR1, LFR2, LFR3 and LFR4 frame regions.

[0051] In some specific embodiments, the antibody or its antigen-binding fragment described in this invention further comprises frame regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4;

[0052] The HFR1 includes SEQ ID NO:1 or an amino acid sequence that has at least 80% identity with it;

[0053] The HFR2 comprises SEQ ID NO:2 or an amino acid sequence having at least 80% identity with it;

[0054] The HFR3 includes SEQ ID NO:3 or an amino acid sequence that has at least 80% identity with it;

[0055] The HFR4 comprises SEQ ID NO:4 or an amino acid sequence having at least 80% identity with it;

[0056] The LFR1 includes SEQ ID NO:5 or an amino acid sequence that has at least 80% identity with it;

[0057] The LFR2 comprises SEQ ID NO:6 or an amino acid sequence having at least 80% identity with it;

[0058] The LFR3 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it;

[0059] The LFR4 includes SEQ ID NO:8 or an amino acid sequence that is at least 80% identical to it.

[0060] It should be noted that, in other embodiments, the amino acid sequences of each frame region of the Ox-ApoB antibody or its antigen-binding fragment provided by the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding frame regions (SEQ ID NO: 1-8) described above.

[0061] Furthermore, the antibody also contains a constant region.

[0062] In an optional implementation, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0063] In an optional implementation, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

[0064] In an optional embodiment, the functional fragment is selected from any one of the antibody’s VHH, F(ab')2, Fab', Fab, Fv and scFv.

[0065] Furthermore, the antibody is a coated antibody.

[0066] The "coated antibody" described in this invention is a coating material capable of capturing antigens.

[0067] The term "antibody" includes various forms of antibody structures, including but not limited to complete antibodies and antibody fragments. Antibodies according to the invention are preferably goat, sheep, mouse, rabbit, or rat antibodies, chimeric antibodies, or further genetically engineered antibodies, provided they retain the characteristic properties according to the invention. An "antibody fragment" comprises a portion of a full-length antibody, preferably its variable domains, or at least its antigen-binding site. Examples of antibody fragments include biantibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)2; biantibodies; and multispecific antibodies formed from antibody fragments.

[0068] In optional embodiments, the coating carrier for the antibody includes, but is not limited to, nitrocellulose membranes, ELISA plates, magnetic beads, latex microspheres, etc.

[0069] In optional embodiments, the magnetic beads include, but are not limited to, carboxyl magnetic beads, methylpropionamide magnetic beads, streptavidin magnetic beads, etc.

[0070] In optional embodiments, the latex microspheres are often polyvinyl alcohol toluene, polystyrene, or microspheres formed by co-polymerization of these two substances as the main components and the addition of other monomers.

[0071] In optional embodiments, the latex microspheres include, but are not limited to, carboxyl latex microspheres, amino latex microspheres, etc.

[0072] On the other hand, the present invention discloses a vector containing a nucleic acid fragment encoding the above-mentioned antibody or its functional fragment.

[0073] On the other hand, the present invention discloses a recombinant cell containing the above-mentioned carrier.

[0074] On the other hand, the present invention discloses a detection reagent, which includes the above-mentioned antibody or its functional fragment.

[0075] In an optional implementation, the detection reagent is used to detect Ox-LDL and / or Ox-ApoB.

[0076] In an optional embodiment, the present invention discloses the application of the above-mentioned antibody and its antigen-binding fragment in an Ox-LDL luminescent detection kit.

[0077] Beneficial effects: Currently, there are few Ox-ApoB related antibody raw materials on the market, and their performance varies. This invention not only provides a monoclonal antibody (wild type) with high affinity for Ox-ApoB, but also performs various high-performance mutations on this wild-type antibody, which has a wider range of applications and higher applicability. It can be used in Ox-ApoB detection kits, providing multiple references for immune detection reactions. Attached Figure Description

[0078] Figure 1 Correlation curve between calibration sample and light value Detailed Implementation

[0079] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0081] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligo Nucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0082] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0083] Example 1: Preparation of Monoclonal Antibodies

[0084] 1. Mouse immunization and antibody detection

[0085] Five 6-8 week old SPF-grade female BALB / c mice were selected. Freund's complete adjuvant and Ox-ApoB protein at a concentration of 2 mg / ml were mixed in equal volumes and emulsified. The emulsified antigen was used to immunize the 6-8 week old SPF-grade female BALB / c mice, with each mouse receiving 50 μg of antigen protein via paw injection or subcutaneous injection in the back. Two weeks after the initial immunization, the antigen protein was mixed with Freund's incomplete adjuvant and emulsified, and each mouse was again injected with 50 μg of antigen protein via paw injection or subcutaneous injection in the back. Two weeks later, blood was collected via tail vein, the supernatant was collected by centrifugation, and serum titer was measured by ELISA. Immunization was repeated every two weeks, and serum titer was measured again. After two immunizations, the serum titer reached over 2.0 after a million-fold dilution. Mice with a serum titer of 10⁶ or higher were selected, and lymphocytes were isolated from lymphocytes for cell fusion.

[0086] 2. Cell fusion and screening of positive hybridoma cells and subcloning

[0087] Lymphocytes from immunized mice were isolated and fused with cultured SP2 / 0 cells via PEG1500-mediated fusion or electrofusion. The fused cells were cultured in HAT-1640 medium containing 20% ​​FBS serum for selection. After one week, the medium was changed, and after another four days of culture, the culture supernatant was used for positive clone selection. Ox-ApoB protein was used for screening positive wells. Wells with a high ELISA positive value to cell number ratio were selected for multiple subcloning. ELISA plates were coated with Ox-ApoB protein. The culture supernatant of the subclones was used to screen for monoclonal clones that showed affinity under antigen-coated conditions. The monoclonal hybridoma cell line with the highest affinity was selected, ultimately yielding a hybridoma cell line with a high antibody titer that secretes Ox-ApoB monoclonal antibody, named #29, which exhibited good stability.

[0088] 3. Production and purification of monoclonal antibodies

[0089] Two groups of 6-8 week old BALB / c mice were selected and injected intraperitoneally with 500 μL of paraffin oil to suppress the immune response. One week after injection, 0.5 ml of Cell 29# was injected intraperitoneally into the mice, with a cell count of approximately 1 × 10⁻⁶. 6 Ascites fluid collection began two weeks later. The collected ascites fluid was precipitated with ammonium sulfate and purified by affinity chromatography of protein A to obtain the target antibody 29#.

[0090] 4. Identification of monoclonal antibody subtypes and cloning of gene sequences

[0091] The heavy and light chain isotypes of monoclonal antibodies were identified using the Southern Biothech SBACOLOnotyping System-HRP kit, following the manufacturer's instructions. The specific procedures were as follows:

[0092] a. Dilute Ox-ApoB antigen to 1 μg / mL with coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), add 100 μL / well to the microplate, and coat overnight at 4°C. Wash the plate three times with PBS buffer containing 0.05% Tween-20 (wash buffer).

[0093] b. Dilute the culture supernatant of the hybridoma cells to be tested 1:1 with diluent (1% BSA, 0.1% PBST), 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, lambda-HRP) 1:3000 with diluent.

[0094] c. After washing the plate three times with washing buffer, add 100 μL of diluted enzyme-labeled antibody to each well and incubate at 37°C for 30 minutes. After washing the plate three more times, add the chromogenic buffer and incubate for approximately 5 minutes (depending on the reaction strength). Then, add 2M sulfuric acid to stop the reaction and read the OD450 absorbance. Identification showed that the heavy chain subtype of antibody 57# was IgG2a, and the light chain subtype was Kappa.

[0095] Based on the antibody subtype results, the antibody gene sequence was cloned using a RACE-based method. Hybridoma cells in good growth condition were collected, and total RNA was obtained from the hybridoma cells using a total RNA extraction kit. The mRNA was reverse transcribed into cDNA according to the Takara SMARTer RACE instruction manual, and the full-length sequence of the target antibody was amplified.

[0096] 5. In vitro expression of antibodies

[0097] Based on the full-length antibody sequence, it was synthesized into the pcDNA3.1(+) vector according to the heavy and light chains, and then transfected into HEK293 for expression verification. The fermentation supernatant was purified by proteinG to obtain antibody #29.

[0098] Example 2: Validation of antibody performance

[0099] 1. ELISA Affinity Test

[0100] Goat anti-mouse antibody was diluted to 1 μg / mL using coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), and 100 μL was added to each well of a 96-well microplate. The plate was incubated overnight at 4°C. The plate was washed three times with PBS buffer containing 0.05% Tween-20. Ox-ApoB antibody #29 from Example 1 was diluted to 1 μg / mL using dilution buffer (1% BSA, 0.1% PBST). 100 μL was added to each well of row A of a 96-well microplate, followed by 50 μL to row B, 25 μL to row C, 12.5 μL to row D, 6.25 μL to row E, and 3.125 μL to row F. The plate was incubated at 37°C for 30 minutes, and then washed three more times. The standard Ox-ApoB protein was diluted to 1000 ng / mL with diluent. The ELISA plate, incubated at 37°C, was washed three times. Then, 100 μL of horseradish peroxidase (HRP)-labeled ApoB antibody (1 μg / mL) diluted 1:5000 was added to the 96-well ELISA plate, and the plate was incubated at 37°C for 30 minutes. The plate was washed three times, and TMB chromogenic reagent was added. The plate was incubated at room temperature for 3 minutes, and the reaction was terminated by adding 0.5 M sulfuric acid. The OD450 value was read using a plate reader. The results are shown in Table 1.

[0101]

[0102] The heavy chain variable region of anti-Ox-ApoB antibody #29 in Example 1 is shown in SEQ ID NO:9. The complementarity-determining regions on the heavy chain variable region have the following amino acid sequences defined by the Kabat system:

[0103] CDR-VH1: GYTF(X1)-TEY(X2)-T

[0104] CDR-VH2: INP(X1)-DN(X2)-GGI

[0105] CDR-VH3: TR(X1)-KGD(X2)-A

[0106] Its light chain variable region is shown in SEQ ID NO:10, wherein the complementarity-determining regions on the light chain variable region are defined by the Kabat system as follows:

[0107] CDR-VL1:ESV(X1)-QY(X2)-YG-X2-SL

[0108] CDR-VL2: AA(X1)-S

[0109] CDR-VL3: QQSR(X1)-KVW(X2)-T

[0110] Based on antibody #29 against Ox-ApoB, mutations were made at sites related to antibody activity in the complementarity-determining region, where X1 and X2 are mutation sites.

[0111] Table 1 Mutation sites related to antibody activity

[0112]

[0113] Table 2 Antibody activity analysis data

[0114] Antibody dilution (ng / mL) WT Mutation 1 Mutation 2 Mutation 3 Mutation 4 Mutation 5 1000.0000 2.061 2.014 2.413 1.586 1.903 1.935 500.0000 1.919 2.008 2.247 1.373 1.747 1.787 250.0000 1.566 1.313 1.754 0.966 1.454 1.302 125.0000 1.114 1.006 1.366 0.514 1.086 1.117 62.5000 0.816 0.845 1.177 0.344 0.767 0.611 31.2500 0.409 0.473 0.763 0.220 0.361 0.334 15.6250 0.202 0.155 0.398 0.144 0.188 0.272 0 0.055 0.063 0.541 0.047 0.024 0.069

[0115] As can be seen from the table above, mutation 2 exhibits the best activity. Therefore, using mutation 2 as the backbone sequence, other mutation sites with good affinity were screened, and some results are shown below:

[0116] Table 3 Mutation sites related to antibody affinity

[0117]

[0118]

[0119]

[0120] Ox-ApoB protein antigen 1000 ng / ml was used, and the absorbance at OD450 of each mutation was measured. It can be seen that the affinity of each mutation is high. The results are as follows:

[0121] Table 4. Affinity test for antibody mutations

[0122] Mutation 2-1 2.2783 Mutation 2-2 2.321 Mutations 2-4 2.385 Mutations 2-4 2.111 Mutations 2-5 2.301 Mutations 2-6 2.361 Mutations 2-7 2.338 Mutations 2-8 2.241 Mutations 2-9 2.231 Mutation 2-10 2.376 Mutation 2-11 2.258 Mutation 2-12 2.233 Mutation 2-13 2.309 Mutation 2-14 2.451 Mutation 2-15 2.404 Mutation 2-16 2.342 Mutation 2-17 2.340 Mutation 2-18 2.469 Mutation 2-19 2.277 Mutation 2-20 2.041 Mutation 2-21 2.140 Mutation 2-22 2.269 Mutation 2-23 2.178 Mutation 2-24 2.411 Mutation 2-25 2.241 Mutation 2-26 2.208 Mutation 2-27 2.207 Mutation 2-28 2.011

[0123] 2. Stability testing

[0124] The antibody was prepared in a predetermined buffer (PBS, 0.05% ProClin). TM The antibodies were accelerated at 37°C for 7 and 14 days (300), and the accelerated antibody was evaluated by ELISA, with a control at 4°C, to determine the long-term stability of the antibody. Additionally, the antibody underwent five freeze-thaw cycles at -20°C, and the results were displayed as the deviation between the values ​​at 4°C and the accelerated values. Mutants 2 to 2-28 were stable at 4°C and remained stable after acceleration at 37°C for 7 and 14 days (a slight decrease in reactivity did not affect reagent performance), ensuring the performance of the reagents after opening and thus guaranteeing the accuracy and stability of the detection results. Specific measurement results are as follows:

[0125] Table 5 Stability Study

[0126]

[0127]

[0128] Example 3: Antibody Application Research

[0129] 1. Application of antibodies in Ox-LDL luminescent detection kit

[0130] (1) Reagent preparation

[0131] Reagent R1: 20 mM PB buffer solution (pH 7.5-9.0).

[0132] R2 reagent: Use a dedicated buffer: 20mM-50Tris buffered physiological saline (TBS, pH 6.0-8.0), 150mM sodium chloride solution. Dilute the ApoB antibody labeled with alkaline phosphatase (Merck & Co., Inc., catalog number: 12352203) to 1ug / ml, prepare 7mL, label it, and place it on a vortex mixer. Mix for at least 60s and set aside.

[0133] Magnetic bead reagent: Dilute the magnetic beads labeled with Ox-ApoB antibody (antibody of mutant 2-7 in Example 2) to 7 mL with 20 mM PB buffer solution (pH 7.5-9.0), label them, place them on a vortex mixer, and mix for at least 60 seconds.

[0134] (2) On-machine test

[0135] Set the instrument according to the "EXI1800 Fully Automated Chemiluminescence Immunoassay Analyzer Operation and Maintenance Manual" and the Ox-LDL project parameters, and load R1, R2, and magnetic bead reagents.

[0136] Test the enterprise reference product and clinical samples: Place the Ox-ApoB enterprise reference product and clinical samples into the sample tray of the EXI1800 (Zhongyuan Huiji Biotechnology Co., Ltd.) instrument, and apply for testing according to the "EXI1800 Fully Automated Chemiluminescence Immunoassay Analyzer Operation and Maintenance Manual".

[0137] (3) Data Results

[0138] a. Calibration experiment

[0139] Table 6 Ox-ApoB Calibration Test Data

[0140]

[0141]

[0142] Calibration was performed using serial dilution calibration, and the results are shown in the table above. The calibration results meet the requirements: the linear regression equation of the standard curve R0... 2≥0.99 (as attached) Figure 1 (As shown).

[0143] b. Clinical testing

[0144] Table 7. Detection data for positive and negative samples

[0145] sample Sample number RLU mean concentration Positive dilution 2 times S1 19082275 143.45 Positive dilution 20-fold S2 2499781 18.2 Positive dilution 200 times S3 282253 1.7 All negative blood S4 2861 0

[0146] The experimental results show that this antibody can be effectively used in the quantitative detection of Ox-LDL and to quantitatively determine positive samples.

[0147] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. An anti-Ox-ApoB antibody or a functional fragment thereof, characterized in that, The antibody or its functional fragment includes the following complementarity-determining regions: CDR-VH1: GYT-X1-TE-X2-T, where X1 is P or F, and X2 is F or Y; CDR-VH2: IN-X1-D-X2-GGI, where X1 is P or F, and X2 is N or Q; CDR-VH3: T-X1-KG-X2-A, where X1 is R or A, and X2 is W or D; CDR-VL1: ES-X1-Q-X2-YG-X3-SL, where X1 is L or V, X2 is F or Y, and X3 is S or T; CDR-VL2: A-X1-S, where X1 is G or A; CDR-VL3: QQS-X1-KV-X2-T, where X1 is H or R, and X2 is Y or W.

2. The anti-Ox-ApoB antibody or its functional fragment as described in claim 1, characterized in that, The complementary determination region has X2 of Y for CDR-VH1, X2 of N for CDR-VH2, X1 of R for CDR-VH3, X1 of V for CDR-VL1, X1 of A for CDR-VL2, and X2 of W for CDR-VL3.

3. The anti-Ox-ApoB antibody or its functional fragment as described in claim 1, characterized in that, In the CDR-VH1, X1 is F; Preferably, in the CDR-VH1, X1 is P; Preferably, X2 in CDR-VH1 is F; Preferably, in the CDR-VH1, X2 is Y; Preferably, in the CDR-VH2, X1 is P; Preferably, in the CDR-VH2, X1 is F; Preferably, in the CDR-VH2, X2 is N; Preferably, X2 in CDR-VH2 is Q; Preferably, in the CDR-VH3, X1 is R; Preferably, in the CDR-VH3, X1 is A; Preferably, in the CDR-VH3, X2 is W; Preferably, in the CDR-VH3, X2 is D; Preferably, in the CDR-VL1, X1 is L; Preferably, in the CDR-VL1, X1 is V; Preferably, X2 in the CDR-VL1 is F; Preferably, in the CDR-VL1, X2 is Y; Preferably, in the CDR-VL2, X1 is G; Preferably, in the CDR-VL2, X1 is A; Preferably, X1 in the CDR-VL3 is H; Preferably, in the CDR-VL3, X1 is R; Preferably, in the CDR-VL3, X2 is Y; Preferably, X2 in the CDR-VL3 is W.

4. The anti-Ox-ApoB antibody or its functional fragment as described in any one of claims 1-3, characterized in that, Each complementarity-determining region is selected from any of the following combinations of mutations:

5. The anti-Ox-ApoB antibody or its functional fragment as described in any one of claims 1-4, characterized in that, The antibody or its antigen-binding fragment further includes the framework regions HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4; The HFR1 includes SEQ ID NO:1 or an amino acid sequence having at least 80% identity with it; The HFR2 comprises SEQ ID NO:2 or an amino acid sequence having at least 80% identity with it; The HFR3 includes SEQ ID NO:3 or an amino acid sequence that has at least 80% identity with it; The HFR4 comprises SEQ ID NO:4 or an amino acid sequence having at least 80% identity with it; The LFR1 includes SEQ ID NO:5 or an amino acid sequence that has at least 80% identity with it; The LFR2 comprises SEQ ID NO:6 or an amino acid sequence having at least 80% identity with it; The LFR3 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity with it; The LFR4 includes SEQ ID NO:8 or an amino acid sequence that is at least 80% identical to it.

6. The anti-Ox-ApoB antibody or its functional fragment as described in claim 5, characterized in that, The antibody also includes a constant region; Preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Preferably, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, or humans.

7. The anti-Ox-ApoB antibody or its functional fragment as described in claim 6, characterized in that, The antibody is a coated antibody; Preferably, the coating carrier for the coated antibody is selected from at least one of nitrocellulose membrane, ELISA plate, magnetic beads, and latex microspheres; Preferably, the magnetic beads are selected from at least one of carboxyl magnetic beads, methacrylamide magnetic beads, and streptavidin magnetic beads; Preferably, the latex microspheres are selected from at least one of polyvinyl alcohol toluene microspheres, polystyrene microspheres, or microspheres polymerized with polyvinyl alcohol toluene and polystyrene as the main components; Preferably, the latex microspheres are selected from at least one of carboxyl latex microspheres and amino latex microspheres.

8. A carrier, characterized in that, It contains a nucleic acid fragment encoding an antibody or a functional fragment thereof as described in any one of claims 1-7.

9. A recombinant cell, characterized in that, It contains a carrier, which is the carrier described in claim 8.

10. A detection reagent, characterized in that, Includes the antibody or its functional fragment as described in any one of claims 1-7; preferably, the reagent is used to detect Ox-ApoB and / or Ox-LDL.

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