Anti-ApoE4 antibody or functional fragment thereof and application thereof

Abstract

The invention discloses an antibody for resisting apolipoprotein E4 (ApoE4) and application of the antibody, and relates to the field of antibodies. The anti-apolipoprotein E4 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 apolipoprotein E4, 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-ApoE4 antibody and application thereof. Background Art

[0002] Apolipoprotein E (ApoE) is an important apolipoprotein in plasma. It participates in lipid metabolism and maintains cholesterol balance. It also plays an important role in the normal growth and development of the nervous system and repair after injury. In addition, ApoE also plays a role in pathological processes such as the deposition and clearance of amyloid proteins, immune regulation, and inhibition of tumor cell proliferation.

[0003] ApoE has three different isoforms, namely ApoE2, ApoE3 and ApoE4, which are encoded by the ε2, ε3 and ε4 alleles of the ApoE gene. Among them, ApoE4 is particularly noteworthy. It is one of the most important genetic risk factors for Alzheimer's disease. It is the risk gene with the strongest association with the onset and prognosis of AD known, and is associated with a higher risk of disease and poorer cognitive function prognosis. Compared with other alleles, people carrying one or two ApoE4 alleles have a higher risk of developing Alzheimer's disease and may develop the disease at an earlier age.

[0004] In clinical practice, ApoE4 genotype testing can be used for research purposes or as an auxiliary diagnostic tool, especially when a doctor suspects that a patient may have early-onset dementia with genetic factors involved. However, the presence of ApoE4 is not a diagnostic criterion for Alzheimer's disease, as there are many other factors that affect the development of the disease, including but not limited to age, lifestyle, and other genetic factors.

[0005] Studies have shown that ApoE4 may affect the development of Alzheimer's disease through multiple mechanisms, including interfering with communication between neurons, promoting the accumulation of beta-amyloid protein, disrupting the function of the blood-brain barrier, and affecting cholesterol metabolism in the brain. All of these factors may lead to neurodegenerative changes and may ultimately lead to the manifestation of Alzheimer's disease symptoms.

[0006] There are many methods for detecting ApoE4, mainly based on the sandwich method of antigen-antibody reaction. This method has the advantages of low cost, simple operation, and suitability for large-scale screening, and is currently the most commonly used detection method. However, in actual testing, the specificity of the currently known ApoE4-specific antibodies is generally low and cannot meet the needs of accurate diagnosis. In clinical testing, chemiluminescent immunoassays and immunochromatographic assays are the mainstream methodologies, and these methods are optimized and upgraded based on the original double antibody sandwich method. Summary of the invention

[0007] In a first aspect, an embodiment of the present invention provides an anti-ApoE4 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: (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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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 Pept. 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.

[0013] 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.

[0014] 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.

[0015] In the present invention, the heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: 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 combinations: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0016] 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; 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.

[0017] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the anti-ApoE4 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: 3-10).

[0018] In a second aspect, an embodiment of the present invention provides an anti-ApoE4 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; 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.

[0019] 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.

[0020] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0021] 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.

[0022] 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.

[0023] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0024] In an alternative embodiment, the light chain constant region is selected from a kappa-type or a lambda-type light chain constant region.

[0025] 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.

[0026] In an alternative embodiment, the species origin of the constant region is mouse.

[0027] 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.

[0028] 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).

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody or antigen-binding fragment thereof.

[0035] 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.

[0036] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.

[0037] 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.

[0038] 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.).

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0044] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0045] In an optional embodiment, the colloidal metal is colloidal gold.

[0046] In an optional embodiment, the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody or antigen-binding fragment thereof.

[0047] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0048] 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.

[0049] 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.

[0050] As mentioned above, the antibodies or antigen-binding fragments thereof in some embodiments or examples of the present invention can effectively bind to ApoE4, and therefore, the reagents or kits containing the ApoE4 antibodies or antigen-binding fragments thereof can effectively detect ApoE4 qualitatively or quantitatively. The reagents or kits provided by the present invention can be used, for example, for detections involving the use of the specific binding properties of ApoE4 and its antibodies, such as 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 a higher binding activity or affinity with ApoE4, and therefore, the reagents or kits containing the antibodies or antigen-binding fragments thereof have a higher detection sensitivity or specificity.

[0051] In a fifth aspect, the present invention provides a method for detecting ApoE4, comprising: a) contacting the above-mentioned antibody or antigen-binding fragment thereof, antibody conjugate, reagent or kit with ApoE4 in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; 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; In an alternative embodiment, the immune complex further comprises a second antibody that binds to the antibody or antigen-binding fragment thereof.

[0052] In an alternative embodiment, the immune complex further comprises a second antibody that binds to ApoE4.

[0053] In a sixth aspect, the present invention provides use of the above-mentioned anti-ApoE4 antibody or antigen-binding fragment thereof, antibody conjugate, or the above-mentioned reagent or kit in the preparation of a product for detecting ApoE4.

[0054] In a seventh aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0055] In an eighth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0056] In a ninth aspect, the present invention provides a cell containing the above-mentioned vector.

[0057] In a tenth aspect, the present invention provides a method for preparing an anti-ApoE4 antibody or an antigen-binding fragment thereof, comprising: culturing the cells as described above.

[0058] Based on the amino acid sequence of the anti-ApoE4 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-ApoE4 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-ApoE4 antibody or its antigen-binding fragment of the present invention, it belongs to the protection scope of the present invention.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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. Specific embodiments

[0066] Example 1 Preparation of ApoE4 Antibody

[0067] (1) Mouse immunization and antibody detection Five 6-8 week old SPF female BALB / c mice were selected, and Freund's complete adjuvant and ApoE4 protein (purchased from LEEbio) at a concentration of 1 mg / ml were mixed and emulsified in equal volumes. 6-8 week old SPF female BALB / c mice were immunized with the emulsified antigen, and 40 μg of antigen protein was injected into each mouse through the sole of the foot. Two weeks after the first immunization, the antigen protein was mixed and emulsified with Freund's incomplete adjuvant, and 40 μg of antigen protein was injected into each mouse through the sole of the foot or subcutaneous injection on the back. Two weeks later, blood was collected through the tail vein, the supernatant was collected by centrifugation, and the serum titer was tested by ELISA. Immunization was performed every two weeks and the serum titer was tested. After two immunizations, the serum titer was as high as 2.0 or above after a million-fold dilution. The screening serum titer was 10 6 Lymph was taken from the above mice to separate lymphocytes for cell fusion.

[0068] (2) Hybridoma antibody screening The antibody screening scheme is the key to successfully developing the antibodies of the present invention. In the antibody screening stage, the ELISA plate is first coated with ApoE4 protein, and after the potential target antibody binds to the ApoE4 protein, a sheep anti-mouse-HRP is used in combination with an ELISA colorimetric solution to develop the color and screen out highly reactive hybridoma clones.

[0069] The target clone obtained by screening has good reactivity to ApoE4 protein. The present invention screens ApoE4-A cell strain for antibody A preparation.

[0070] (3) Production and purification of monoclonal antibodies 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 ApoE4-A hybridoma cells were injected intraperitoneally into one group of mice. The number of cells was about 1×10 6 After two weeks, the ascites was collected. The collected ascites was precipitated with ammonium sulfate and purified with protein G affinity to obtain the target antibody.

[0071] (4) Monoclonal antibody subtype identification and gene sequence cloning 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: a. Dilute the capture antibody to 1 μg / mL with coating solution (0.05 M carbonate and bicarbonate buffer at pH 9.5), 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; 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; After washing the plate three times with the plate washing solution, 100 μL of diluted enzyme-labeled antibody was added to each well and incubated at 37°C for 30 minutes. After washing the plate three times again, the color developing solution was added. After about 5 minutes (depending on the strength of the reaction), 2 M sulfuric acid was added to terminate the reaction and the OD450 absorbance was read. After identification, the heavy chain subtype of the ApoE4-A antibody was IgG1 and the light chain was 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 the SMARTer RACE manual of Takara, the mRNA was reversely transcribed into cDNA, and the full-length sequence of the target antibody was amplified.

[0072] The obtained antibody was named ApoE4-A. ApoE4-A was mutated and 5 mutant antibodies with equivalent performance were screened. The antibody sequences are shown in Table 1 below (wherein CDR is defined by the Kabat system):

[0073] Example 2 Performance testing of antibodies

[0074] ApoE4 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, wash twice with PBST and pat dry; add blocking solution (20% BSA + 80% PBS), 120 μL per well, 37°C, 1h, pat dry; add the above antibodies, starting from 1000 ng / ml, and dilute 3 times, load, 100 μL / well, 37°C, 30min (part of the supernatant 1h ); wash 5 times with PBST washing solution, pat dry; add horseradish peroxidase labeled sheep anti-mouse IgG, 100 μL per well, 37°C, 30 min; wash 5 times with PBST washing solution, pat dry; add urea peroxide (50 μL / well), add tetramethylbenzidine (50 μL / well), 10 min; add dilute hydrochloric acid to terminate the reaction, 50 μL / well; read the OD value at 450nm (reference 630nm) on the microplate reader; the above antibody reactivity identification is shown in Table 2 below:

[0075] It can be seen from the experimental results that the above 6 strains of anti-ApoE4 antibodies all have good reactivity.

[0076] 2. Functional testing A. Biotinylated antibody ApoE4 AF Weigh biotin (NHS-LC-LC-Biotin, thermo scientific, 21343), dissolve biotin into 5.677 mg / mL with DMSO, take 0.3 mg ApoE4 AF, a total of 6 antibodies, the volume of biotin solution required for each 1 mg antibody is 3.3 uL, add biotin to the 6 antibodies respectively and mix well, connect the antibody and biotin, react at 25°C in the dark for 4 hours, and prepare biotin-labeled ApoE4-A / B / C / D / E / F antibodies.

[0077] B. Alkaline phosphatase labeled antibody 0.3 mg of ApoE4 A-F6 strain antibody was replaced in TSE (pH 8.5) buffer, and 0.66 mg of ALP (from Sigma, ALPI12G) was replaced in ALP dialysis buffer (pH 7.6) solution, mixed and measured the concentration. The weighed Traut's Reagent 2-Iminothiolane·HCl (2-IT, Thermo scientific, 26101) was dissolved to 13.76 mg / mL in TSE (pH 8.5) solution to prepare activator 1, and the weighed Sulfo-SMCC (Thermoscientific, PG82085) was dissolved to 3.7 mg / mL in purified water to prepare activator 2. The two activator solutions were prepared and used immediately, and used within 10 minutes after preparation. The volume of 2-IT solution required for each 1 mg of antibody is 5uL. Add 2-IT solution to the antibody, mix well, and place it at (25±2)℃ for 20 minutes. This operation is antibody activation. After activation, replace the activated antibody in TSE (pH7.3) solution and measure the antibody concentration. At the same time, calculate the required volume of Sulfo-SMCC solution based on the volume of SMCC solution required for each 1mg ALP as 10uL. Add Sulfo-SMCC solution to the desalted ALP, mix well, and place it at (25±2)℃ for 20 minutes. After activation, replace the activated ALP in TSMZ (pH7.3) solution and measure the ALP concentration. Dilute the desalted antibody with TSE (pH7.3) solution to 0.3 mg / mL, and dilute the desalted ALP with TSMZ (pH7.3) solution to 0.4 mg / mL. Calculate the antibody volume and ALP volume for connection based on the mass ratio of antibody to ALP of 1:0.91. Mix the antibody and ALP according to the calculated volume, and place at 2~8℃ for 12-20h. Dissolve maleimide in DMSO to 9.7mg / mL, and dilute it 10 times with TSMZ (pH7.3) to obtain the stop solution. Add 20uL of the stop solution required for 1mL of antibody-ALP conjugate to terminate the reaction, and prepare alkaline phosphatase-labeled ApoE4-A / B / C / D / E / F antibodies respectively.

[0078] C. Performance Evaluation (1) Linear experiment Assay reagents, including R1, R2 and streptavidin magnetic bead solution: The R1 includes: biotin-labeled ApoE4-A / B / C / D / E / F antibody, used at a concentration of 1 ug / mL; The R2 includes: alkaline phosphatase-labeled ApoE4 antibody, with a concentration of 1 ug / mL.

[0079] The specific experimental steps are as follows: a. Experimental preparation: 5 mL each of R1 and R2 working solutions, 4 mL of magnetic bead solution, and calibrators WRS A~WRS F.

[0080] b. Place each component into the kit respectively, and then place the kit into the fully automatic chemiluminescence immunoassay analyzer EXI1800 (Zhongyuan Huiji Biotechnology Co., Ltd.), and select the anti-ApoE4 project (sample 30uL, R1 45uL, R2 45uL, magnetic bead solution 30uL) for the experiment.

[0081]

[0082] Therefore, as shown in Table 3, the above 6 anti-ApoE4 antibodies all have good linear effects when used as labeled antibodies in ApoE4 chemiluminescence detection.

[0083] 3. Clinical trials The specific experimental steps are as follows: (1) Experimental preparation: Take out 20 ApoE4 samples, thaw at room temperature and mix thoroughly on a vortex mixer; (2) Testing: ApoE4-A and mutant ApoE4-B antibodies were collected and tested in 20 clinical samples. The clinical results are shown in Table 4 below:

[0084] From the above clinical data, it can be seen that the clinical measurement values ​​of ApoE4-A and mutant strain ApoE4-B are both ≥0.99, which meets the requirements.

[0085] All data, reagents and steps herein should be understood as illustrative and non-restrictive, although described the present invention in conjunction with above-mentioned specific embodiment, many modifications and other variations are apparent to those skilled in the art.All such modifications and other variations also fall within the scheme of the present invention and protection scope.

Claims

1. An anti-ApoE4 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-ApoE4 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 ApoE4, 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 ApoE4 in a sample to be detected under conditions sufficient for an antibody / antigen binding reaction to form an immune complex; 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 ApoE4.

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 ApoE4.

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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