Anti-hepatitis A virus antibody and application thereof

Abstract

The invention discloses an anti-hepatitis A virus antibody and application thereof, and relates to the field of antibodies. The anti-hepatitis A virus 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 the hepatitis A virus antibody, and has good affinity or activity.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to an anti-hepatitis A virus antibody and an application thereof. Background Art

[0002] Hepatitis A virus (HAV) belongs to the Picornaviridae family, a new type of enterovirus 72, has only one serotype, is non-enveloped, has a diameter of 27 to 32 nm, and is in the form of spherical particles with icosahedral symmetry.

[0003] Hepatitis A, also known as hepatitis A or hepatitis A for short, is an infectious disease caused by hepatitis A virus (HAV) and is characterized by inflammation of the liver. Hepatitis A virus infection is mostly self-limiting, but severe cases can form fulminant hepatitis and lead to death. The clinical symptoms of hepatitis A are related to the age of the infected person. In children under 6 years old, 70% of infections are asymptomatic. In older children and adults, the infected people often show clinical symptoms, most of which will develop jaundice. The average incubation period of hepatitis A virus is 28 days (ranging from 15 to 50 days). Typical clinical symptoms include fever, malaise, anorexia, vomiting, abdominal discomfort, etc. These symptoms usually do not exceed 2 months. The most obvious clinical symptom is jaundice, which occurs in 70% of cases.

[0004] Anti-HAV IgM and IgG can be detected in the serum of patients in the acute stage of hepatitis A. The anti-HAV IgM positivity rate of clinical patients is 100% 2 weeks after the onset of the disease, which lasts for 2 to 3 weeks and then drops rapidly. HAV antibodies can be detected before the onset of symptoms. Once hepatitis A is infected, total HAV antibodies are positive. After natural infection, HAV IgG antibodies are usually detected throughout life and can have a secondary immune effect when infected again. Nowadays, it can be prevented by vaccination with hepatitis A vaccine, and HAV IgG antibodies can be detected two weeks after vaccination with hepatitis A vaccine. HAV total antibody IgG / IgM detection is used to diagnose past or current hepatitis A virus infection, and can also observe the immune effect after HAV vaccination.

[0005] At present, the detection method of HAV antibody is mainly immunological detection method, such as enzyme-linked immunosorbent assay, the principle of which is 1) to bind the antigen or antibody to the surface of a solid phase carrier and maintain its immune activity 2) to connect the antigen or antibody with a certain enzyme to form an enzyme-labeled antigen or antibody, which retains both its immune activity and enzyme activity. The specimen to be tested (the antibody or antigen to be measured) and the enzyme-labeled antigen or antibody are reacted with the antigen or antibody on the surface of the solid phase carrier in different steps. The antigen-antibody complex formed on the solid phase carrier is separated from other substances by washing. After the substrate of the enzyme reaction is added, the substrate is catalyzed by the enzyme to become a colored product. The amount of the product is directly related to the amount of the substance to be tested in the specimen, so qualitative or quantitative analysis can be performed based on the depth of the color reaction. Similar immunological detection methods include radioimmunoassay, fluorescent immunochromatography, chemiluminescence, etc.

[0006] The above immunological detection methods all require antibodies against hepatitis A virus. Therefore, there is a strong demand in the art for antibodies against hepatitis A virus with good performance. Summary of the invention

[0007] The present application provides an anti-hepatitis A virus antibody, which provides an important source of raw materials for the detection of hepatitis A virus antibodies and has good activity or affinity.

[0008] In order to achieve the above-mentioned object, according to one aspect of the present invention, an anti-hepatitis A virus antibody is provided, which comprises three complementary determining regions of a heavy chain variable region having an amino acid sequence of any one of SEQ ID NOs: 18 and 19 and three complementary determining regions of a light chain variable region having an amino acid sequence of SEQ ID NO: 22.

[0009] In order to achieve the above object, according to a second aspect of the present invention, an anti-hepatitis A virus antibody is provided, wherein the antibody comprises the following complementary determining regions:

[0010] HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1;

[0011] HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2;

[0012] HCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 3;

[0013] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0014] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and

[0015] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6.

[0016] In order to achieve the above-mentioned object, according to the third aspect of the present invention, an anti-hepatitis A virus antibody is provided, 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: 18 and 19; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22.

[0017] In order to achieve the above-mentioned object, according to the fourth aspect of the present invention, an anti-hepatitis A virus antibody is provided, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NO:20 and 21; and the amino acid sequence of the light chain is shown in SEQ ID NO:23.

[0018] In order to achieve the above object, according to the fifth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises the above antibody.

[0019] In order to achieve the above object, according to the sixth aspect of the present invention, a reagent or a kit is provided, wherein the reagent or the kit comprises the above antibody or the above antibody conjugate.

[0020] In order to achieve the above object, according to the eighth aspect of the present invention, there is provided a use of the above-mentioned antibody or antibody conjugate in the preparation of a product for detecting hepatitis A virus antibodies.

[0021] In order to achieve the above object, the present invention also provides a nucleic acid, a vector, a cell and a method for preparing the above antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 The results of reducing SDS-PAGE of Anti-HAV 7A12 Rmb1 to Anti-HAV 7A12 Rmb2 are shown. DETAILED DESCRIPTION

[0024] In a first aspect, an embodiment of the present invention provides an anti-hepatitis A virus antibody, which comprises three complementary determining regions of a heavy chain variable region having the amino acid sequence of any one of SEQ ID NOs: 18 and 19 and three complementary determining regions of a light chain variable region having the amino acid sequence of SEQ ID NO: 22.

[0025] 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 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 described in the first aspect.

[0026] For example, the HCDR1, HCDR2, and HCDR3 are amino acid sequences consistent with HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:18; the LCDR1, LCDR2, and LCDR3 are amino acid sequences consistent with LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:22.

[0027] In the present invention, the term "antibody" is used in the broadest sense and may include full-length monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies or antigen-binding fragments, so long as they exhibit the desired biological activity.

[0028] The above antigen-binding fragments usually have the same binding specificity as the antibody from which they are derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the above antigen-binding fragments 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, a person skilled in the art can easily obtain the above antigen-binding fragments.

[0029] The above antigen-binding fragments 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.

[0030] 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.

[0031] 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.

[0032] The definition methods of CDR are well known in the art, and the CDR definition methods include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, "Kabat definition" refers to the definition system described in 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 region defined by Kabat, although they may be shortened or extended based on predictions or experimental results of specific residues or residue groups. Exemplary defined CDRs are listed in Table 1 below, and the definitions in different documents are slightly different. Given the variable region amino acid sequence of an antibody, a person skilled in the art can routinely determine which residues contain a specific CDR. It should be noted that CDRs defined by other methods not limited to Table 1 also belong to the scope of protection of the present disclosure.

[0033] Table 1: CDR Definition 1

[0034]

[0035]

[0036] 1 The numbering of all CDR definitions in Table 1 is based on the Kabat numbering system (see below), with the amino acid numbering on the heavy chain represented by "H+number" and the amino acid numbering on the light chain represented by "L+number". One of ordinary skill in the art can unambiguously map the Kabat numbering system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Patent. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0037] 2"AbM" as used in Table 1 with a lowercase "b" refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0038] 3 If both H35A and H35B are absent, CDR-H1 ends at position 35; if only H35A is present, CDR-H1 ends at position 35A; if both H35A and H35B are present, CDR-H1 ends at position 35B.

[0039] 4 If both H35A and H35B are absent, CDR-H1 ends at position 32; if only H35A is present, CDR-H1 ends at position 33; if both H35A and H35B are present, CDR-H1 ends at position 34.

[0040] 5 If both H35A and H35B are absent, CDR-H1 ends at position 33; if only H35A is present, CDR-H1 ends at position 34; if both H35A and H35B are present, CDR-H1 ends at position 35.

[0041] 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.

[0042] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Kabat system.

[0043] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the Chothia system.

[0044] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT system.

[0045] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the AbM system.

[0046] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a Contact system.

[0047] In some optional embodiments of the present invention, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by a combination of Kabat, Chothia, IMGT, AbM or Contact systems.

[0048] In a second aspect, an embodiment of the present invention provides an anti-hepatitis A virus antibody, wherein the antibody comprises the following complementary determining regions:

[0049] HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1;

[0050] HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2;

[0051] HCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 3;

[0052] LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4;

[0053] LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and

[0054] LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:6.

[0055] According to an embodiment of the present invention, the HCDRs and LCDRs are defined by the Kabat system.

[0056] 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.

[0057] 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.

[0058] In an alternative embodiment, the antibody of the first aspect or the second aspect further has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

[0059] In an alternative embodiment, the HFR1 comprises / is SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

[0060] The HFR2 comprises / is SEQ ID NO:8 or an amino acid sequence having at least 80% identity thereto;

[0061] The HFR3 comprises / is SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto;

[0062] The HFR4 comprises / is SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

[0063] The LFR1 comprises / is SEQ ID NO:11 or an amino acid sequence having at least 80% identity thereto;

[0064] The LFR2 comprises / is SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

[0065] The LFR3 comprises / is SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and

[0066] The LFR4 comprises / is SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

[0067] It should be noted that, in other embodiments, the amino acid sequences of the framework regions of the anti-hepatitis A virus antibodies 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: 7, 8, 9, 10, 11, 12, 13 or 14).

[0068] In an optional embodiment, the HFR2 comprises / is represented by the amino acid sequence shown in SEQ ID NO:17.

[0069] In a third aspect, an embodiment of the present invention provides an anti-hepatitis A virus antibody, 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: 18 and 19, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22.

[0070] In an optional embodiment, the antibody described in the first aspect, the second aspect, and the third aspect further comprises a constant region.

[0071] In an alternative embodiment, the constant region includes a heavy chain constant region and / or a light chain constant region.

[0072] 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.

[0073] 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.

[0074] In an alternative embodiment, the IgG is selected from IgG1, IgG2, IgG3 or IgG4.

[0075] In an alternative embodiment, the light chain constant region is selected from a kappa-type or a lambda-type light chain constant region.

[0076] In an alternative embodiment, the species of origin of the constant region is cattle, horses, dairy cows, pigs, sheep, rats, mice, dogs, camels, cats, rabbits, donkeys, deer, minks, chickens, ducks, geese, turkeys, fighting cocks or humans.

[0077] In an alternative embodiment, the species origin of the constant region is mouse.

[0078] In an optional embodiment, the heavy chain constant region sequence (CH) is as shown in SEQ ID NO:15, and the light chain constant region (CL) sequence is as shown in SEQ ID NO:16.

[0079] 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: 15 or 16).

[0080] In an alternative embodiment, the antibody comprises any one of F(ab)2, F(ab')2, Fab', Fab, Fv and scFv.

[0081] In a fourth aspect, the present invention provides an anti-hepatitis A virus antibody, comprising a heavy chain and / or a light chain, wherein the amino acid sequence of the heavy chain is shown in any one of SEQ ID NO: 20 and 21, and the amino acid sequence of the light chain is shown in SEQ ID NO: 23.

[0082] In a fifth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the above-mentioned antibody.

[0083] In an optional embodiment, the above-mentioned antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody.

[0084] In an optional embodiment, the antibody conjugate further comprises a label coupled to the antibody.

[0085] 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.

[0086] In an alternative embodiment, the label includes but is not limited to fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.

[0087] 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.

[0088] 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.).

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, colloidal carbon, disperse dyes, dye-labeled microspheres, and latex.

[0094] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0095] In an optional embodiment, the colloidal metal is colloidal gold.

[0096] In an optional embodiment, the above-mentioned antibody conjugate further includes a solid phase carrier coupled to the antibody.

[0097] In an alternative embodiment, the solid support is selected from microspheres, plates and membranes.

[0098] 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.

[0099] In a sixth aspect, the present invention provides a reagent or a kit, wherein the reagent or the kit comprises the above-mentioned antibody or the above-mentioned antibody conjugate.

[0100] As mentioned above, the antibodies in some embodiments or examples of the present invention can effectively bind to the hepatitis A virus, and therefore, the reagents or kits containing the hepatitis A virus antibodies can effectively detect the hepatitis A virus antibodies qualitatively or quantitatively. The reagents or kits provided by the present invention can be used, for example, for detections involving the specific binding properties of hepatitis A virus and its antibodies, such as immunoblotting and immunoprecipitation. As mentioned above, the antibodies in some embodiments or examples of the present invention have higher binding activity or affinity with the hepatitis A virus, and therefore the reagents or kits containing the antibodies have higher detection sensitivity or specificity.

[0101] In a seventh aspect, the present invention provides a method for detecting hepatitis A virus, comprising: a) contacting the above-mentioned antibody or antibody conjugate with a hepatitis A virus antigen and a hepatitis A virus antibody 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 hepatitis A virus antibodies in the test sample.

[0102] In an eighth aspect, the present invention provides use of the above-mentioned anti-hepatitis A virus antibodies and antibody conjugates in the preparation of products for detecting hepatitis A virus.

[0103] It should be noted that the products of the present invention include but are not limited to reagents, test kits, test strips or test plates.

[0104] In a ninth aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned antibody.

[0105] In a tenth aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0106] In an eleventh aspect, the present invention provides a cell containing the above-mentioned vector.

[0107] In a twelfth aspect, the present invention provides a method for preparing anti-hepatitis A virus antibodies, comprising: culturing the cells as described above.

[0108] Based on the amino acid sequence of the anti-hepatitis A virus antibody 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-hepatitis A virus antibody, for example, separating and purifying the antibody from the culture product of a recombinant cell that can recombinantly express the antibody 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-hepatitis A virus antibody of the present invention, it belongs to the protection scope of the present invention.

[0109] 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.

[0110] 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.

[0111] 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., 1987). 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.

[0112] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0113] Example 1 Preparation of Anti-HAV 7A12 Monoclonal Antibody

[0114] In this example, restriction endonucleases and Prime Star DNA polymerase were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO. BD SMART TMThe RACE cDNA Amplification Kit was purchased from Takara. The pMD-18T vector was purchased from Takara. The plasmid extraction kit was purchased from Tiangen. Primer synthesis and gene sequencing were completed by Invitrogen. The hybridoma cell line secreting the anti-HAV 7A12 monoclonal antibody was prepared in this laboratory and revived for future use.

[0115] (1) Antibody gene preparation

[0116] mRNA was extracted from the hybridoma cell line secreting Anti-HAV 7A12 monoclonal antibody, and the DNA product was obtained by RT-PCR. The product was inserted into the pMD-18T vector after A addition reaction with rTaq DNA polymerase and transformed into DH5α competent cells. After the colonies grew, the Heavy Chain and Light Chain gene clones were taken respectively, and 4 clones each were sent to a gene sequencing company for sequencing.

[0117] (2) Sequence analysis of the variable region gene of Anti-HAV 7A12 antibody

[0118] The gene sequences obtained by the above sequencing were placed in the kabat antibody database for analysis, and the VNTI11.5 software was used for analysis to determine that the genes amplified by the heavy chain and light chain primer pairs were correct. Among the gene fragments amplified by the Light Chain, the VL gene sequence was 318bp, preceded by a 57bp leader peptide sequence; among the gene fragments amplified by the Heavy Chain primer pair, the VH gene sequence was 360bp, belonging to the VH1 gene family, preceded by a 57bp leader peptide sequence.

[0119] (3) Construction of recombinant antibody expression plasmid

[0120] pcDNA TM 3.4 vector is a recombinant antibody eukaryotic expression vector constructed, which has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and is named pcDNA3.4A expression vector, hereinafter referred to as 3.4A expression vector; according to the sequencing results of the antibody variable region genes in the above pMD-18T, the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, respectively, and the 0.70kb Light Chain gene fragment and the 1.43kb Heavy Chain gene fragment were amplified by the PCR amplification method.

[0121] The Heavy Chain and Light Chain gene fragments were double-digested with HindIII / EcoRI, and the 3.4A vector was double-digested with HindIII / EcoRI. After the fragments and vectors were purified and recovered, the Heavy Chain gene and the Light Chain gene were respectively connected to the 3.4A expression vector to obtain the recombinant expression plasmids of Heavy Chain and Light Chain, respectively.

[0122] 2. Recombinant Antibody Production

[0123] Resuscitate HEK293 cells in advance and subculture them to 200 ml system to make the cell density reach 3-5×10 6 cells / ml, the cell density reaches the selected antibody concentration and cells, and the cell viability is >95%; the cells are washed by centrifugation and re-dissolved with culture medium, and the cell density is adjusted to 2.9×10 6 cells / ml, wash the cells, and re-dissolve them with culture medium, and use it as a cell diluent. Prepare plasmid DNA and transfection reagent diluents with culture medium respectively. Add the transfection reagent diluent to the plasmid DNA diluent, mix well and let stand at room temperature for 15 minutes; slowly add the mixture to the cell diluent within 1 minute, mix well and take samples to count, record and observe the viability of the cells after transfection, and place them in a 35℃ constant temperature incubator for culture, with a speed of 120rmp and a CO2 content of 8%. Centrifuge and collect samples after 13 days. The centrifuged supernatant was affinity purified using a proteinA affinity chromatography column. Take 6ug layer of purified antibodies for reducing SDS-PAGE, and the electrophoresis is shown in the figure. After reducing SDS-PAGE, two bands are shown, one Mr is 50KD (heavy chain) and the other Mr is 28KD (light chain).

[0124] The obtained antibody was named Anti-HAV 7A12Rmb1. Anti-HAV 7A12Rmb1 was mutated to obtain a mutant antibody. The sequences of the heavy chain (H) and light chain (L) of the above antibody are shown in the following table:

[0125] Table 2 Antibody sequences

[0126] Antibody Name Heavy chain Light chain Anti-HAV 7A12Rmb1 SEQ ID NO:20 SEQ ID NO:23 Anti-HAV 7A12Rmb2 SEQ ID NO:21 SEQ ID NO:23

[0127] Example 2 Performance testing of antibodies

[0128] 1. Performance Evaluation

[0129] The coating solution (main component NaHCO3) was used to dilute the mouse anti-human IgM monoclonal antibody (from Feipeng Biotechnology) to 1ug / ml, 100uL per well, and incubated at 4°C overnight; the next day, the cells were washed twice with the washing solution (main component Na2HPO4+Nacl), and patted dry; the blocking solution (20% BSA+80% PBS) was added, 120uL per well, and incubated at 37°C for 1h, and patted dry; the diluted HAV positive specimen was added, 100uL / well, and incubated at 37°C for 30min; the cells were washed 5 times with the washing solution, and patted dry; 50uL / well of HAV antigen (from Feipeng Biotechnology) was added, and then HRP-labeled Anti-HAV 7A12Rmb1, Anti-HAV 7A12Rmb2 and HRP-labeled mainstream control antibodies in the market, 100uL per well, 37°C, 30min; wash 5 times with detergent and pat dry; add colorimetric solution A (50uL / well), add colorimetric solution B (50uL / well), 10min; add stop solution, 50uL / well; read OD value at 450nm (reference 630nm) on the microplate reader.

[0130] Note: Solution A (main ingredients: citric acid + sodium acetate + acetanilide + urea peroxide); Solution B (main ingredients: citric acid + EDTA·2Na + TMB + concentrated HCL); Stop solution (EDTA·2Na + concentrated H2SO4)

[0131] Table 3 Performance evaluation data

[0132]

[0133] 2. Stability assessment

[0134] The above antibodies were placed at 4°C (refrigerator), -80°C (refrigerator), and 37°C (constant incubator) for 21 days, and samples were taken for 7 days, 14 days, and 21 days for status observation, and HRP was labeled for samples under different conditions for 21 days, and then the activity was tested by the above performance evaluation method. The results showed that there was no obvious protein state change in the antibodies under the three assessment conditions for 21 days, and the activity after HRP labeling did not show a downward trend with the increase of the assessment temperature, indicating that the expressed antibodies were stable. The following table shows the OD results of enzyme immunoassay activity detection of antibody Anti-HAV7A12Rmb2 labeled with HRP at different temperatures after 21 days of assessment, at the dosage of positive sample 2 (1:24k).

[0135] Table 4 Stability data

[0136]

[0137]

[0138] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0139] Some of the amino acid sequences involved in this application are shown in Table 5:

[0140]

[0141]

Claims

1. An anti-hepatitis A virus antibody, comprising three complementary determining regions of a heavy chain variable region having the amino acid sequence of any one of SEQ ID NOs: 18 and 19 and three complementary determining regions of a light chain variable region having the amino acid sequence of SEQ ID NO:

22.

2. The antibody according to claim 1, It is characterized in that The complementarity determining regions of the variable regions are defined by any one of the Kabat, Chothia, IMGT, AbM or Contact systems or a combination of multiple systems.

3. An antibody against hepatitis A virus, It is characterized in that The antibody comprises the following complementarity determining regions: HCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 1; HCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 2; HCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 3; LCDR1, which comprises or consists of the amino acid sequence shown in SEQ ID NO:4; LCDR2, which comprises or consists of the amino acid sequence shown in SEQ ID NO:5; and LCDR3, which comprises or consists of the amino acid sequence shown in SEQ ID NO:6; Optionally, the HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto; The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto; The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto; The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto; The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto; The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto; The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto; and The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

4. An anti-hepatitis A virus antibody comprising a heavy chain variable region and / or a light chain variable region, It is characterized in that The amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID NOs: 18 and 19; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 22; Optionally, the antibody further comprises a constant region; Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region; Optionally, 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; Optionally, the heavy chain constant region includes CH1 of IgG, hinge region of IgG, CH2 of IgM, CH3 of IgM and / or CH4 of IgM; Optionally, the species of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, camel, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the species origin of the constant region is mouse; Optionally, the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto; Optionally, the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto; Optionally, the antibody comprises any one of F(ab')2, Fab', Fab, Fv and scFv.

5. An anti-hepatitis A virus antibody comprising a heavy chain and / or a light chain, It is characterized in that The amino acid sequence of the heavy chain is shown in any one of SEQ ID NOs: 20 and 21; the amino acid sequence of the light chain is shown in SEQ ID NO:

23.

6. An antibody conjugate, It is characterized in that The antibody conjugate comprises the antibody according to any one of claims 1 to 5; Optionally, the antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody; Optionally, the antibody conjugate further comprises a label coupled to the antibody; Optionally, the label is selected from fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents and nanoparticle labels; Optionally, the antibody conjugate further comprises a solid phase carrier coupled to the antibody.

7. A reagent or a kit, It is characterized in that The reagent or kit comprises the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6.

8. Use of the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6 in the preparation of an antibody product for detecting hepatitis A virus; Optionally, the use includes: a) contacting the antibody according to any one of claims 1 to 5 or the antibody conjugate according to claim 6 with a hepatitis A virus antigen and a hepatitis A virus antibody in a sample to be tested 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 hepatitis A virus antibodies in the test sample.

9. A nucleic acid, a vector, a cell or a method for preparing the antibody according to any one of claims 1 to 5, wherein the nucleic acid encodes the antibody according to any one of claims 1 to 5; the vector contains a nucleic acid encoding the antibody according to any one of claims 1 to 5; the cell contains the above nucleic acid or vector; the method comprises the above cell.

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