A monoclonal antibody of VCE and preparation method thereof

Abstract

The present invention provides a monoclonal antibody binding to VCE and a preparation method thereof, belonging to the field of immunity. The monoclonal antibody binding to VCE provided by the present invention can be used to detect residual VCE in the production process of mRNA vaccines.

Description

[0001] This application is a divisional application of invention patent application No. 2021115608109, filed on December 20, 2021, entitled “A monoclonal antibody for VCE and a method for preparing the same”. Technical Field

[0002] The present invention belongs to the field of immunology, and specifically relates to a monoclonal antibody of VCE. The present invention also relates to a preparation method of the monoclonal antibody and its application. Background Art

[0003] mRNA is "messenger ribonucleic acid". As a "messenger", the main function of mRNA is to transmit transcription DNA information and pass on the template instructions for producing various proteins. When the "factory" that processes proteins in human cells receives this instruction, the protein synthesis process will be started. At the end of 1987, Robert Malone mixed mRNA with fat droplets and found that human cells could absorb these mRNAs added to fat droplets and produce proteins (Malone et al Sci. USA86, 6077-6081 (1989)). This discovery became the first successful attempt to express mRNA in vivo, and also proved the feasibility of mRNA vaccines. Since then, after 30 years of research and development, major technological innovations and research investments have made mRNA an important tool in the field of vaccine development and cancer treatment.

[0004] After entering the human body through a specific delivery system, mRNA vaccines use the body's transcription and translation systems to generate proteins with specific immunogenicity. After these proteins are recognized by antigen presenting cells (APCs), they induce the maturation of dendritic cells and then activate B cells and T cells to produce a strong immune response, triggering both humoral and cellular immune responses and forming immune memory. However, there are few reports that traditional vaccines can produce cellular immunity. In terms of production technology, mRNA vaccines have significant advantages such as simple production process, fast development speed, no need for cell culture, and low cost.

[0005] In practical applications, on the one hand, mRNA and cell membranes are both negatively charged, so a delivery system is needed to effectively encapsulate mRNA so that it will not be degraded by RNA enzymes present in the blood and tissues. On the other hand, mRNA in the eukaryotic system has a cap structure to allow mRNA to be stably expressed in cells. mRNA in the vaccine IVT process requires such modification, so Vaccine Capping Enzyme (VCE) can cap exogenous antigen mRNA in mRNA vaccines to enable it to be stably expressed in cells. There are three forms of cap structures in organisms: cap0, cap1, and cap2. The mRNA of unicellular eukaryotic organisms is mainly cap0, that is, the first methyl group appears in the mRNA of all eukaryotic cells, catalyzed by guanylate-7 methyltransferase; the mRNA in eukaryotic cells is mainly cap1, and another methyl group is added to the 2'-OH position of the second nucleotide, which is completed by 2'-O-methyltransferase. The vaccinia virus capping enzyme is composed of two subunits, D12 and D1. The D1 subunit has RNA triphosphatase and guanosine transferase activities, and the D12 subunit has guanine methyltransferase activity. Therefore, the vaccinia virus capping enzyme can add the cap0 cap to the 5' end of mRNA with an efficiency of 100% and in the correct direction.

[0006] Detecting VCE residues is also an important step. Therefore, it is urgent to develop a highly sensitive and specific monoclonal antibody for detecting anti-VCE residues. Summary of the invention

[0007] The present invention aims to provide an isolated antibody or an antigen-binding fragment thereof that specifically binds to VCE (vaccinia virus capping enzyme) for use in detecting the residue of VCE.

[0008] In a first aspect of the present invention, there is provided an isolated antibody or antigen-binding fragment thereof, comprising:

[0009] (a) a heavy chain variable domain (VH), the heavy chain variable domain comprising:

[0010] The heavy chain complementary determining region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO:1, the HCDR2 of the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 of the amino acid sequence shown in SEQ ID NO:3; and

[0011] (b) a light chain variable domain (VL), the light chain variable domain comprising:

[0012] light chain complementary determining region 1 (LCDR1) of the amino acid sequence shown in SEQ ID NO:4, LCDR2 of the amino acid sequence shown in SEQ ID NO:5, and LCDR3 of the amino acid sequence shown in SEQ ID NO:6;

[0013] The antibody or antigen-binding fragment thereof can specifically bind to VCE, and preferably the VCE comprises the amino acid sequence shown in SEQ ID NO:11.

[0014] In some embodiments, the isolated antibody or antigen-binding fragment thereof, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:7, and the VL comprises the amino acid sequence shown in SEQ ID NO:8.

[0015] In some embodiments, the isolated antibody or antigen-binding fragment thereof is rodent, chimeric, human, partially humanized, or fully humanized.

[0016] In some embodiments, the isolated antibody or antigen-binding fragment thereof is murine.

[0017] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain constant domain (CH) with the amino acid sequence shown in SEQ ID NO:9 and a light chain constant domain (CL) with the amino acid sequence shown in SEQ ID NO:10.

[0018] In a second aspect, the present invention provides a method for preparing the isolated antibody or antigen-binding fragment thereof according to the first aspect.

[0019] In some embodiments, the method comprises immunizing mice with VCE as an antigen to obtain mouse spleen cells, performing hybridoma fusion to obtain hybridoma cells, and screening hybridoma cells that can secrete and express antibodies that bind to VCE; sequencing the hybridoma cells to obtain the variable domain and constant domain amino acid sequences and nucleic acid sequences of the antibodies, and recombinantly expressing the desired antibodies based on the above sequence information.

[0020] In some embodiments, the amino acid sequence of the VCE is as shown in SEQ ID NO:11.

[0021] The third aspect of the present invention provides a kit, which comprises the isolated antibody or antigen-binding fragment thereof according to the first aspect of the present invention, and a second isolated antibody or antigen-binding fragment thereof capable of binding to the VCE; wherein the second isolated antibody or antigen-binding fragment thereof binds to a different epitope on the VCE than the isolated antibody or antigen-binding fragment thereof.

[0022] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable domain (VH), the heavy chain variable domain comprising: a heavy chain complementary determining region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO:1, a HCDR2 of the amino acid sequence shown in SEQ ID NO:2, and a HCDR3 of the amino acid sequence shown in SEQ ID NO:3; and (b) a light chain variable domain (VL), the light chain variable domain comprising: a light chain complementary determining region 1 (LCDR1) of the amino acid sequence shown in SEQ ID NO:4, a LCDR2 of the amino acid sequence shown in SEQ ID NO:5, and a LCDR3 of the amino acid sequence shown in SEQ ID NO:6; wherein the antibody or antigen-binding fragment thereof can specifically bind to VCE, and preferably the VCE comprises a VCE of the amino acid sequence shown in SEQ ID NO:11.

[0023] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:7, and the VL comprises the amino acid sequence shown in SEQ ID NO:8.

[0024] In some embodiments, the isolated antibody or antigen-binding fragment thereof further comprises a heavy chain constant domain (CH) with the amino acid sequence shown in SEQ ID NO:9 and a light chain constant domain (CL) with the amino acid sequence shown in SEQ ID NO:10.

[0025] The fourth aspect of the present invention provides another kit, which comprises the isolated antibody or antigen-binding fragment thereof described in the first aspect, and a second isolated antibody or antigen-binding fragment thereof capable of binding to the VCE; wherein the second isolated antibody or antigen-binding fragment thereof binds to the VCE non-competitively with the isolated antibody or antigen-binding fragment thereof.

[0026] In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable domain (VH), the heavy chain variable domain comprising: a heavy chain complementary determining region 1 (HCDR1) of the amino acid sequence shown in SEQ ID NO:1, a HCDR2 of the amino acid sequence shown in SEQ ID NO:2, and a HCDR3 of the amino acid sequence shown in SEQ ID NO:3; and (b) a light chain variable domain (VL), the light chain variable domain comprising: a light chain complementary determining region 1 (LCDR1) of the amino acid sequence shown in SEQ ID NO:4, a LCDR2 of the amino acid sequence shown in SEQ ID NO:5, and a LCDR3 of the amino acid sequence shown in SEQ ID NO:6; wherein the antibody or antigen-binding fragment thereof can specifically bind to VCE, and preferably the VCE comprises a VCE of the amino acid sequence shown in SEQ ID NO:11.

[0027] In some embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:7, and the VL comprises the amino acid sequence shown in SEQ ID NO:8.

[0028] In some embodiments, the isolated antibody or antigen-binding fragment thereof further comprises a heavy chain constant domain (CH) with the amino acid sequence shown in SEQ ID NO:9 and a light chain constant domain (CL) with the amino acid sequence shown in SEQ ID NO:10.

[0029] The fifth aspect of the present invention provides a method for detecting VCE residues during the preparation of mRNA vaccines, which uses the above-mentioned separated antibodies or antigen-binding proteins and the above-mentioned kit to detect VCE residues.

[0030] The sixth aspect of the present invention provides the use of the above-mentioned antibodies or antigen-binding proteins, and kits containing the above-mentioned antibodies or antigen-binding fragments in the production of mRNA vaccines. The application of the mRNA vaccine production described in the present invention refers to the application in various links of vaccine production, including but not limited to the production process and the end of production. Any production link involving VCE residues can be removed by using the antibodies or antigen-binding fragments and their kits of the present invention.

[0031] The seventh aspect of the present invention provides use of the aforementioned kit in an enzyme-linked immunosorbent assay.

[0032] The present invention also provides a nucleic acid encoding the isolated antibody or antigen-binding fragment thereof; the present invention also provides a vector comprising the nucleic acid, which can express the nucleic acid of the isolated antibody or antigen-binding fragment thereof; the present invention also provides a host cell comprising the vector.

[0033] The amino acid sequence information of the present invention is shown in Table 1:

[0034] Table 1 Amino acid sequence

[0035]

[0036]

[0037]

[0038] the term

[0039] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0040] The term "epitope" means a protein determinant that is capable of specific binding to an antibody. An epitope is usually composed of chemically active surface groups of a molecule (such as amino acids or sugar side chains) and usually has specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between a conformational epitope and a non-conformational epitope is that in the presence of a denaturing solvent, the binding to the former but not the latter is lost.

[0041] The terms "antibody," "antibody portion," "antigen-binding fragment," or "antibody construct" are used in the broadest sense and encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.

[0042] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of 5 basic heterotetrameric units and another polypeptide called J chain, and contain 10 antigen binding sites, while IgA antibodies contain 2-5 basic 4-chain units, which can be polymerized to form a multivalent aggregate combined with J chain. In the case of IgG, the 4-chain unit is usually about 150,000 Daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds according to the H chain isotype. Each H and L chain also has a regularly spaced intrachain disulfide bridge. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) of each α and γ chain and four CH domains of μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain at its other end. VL is aligned with VH, and CL is aligned with the first constant domain (CH1) of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. VH and VL are paired together to form a single antigen binding site. For the structure and characteristics of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P.Sties, Abba I.Terr and Tristram G.Parsolw (editor), Appleton & Lang e, Norwalk, Conn., 1994, page 71 and chapter 6. According to the amino acid sequence of its constant domain, the L chain from any vertebrate species can be assigned to one of two significantly different types (called κ and λ). Depending on the amino acid sequence of its heavy chain constant domain (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, which have heavy chains named α, δ, ε, γ and μ, respectively. The gamma and alpha classes are further divided into subclasses based on relatively small differences in CH sequence and function.

[0043] "Isolated" antibodies are antibodies that have been identified, separated and / or recovered from components (e.g., natural or recombinant) of their production environment. Preferably, the isolated polypeptide is not associated with all other components from its production environment. The contaminating components of its production environment (such as those produced by recombinant transfected cells) are substances that usually interfere with the research, diagnosis or therapeutic use of the antibody, and may include enzymes, hormones and other proteins or non-protein solutes. The isolated antibody includes the in situ antibody in the recombinant cell, and at least one component of the antibody's natural environment will not be present. However, usually, isolated polypeptides, antibodies or constructs will be prepared by at least one purification step.

[0044] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of an antibody heavy or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen binding site.

[0045] The term "variable" refers to the fact that certain segments of the variable domain differ greatly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed throughout the entire range of the variable domain. Instead, it is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions (HVRs) in the light chain variable domain and the heavy chain variable domain. The more highly conserved parts of the variable domain are referred to as framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions connected by three CDRs, mainly in a β-folded configuration, and the three CDRs form a loop that connects the β-folded structure and forms a part of the β-folded structure in some cases. The CDRs in each chain are tightly bound together by the FR region and contribute to the formation of the antigen binding site of the antibody together with the CDRs in the other chain (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0046] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, deamidation) that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Contrary to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" represents the characteristics of the antibody obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the production of antibodies by any ad hoc method. For example, monoclonal antibodies for use in accordance with the present application can be prepared by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)); recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567); phage display technology (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat'l. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)).

[0047] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of the immunoglobulin (i.e., the variable domain), which contains an antigen binding site. The constant domain contains the CH1, CH2, and CH3 domains (collectively referred to as CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0048] The term "complementarity determining region" or "CDR" is used to refer to the hypervariable regions defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0049] As used herein, the terms "specifically bind", "specifically recognize" or "specific for" refer to a measurable and reproducible interaction such as binding between a target and an antigen binding protein (such as mAb), which determines the presence of the target in the presence of a heterogeneous population of molecules including biomolecules. For example, an antigen binding protein (such as mAb) that specifically binds to a target (which may be an epitope) is an antigen binding protein (such as mAb) that binds to this target with greater affinity, avidity, more readily and / or for a longer duration than it binds to other targets. In some embodiments, the extent of binding of an antigen binding protein (such as mAb) to an unrelated target is less than about 10% of the binding of the antigen binding protein (such as mAb) to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antigen binding protein (such as mAb) that specifically binds to a target has the following dissociation constant (KD): ≤10-5M, ≤10-6M,

[0050] ≤10-7M, ≤10-8M, ≤10-9M, ≤10-10M, ≤10-11M, or ≤10-12M. In some embodiments, the antigen binding protein specifically binds to an epitope on a protein that is conserved in proteins from different species. In some embodiments, specific binding may include but does not require exclusive binding. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The results of SDS-PAGE electrophoresis of full-length anti-VCE monoclonal antibody are shown.

[0052] Figure 2 The results of the best functional VCE monoclonal antibody binding to VCE are shown. DETAILED DESCRIPTION

[0053] Example 1 Preparation of VCE Antibody

[0054] Mice were immunized with VCE, with 100 ug for the first immunization, 50 ug for the second immunization, 50 ug for the third immunization, and 50 ug for the fourth immunization. Hybridoma cells were then prepared and hybridomas numbered E7-2, E7-3, E7-6, and E7-7 were obtained by screening. The hybridoma cells were then cloned. For details, see (Proetzel, Gabriele; Ebersbach, Hilmar (2012). [Methods in Molecular Biology] Antibody Methods and Protocols Volume 901|| Hybridoma Technology for the Generation of Monoclonal Antibodies., 10.1007 / 978-1-61779-931-0 (Chapter 7), 117–135).

[0055] VCE amino acid sequence:

[0056] MDANVVSSSTIATYIDALAKNASELEQRSTAYEINNELELVFIKPPLITLTNVV

[0057] NISTIQESFIRFTVTNKEGVKIRTKIPLSKVHGLDVKNVQLVDAIDNIVWEKKS

[0058] LVTENRLHKECLLRLSTEERHIFLDYKKYGSSIRLELVNLIQAKTKNFTIDFKL

[0059] KYFLGSGAQSKSSLLHAINHPKSRPNTSLEIEFTPRDNETVPYDELIKELTTLS

[0060] RHIFMASPENVILSPPINAPIKTFMLPKQDIVGLDLENLYAVTKTDGIPITIRVTSNGLYCYFTHLGYIIRYPVKRIIDSEVVVFGEAVKDKNWTVYLIKLIEPVNAINDRLEESKYVESKLVDICDRIVFKSKKYEGPFTTTSEVVDMLSTYLPKQ PEGVILFYSKGPKSNIDFKIKKENTIDQTANVVFRYMSSEPIIFGESSIFVEYKKFSNDKGFPKEYGSGKIVLYNGVNYLNNIYCLEYINTHNEVGIKSVVVPIKFIAEFLVNGEILKPRIDKTMKYINSEDYYGNQHNIIVEHLRDQSIKIGDIFN EDKLSDVGHQYANNDKFRLNPEVSYFTNKRTRGPLGILSNYVKTLLISMYCSKTFLDDSNKRKVLAIDFGNGADLEKYFYGEIALLVATDPDADAIARGNERYNKLNSGIKTKYYKFDYIQETIRSDTFVSSVREVFYFGKFNIIDWQFAIHYSFHP RHYATVMNNLSELTASGGKVLITTMDGDKLSKLTDKKTFIIHKNLPSSENYMSVEKIADDRIVVYNPSTMSTPMTEYIIKKNDIVRVFNEYGFVLVDNVDFATIIERSKKFINGASTMEDRPSTRNFFELNRGAIKCEGLDVEDLLSYYVVYVFSKR

[0061] Example 2 Expression and purification of monoclonal antibodies

[0062] The hybridoma cells screened in Example 1 were lysed and mRNA was extracted, and then the mRNA was reverse transcribed to form cDNA. After a round of cDNA amplification, the IgG heavy and light chain gene fragments were retrieved, and then the retrieved IgG heavy and light chain gene fragments were spliced ​​into the expression vector to construct a plasmid. The plasmid was prepared using ExpiCHO TM Reagent was co-transfected into HEK293 suspension culture cells for transient expression. During transfection, the cell density was maintained at 6×10 6 Cells / mL, ExpiCHO TM The ratio of Reagent: DNA (plasmid) was 4:1. The cells were cultured in a 37°C, 8% CO2 incubator with shaking at 120 rpm. 16-18 hours after transfection, 180 μL ExpressiFectamine TMCHO Enhance and 4.8mL ExpiCHO TM Feed, then transfer to 32°C, 5%, 120rpm shaker culture. Collect cell supernatant 11 days after transfection. Purify after filtration with a 0.22μm filter. Before purification, the pipeline and protein A column were depyrogenated with 0.2M NaOH. The column was rebalanced with a buffer containing 0.05M Tris and 1.5M NaCl (pH8.0). The harvested cell culture supernatant was then diluted 1:1 with 2× the above buffer and sterilized by filtration. The filtered supernatant and protein A column were incubated at room temperature for 2 hours, and after washing the column with 1× the above buffer, IgG was eluted with sterile 0.1M sodium citrate (pH3.5), and the eluate was collected and neutralized with one-ninth volume of sterile 1M Tris-HCl (pH9). Under sterile conditions, the product buffer was exchanged with PBS (pH7.4) to remove any elution buffer and concentrate the sample. After concentration, the antibody was quantified by OD280nm using an extinction coefficient Ec of 1.43 (0.1%).

[0063] The antibodies were numbered according to the number of the hybridoma, and the purified E7-2, E7-3, E7-6, and E7-7 antibodies were analyzed by SDS-PAGE using a BioRad electrophoresis system with a 10% precast gel (GenScript) under 50 mM dithiothreitol reducing conditions. The gel was stained with Estain 2.0 (GenScript) and the molecular size was estimated by comparing the stained bands with Protein Ladder (GenScript). Figure 1 The results showed that E7-2, E7-3, E7-6, and E7-7 antibodies all showed two bands with molecular weights of 50 kDa and 25 kDa, which were the heavy chain and light chain of the antibody, respectively. Lane 1: E7-2; Lane 2: 7-3; Lane 3: E7-6; Lane 4: 7-7; Lane 5: protein maker.

[0064] Example 3: Verification of Binding Activity of VCE Monoclonal Antibody

[0065] 3.1 Coating antigen: Use coating solution (1.59g anhydrous sodium carbonate, 2.93g anhydrous sodium bicarbonate dissolved in 1000ml distilled water, purchased from Sinopharm) to add 2ug / ml VCE to the enzyme-labeled wells of the reaction plate, 100μL per well, cover the reaction plate and place it at 4℃ overnight for coating. The upper layer is liquid, and the coated VCE is adsorbed on the reaction plate.

[0066] 3.2 Blocking: Rinse the reaction plate once with 350 μL of washing solution per well to wash off the coating solution, pat dry the washing solution (8 g sodium chloride, 0.5 g potassium dihydrogen phosphate, 2.9 g disodium hydrogen phosphate, 0.5 ml Tween20 dissolved in 1000 ml distilled water, purchased from Sinopharm) in the reaction plate on absorbent paper, quickly add 100-200 μL of blocking solution to each well and place in a 37°C incubator for incubation for 2 hours; pour out the blocking solution (5 g bovine serum albumin dissolved in 100 ml washing buffer, purchased from Sinopharm), spin dry with a plate spinner or pat dry the blocking solution on absorbent paper, and then use the reaction plate. If not used immediately, the reaction plate should be placed in a sealed bag and stored at 2-8°C with a drying bag. The drying bag should be located at the bottom of the reaction plate and cannot touch the reaction plate holes.

[0067] 3.3 Add the samples to be tested: After the reaction plate is equilibrated to room temperature, take it out of the sealed bag and add 100ul E7-2, E7-3, E7-6, E7-7 to each well. The initial concentration is 5000ng / ml, and the sample is diluted 2-fold to 0.3ng / ml. Oscillate on a micro-oscillator for 60 seconds to mix the liquid in the well evenly. Place the plate in a 37℃ incubator and incubate for 1 hour.

[0068] 34 Add enzyme-labeled conjugate: Use sample diluent to dilute secondary antibody-HRP at 1:20000, carefully peel off the plate sticker, add 100 μL diluted secondary antibody-HRP (Jackson Immuno Research, 115-005-008 self-produced) to each well, replace the plate sticker and stick it well, oscillate on a micro-oscillator for 60 seconds to mix the liquid in the well evenly, place in a 37°C incubator, and incubate for 1 hour.

[0069] 3.5 Wash the plate: Carefully peel off the plate sticker and rinse the reaction plate six times with 1x washing solution (it is recommended to use a plate washer, with 350μL of solution added to each well). Finally, pat dry on absorbent paper or spin dry with a plate spinner.

[0070] 3.6 Color development: Mix color development solution A (13.6 g sodium acetate, 1.6 g citric acid, 0.3 ml 30% hydrogen peroxide, distilled water added to 500 ml, purchased from Sinopharm) and solution B (0.2 g disodium ethylenediaminetetraacetic acid, 0.95 g citric acid, 50 ml glycerol, 0.15 g TMB, distilled water added to 500 ml, purchased from Sinopharm) in a volume ratio of 1:1 to form a color development working solution, add 100 μL of the color development working solution to each well, replace the plate sticker and stick it well, and place it at 37°C in the dark for 10 minutes.

[0071] 3.7 Reading:

[0072] Carefully peel off the plate sticker, add 50 μL of stop solution to each well to stop the reaction, and use a microplate reader to detect at a single wavelength of 450 nm to measure the absorbance of each well. The reaction data of each group are shown in Table 2:

[0073] Table 2 Absorbance values ​​of each antibody at a single wavelength of 450 nm

[0074] Antibody concentration ng / ml E7-2 E7-3 E7-6 E7-7 5000 2.6623 2.567 2.8826 2.9077 2500 2.6645 2.5752 2.8877 2.7799 1250 2.5022 2.1877 2.8241 2.6012 625 2.7006 2.2257 2.9298 2.8753 312.5 2.5366 2.0259 2.8005 2.5302 156.25 2.4027 1.7638 2.673 2.3204 78.125 1.8704 0.8398 2.1693 1.728 39.0625 1.3715 0.5012 1.692 1.2285 19.53125 0.6919 0.4341 0.8616 0.6503 9.765625 0.3526 0.2306 0.4557 0.3005 4.8828125 0.2269 0.1322 0.2442 0.2087 2.44140625 0.1405 0.0917 0.1713 0.1448 1.220703125 0.0861 0.0662 0.1069 0.0977 0.610351563 0.063 0.0546 0.0715 0.065 0.305175781 0.0529 0.0561 0.0619 0.0583 0 0.052 0.0715 0.0542 0.0542 <![CDATA[EC 50 ]]> 40.78 110.7 34.79 53.09 <![CDATA[R 2 ]]> 0.9977 0.9886 0.9987 0.9961

[0075] Elisa plates were coated with VCE antigen, different concentrations of E7-2 antibody were used to bind to the antigen molecules coated on the plates, and the bound antibodies were detected with HRP-labeled goat anti-mouse IgG Fc antibody. Figure 2 The results showed that E7-2 antibody could bind to VCE in a concentration-dependent and saturable manner.

Claims

1. An isolated antibody, the amino acid sequence of its heavy chain variable domain is shown in SEQ ID NO: 7, the amino acid sequence of its light chain variable domain is shown in SEQ ID NO: 8, the amino acid sequence of its heavy chain constant domain is shown in SEQ ID NO: 9, and the amino acid sequence of its light chain constant domain is shown in SEQ ID NO: 10, wherein the antibody can specifically bind to VCE.

2. The isolated antibody of claim 1, wherein the VCE comprises the amino acid sequence shown in SEQ ID NO:

11.

3. A nucleic acid encoding the isolated antibody of claim 1.

4. A vector comprising the nucleic acid of claim 3.

5. A host cell comprising the vector according to claim 4.

6. A kit comprising the isolated antibody of claim 1 and a second isolated antibody or antigen-binding fragment thereof capable of binding to the VCE of claim 1; wherein: The second isolated antibody or antigen-binding fragment thereof binds to a different epitope on the VCE than the isolated antibody.

7. A kit comprising the isolated antibody of claim 1 and a second isolated antibody or antigen-binding fragment thereof capable of binding to the VCE of claim 1; wherein: The second isolated antibody or antigen-binding fragment thereof binds to the VCE non-competitively with the isolated antibody.

8. A method for detecting VCE residues during the preparation of mRNA vaccines, the method comprising using the isolated antibody described in claim 1 and the kit described in claim 6 or 7 to detect VCE residues.

9. Use of the isolated antibody according to claim 1 or the kit according to claim 6 or 7 in the production of mRNA vaccines.

10. Non-diagnostic use of the kit according to claim 6 or 7 in enzyme-linked immunosorbent assay.

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