CN114213541B

CN114213541B - Monoclonal antibody of totipotent nuclease and preparation method thereof

Info

Publication number: CN114213541B
Application number: CN202111560816.6A
Authority: CN
Inventors: 曹林; 徐晓昱; 徐灵杰; 鲜婷婷; 李悦
Original assignee: Nanjing Novozan Biotechnology Co ltd
Current assignee: Nanjing Novozan Biotechnology Co ltd
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2022-12-30
Anticipated expiration: 2041-12-20
Also published as: CN114213541A

Abstract

The invention provides a monoclonal antibody combined with totipotent nuclease and a preparation method thereof, belonging to the field of immunity. The monoclonal antibody combined with the totipotent nuclease can be used for detecting the totipotent nuclease remained in the production process of mRNA vaccines.

Description

Monoclonal antibody of totipotent nuclease and preparation method thereof

Technical Field

The invention belongs to the field of immunology, and particularly relates to a monoclonal antibody of a totipotent nuclease. The invention also relates to a preparation method and application of the monoclonal antibody.

Background

SuperNuclease is a genetically engineered endonuclease derived from Serratia marcescens (Serratia marcescens). Supernucleic can degrade double-stranded, single-stranded, linear, and circular DNA and RNA, and completely degrade nucleic acid into 5' -monophosphate oligonucleotide 3-5 bases in length. It is also called "totipotent nuclease" (SM 2) because it is highly efficient at degrading any form of DNA and RNA.

The totipotent nuclease monomer contains 245 amino acid residues, has the molecular weight of 30kDa, and is connected into an active dimer structure by two disulfide bonds. The totipotent nuclease is an endonuclease, which can efficiently degrade DNA and RNA in any form (including double-stranded, single-stranded, linear and circular) into 5' monophosphate nucleotide with 2-5 bases. The totipotent nuclease can effectively reduce the viscosity of the protein sample and remove the pollution of nucleic acid in the protein sample. While reducing tack, there are a number of other uses, such as: the sample processing time is reduced, the protein yield is increased, the precipitation and the supernatant are separated more thoroughly during centrifugation, and the solution is more convenient to centrifuge (especially ultrafiltration); improving the efficiency of chromatographic purification, and the like.

The detection of the residue of the totipotent nuclease is also an important link, so that the development of a monoclonal antibody with high sensitivity and high specificity for detecting the totipotent nuclease is urgent.

Disclosure of Invention

The present invention aims to provide an isolated antibody or an antigen-binding fragment thereof that specifically binds to a pluripotent nuclease, for detecting the residue of the pluripotent nuclease.

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

(a) A heavy chain variable domain (VH) comprising:

heavy chain complementarity determining region 1 (HCDR 1) of the amino acid sequence shown in SEQ ID NO. 1, HCDR2 of the amino acid sequence shown in SEQ ID NO. 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO. 3; and

(b) A light chain variable domain (VL) comprising:

light chain complementarity determining region 1 (LCDR 1) 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;

wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to a pluripotent nuclease, preferably the pluripotent nuclease comprises the amino acid sequence shown in SEQ ID NO. 11.

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

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

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

In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain constant domain (CH) of the amino acid sequence set forth in SEQ ID NO. 9 and a light chain constant domain (CL) of the amino acid sequence set forth in SEQ ID NO. 10.

In a second aspect of the invention, there is provided a method of making the isolated antibody or antigen-binding fragment thereof of the first aspect.

In some embodiments, the hybridoma cell is obtained by performing hybridoma fusion after obtaining a mouse spleen cell by immunizing a mouse using a pluripotent nuclease as an antigen, and screening for a hybridoma cell capable of secreting an antibody expressing a binding to the pluripotent nuclease; sequencing the hybridoma cells to obtain the amino acid sequences and the nucleic acid sequences of the variable domains and the constant domains of the antibodies, and recombining and expressing the required antibodies according to the sequence information.

In some embodiments, the amino acid sequence of the pluripotent nuclease is shown as SEQ ID NO. 11.

In a third aspect of the invention, there is provided a kit comprising an isolated antibody or antigen-binding fragment thereof according to the first aspect of the invention, and comprising a second isolated antibody or antigen-binding fragment thereof capable of binding to the pluripotent nuclease; wherein the second isolated antibody or antigen binding fragment thereof binds to a different epitope of the pluripotent nuclease than the isolated antibody or antigen binding fragment thereof.

In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) A heavy chain variable domain (VH) comprising: heavy chain complementarity determining region 1 (HCDR 1) of the amino acid sequence shown in SEQ ID NO. 1, HCDR2 of the amino acid sequence shown in SEQ ID NO. 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO. 3; and (b) a light chain variable domain (VL) comprising: light chain complementarity determining region 1 (LCDR 1) 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; wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to a totipotent nuclease, preferably the totipotent nuclease comprises the amino acid sequence shown in SEQ ID NO. 11.

In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO. 7 and the VL comprises the amino acid sequence set forth in SEQ ID NO. 8.

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

In a fourth aspect of the invention, there is provided a further kit comprising the isolated antibody or antigen-binding fragment thereof of the first aspect and comprising a second isolated antibody or antigen-binding fragment thereof capable of binding to the pluripotent nuclease; wherein the second isolated antibody or antigen-binding fragment thereof binds the pluripotent nuclease non-competitively to the isolated antibody or antigen-binding fragment thereof.

In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises: (a) A heavy chain variable domain (VH) comprising: heavy chain complementarity determining region 1 (HCDR 1) of the amino acid sequence shown in SEQ ID NO. 1, HCDR2 of the amino acid sequence shown in SEQ ID NO. 2, and HCDR3 of the amino acid sequence shown in SEQ ID NO. 3; and (b) a light chain variable domain (VL) comprising: light chain complementarity determining region 1 (LCDR 1) 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; wherein the antibody or antigen-binding fragment thereof is capable of specifically binding to a pluripotent nuclease, preferably the pluripotent nuclease comprises the amino acid sequence shown in SEQ ID NO. 11.

In the fifth aspect of the invention, a method for detecting the residual totipotent nuclease in the preparation process of mRNA vaccine is provided, and the separated antibody or antigen binding protein and the kit are adopted to detect the residual totipotent nuclease.

In a sixth aspect of the invention, there is provided the use of the above antibody or antigen binding protein, and a kit comprising the above antibody or antigen binding fragment in the production of an mRNA vaccine. The application of the mRNA vaccine in the production of the vaccine refers to the application in each link of the vaccine production, including but not limited to the production process and the production end, and the antibody or the antigen binding fragment and the kit thereof can be applied to remove residual totipotent nuclease under the condition that the production link relates to the residue of the totipotent nuclease.

In a seventh aspect of the invention, the use of the aforementioned kit in enzyme-linked immunosorbent assay is provided.

The invention also provides nucleic acids encoding the isolated antibodies or antigen-binding fragments thereof; the invention also provides a vector comprising said nucleic acid, capable of expressing said nucleic acid of the isolated antibody or antigen-binding fragment thereof; the invention also provides a host cell comprising the vector.

The amino acid sequence information of the present invention is shown in table 1:

TABLE 1 amino acid sequence

Term(s) for

It should 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.

The term "epitope" means a protein determinant capable of specifically binding to an antibody. Epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

The terms "antibody," "antibody portion," "antigen-binding fragment," or "antibody construct" are used in the broadest sense and encompass a variety of 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.

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 consist of 5 elementary heterotetramer units and an additional polypeptide called the J chain and contain 10 antigen binding sites, while IgA antibodies comprise 2-5 elementary 4 chain units that can polymerize to form multivalent aggregates in combination with the J chain. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has one variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each alpha and gamma chain and four CH domains for the mu and epsilon isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain at its other terminus. VL is aligned with VH, and CL is aligned with the first constant domain (CH 1) of the heavy chain. It is believed that particular amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. VH and VL pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies see, for example, basic and Clinical Immunology, 8 th edition, daniel p.sties, abba i.terr and tristramg.parsolw (ed.), appleton & lange, norwalk, conn.,1994, page 71 and chapter 6. The L chain from any vertebrate species can be assigned to one of two distinctly different types (termed κ and λ) depending on the amino acid sequence of its constant domain. 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 designated α, δ, ε, γ and μ, respectively. The γ and α classes are further divided into subclasses based on the relatively small differences in CH sequence and function.

An "isolated" antibody is one that has been identified, isolated, and/or recovered from a component of its production environment (e.g., naturally occurring or recombinant). Preferably, an isolated polypeptide is not associated with all other components from its production environment. Contaminating components of their production environment (such as those produced by recombinant transfected cells) are substances that generally interfere with the research, diagnostic, or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Isolated antibodies include antibodies in situ within recombinant cells, in which at least one component of the antibody's natural environment will not be present. Typically, however, an isolated polypeptide, antibody or construct will be prepared by at least one purification step.

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

The term "variable" refers to the fact that certain segments of a variable domain differ greatly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable domains. Instead, it is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs) in the light chain variable domain and the heavy chain variable domain. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, predominantly in a β -sheet configuration, connected by three CDRs, which form loops connecting and in some cases forming part of the β -sheet structure. The CDRs in each chain are held together tightly by the FR regions and, together with the CDRs in the other chain, contribute to the antigen-binding site of the antibody (see Kabat et al, sequences of Immunological Interest, fifth edition, national Institute of Health, bethesda, md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, deamidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma cultures and are free of contamination by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal Antibodies for use according to 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, 2 nd edition 1988); hammerling et al, monoclonal Antibodies and T-Cell hybrids 563-681 (Elsevier, N.Y., 1981)); recombinant DNA methods (see, e.g., U.S. patent No. 4,816,567); phage display techniques (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.Methods284 (1-2): 119-132 (2004)).

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., a variable domain), which contains an antigen binding site. The constant domain contains the CH1, CH2 and CH3 domains of the heavy chain (collectively referred to as CH) and the CHL (or CL) domain of the light chain.

The term "complementarity determining regions" or "CDRs" is used to refer to hypervariable regions defined by the Kabat system. See Kabat et al, sequences of Proteins of Immunological Interest, published Health Service 5, national Institutes of Health, bethesda, md. (1991).

As used herein, the terms "specifically binds," "specifically recognizes," or "specific for … …" refer to a measurable and reproducible interaction such as binding between a target and an antigen binding protein (such as a mAb) that 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 a mAb) that specifically binds a target (which may be an epitope) is one that binds this target with greater affinity, avidity, more easily, and/or for a longer duration than it binds other targets (such as a mAb). In some embodiments, the extent of binding of an antigen binding protein (such as a mAb) to an unrelated target is less than about 10% of the binding of the antigen binding protein (such as a mAb) to the target, as measured, for example, by a Radioimmunoassay (RIA). In some embodiments, an antigen binding protein, such as a mAb, that specifically binds a target has the following dissociation constant (KD): less than or equal to 10-5M, less than or equal to 10-6M, less than or equal to 10-7M, less than or equal to 10-8M, less than or equal to 10-9M, less than or equal to 10-10M, less than or equal to 10-11M, or less than or equal to 10-12M. In some embodiments, the antigen binding protein specifically binds to an epitope on the protein that is conserved among proteins from different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding.

Brief Description of Drawings

FIG. 1 shows the result of SDS-PAGE electrophoresis of a full-length anti-totipotent nuclease monoclonal antibody.

FIG. 2 shows the binding results of the best-functioning monoclonal antibody against a pluripotent nuclease.

Detailed Description

Example 1 preparation of a pluripotent nuclease antibody

Mice were immunized with the nuclease at 100ug, 50ug, and 50ug, then Hybridoma cells were prepared, and the hybridomas E6-7, E6-9, E6-14, and E6-15 were selected, followed by Hybridoma cell cloning (Proetzel, gabrile; ebersbach, hilmar (2012) [ Methods in Molecular Biology ] Antibody Methods and Protocols Volume 901| Hybridoma Technology for the Generation of Monoclonal antibodies, 8978 zft 8978/978-1-61779-931-0 (Chapter 7), 117-135.).

The amino acid sequence of the totipotent nuclease:

MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN

EXAMPLE 2 expression and purification of monoclonal antibodies

The hybridoma selected in example 1 is lysed to extract mRNA, the mRNA is subjected to reverse transcription to form cDNA, after one round of amplification of the cDNA, igG heavy and light chain gene segments are taken, and then the taken IgG heavy and light chain gene segments are spliced into an expression vector to construct a plasmid. The plasmid adopts ExpicHO ^TM Reagent co-transfects HEK293 suspension culture cells for transient expression. Cell density was maintained at 6X 10 at transfection ⁶ cells/mL, expicHO ^TM Reagent DNA (plasmid) ratio of 4:1. Cell at 37 ℃ 8% CO ₂ Shaking culture is carried out in an incubator at 120 r/min. After transfection for 16-18h, 180. Mu.L Expifeacylamine was added ^TM CHO Enhance and 4.8mL ExpicHO ^TM Feed was then transferred to 32 ℃,5%,120rpm shake culture. Cell supernatants were collected 11 days after transfection. The purification was carried out after filtration through a 0.22 μm filter. Prior to purification, tubing and protein A columns were depyrogenated with 0.2M NaOH. The column was re-equilibrated with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). The harvested cell culture supernatant was then diluted with 2 × buffer 1:1 as described above and filter sterilized. The filtered supernatant and the protein A column were incubated at room temperature for 2 hours, and after washing the column with 1 × the above buffer, igG was eluted using sterile 0.1M sodium citrate (pH 3.5), and the eluate was collected and neutralized with one-ninth volume of sterile 1M Tris-HCl (pH 9). Under sterile conditions, the product buffer was exchanged for PBS (pH 7.4) to remove any elution buffer and the product was concentratedAnd (3) sampling. After concentration, the antibody was quantified by OD280nm using an extinction coefficient Ec of 1.43 (0.1%).

Antibodies were numbered along with the number of hybridomas and purified E6-7, E6-9, E6-14, E6-15 antibodies were analyzed by SDS-PAGE using a BioRad electrophoresis system with 10% pre-gel (GenScript) under 50mM dithiothreitol reduction conditions. The gel was stained with estain2.0 (GenScript) and the molecular size was estimated by comparing the stained band to Protein Ladder (GenScript). The results in FIG. 1 show that the E6-7, E6-9, E6-14, and E6-15 antibodies all present two bands with molecular weights of 50kDa and 25kDa, respectively, the heavy and light chains of the antibodies. Lane 1: e6-7; lane 2: e6-9; lane 3: e6-14; lane 4: e6-15; lane 5: and (3) protein marker.

Example 3 binding Activity verification of a pluripotent nuclease monoclonal antibody

3.1 coating antigen: using a coating solution (1.59 g of anhydrous sodium carbonate, 2.93g of anhydrous sodium bicarbonate dissolved in 1000ml of distilled water, purchased from national medicine), 2ug/ml of a universal nuclease was added to the wells of the reaction plate labeled with 100. Mu.L per well, the reaction plate was coated overnight at 4 ℃ with a lid, and the upper layer was a liquid, and the coated universal nuclease was adsorbed on the reaction plate.

3.2 sealing: washing the reaction plate once by using a washing solution (8 g of sodium chloride, 0.5g of monopotassium phosphate, 2.9g of disodium hydrogen phosphate and 0.5ml of Tween20, wherein the washing solution is dissolved in 1000ml of distilled water and purchased from Chinese medicaments) with 350 mu L per hole to wash out coating solution, beating the washing solution in the reaction plate on absorbent paper to be dry, quickly adding 100-200 mu L of sealing solution into each hole, placing the reaction plate in an incubator at 37 ℃, and incubating for 2 hours; the reaction plate can be used by pouring off the blocking solution (bovine serum albumin 5g dissolved in 100ml washing buffer solution purchased from Chinese medicine) and spin-drying with a plate-throwing machine or patting dry the blocking solution on absorbent paper, if the reaction plate is not used immediately, the reaction plate is placed in a sealed bag and added with a drying bag to be stored at 2-8 ℃, and the drying bag is required to be positioned at the bottom of the reaction plate and can not contact with the holes of the reaction plate.

3.3 adding the sample to be tested: the reaction plate was equilibrated to room temperature and removed from the sealed bag, 100ul E6-7, E6-9, E6-14, E6-15 antibody against the holonuclease was added to each well at an initial concentration of 5000ng/ml, diluted 2-fold in a gradient to 0.3ng/ml, shaken on a micro-shaker for 60 seconds to mix the liquids in the wells uniformly, placed in a 37 ℃ incubator, and incubated for 1 hour.

3.4 addition of enzyme-labeled conjugates: using a sample diluent at 1:20000 diluted secondary antibody-HRP (Jackson Immuno Research, 115-005-008), carefully remove the plate, add 100. Mu.L of diluted secondary antibody-HRP to each well, replace the plate and stick, shake on a micro-shaker for 60 seconds to mix the liquid in the wells uniformly, place in an incubator at 37 ℃ and incubate for 1 h.

3.5 plate washing: carefully remove the plate, wash the plate six times with 1 Xwashing solution (recommended plate washer, 350. Mu.L of solution per well), and finally dry on absorbent paper or spin-dry with a plate-throwing machine.

3.6 color development: mixing the color developing solution A (13.6 g of sodium acetate, 1.6g of citric acid, 0.3ml of 30% hydrogen peroxide and distilled water to 500ml, which are purchased from national medicines.) and the solution B (0.2 g of disodium ethylenediamine tetraacetic acid, 0.95g of citric acid, 50ml of glycerol, 0.15g of TMB, distilled water to 500ml, which are purchased from national medicines.) according to the volume ratio of 1:1 to obtain a color developing working solution, adding 100 mu L of the color developing working solution into each hole, replacing plates, sticking, and placing at 37 ℃ for a dark reaction for 10 minutes.

3.7 reading: carefully uncovering the plate, adding 50 mu L of stop solution into each hole to stop the reaction, detecting with 450nm single wavelength of an enzyme-labeling instrument, and measuring the light absorption value of each hole. The data for each set of reactions are shown in table 2:

TABLE 2 binding Capacity of different antibodies to antigen at different concentrations

Antibody concentration (ng/ml)	E6-7	E6-9	E6-14	E6-15
					5000	2.8694	2.656	2.7863	2.7917
2500	2.8466	2.6458	2.6736	2.6544
					1250	2.6517	2.6593	2.5151	2.6866
625	2.4394	2.4706	2.482	2.6664
					312.5	1.9397	2.1555	2.1675	2.1933
156.25	1.3931	1.6939	1.6689	1.74
					78.125	0.7178	1.1179	1.1232	1.1227
39.0625	0.3608	0.6193	0.654	0.6428
					19.53125	0.2388	0.3091	0.3228	0.2874
9.765625	0.1524	0.2171	0.2038	0.2154
					4.882812	0.0857	0.1468	0.1416	0.154
2.441406	0.0633	0.0935	0.0939	0.0925
					1.220703	0.0479	0.0615	0.058	0.0617
0.610351	0.0472	0.0497	0.0469	0.0515
					0.305175781	0.0499	0.0467	0.0464	0.0494
Negative of	0.0489	0.0475	0.0472	0.0474
					EC ₅₀	182.9	108.2	110.9	109.3
R ²	0.9993	0.9995	0.999	0.9982

Table 2 shows the binding results of 4 excellent-function monoclonal antibodies against the pluripotent nuclease. An Elisa plate is coated with a totipotent nuclease antigen, E6-14 antibodies with different concentrations are combined with antigen molecules coated on the plate, and the combined antibodies are measured by an HRP-labeled goat anti-mouse IgG Fc antibody. The results show that these 4 groups of antibodies can bind to a totipotent nuclease and exhibit concentration dependence and saturability.

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Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 10

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 11

<211> 266

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Arg Phe Asn Asn Lys Met Leu Ala Leu Ala Ala Leu Leu Phe Ala

1 5 10 15

Ala Gln Ala Ser Ala Asp Thr Leu Glu Ser Ile Asp Asn Cys Ala Val

20 25 30

Gly Cys Pro Thr Gly Gly Ser Ser Asn Val Ser Ile Val Arg His Ala

35 40 45

Tyr Thr Leu Asn Asn Asn Ser Thr Thr Lys Phe Ala Asn Trp Val Ala

50 55 60

Tyr His Ile Thr Lys Asp Thr Pro Ala Ser Gly Lys Thr Arg Asn Trp

65 70 75 80

Lys Thr Asp Pro Ala Leu Asn Pro Ala Asp Thr Leu Ala Pro Ala Asp

85 90 95

Tyr Thr Gly Ala Asn Ala Ala Leu Lys Val Asp Arg Gly His Gln Ala

100 105 110

Pro Leu Ala Ser Leu Ala Gly Val Ser Asp Trp Glu Ser Leu Asn Tyr

115 120 125

Leu Ser Asn Ile Thr Pro Gln Lys Ser Asp Leu Asn Gln Gly Ala Trp

130 135 140

Ala Arg Leu Glu Asp Gln Glu Arg Lys Leu Ile Asp Arg Ala Asp Ile

145 150 155 160

Ser Ser Val Tyr Thr Val Thr Gly Pro Leu Tyr Glu Arg Asp Met Gly

165 170 175

Lys Leu Pro Gly Thr Gln Lys Ala His Thr Ile Pro Ser Ala Tyr Trp

180 185 190

Lys Val Ile Phe Ile Asn Asn Ser Pro Ala Val Asn His Tyr Ala Ala

195 200 205

Phe Leu Phe Asp Gln Asn Thr Pro Lys Gly Ala Asp Phe Cys Gln Phe

210 215 220

Arg Val Thr Val Asp Glu Ile Glu Lys Arg Thr Gly Leu Ile Ile Trp

225 230 235 240

Ala Gly Leu Pro Asp Asp Val Gln Ala Ser Leu Lys Ser Lys Pro Gly

245 250 255

Val Leu Pro Glu Leu Met Gly Cys Lys Asn

260 265

Claims

1. An isolated antibody or antigen-binding fragment thereof, comprising:

(a) A heavy chain variable domain (VH) comprising:

(b) A light chain variable domain (VL) comprising:

wherein the antibody or the antigen binding fragment thereof can be specifically bound to totipotent nuclease, and the totipotent nuclease has an amino acid sequence shown in SEQ ID NO. 11.

2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises the amino acid sequence set forth in SEQ ID No. 7 and the VL comprises the amino acid sequence set forth in SEQ ID No. 8.

3. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 or 2, further comprising a heavy chain constant domain (CH) of the amino acid sequence set forth in SEQ ID NO. 9 and a light chain constant domain (CL) of the amino acid sequence set forth in SEQ ID NO. 10.

4. The isolated antibody or antigen-binding fragment thereof of claim 1, which is rodent, partially humanized, or fully humanized.

5. The isolated antibody or antigen-binding fragment thereof of claim 4, which is murine.

6. A nucleic acid encoding the isolated antibody or antigen-binding fragment thereof of any one of claims 1-5.

7. A vector comprising the nucleic acid of claim 6.

8. A host cell comprising the vector of claim 7.

9. A method of making the isolated antibody or antigen-binding fragment thereof of any one of claims 1-5.

10. A method of detecting a pluripotent nuclease residual during mRNA vaccine production, the method comprising detecting the pluripotent nuclease residual using the isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 5.

11. Use of the isolated antibody or antigen-binding fragment thereof of any one of claims 1-5 in the manufacture of an mRNA vaccine.