CN116874594B - Novel coronavirus mutant XBB.1.5 specific antibody and application thereof - Google Patents
Novel coronavirus mutant XBB.1.5 specific antibody and application thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- C07K2317/565—Complementarity determining region [CDR]
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Abstract
The invention discloses a novel coronavirus mutant XBB.1.5 specific antibody and application thereof. The invention provides an antibody specifically combined with a novel coronavirus mutant strain XBB.1.5, which can be used as a specific antibody for detecting an antigen of the mutant strain XBB.1.5, is used for identifying a novel coronavirus vaccine designed for the mutant strain XBB.1.5, quantitatively detecting the Spike RBD protein content expressed by the vaccine, or is used for quality control of the novel coronavirus vaccine and immunogenicity detection of clinical and preclinical researches, and can also be used as a quality control antibody for detecting protective antibodies in serum after vaccination.
Description
Technical Field
The invention relates to the technical fields of cytoimmunology, molecular biology and antibody preparation, in particular to a novel coronavirus mutant XBB.1.5 specific antibody and application thereof.
Background
The novel coronavirus mutant XBB.1.5 is a recombinant strain consisting of BJ.1 (BA.2.10.1.1) and BM.1.1.1 (BA.2.75.3.1.1.1), the recombination site of which is located in the RBD domain, after which the XBB mutant gradually evolves into the Omik Rong Ya branch, including XBB.1 and XBB.1.5, etc. XBB.1.5 is the novel coronavirus strain with the fastest transmission rate and the strongest immune evasion and infection capacity. The virologist andelu pecies at johns hopkins university, xbb.1.5, unlike the same strain, has an additional key mutation F486P that binds better to human cells, which is the most critical site for antibody neutralization, and thus mutations at this site may be closely related to immune escape. The David d, ho et al article published in Cell indicates that vaccinators and infected individuals' sera have significantly reduced neutralizing capacity against XBB and BQ strains, and that all currently marketed monoclonal antibodies fail to XBB, indicating XBB has the strongest immune escape capacity to date. The Cao Yunlong group of recent experimental results show that the immune escape capacity of XBB.1.5 is equivalent to that of XBB.1, but the binding capacity of XBB.1.5 to hACE2 receptor (compared with XBB.1) is greatly improved. Neutralization experiments show that antibodies formed by the three-needle inactivated vaccine +BF7 breakthrough infection or the mrna vaccine +B5 breakthrough infection have no protection capability on XBB1.5 basically. In order to cope with widely popular XBB.1.5 and emerging variants, the development of new generation vaccines is considered to be the most effective means for resisting the mutant viruses, and the development of vaccines has strict requirements on immunogenicity and effectiveness, so that the detection of antigen content is an indispensable key step in the development process of vaccines. In contrast, for the development of multivalent vaccines, it is also necessary to establish quantitative detection methods for various mutant strain antigens.
The recommendations of the World Health Organization (WHO) consultation group-new vaccines should be directed mainly against the currently dominant variant XBB of the omnix Rong Ya type. Internationally, new coronal vaccine manufacturers such as duel/BioNtech, moderna, novavax are developing newer vaccine versions against xbb.1.5 and other currently popular new coronal strains. In China, two vaccines against mutant XBB have recently been obtained as clinical trial batches. On day 5 and 17, two products developed by Chengdu Weissex/Chuan Dahua Western medicine institute aiming at new crown variant strains such as the latest popular XBB are obtained from the "pharmaceutical clinical test parts" issued by the Chinese national drug administration, and related clinical test researches are performed as soon as possible. The two products are recombinant bivalent novel coronavirus protein vaccine (Sf 9 cells) and recombinant trivalent novel coronavirus trimer protein vaccine (Sf 9 cells). The Wilski organism aspect shows that the two vaccines are novel crown vaccines aiming at variant strains such as XBB and the like, which enter clinical trials in the global first place. It is known that the two vaccine products are added with XBB and BA.5 variant antigens on vaccine products aiming at prototype strains and Delta variant strains, and have universal broad-spectrum cross protection. Wherein the recombinant bivalent new crown vaccine is an upgrade version vaccine developed on the basis of the recombinant new crown vaccine Wilkin.
Development of specific antibody against XBB.1.5 to detect and identify the specificity of vaccine designed by novel mutant strain XBB.1.5 of coronavirus. In order to promote more effective vaccines to come out at a faster speed, a convenient and efficient vaccine development tool will play an important role.
Disclosure of Invention
The invention aims to provide a novel coronavirus mutant XBB.1.5 specific antibody and application thereof.
The invention uses novel coronavirus XBB.1.5 mutant strain Spike RBD protein as immunogen to perform mouse immunity, and obtains hybridoma cell strain for expressing antibody through cell fusion and screening and hybridoma cell subcloning. Experiments prove that the antibody can specifically identify the antigen recognition site of the novel coronavirus XBB.1.5 mutant strain. Further, the invention obtains the amino acid sequence of the antibody and the nucleotide sequence of the encoding gene thereof through hybridoma sequencing.
To achieve the object of the present invention, in a first aspect, the present invention provides a novel coronavirus mutant xbb.1.5-specific antibody or antigen-binding fragment thereof, characterized in that said antibody is selected from any one of 1D6A8, 7a12D9, 9A6a11, 5F8B11, 2C9B4, 5C9F4, 1B5C11, 8E10F 4;
wherein, the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of antibody 1D6A8 are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4. 5 and 6;
the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of antibody 7A12D9 are shown in SEQ ID NO: 7. 8 and 9; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 10. 11, 12;
the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of antibody 9A6A11 are shown in SEQ ID NO: 13. 14, 15; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 16. 17, 18;
the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of antibody 5F8B11 are shown in SEQ ID NO: 19. 20, 21; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 22. 23, 24;
the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of antibody 2C9B4 are shown in SEQ ID NO: 25. 26, 27; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 28. 29, 30;
the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the antibody 5C9F4 heavy chain variable region are shown in SEQ ID NO: 31. 32, 33; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 34. 35, 36;
the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the antibody 1B5C11 heavy chain variable region are shown in SEQ ID NO: 37. 38, 39; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 40. 41, 42;
the amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region of antibody 8E10F4 are shown in SEQ ID NO: 43. 44, 45; the amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ ID NO: 46. 47, 48.
Further, the amino acid sequence of the heavy chain variable region of antibody 1D6A8 is as set forth in SEQ ID NO: shown at 49; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 50;
the amino acid sequence of the heavy chain variable region of antibody 7A12D9 is shown in SEQ ID NO: 51; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 52;
the amino acid sequence of the heavy chain variable region of antibody 9A6A11 is shown in SEQ ID NO: 53; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: indicated at 54;
the amino acid sequence of the heavy chain variable region of the antibody 5F8B11 is shown in SEQ ID NO: indicated at 55; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 56;
the amino acid sequence of the heavy chain variable region of the antibody 2C9B4 is shown in SEQ ID NO: 57; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: indicated at 58;
the amino acid sequence of the antibody 5C9F4 heavy chain variable region is shown in SEQ ID NO: 59; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 60;
the amino acid sequence of the heavy chain variable region of the antibody 1B5C11 is shown in SEQ ID NO: indicated at 61; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: indicated at 62;
the amino acid sequence of the heavy chain variable region of the antibody 8E10F4 is shown in SEQ ID NO: indicated at 63; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 64.
The antibody or antigen binding fragment thereof of the present invention may be selected from the group consisting of Fab, fab ', F (ab') 2, fd, fv, dAb, complementarity determining region fragments, single chain antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies, and the like.
In a second aspect, the invention provides a nucleic acid molecule which encodes said antibody or antigen binding fragment thereof.
Based on the amino acid sequences of the above antibodies or antigen binding fragments thereof, the skilled artisan can obtain nucleotide sequences of nucleic acid molecules encoding the above antibodies or antigen binding fragments thereof. Because of the degeneracy of the codons, the nucleotide sequences of the nucleic acid molecules encoding the antibodies or antigen binding fragments thereof are not unique, and all nucleic acid molecules capable of encoding the production of the antibodies or antigen binding fragments thereof are within the scope of the invention.
In a third aspect, the invention provides biological materials comprising the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria, or transgenic cell lines.
Such cells include, but are not limited to, microbial cells, insect cells, or other animal cells.
In a fourth aspect, the present invention provides an antibody conjugate obtained by coupling the antibody or antigen binding fragment thereof to a label selected from one or more of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label, and a radioactive label.
In a fifth aspect, the invention provides any of the following uses of the antibody or antigen binding fragment thereof or the nucleic acid molecule or the biological material or the antibody conjugate:
(1) Identification or immunogenicity detection for novel coronavirus vaccines;
(2) Quality control for novel coronavirus vaccine products;
(3) For preparing novel coronavirus detection reagents;
(4) Detection reagents for preparing novel coronavirus or vaccine-expressed Spike RBD proteins thereof;
(5) Used as a quality control antibody for detecting protective antibodies in serum after immunization of a novel coronavirus vaccine.
In the application, the identification of the novel coronavirus vaccine is specifically to identify whether the novel coronavirus vaccine contains an antigen of the novel coronavirus (especially the Spike RBD protein of the novel coronavirus XBB.1.5 mutant strain) and the content level thereof or whether the novel coronavirus vaccine is true or false, namely whether the vaccine is a vaccine aiming at the novel coronavirus (especially the novel coronavirus XBB.1.5 mutant strain) or not by utilizing the antibody or the antigen binding fragment thereof provided by the invention.
The identification method comprises detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography and the like.
In the application, the immunogenicity detection is specifically to detect the immune response performance of the novel coronavirus vaccine to the animal organism, including the evaluation of the humoral immune function (such as neutralizing antibody, neutralizing antibody level and antibody affinity) of the immunized animal, etc., and the antibody or the antigen binding fragment thereof provided by the invention can be used as a standard control antibody for the immunogenicity detection of the vaccine.
In the application, the quality control of the novel coronavirus vaccine is specifically to detect whether the quality, the content, the stability and the like of the antigen in the novel coronavirus vaccine are qualified, and the antibody or the antigen binding fragment provided by the invention can be used as a binding antibody of an antigen detection method (detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography and the like) for detecting the quality, the content and the stability of the antigen in the vaccine.
In the application, the novel coronavirus detection specifically comprises the step of detecting whether the novel coronavirus (especially the novel coronavirus XBB.1.5 mutant strain) or the Spike protein thereof or the RBD of the Spike protein exists in a sample or the content level of the RBD is detected by using the antibody or the antigen binding fragment thereof provided by the invention. Detection includes diagnostic purposes (the sample is from a subject, including subject's excretions, oral nasal secretions, etc.) or non-diagnostic purposes (the sample is a cell sample cultured in vitro). The detection method can be enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography and the like.
Reagents for the detection of novel coronaviruses include the antibodies or antigen-binding fragments thereof of the invention, preferably the antibodies or antigen-binding fragments thereof further include a detectable label, and may further include a second antibody carrying a detectable label to detect the antibodies or antigen-binding fragments thereof of the invention.
In such applications, the detection of the content of Spike RBD protein expressed by the novel coronavirus or vaccine thereof is specifically to detect the level of Spike protein or RBD of Spike protein in a sample (the sample is derived from a subject, including excreta of the subject, oronasal secretions, etc.) or non-diagnostic (the sample is a cell sample cultured in vitro) using the antibodies or antigen binding fragments thereof provided by the present invention. As the detection method, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competition method and the like can be used.
In the application, the antibody or the antigen binding fragment thereof provided by the invention can also be used as a quality control antibody for detecting protective antibodies in serum after immunization of a novel coronavirus vaccine, and particularly used as a standard control antibody in the process of detecting the protective antibodies by using detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography and the like.
Preferably, in the application, the novel coronavirus is a novel mutant strain of coronavirus xbb.1.5.
In a sixth aspect, the present invention provides a novel coronavirus or vaccine detection kit comprising said antibody or antigen binding fragment thereof, or comprising said antibody conjugate.
In a seventh aspect, the invention provides a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof.
The pharmaceutical composition is useful for preventing or treating a novel coronavirus infection or a disease associated with a novel coronavirus infection (e.g., a novel coronavirus pneumonia). The antibodies or antigen binding fragments thereof provided herein may be used as the sole active ingredient of a pharmaceutical composition, or in combination with other pharmaceutically active ingredients. The pharmaceutical composition may further comprise pharmaceutically acceptable excipients.
In an eighth aspect, the invention provides the use of said antibody or antigen binding fragment thereof in the diagnosis of infection by a novel coronavirus mutant xbb.1.5.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the invention provides an antibody specifically binding to a novel mutant strain XBB.1.5, which binds to a special spatial epitope, specifically binds to Spike RBD of the novel mutant strain XBB.1.5 only, has higher affinity, does not bind to wild type and other mutant antigens (Alpha, beta, gamma, delta, omicron BA.1, BA.2, BA.3, BA.4/5, BQ.1.1), is an ideal antigen detection antibody of the mutant strain XBB.1.5, can be used for detecting and identifying the mutant strain XBB.1.5 or a novel coronavirus vaccine or multivalent vaccine designed for the mutant strain XBB.1.5, quantitatively detects the content of Spike RBD protein expressed by the vaccine, or is used for quality control of the novel coronavirus vaccine or multivalent vaccine and immunogenicity detection in clinical and preclinical researches, can be used as a quality control antibody for detecting protective antibodies in serum after vaccination, can be used as a high-efficiency vaccine development tool, and is beneficial to accelerating the development of a new generation vaccine.
Drawings
FIG. 1 is a SDS-PAGE identification of a specific novel coronavirus XBB.1.5 mutant antibody in example 3 of the present invention; wherein the molecular weight Marker bands of the protein are 180, 130, 95, 375, 55, 43, 33, 25 and 17kDa from top to bottom.
FIG. 2 shows the SEC-MALS assay of the specific novel coronavirus XBB.1.5 mutant antibody of example 3 of the present invention.
FIG. 3 shows the results of ELISA specific binding assay for the specific novel coronavirus XBB.1.5 mutant antibody of example 3 of the present invention.
FIG. 4 shows the BLI analysis result of the specific novel coronavirus XBB.1.5 mutant antibody in example 3 of the present invention.
FIG. 5 shows the quantitative analysis result of vaccine Spike RBD protein by using the specific novel coronavirus XBB.1.5 mutant antibody in example 3 of the present invention.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
EXAMPLE 1 preparation of novel coronavirus XBB.1.5 antibody specific
In this example, a novel coronavirus xbb.1.5 mutant-specific antibody was prepared as follows:
1. immunization of mice: the novel coronavirus XBB.1.5 mutant Spike RBD protein (purchased from Acrobiosystems) was used as an immunogen, and the novel coronavirus XBB.1.5 mutant Spike RBD protein was used to immunize mice (Balb/c, 6-7 weeks old, body weight 20 g.+ -. 2 g) using a fast immune procedure. After the immunization, the serum of the immunized animal is detected by ELISA method. After immunization, if the immunized animal is able to reach the level of immune response against the immunogen, cell fusion is performed.
2. Screening: the supernatant of the fused cells was screened by ELISA to select out cell clones positive for the specific binding of the novel coronavirus XBB.1.5 mutant Spike RBD protein and not binding to the wild, alpha, beta, gamma, delta, BA.1, BA.2, BA.3, BA.4/5,BQ.1.1Spike RBD proteins.
3. Cloning and expanding culture: positive master clone cells were transferred to 24 well plates for expansion culture. Supernatants were collected from each of the expanded clones and tested by ELISA.
4. Subcloning: subcloning positive parent clone by limiting dilution method and subcloning screening by ELISA method.
5. Hybridoma cell antibody gene sequencing: total RNA of the hybridoma cells is extracted, and the RNA is reversely transcribed into cDNA through an RT-PCR reaction. Cloning antibody light chain and heavy chain sequences, constructing the antibody light chain and heavy chain sequences on a T vector, and then carrying out DNA sequencing analysis to obtain antibody gene sequences.
6. Antibody production and purification: cloning the antibody gene sequence obtained in the step 5 into an expression vector, and transferring the expression vector into HEK293 cells, performing amplification culture, purifying the antibody by adopting a protein A/G affinity chromatography method, and storing the purified antibody in Phosphate Buffer (PBS) by adopting a dialysis method.
EXAMPLE 2 novel coronavirus XBB.1.5 mutant antibody-specific assay
8 monoclonal antibodies of different sequences were obtained as in example 1 and specifically analyzed for the novel coronavirus XBB.1.5 mutant antibody by ELISA as follows:
1. with CBS (0.015 mol/L Na 2 CO 3 ，0.035mol/L NaHCO 3 , 0.0077mol/L NaN 3 pH 9.59) will be novel crownThe Spike RBD proteins of the Rhabdoviral wild type, alpha, beta, gamma, delta, omicron BA.1, BA.2, BA.3, BA.4/5, XBB.1.5 and BQ.1.1 were diluted to 2. Mu.g/mL and added to the wells of the microplate at 100. Mu.L per well. The plates were sealed with a plate and left at 4℃overnight.
2. The wells were discarded, the ELISA plates were dried, washed with PBST wash solution, 300. Mu.L/Kong Jinpao, and the ELISA plates were dried and washed 3 times.
3. mu.L of blocking agent (PBST wash containing 1.5% BSA) was added to each well, the plates were sealed with a plate, incubated at 37℃and then washed.
4. The novel coronavirus XBB.1.5 mutant antibody was diluted to 1. Mu.g/mL with a sample dilution (PBST wash containing 0.5% BSA). Add to the elisa plate 100 μl per well. And (3) sealing the plates by using sealing plates, incubating at 37 ℃ and then cleaning.
5. HRP-Anti-Mouse IgG was diluted to 0.05. Mu.g/mL with sample dilution, 100. Mu.L was added to each well, membrane sealed with sealing plate, incubated at 37℃and then washed.
6. The wells were then filled with 100. Mu.L of color development solution, and the wells were covered with a plate membrane and incubated at 37℃in the absence of light.
7. 50 mu L of stop solution is added into each hole, and the ELISA plate is gently shaken until color development is uniform.
8. Absorbance values at 450nm and 630nm were read with an ELISA reader using OD 450 Knot subtracts OD 630 The values give absorbance values (OD values).
The OD value of each monoclonal antibody is shown in table 1.
TABLE 1 ELISA detection of different antibodies OD values
The above experimental results show that among the 8 novel coronavirus antibodies, clone numbers 9A6a11, 2C9B4, 7a12D9 show high specificity for the novel coronavirus xbb.1.5 mutant strain and have no cross reaction with other mutant strains.
EXAMPLE 3 analytical identification and functional analysis of novel coronavirus XBB.1.5 mutant-specific antibodies
In this example, the novel coronavirus XBB.1.5 mutant-specific antibody (clone No. 9A6A 11) screened in example 2 was analyzed, identified and functionally analyzed by methods known in the art, as follows:
1. SDS-PAGE identification result (figure 1) shows that the molecular weight of two bands of 9A6A11 clone number antibody reduction electrophoresis is about 25kDa and 50kDa respectively, and the purity is more than 95%.
2. The SEC-MALS assay (FIG. 2) showed that the 9A6A11 clone number antibody had a purity of greater than 94.74% and a molecular weight of 143kDa.
3. ELISA binding experiments (FIG. 3) showed that antibody clone 9A6A11 was able to specifically recognize the novel mutant antigen (Spike RBD protein of the novel mutant strain XBB.1.5) of coronavirus but did not bind to the Spike RBD protein of the novel mutant strain XBB.1.1 of coronavirus wild-type and Alpha, beta, gamma, delta, omicronBA.1,2/4/5,3, BQ.1.1.
4. BLI analysis data (FIG. 4) shows that the fitted line represents the law of change over time of affinity and dissociation between XBB.1.5 mutant antigen and 9A6A11 clone number antibody at 1000nM concentration, and the result shows that 9A6A11 clone number antibody binds to novel coronavirus XBB.1.5 mutant antigen with an affinity as high as 2.38 nM.
5. The quantitative detection experimental result (figure 5) of the novel coronavirus XBB.1.5 mutant antigen shows that the antibody of clone number 9A6A11 can quantitatively detect the novel coronavirus XBB.1.5 mutant antigen by adopting a double-antibody sandwich method, so that the content of vaccine Spike RBD protein is obtained, and the sensitivity reaches 1.56ng/ml.
6. Subtype identification of the 9A6A11 clone number antibody showed that the antibody was subtype IgG1 kappa by Ig Isotyping Mouse Instant ELISA Kit (cat 88-50660, invitrogen).
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. Novel coronavirus mutant xbb.1.5-specific antibody or antigen-binding fragment thereof, characterized in that said antibody is 9A6a11;
the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2, CDR3 of antibody 9A6A11 are shown in SEQ ID NO: 13. 14, 15; the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2, CDR3 of antibody 9A6A11 are shown in SEQ ID NO: 16. 17, 18.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region of antibody 9A6a11 is as set forth in SEQ ID NO: 53; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: indicated at 54.
3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, fab ', F (ab') 2, fd, fv, dAb, complementarity determining region fragment, single chain antibody, animal-derived antibody, chimeric antibody, humanized antibody, bispecific antibody, or multispecific antibody.
4. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3.
5. A biological material comprising the nucleic acid molecule of claim 4, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineering bacterium or a transgenic cell line.
6. An antibody conjugate, characterized in that it is obtained by coupling the antibody or antigen-binding fragment thereof according to any one of claims 1-3 to a label; the marker is selected from one or more of enzyme markers, biotin markers, fluorescent dye markers, chemiluminescent dye markers and radioactive markers.
7. The antibody or antigen binding fragment thereof of any one of claims 1-3 or the nucleic acid molecule of claim 4 or the biological material of claim 5 or any one of the following uses of the antibody conjugate of claim 6:
(1) Identification or immunogenicity detection for novel coronavirus vaccines;
(2) Quality control for novel coronavirus vaccine products;
(3) For preparing novel coronavirus detection reagents;
(4) Detection reagents for preparing novel coronavirus or vaccine-expressed Spike RBD proteins thereof;
(5) Used as a quality control antibody for detecting protective antibodies in serum after immunization of a novel coronavirus vaccine.
8. The use according to claim 7, characterized in that the novel coronavirus is a novel coronavirus mutant xbb.1.5.
9. A novel coronavirus or vaccine detection kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-3, or comprising the antibody conjugate according to claim 6.
10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3.
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