CN117683121B - Anti-varicella-zoster virus antibodies and uses thereof - Google Patents
Anti-varicella-zoster virus antibodies and uses thereof Download PDFInfo
- Publication number
- CN117683121B CN117683121B CN202410126538.0A CN202410126538A CN117683121B CN 117683121 B CN117683121 B CN 117683121B CN 202410126538 A CN202410126538 A CN 202410126538A CN 117683121 B CN117683121 B CN 117683121B
- Authority
- CN
- China
- Prior art keywords
- varicella
- zoster virus
- antibody
- amino acid
- acid sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
- C07K16/088—Varicella-zoster virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/56983—Viruses
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of antibodies, in particular to an anti-varicella-zoster virus antibody and application thereof. The antibody or the antigen binding fragment thereof provided by the invention can be specifically combined with varicella-zoster virus or gE protein thereof, has higher affinity, and can realize specific, sensitive and accurate detection of varicella-zoster virus or gE protein thereof. The detection reagent provided by the invention based on the antibody or the antigen binding fragment thereof can realize detection of varicella-zoster virus or gE protein thereof in a sample, has higher sensitivity, specificity, accuracy and precision, has low detection cost, can be used for quantitative detection of gE protein in varicella-zoster virus vaccine, detection of varicella-zoster virus vaccine humoral immunity titer in mice, detection of varicella-zoster virus vaccine humoral immunity titer in human bodies and the like, and has better application value.
Description
Technical Field
The invention relates to the technical field of antibodies, in particular to an anti-varicella-zoster virus antibody and application thereof.
Background
Varicella-zoster virus (VZV), also known as human herpesvirus type 3, is a double stranded DNA virus belonging to the family human herpesviridae. The virus may be infected at any stage of human life, and the older the population, the higher the infection rate of varicella-zoster virus. As the problem of aging population becomes more serious worldwide, the study of varicella-zoster virus is receiving more and more attention.
In addition to encoding biologically active related proteins involved in the replication transcription of the virus, VZV has a total of 8 glycoproteins gB, gC, gH, gI, gL, gK, gM and gE, respectively. gB. The gH and gE exist on the surface of the virus in a relatively abundant way, so that the related research of the VZV antibody and vaccine is mainly carried out on the three glycoproteins at present, wherein the gE protein is one of the glycoproteins with the strongest antigenicity of the VZV and is also the glycoprotein with the highest expression on the surface of the virus and infected cells, and the gH and gE can induce the cellular immunity. Thus, most of the current VZV vaccines are developed against gE proteins.
Up to now, only 2 versions of the herpes zoster virus vaccine are marketed worldwide in batches, zostavax for moxadong and Shingrix for GSK, respectively. In addition, recombinant protein vaccines have been studied that employ the extracellular domain of recombinant VZVgE protein expressed by CHO cells. Attenuated, adenovirus vectors, and mRNAs are also in the development process. At present, the development of the VZV vaccine is still in an early stage, and vaccine related detection means have not been formed and standardized yet.
In the development of vaccines, the amount of VZVgE protein produced needs to be determined, and the immune titer needs to be detected in animal experiments as well as clinical experiments. Therefore, it is necessary to develop antibodies that bind VZVgE proteins efficiently and to provide an effective detection method for the development and application of VZV vaccines based on the detection reagents associated with antibody development.
Disclosure of Invention
The present invention provides anti-varicella zoster virus antibodies and uses thereof.
Specifically, the invention provides the following technical scheme:
The present invention provides an anti-varicella zoster virus antibody or antigen binding fragment thereof which is any one of the following (1) to (2):
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of the CDR2 is KVS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:5 is shown in the figure;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 6. 7 and 8; the amino acid sequence of CDR1 is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is ATS, the amino acid sequence of CDR3 is shown in SEQ ID NO: shown at 10.
Preferably, the antibody or antigen binding fragment thereof is any one of the following (1) - (2):
(1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 12;
(2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14.
Preferably, the antibody or antigen binding fragment thereof is selected from the group consisting of monoclonal antibodies, fab ', F (ab') 2, fd, fv, complementarity determining region fragments, single chain antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies, or multispecific antibodies.
In some embodiments of the invention, an antibody clone 1B10 is provided whose amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region are set forth in SEQ ID NOs: 1.2 and 3; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of the CDR2 is KVS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:5 is shown in the figure; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 12.
The amino acid sequence of the antibody clone No. 1B10 is specifically as follows:
amino acid sequence of heavy chain variable region CDR 1-3:
CDR1：DTEFPSHD；
CDR2：ITNDGGST；
CDR3：ARHYGNYAWFPY；
Amino acid sequence of heavy chain variable region:
EVQLVESGGGLVQPGESLKLSCASNDTEFPSHDMSWVRKTPEKRLELVAAITNDGGSTYYPDTMERRFIISRDNTKKTLYLQMSSLRSEDTALYYCARHYGNYAWFPYWGQGTLVTVSA.
amino acid sequence of CDR1-3 of the light chain variable region:
CDR1：QSIVHSNGNTY；
CDR2：KVS；
CDR3：FQGSHVPRT；
Amino acid sequence of the light chain variable region:
DVLLTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGLSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPRTFGGGTKLEIK.
In some embodiments of the invention, an antibody clone number 4G2 is provided whose amino acid sequences of complementarity determining regions CDR1, CDR2, CDR3 of the heavy chain variable region are set forth in SEQ ID NOs: 6. 7 and 8; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is ATS, the amino acid sequence of CDR3 is shown in SEQ ID NO:10 is shown in the figure; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14.
The amino acid sequence of the antibody clone No. 4G2 is specifically as follows:
amino acid sequence of heavy chain variable region CDR 1-3:
CDR1：GYSIYSDYA；
CDR2：ISYSGST；
CDR3：ARDDRFLSYFDY；
Amino acid sequence of heavy chain variable region:
DVQLQESGPGLVKPSQSLSLSCTVTGYSIYSDYACNWFRQFPGNKLEWMGFISYSGSTSYHPSLKSRISITRDTSKNQFFLRLNSVTTEDTATYYCARDDRFLSYFDYWGQGTTLTVSS.
amino acid sequence of CDR1-3 of the light chain variable region:
CDR1：SSVSY；
CDR2：ATS；
CDR3：QWSSSPPT；
Amino acid sequence of the light chain variable region:
QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPTRFSGSGSGTSYSLTISRVETEDAATYYCQQWSSSPPTFGGGTKLEMK.
The antibody or the antigen binding fragment thereof provided by the invention can specifically bind to varicella-zoster virus or gE protein thereof, and has higher affinity.
The present invention provides a nucleic acid molecule encoding the varicella-zoster virus antibody or antigen binding fragment thereof as described above.
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.
The invention also provides biological materials containing the nucleic acid molecules; the biological material is an expression cassette, a vector or a host cell.
The above-mentioned expression cassette can be obtained by ligating a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and/or ligating a transcription or translation regulatory element such as a terminator downstream thereof.
Such vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, artificial chromosome vectors, and the like.
The host cells include microbial cells, insect cells, or other animal cells.
The invention provides an antibody conjugate, which is obtained by coupling the anti-varicella-zoster virus antibody or antigen binding fragment thereof with a marker, wherein the marker is selected from one or more of enzyme markers, biotin markers, fluorescent dye markers, chemiluminescent dye markers and radioactive markers.
The present invention provides an antibody composition for varicella-zoster virus comprising the antibodies in (1) and (2) below:
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of the CDR2 is KVS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:5 is shown in the figure;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 6. 7 and 8; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is ATS, the amino acid sequence of CDR3 is shown in SEQ ID NO: shown at 10.
The antibodies of (1) - (2) above bind to different epitopes of the antigen space.
The antibody composition can be used as a pairing antibody for detecting varicella-zoster virus or gE protein thereof by a double antibody sandwich ELISA (enzyme linked immunosorbent assay) method, and two antibodies in the antibody composition are respectively used as a coating antibody and a detection antibody in the double antibody sandwich ELISA method. Wherein the detection antibody may also carry a detectable label.
In some embodiments of the invention, the antibody described in (1) is used as a coating antibody and the antibody described in (2) is used as a detection antibody.
The present invention provides a detection kit comprising the antibody or antigen-binding fragment thereof for varicella-zoster virus described above, or comprising the antibody conjugate, or an antibody composition comprising the varicella-zoster virus.
The kit can be used as a detection kit for varicella-zoster virus or gE protein thereof.
In some embodiments of the invention, the detection kit is a double antibody sandwich ELISA detection kit.
Based on the antibody or the antigen binding fragment thereof or the antibody conjugate or the antibody composition, the content of varicella-zoster virus gE protein in the sample can be specifically detected by a double-antibody sandwich ELISA method, and the antibody has higher sensitivity, specificity and accuracy.
In other embodiments of the invention, the detection kit is an indirect ELISA detection kit.
The antibodies or antigen binding fragments thereof or antibody conjugates of the invention can also be used as a positive reference antibody for detecting the antibody level of anti-varicella-zoster virus or the gE protein thereof by an indirect ELISA method.
For ease of detection, the kit may also contain other reagents for ELISA detection including, but not limited to, secondary antibodies carrying a detectable label, varicella-zoster virus or gE protein standards thereof, PBST wash, blocking solution, chromogenic solution, stop solution, and the like.
Based on the function of the antibody or antigen binding fragment thereof of the present invention, the present invention provides any one of the following uses of the antibody or antigen binding fragment thereof of the varicella-zoster virus or the nucleic acid molecule or the biological material or the antibody conjugate or the antibody composition or the detection kit:
(1) Use of varicella-zoster virus or gE protein thereof in the detection of the presence or level of same in a sample for non-disease diagnostic and therapeutic purposes;
(2) Use in the preparation of a reagent for detecting the presence or level of varicella-zoster virus or a gE protein thereof in a sample;
(3) Use in the preparation of a reagent for diagnosing varicella-zoster virus infection;
(4) Use in the identification of varicella zoster virus vaccines;
(5) Use in the preparation of a product for immunogenicity detection of varicella-zoster virus vaccine;
(6) Use in quality control of varicella-zoster virus vaccine;
(7) Use of the antibody as a positive reference antibody for the detection of varicella-zoster virus antibody levels in serum after immunization with varicella-zoster virus vaccine.
In the above (1), the use comprises detecting the presence or the level of varicella-zoster virus or the gE protein thereof in a sample using the antibody or the antigen binding fragment thereof or the antibody conjugate or the antibody composition or the detection kit. In the detection of non-disease diagnosis and treatment purposes, the sample may be a virus liquid or vaccine prepared by in vitro culture, a gE protein prepared in vitro, or the like, which is not derived from a human or animal.
The method for detecting varicella-zoster virus or the gE protein thereof using the antibody or the antigen binding fragment thereof or the antibody conjugate or the antibody composition or the detection kit may use detection methods such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography and the like.
In some embodiments of the invention, the varicella-zoster virus or the gE protein thereof is detected by a double antibody sandwich ELISA method (as coated antibody and/or detection antibody) using the antibody or antigen binding fragment thereof, antibody conjugate, antibody composition or detection kit.
In the above (2), the product may be a detection reagent or kit for the purpose of non-disease diagnosis and treatment or a detection reagent or kit for the purpose of disease diagnosis and treatment.
In the above (3), the agent diagnoses varicella-zoster virus infection by detecting the presence or level of varicella-zoster virus in the sample.
In the above (4) and (6), the identification or quality control of the vaccine is performed by detecting the presence or the content of varicella-zoster virus or the gE protein thereof in the vaccine.
The above-mentioned item (5) is an item for detecting the immunogenicity of varicella-zoster virus vaccine, specifically, the ability of the varicella-zoster virus vaccine to induce immune response in animal body, including evaluation of humoral immune function (such as neutralizing antibody and level thereof, affinity of antibody) of immunized animal, etc., and the antibody or antigen binding fragment thereof provided by the invention can be used as positive reference antibody for detecting the immunogenicity of vaccine.
In the above (7), the antibody or antigen-binding fragment thereof of the present invention can be used as a positive reference antibody for detecting the level of varicella-zoster virus antibodies in serum after immunization of varicella-zoster virus vaccine (for example, by indirect ELISA), i.e., for detecting the humoral immune titer of varicella-zoster virus vaccine. Wherein the antibody or antigen binding fragment thereof can be linked to Fc segments of different species for use in the detection of the humoral immunity potency of varicella-zoster virus vaccine of the corresponding animal, for example, a murine monoclonal antibody obtained by linking to a mouse Fc (mFc) segment can be used in the detection of the mouse humoral immunity potency of varicella-zoster virus vaccine, and a monoclonal antibody obtained by linking to human Fc (hFc) can be used in the detection of the in vivo humoral immunity potency of varicella-zoster virus vaccine.
The present invention provides a method for detecting varicella-zoster virus or the gE protein thereof for non-disease diagnostic and therapeutic purposes, comprising: detecting varicella-zoster virus or the gE protein thereof in a sample to be detected by using the varicella-zoster virus antibody or the antigen binding fragment thereof or the antibody conjugate or the antibody composition of varicella-zoster virus or the detection kit.
The detection can be performed by enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography and other detection methods.
The beneficial effects of the invention at least comprise: the antibody or the antigen binding fragment thereof provided by the invention can be specifically combined with varicella-zoster virus or gE protein thereof, has higher affinity, and can realize specific, sensitive and accurate detection of varicella-zoster virus or gE protein thereof.
The detection reagent provided by the invention based on the antibody or the antigen binding fragment thereof can realize detection of varicella-zoster virus or gE protein thereof in a sample, has higher sensitivity, specificity, accuracy and precision, has low detection cost, can be used for quantitative detection of gE protein in varicella-zoster virus vaccine, detection of varicella-zoster virus vaccine humoral immunity titer in mice and varicella-zoster virus vaccine humoral immunity titer in human bodies, can also be used for preparation of diagnostic reagent of varicella-zoster virus infection and the like, provides effective detection means for development and application of varicella-zoster virus vaccine and varicella-zoster virus infection detection, and has better application value.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the affinity detection results of monoclonal antibody 4G2 in example 3 of the present invention.
FIG. 2 shows the affinity assay results of monoclonal antibody 1B10 of example 3 of the present invention.
FIG. 3 is a standard curve of the detection of Varicella Zoster Virus (VZV) gE protein content in example 4 of the invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention uses varicella-zoster virus gE protein as immunogen to carry out mouse immunity, obtains hybridoma cell strain for expressing mouse antibody through cell fusion and maternal clone screening and cell subcloning, and screens proper cell strain for antibody production and expression by establishing an activity analysis method. Experiments prove that the antibody can specifically identify varicella-zoster virus gE protein, has higher affinity, can detect varicella-zoster virus or gE protein thereof, and can also detect the humoral immunity titer of varicella-zoster virus vaccine in mice and the humoral immunity titer of varicella-zoster virus vaccine in human bodies. Further, the invention obtains the amino acid sequence of the antibody and the nucleotide sequence of the encoding gene thereof through hybridoma sequencing.
EXAMPLE 1 preparation of varicella zoster Virus gE protein specific antibody
The varicella-zoster virus gE protein specific antibody of the invention is obtained by the following steps:
1. Immunization of mice: mice were immunized with varicella zoster virus gE protein (purchased from Acrobiosystems under accession number Q9J3M 8-1) as immunogen. After the immunization, the immune response level was determined by detecting serum of the immunized animal by ELISA. After the conventional immunization is finished, if the immunized animal can reach the immune response level aiming at the immunogen, cell fusion is carried out.
2. Screening: the supernatant of the fused cells was screened by ELISA, and positive cells specifically binding to varicella-zoster virus gE protein were selected.
3. Cloning and expanding culture: transferring positive maternal clone cells to a 24-well plate for expansion culture; supernatants were collected from each of the expanded clones and assayed by ELISA.
4. Subcloning: subcloning positive parent clone by limiting dilution method and subcloning screening by ELISA method.
5. Hybridoma cell antibody gene sequencing: extracting total RNA of the hybridoma cells, and reversely transcribing the RNA into cDNA through 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: and (3) transfecting the antibody gene sequence obtained in the step (5) 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.
Two monoclonal antibodies, designated monoclonal antibodies 1B10 and 4G2, were obtained by cell fusion and screening according to the methods described above.
Example 2 specific detection of antibodies
Specific assays for monoclonal antibodies 1B10 and 4G2 were performed as follows:
1. the VZV gE protein and other related proteins (respiratory syncytial virus pre-fusion glycoprotein F0, novel coronavirus XBB.1.5 strain spike protein, influenza A virus protein ([ A/hong Kong/483/97 (H5N 1) ] Hemagglutinin (HA))) were diluted to 1 μg/mL with PBS, added to the wells of the enzyme-labeled plate, 100 μl per well, sealed with A sealing plate membrane, and left overnight at 4deg.C.
2. The wells were discarded, the ELISA plates were dried, washed with PBST wash solution, 300. Mu.L/Kong Jinpao, the ELISA plates were dried, and the plates were washed 3 times.
3. Mu.L of blocking agent (PBST wash containing 5% BSA) was added to each well, membrane-sealed with a plate, incubated at 37℃and then washed.
4. Monoclonal antibodies 4G2 and 1B10 were each diluted in a gradient with a sample diluent at a concentration of 10. Mu.g/mL, added to an ELISA plate at 100. Mu.L per well, membrane sealed with a seal plate, incubated at 37℃and then washed.
5. Anti-Mouse IgG secondary antibody was diluted with sample according to 1: diluted 20000, 100. Mu.L of each well was added, membrane sealed with a sealing plate, incubated at 37℃and then washed.
6. 100 Mu L of color development liquid is added into each hole, the plates are sealed by sealing plates, and the plates are placed at 37 ℃ for light-proof incubation.
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. The absorbance values of 450 nm and 630 nm were read with a microplate reader, and the OD 630 value was subtracted by OD 450 (OD value), and the results are shown in table 1.
TABLE 1 anti-VZV gE antibody specificity analysis
Example 3 affinity detection of antibodies to varicella zoster Virus gE protein
The monoclonal antibodies 1B10 and 4G2 were subjected to affinity detection, in particular by the following methods and results:
1. Capture of ligand: anti-VZV gE protein antibodies 1B10 and 4G2 were diluted to 5. Mu.g/mL with running buffer (1 XHEPES, containing 0.005% Tween-20) and captured on a protein A chip of Biacore 8K (Cytiva), respectively. The capture flow rate was 10 μl/min, reaching a capture level of about 150 RU, and a reference channel was set simultaneously, which did not require a ligand capture step. Each experimental cycle of analytes requires separate capture of the ligand onto the experimental channel.
2. Analysis of analytes: the VZV gE protein was diluted to 9 concentrations (250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953 and 0.0 nM) with the same running buffer (1 XHEPES, containing 0.005% Tween-20). The VZV gE protein was injected into the experimental and reference channels at a flow rate of 30. Mu.L/min with a binding time of 120 s and a dissociation time of 300 s. Both the binding and dissociation processes were treated in running buffer. The analyte was repeated at the lowest to highest concentration. After each cycle of interaction analysis, the chip surface was regenerated with 10mM glycine-hydrochloric acid, pH 1.5, flow rate 20 μl/min for 30: 30 s to remove ligand and any bound analyte, and then the next concentration cycle of analysis required repeated ligand capture, analyte injection and regeneration steps.
The affinity detection result of monoclonal antibody 4G2 is shown in fig. 1, kd=4.81E-09M, and the affinity detection result of monoclonal antibody 1B10 is shown in fig. 2, kd=5.63E-09M.
Example 4 varicella zoster Virus gE protein content assay
In this example, the monoclonal antibody obtained by the above screening is used to detect varicella-zoster virus gE protein by a double antibody sandwich ELISA method, and the specific method is as follows:
1. Monoclonal antibodies were diluted to 1. Mu.g/mL with PBS, added to wells of an enzyme-labeled plate, 100. Mu.L per well, membrane-sealed with a seal 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 30, 30 s, dried, and washed 3 times.
3. Mu.L of blocking agent (PBST wash containing 5% BSA) was added to each well, membrane-sealed with a plate, incubated at 37℃and then washed.
4. The varicella-zoster virus gE protein is subjected to gradient dilution by a sample diluent with the concentration range of 0.9375-60 ng/mL, added into an ELISA plate, 100 mu L of each hole is sealed by a sealing plate membrane, incubated at 37 ℃ and then washed.
5. Monoclonal antibodies were HRP-labeled, diluted to 0.05 μg/mL with sample dilution, added 100 μl per well, membrane sealed with sealing plate, incubated at 37 ℃, and then washed.
6. 100 Mu L of color development liquid is added into each hole, the plates are sealed by sealing plates, and the plates are placed at 37 ℃ for light-proof incubation.
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. The absorbance values of 450 nm and 630 nm were read with a microplate reader, and the OD 630 value was subtracted by OD 450 (OD value).
The results of the antibody pairing detection screening show that monoclonal antibodies 1B10 and 4G2 can accurately and sensitively detect the content of varicella-zoster virus gE protein, wherein the monoclonal antibody 1B10 is used as a coating antibody, and the monoclonal antibody 4G2 is used as a detection antibody after being marked by HRP.
A standard curve (FIG. 3, table 2) was established by a double antibody sandwich ELISA method, and the content of varicella-zoster virus gE protein in the sample was quantified according to the standard curve. The calculation formula is y=0.0315 x-0.0535, where y is the OD value, and the OD value of the sample is taken into the formula to calculate x, i.e. the concentration value of the sample. The results showed that the sensitivity of the method for detecting varicella-zoster virus gE protein was 3.75ng/mL.
TABLE 2 varicella zoster Virus gE protein content detection Standard Curve
Note that: in Table 2, OD values-1 and OD value-2 are the results of two parallel tests.
Example 5 varicella zoster Virus vaccine mouse humoral immune titre assay
In this example, the level of varicella-zoster virus gE antibody in the serum of the varicella-zoster virus vaccine immunized mice is analyzed by indirect ELISA using monoclonal antibody 1B10 as a positive reference antibody, and the analysis method is as follows:
1. The VZV gE protein was diluted to 2. Mu.g/mL with PBS, added to the wells of the microplate, 100. Mu.L per well, membrane sealed with a seal plate, and left overnight at 4 ℃.
2. The wells were discarded, the ELISA plates were dried, washed with PBST wash solution, 300. Mu.L/Kong Jinpao 30, 30 s, dried, and washed 3 times.
3. Mu.L of blocking agent (PBST wash containing 5% BSA) was added to each well, membrane-sealed with a plate, incubated at 37℃and then washed.
4. The monoclonal antibody 1B10 is subjected to gradient dilution by using a sample diluent with the concentration range of 0.39-12.5 ng/mL, added into an ELISA plate, and incubated at 37 ℃ by using a sealing plate membrane sealing plate at 100 mu L of each hole, and then cleaned.
5. Anti-Mouse IgG secondary antibody was diluted with sample according to 1: diluted 20000, 100. Mu.L of each well was added, membrane sealed with a sealing plate, incubated at 37℃and then washed.
6. 100 Mu L of color development liquid is added into each hole, the plates are sealed by sealing plates, and the plates are placed at 37 ℃ for light-proof incubation.
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. The absorbance values of 450 nm and 630 nm were read with a microplate reader, and the OD 630 value was subtracted by OD 450 (OD value), and the results are shown in table 3.
9. The serum samples of the mice immunized by the VZV vaccine are diluted by sample diluents in different proportions, and the titers of the VZV antibodies in the serum samples of the mice can be obtained by detecting the serum samples of the mice according to the same method.
TABLE 3 detection data for positive reference antibodies in mouse humoral immune titer detection
EXAMPLE 6 varicella-zoster Virus vaccine human humoral immune potency detection
In this example, the Fc segment of the monoclonal antibody 1B10 was modified and expressed as a sequence of human Fc by recombinant expression to become a human mouse chimeric antibody, and the level of varicella-zoster virus gE antibody in varicella-zoster virus vaccine immune human serum was analyzed by indirect ELISA using the human mouse chimeric antibody as a positive reference antibody as follows:
1. The VZV gE protein was diluted to 1. Mu.g/mL with PBS, added to the wells of the microplate, 100. Mu.L per well, membrane sealed with a seal plate, and left overnight at 4 ℃.
2. The wells were discarded, the ELISA plates were dried, washed with PBST wash solution, 300. Mu.L/Kong Jinpao, the ELISA plates were dried, and the plates were washed 3 times.
3. Mu.L of blocking agent (PBST wash containing 5% BSA) was added to each well, membrane-sealed with a plate, incubated at 37℃and then washed.
4. The human mouse chimeric antibody of monoclonal antibody 1B10 is subjected to gradient dilution with sample diluent with the concentration range of 0.0488-3.125 ng/mL, added into an ELISA plate, and subjected to membrane sealing plate, placed at 37 ℃ for incubation, and then washed, wherein each hole is 100 mu L.
5. Anti-Human IgG secondary antibodies were diluted with sample according to 1: dilution was 40000, 100. Mu.L was added to each well, membrane sealed with a sealing plate, incubated at 37℃and then washed.
6. 100 Mu L of color development liquid is added into each hole, the plates are sealed by sealing plates, and the plates are placed at 37 ℃ for light-proof incubation.
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. The absorbance values of 450 nm and 630 nm were read with a microplate reader, and the absorbance value (OD value) was subtracted from the OD 630 value by deduction from OD 450, and the results are shown in table 4.
9. The human serum sample after the immunization of the VZV vaccine is diluted with sample diluents in different proportions, and the titer of the VZV antibody in different human serum samples can be obtained by detecting according to the same method.
TABLE 4 detection data for positive reference antibodies in human hydrodynamic titers
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. An anti-varicella zoster virus antibody or antigen binding fragment thereof, characterized in that said antibody or antigen binding fragment thereof is any one of the following (1) - (2):
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of the CDR2 is KVS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:5 is shown in the figure;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 6. 7 and 8; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is ATS, the amino acid sequence of CDR3 is shown in SEQ ID NO: shown at 10.
2. The anti-varicella zoster virus antibody or antigen binding fragment thereof according to claim 1 which is any one of the following (1) to (2):
(1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: shown at 12;
(2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14.
3. The varicella-zoster virus antibody or antigen binding fragment thereof according to claim 1 or 2, which is selected from monoclonal antibodies, fab ', F (ab') 2, fd, fv, complementarity determining region fragments, single chain antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies or multispecific antibodies.
4. A nucleic acid molecule encoding an anti-varicella zoster virus antibody or antigen binding fragment thereof according to any one of claims 1 to 3.
5. A biological material comprising the nucleic acid molecule of claim 4, wherein the biological material is an expression cassette, a vector or a host cell.
6. An antibody conjugate, which is obtained by coupling the anti-varicella-zoster virus antibody or antigen binding fragment thereof according to any one of claims 1 to 3 with a label, wherein the label is one or more selected from the group consisting of an enzyme label, a biotin label, a fluorescent dye label, a chemiluminescent dye label and a radioactive label.
7. An antibody composition for varicella-zoster virus, characterized in that the antibody composition comprises an antibody in the following (1) and (2):
(1) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1. 2 and 3; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:4, the amino acid sequence of the CDR2 is KVS, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:5 is shown in the figure;
(2) The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 6. 7 and 8; the amino acid sequence of CDR1 of the complementarity determining region of the light chain variable region is shown in SEQ ID NO:9, the amino acid sequence of CDR2 is ATS, the amino acid sequence of CDR3 is shown in SEQ ID NO: shown at 10.
8. A test kit comprising the anti-varicella-zoster virus antibody or antigen binding fragment thereof as claimed in any one of claims 1 to 3, or comprising the antibody conjugate as claimed in claim 6, or comprising the varicella-zoster virus antibody composition as claimed in claim 7.
9. Use of any one of the anti-varicella-zoster virus antibody or antigen binding fragment thereof as defined in any one of claims 1 to 3 or the nucleic acid molecule as defined in claim 4 or the biological material as defined in claim 5 or the antibody conjugate as defined in claim 6 or the varicella-zoster virus antibody composition as defined in claim 7 or the detection kit as defined in claim 8:
(1) Use of varicella-zoster virus or gE protein thereof in the detection of the presence or level of same in a sample for non-disease diagnostic and therapeutic purposes;
(2) Use in the preparation of a reagent for detecting the presence or level of varicella-zoster virus or a gE protein thereof in a sample;
(3) Use in the preparation of a reagent for diagnosing varicella-zoster virus infection;
(4) Use in the identification of varicella zoster virus vaccines;
(5) Use in the preparation of a product for immunogenicity detection of varicella-zoster virus vaccine;
(6) Use in quality control of varicella-zoster virus vaccine;
(7) Use of the antibody as a positive reference antibody for the detection of varicella-zoster virus antibody levels in serum after immunization with varicella-zoster virus vaccine.
10. A method for detecting varicella-zoster virus or gE protein thereof for non-disease diagnostic and therapeutic purposes, comprising: detecting varicella-zoster virus or the gE protein thereof in a sample to be detected by using the anti-varicella-zoster virus antibody or antigen binding fragment thereof as defined in any one of claims 1 to 3, the antibody conjugate as defined in claim 6, the antibody composition of varicella-zoster virus as defined in claim 7, or the detection kit as defined in claim 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410126538.0A CN117683121B (en) | 2024-01-30 | 2024-01-30 | Anti-varicella-zoster virus antibodies and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410126538.0A CN117683121B (en) | 2024-01-30 | 2024-01-30 | Anti-varicella-zoster virus antibodies and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117683121A CN117683121A (en) | 2024-03-12 |
| CN117683121B true CN117683121B (en) | 2024-04-16 |
Family
ID=90135567
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410126538.0A Active CN117683121B (en) | 2024-01-30 | 2024-01-30 | Anti-varicella-zoster virus antibodies and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117683121B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118671341B (en) * | 2024-08-01 | 2025-01-28 | 简达生物科技(嘉兴)有限公司 | Anti-herpes zoster virus glycoprotein E monoclonal antibody, binding epitope analysis method |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019225962A1 (en) * | 2018-05-23 | 2019-11-28 | 재단법인 목암생명과학연구소 | Antigen variant of varicella zoster virus and use thereof |
| CN112778413A (en) * | 2019-11-11 | 2021-05-11 | 珠海泰诺麦博生物技术有限公司 | Anti-varicella-zoster virus antibody |
| CN113272421A (en) * | 2018-11-05 | 2021-08-17 | 艾欧凡斯生物治疗公司 | Method for generating tumor infiltrating lymphocytes and use thereof in immunotherapy |
| DE102021204116A1 (en) * | 2020-04-27 | 2021-10-28 | Tanaka Kikinzoku Kogyo K.K. | ANTI-VARICELLA ZOSTER VIRUS ANTIBODIES, IMMUNLOGICAL MEASURING METHOD AND IMMUNOLOGICAL MEASURING DEVICE |
| CN114621343A (en) * | 2022-05-17 | 2022-06-14 | 首都医科大学 | Japanese encephalitis virus antibody 2G1 and application thereof |
| WO2022188565A1 (en) * | 2021-03-10 | 2022-09-15 | 北京智仁美博生物科技有限公司 | Anti-varicella-zoster virus antibody and use thereof |
| WO2022261846A1 (en) * | 2021-06-16 | 2022-12-22 | 上海鑫湾生物科技有限公司 | Antibody targeting axl protein and antigen binding fragment thereof, and preparation method therefor and application thereof |
-
2024
- 2024-01-30 CN CN202410126538.0A patent/CN117683121B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019225962A1 (en) * | 2018-05-23 | 2019-11-28 | 재단법인 목암생명과학연구소 | Antigen variant of varicella zoster virus and use thereof |
| CN113272421A (en) * | 2018-11-05 | 2021-08-17 | 艾欧凡斯生物治疗公司 | Method for generating tumor infiltrating lymphocytes and use thereof in immunotherapy |
| CN112778413A (en) * | 2019-11-11 | 2021-05-11 | 珠海泰诺麦博生物技术有限公司 | Anti-varicella-zoster virus antibody |
| WO2021093639A1 (en) * | 2019-11-11 | 2021-05-20 | 珠海泰诺麦博生物技术有限公司 | Anti-varicella-zoster virus antibody |
| CN117106068A (en) * | 2019-11-11 | 2023-11-24 | 珠海泰诺麦博制药股份有限公司 | Antibodies against varicella-zoster virus |
| DE102021204116A1 (en) * | 2020-04-27 | 2021-10-28 | Tanaka Kikinzoku Kogyo K.K. | ANTI-VARICELLA ZOSTER VIRUS ANTIBODIES, IMMUNLOGICAL MEASURING METHOD AND IMMUNOLOGICAL MEASURING DEVICE |
| WO2022188565A1 (en) * | 2021-03-10 | 2022-09-15 | 北京智仁美博生物科技有限公司 | Anti-varicella-zoster virus antibody and use thereof |
| WO2022261846A1 (en) * | 2021-06-16 | 2022-12-22 | 上海鑫湾生物科技有限公司 | Antibody targeting axl protein and antigen binding fragment thereof, and preparation method therefor and application thereof |
| CN114621343A (en) * | 2022-05-17 | 2022-06-14 | 首都医科大学 | Japanese encephalitis virus antibody 2G1 and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 水痘带状疱疹病毒及其疫苗的研究进展;杨吉星;居丽雯;;上海预防医学杂志;20080120(第01期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117683121A (en) | 2024-03-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112250763B (en) | Antibody targeting SARS-CoV-2 coronavirus and its diagnosis and detection use | |
| CN117683121B (en) | Anti-varicella-zoster virus antibodies and uses thereof | |
| CN110352352B (en) | Kit for rapid diagnosis of asthma or allergic diseases | |
| CN111018971B (en) | Monoclonal antibody of Coxsackie virus A6 type solid virus and application thereof | |
| CN117720650B (en) | Anti-human respiratory syncytial virus antibody and application thereof | |
| CN114702578B (en) | Novel coronavirus Omicron mutant strain specific antibody and application thereof | |
| CN115286712B (en) | Novel coronavirus Delta mutant strain-specific antibodies and their applications | |
| CN117229393B (en) | Antibodies that specifically bind adeno-associated virus 5 and uses thereof | |
| JP7359390B2 (en) | Antibodies or antigen-binding fragments thereof against chikungunya virus and their uses | |
| KR20220167286A (en) | Adenovirus immunoassay method and immunoassay instrument | |
| WO2023088443A1 (en) | Anti-human igm antibody and preparation method therefor and use thereof | |
| KR20230039786A (en) | An antibody specific to C-terminal region of coronavirus nucleocapsid protein and uses thereof | |
| CN116514989B (en) | Omnipotent nuclease antibody and application thereof | |
| CN116143929B (en) | Antibody against recombinant human coagulation factor VIIa-Fc fusion protein and application thereof | |
| CN119350501B (en) | An antibody against REG3A protein or its antigen-binding fragment and its use | |
| CN116987184B (en) | Novel coronavirus BQ.1.1 mutant specific antibody and application thereof | |
| CN117304327B (en) | Anti-goat IgG rabbit monoclonal antibody and application thereof | |
| CN119431565B (en) | Antibody for resisting HPV16 type E6 protein and E7 protein, detection product and application thereof | |
| CN116284411B (en) | Antibody against recombinant human coagulation factor VIII-Fc fusion protein and application thereof | |
| CN115677853B (en) | anti-HBeAg antibody or antigen binding fragment thereof and application thereof | |
| CN112646040B (en) | Protein specifically binding to human IgG4 and application thereof | |
| EP4116322A1 (en) | Adenovirus immunoassay method and immunoassay instrument | |
| US20230296605A1 (en) | Antibody for porcine reproductive and respiratory syndrome virus and uses thereof | |
| JP5448424B2 (en) | Reagent for measuring protein containing Fc of human IgG | |
| CN119505004A (en) | Anti-RF antibody or its functional fragment and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |