Monoclonal antibody aiming at varicella-zoster virus and application thereof

Abstract

The invention provides a monoclonal antibody aiming at varicella-zoster virus and application of the monoclonal antibody. The monoclonal antibody comprises a light chain complementarity determining region 1, a light chain complementarity determining region 2 and a light chain complementarity determining region 3, and / or a heavy chain complementarity determining region 1, a heavy chain complementarity determining region 2 and a heavy chain variable region complementarity determining region 3. The present invention provides a monoclonal antibody or an antigen-binding fragment thereof, which has specificity against a varicella-zoster virus and is capable of binding to the varicella-zoster virus with high affinity. The detection kit prepared based on the antibody or the antigen binding fragment thereof is simple and convenient to use, high in sensitivity and precise and accurate in detection result.

Description

Technical Field

[0001] The present invention belongs to the field of biology and immunology, and specifically relates to a monoclonal antibody or an antigen-binding fragment thereof against varicella-zoster virus. The present invention also relates to a preparation method and use of the antibody or the antigen-binding fragment thereof. Background Art

[0002] Herpes zoster is a viral skin disease caused by varicella-zoster virus (VZV). During primary infection, VZV enters local lymph nodes through the respiratory mucosal epithelium and replicates. Lymphocytes infected with the virus then enter the blood circulation through the lymphatic circulation and infect peripheral blood mononuclear leukocytes. The virus spreads to the skin through the bloodstream, and the clinical manifestation is chickenpox. After chickenpox is cured, the virus lurks in the cranial ganglia and replicates, and migrates along the peripheral nerves to the skin, and the clinical manifestation is herpes zoster. The most important complication of herpes zoster is post-herpetic neuralgia (PHN). About 20% of people over 50 years old will develop PHN, and the possibility of PHN increases with age, and the pain lasts for months or even years.

[0003] Vaccination against herpes zoster is an effective means to prevent herpes zoster and reduce complications such as PHN. Currently, the main herpes zoster vaccines on the market are Merck's Zostavax and GlaxoSmithKline's Shingrix. Among them, Zostavax was launched in 2006 as a live attenuated vaccine; Shingrix was launched in October 2017 as a recombinant herpes zoster vaccine. The treatment method is mainly antiviral drugs, including acyclovir, famciclovir, valacyclovir, acyclovir, acyclovir, vidarabine, etc. Therefore, there is currently no effective and specific treatment. Therefore, preventing herpes zoster is the most important means to deal with herpes zoster virus infection.

[0004] In the development of shingles vaccine, antigen detection is a key quality control technology. Commonly used methods include enzyme-linked immunosorbent assay (ELISA), membrane antigen fluorescent antibody assay (FAMA), complement fixation test (CF), etc. ELISA detection is the most widely used method because of its simple and fast operation, high accuracy, no need for special instruments, and the ability to perform large-scale testing.

[0005] Given that herpes zoster antigens involve many post-translational modifications such as glycosylation, sialylation, and phosphorylation during expression, gE antigens expressed by different stable strains differ in terms of glycoforms and sialic acid content, which in turn affects the specific binding to antibodies. Therefore, it is necessary to screen monoclonal antibodies with high-efficiency binding levels, which is an important technical issue in this field. Summary of the invention

[0006] Based on the above research status, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that has specificity for varicella-zoster virus and binds to varicella-zoster virus with high affinity, which can be used to prepare a kit for detecting herpes zoster virus to detect the presence of herpes zoster virus, or to assist in the development of immune preparations or products against herpes zoster virus.

[0007] In one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof against varicella-zoster virus (VZV), comprising:

[0008] A light chain complementary determining region 1, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1; a light chain complementary determining region 2, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 2; and a light chain complementary determining region 3, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: NO:3 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and / or

[0009] A heavy chain complementary determining region 1, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:4; a heavy chain complementary determining region 2, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:5; and a heavy chain complementary determining region 3, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:5. The sequence shown in NO:6 has a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0010] According to some preferred embodiments of the present invention, the amino acid sequence of light chain complementary determining region 1 (LCDR1) is shown as SEQ ID No. 1; the amino acid sequence of light chain complementary determining region 2 (LCDR2) is shown as SEQ ID No. 2; and the amino acid sequence of light chain complementary determining region 3 (LCDR3) is shown as SEQ ID No. 3; and / or

[0011] The amino acid sequence of the heavy chain complementary determining region 1 (HCDR1) is shown in SEQ ID No.4; the amino acid sequence of the heavy chain complementary determining region 2 (HCDR2) is shown in SEQ ID No.5; and the amino acid sequence of the heavy chain variable region complementary determining region 3 (HCDR3) is shown in SEQ ID No.6.

[0012] According to some preferred embodiments of the present invention, the varicella-zoster virus monoclonal antibody or its antigen-binding fragment comprises:

[0013] A light chain complementary determining region 1, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1; a light chain complementary determining region 2, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 2; and a light chain complementary determining region 3, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: NO:3 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and

[0014] A heavy chain complementary determining region 1, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:4; a heavy chain complementary determining region 2, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:5; and a heavy chain complementary determining region 3, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:5. The sequence shown in NO:6 has a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0015] According to some preferred embodiments of the present invention, the varicella-zoster virus monoclonal antibody or its antigen-binding fragment comprises:

[0016] A light chain complementary determining region 1, whose amino acid sequence is shown in SEQ ID No. 1; a light chain complementary determining region 2, whose amino acid sequence is shown in SEQ ID No. 2; and a light chain complementary determining region 3, whose amino acid sequence is shown in SEQ ID No. 3; and

[0017] The heavy chain complementary determining region 1, whose amino acid sequence is shown in SEQ ID No.4; the heavy chain complementary determining region 2, whose amino acid sequence is shown in SEQ ID No.5; and the heavy chain complementary determining region 3, whose amino acid sequence is shown in SEQ ID No.6.

[0018] In the varicella-zoster virus (VZV) monoclonal antibody or its antigen-binding fragment according to the present invention, LCDR1, LCDR2 and LCDR3 of the amino acid sequences shown in SEQ ID No.1 to 3, respectively, and HCDR1, HCDR2 and HCDR3 of the amino acid sequences shown in SEQ ID No.4 to 6, respectively, are complementarity determining regions (CDRs) according to the Kabat definition.

[0019] According to some embodiments of the present invention, the antigen binding fragment is selected from one or more of Fab, Fab′, (Fab′)2, Fv, disulfide-bonded Fv, scFv, diabody or single domain antibody (sdAb).

[0020] According to some embodiments of the present invention, the monoclonal antibody or antigen-binding fragment thereof is selected from one or more of a murine antibody, a chimeric antibody or a humanized antibody.

[0021] According to some preferred embodiments of the present invention, the monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody; preferably, at least a portion of the constant region of the chimeric antibody is a human common constant region, specifically IgG1.

[0022] According to some preferred embodiments of the present invention, the amino acid sequence of the light chain variable region of the monoclonal antibody or its antigen-binding fragment is as shown in SEQ ID NO: 7, and / or the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8; more preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody or its antigen-binding fragment is as shown in SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8.

[0023] According to some preferred embodiments of the present invention, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody or its antigen-binding fragment is shown as SEQ ID NO.9, and / or the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment is shown as SEQ ID NO.10.

[0024] On the other hand, the present invention provides a nucleic acid encoding a monoclonal antibody or an antigenic fragment thereof according to the present invention, which comprises a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO.9, and / or a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO.10.

[0025] Preferably, the nucleic acid encoding the monoclonal antibody or antigenic fragment thereof according to the present invention comprises a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO.9, and a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO.10.

[0026] In yet another aspect, the present invention provides a vector comprising a nucleic acid encoding the monoclonal antibody or antigenic fragment thereof according to the present invention.

[0027] According to some embodiments of the present invention, the nucleic acid comprises a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO.9, and / or a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO.10.

[0028] According to some embodiments of the present invention, the vector is an expression vector, wherein the expression vector for the heavy chain may be pEE12.4, and the expression vector for the light chain may be pEE6.4.

[0029] In yet another aspect, the present invention provides a host cell comprising the vector according to the present invention.

[0030] According to some embodiments of the present invention, the host cell is a prokaryotic cell or a eukaryotic cell. Preferably, the host cell is selected from one or more of a yeast cell and a mammalian cell, more preferably a human embryonic kidney cell 293F.

[0031] On the other hand, the present invention provides use of a monoclonal antibody or an antigen-binding fragment thereof according to the present invention, a nucleic acid according to the present invention, a vector according to the present invention and / or a host cell according to the present invention in the preparation of a product for detecting varicella-zoster virus infection.

[0032] According to some embodiments of the present invention, the product is a detection kit.

[0033] In yet another aspect, the present invention provides a varicella-zoster virus detection kit comprising the monoclonal antibody or antigen-binding fragment thereof according to the present invention.

[0034] According to some embodiments of the present invention, the kit further comprises one or more of a coating solution, a washing solution, a blocking solution, a diluent, a stop solution or a standard; preferably, the kit further comprises an ELISA plate and / or a sealing film.

[0035] In yet another aspect, the present invention provides a method for detecting varicella-zoster virus, comprising using the kit according to the present invention to detect a sample from a subject.

[0036] The term "antibody" as used herein refers to an immunoglobulin molecule, which is usually a tetramer composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. According to the conservative differences in the amino acid sequences, the heavy chain and the light chain are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. In the variable regions of the heavy chain and the light chain, there are three local regions with a higher degree of variation in the amino acid composition and arrangement order, which are the key positions for the binding of antibodies to antigens, and are therefore also referred to as complementary determining regions (CDRs). In this article, the three heavy chain complementary determining regions are referred to as HCDR1, HCDR2 and HCDR3, respectively, and the three light chain complementary determining regions are referred to as LCDR1, LCDR2 and LCDR3, respectively. The variable regions of a heavy chain and a light chain interact to form an antigen binding site (Fv). Antibodies can be divided into different categories according to the amino acid sequences of their heavy chain constant regions. There are five major types of intact antibodies: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are known in the art. The present invention is intended to include antibodies of any of the aforementioned classes or subclasses.

[0037] The term "antibody" as used herein is also intended to cover digested fragments or functional variants thereof, for example, antibody fragments capable of binding to a herpes zoster antigen or a portion thereof, including but not limited to Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody or single domain antibody (sdAb).

[0038] The term "monoclonal antibody" as used herein refers to a homogeneous antibody directed only against a specific antigenic epitope. Compared to common polyclonal antibody preparations that typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. The modifier "monoclonal" indicates the uniform character of the antibody and is not to be construed as requiring the antibody to be produced by any particular method.

[0039] The term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus, etc.) used to transfer nucleotide coding information into a host cell. The term "expression vector" refers to a vector suitable for expressing a target gene (nucleotide sequence to be expressed) in a host cell, and generally includes parts such as the target gene, a promoter, a terminator, and a marker gene.

[0040] The term "host cell" as used herein refers to a cell that has been or can be transformed with a nucleic acid sequence and thereby expresses a selected target gene. The term includes the offspring of a parent cell, whether or not the offspring is identical to the original parent cell in morphology or genetic composition, as long as the offspring has the selected target gene. Commonly used host cells include bacteria, yeast, mammalian cells, etc.

[0041] The present invention provides a monoclonal antibody or an antigen-binding fragment thereof that is specific to varicella-zoster virus (VZV) and can bind to varicella-zoster virus with high affinity, and a preparation method for producing such an antibody or an antigen-binding fragment thereof. The detection kit prepared based on the above-mentioned antibody or antigen-binding fragment thereof is easy to use, has high sensitivity, and the detection result is precise and accurate. DETAILED DESCRIPTION

[0042] The present invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0043] The experimental methods used in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0044] Example 1 Preparation of monoclonal antibodies against herpes zoster virus

[0045] 1. Preparation of Herpes gE Protein

[0046] Refer to the report by Thomsson E, Persson L et al. in Journal of Virological Methods, 2011, Vol. 175, No. 1, pp. 53-59. For specific steps, refer to Chinese invention patent application No. 202011456435.9.

[0047] 2. Mouse immunization and tail blood test

[0048] 2.1 Immunization of mice

[0049] The herpes gE protein prepared in step 1 was used to immunize three NIH mice (numbered 1#, 2#, and 3#, respectively). Tail blood was collected on the 14th day after immunization to prepare serum.

[0050] 2.2 Evaluation of antibody titer in tail blood

[0051] The ELISA plate was coated with 1 μg / mL herpes gE protein, 100 μL was added to each well, and it was kept at 4°C overnight; the plate was washed 3 times with PBS solution, and blocked with 5% milk-PBS at room temperature for 1 hour; the plate was washed once with PBS solution, and gradiently diluted mouse tail blood serum was added, and the reaction was carried out at room temperature for 1 hour; the plate was washed 3 times with PBS solution, and 1:2000 diluted HRP-labeled goat anti-mouse Fc secondary antibody (purchased from Sigma) was added, and the reaction was carried out at room temperature for 1 hour; the plate was washed 5 times with PBS solution, and an equal volume of TMB colorimetric solution was added, and the reaction was carried out at room temperature for 20 minutes; 50 μL of stop solution was added, and the OD450 value was read on the ELISA reader after mixing.

[0052] The results of indirect ELISA evaluation of mouse tail blood serum on the 14th day after immunization are shown in Table 1. The titer of 1# mouse tail blood serum exceeded 1:50000.

[0053] Table 1 Evaluation of antibody titers in mouse tail blood serum on day 14 after immunization

[0054]

[0055] Note: No. 2 and No. 3 mice died, and tail blood was not collected. NC is the negative control, 5% milk-PBS. PC is the tail blood serum of No. 4 mouse (BALB / c mouse, 1 / 500 dilution). NaN means OD ≥ 4.0.

[0056] 2.3 Cell fusion and monoclonal screening

[0057] Mouse spleen cells #1 were fused with SP2 / 0 cells, and clones were picked after culturing into monoclonal cell lines in the selection medium. A total of 5 clones in 96-well plates were picked.

[0058] The ELISA plate was coated with herpes gE protein (1 μg / mL), 100 μL was added to each well, and the reaction was allowed to proceed overnight at 4°C; the plate was washed 3 times with PBS solution, and blocked with 5% milk-PBS at room temperature for 1 hour; the plate was then washed once with PBS solution; the monoclonal cell supernatant was mixed with 5% milk-PBS at a ratio of 1:1 and added to the wells, and the reaction was allowed to proceed for 1 hour at room temperature; the plate was then washed 3 times with PBS solution, and after patting dry, a 1:2000 diluted HRP-labeled goat anti-mouse Fc secondary antibody was added, and the reaction was allowed to proceed for 1 hour at room temperature; the plate was washed 5 times with PBS solution, and after patting dry, an equal volume of A solution and B solution was added, and the reaction was allowed to proceed for 20 minutes at room temperature in the dark; then 50 μL of stop solution was added, and the OD450 value was read on the ELISA reader after mixing. A total of 21 strong positive clones with OD ≥ 1.0 were retained from a total of 470 clones in 5 96-well plates.

[0059] Table 2 Positive clone detection results

[0060]

[0061]

[0062] Note: NC is the negative control, PC is the heart blood of mouse #1, and NaN indicates OD ≥ 4.0.

[0063] 2.4 Antibody Production

[0064] The 21 strong positive clones were cultured in shake flasks and purified, and antibodies from 19 strains were obtained. The antibody detection results are shown in Table 3. The detection method is the same as 2.3. The results show that the antibody sensitivity of clone 4G8 is the highest, reaching 0.0005 μg / mL.

[0065] Table 3 Antibody sensitivity test

[0066]

[0067] Note: NC is the negative control, NaN means OD ≥ 4.0.

[0068] The 4G8 antibody was sequenced. The monoclonal antibody or its antigenic fragment comprises a light chain (LC) and a heavy chain (HC), the light chain comprises a light chain variable region (LCVR) and the heavy chain comprises a heavy chain variable region (HCVR), the LCVR has the amino acid sequence shown in SEQ ID NO.7, and the HCVR has the amino acid sequence shown in SEQ ID NO.8.

[0069] According to the Kabat rule (reference: Kabat EA, Wu TT, Bilofsky H. Attempts to locate residues in complementarity-determining regions of antibody ombining sites that make contact with antigen. Proc. Natl. Acad. Sci. USA. 1976; 73: 617-619.), the CDR sequences shown as SEQ ID No. 1 to SEQ ID No. 3 in the LCVR and the CDR sequences shown as SEQ ID No. 4 to SEQ ID No. 6 in the HCVR were obtained respectively.

[0070] Example 2 Herpes zoster virus detection kit

[0071] 1. Kit Materials

[0072] The skim milk used in the present invention was purchased from BD Biosciences, the TMB colorimetric solution was purchased from Thermo, and other chemical reagents were domestic analytical grade or above.

[0073] 2. Kit Assembly

[0074] The kit includes the following antibodies:

[0075] Herpes gE protein rabbit polyclonal antibody (commissioned by Jingtiancheng to prepare)

[0076] Horseradish peroxidase-labeled goat anti-mouse monoclonal antibody (hereafter referred to as goat anti-mouse-HRP, SIGMA)

[0077] Murine anti-herpes zoster virus monoclonal antibody 4G8 (prepared in Example 1).

[0078] The kit also includes the following reagents:

[0079] 1) Coating solution reagent: 0.5% (g / 100mL) carbonate solution;

[0080] 2) Washing solution reagent: 10g / L PBS + 0.5mL / L Tween 20 (PBST);

[0081] 3) Blocking reagent: 5% skim milk PBST;

[0082] 4) Diluent reagent: 2% skim milk PBST;

[0083] 5) Stop solution reagent: 2M H2SO4;

[0084] 6) Standard: Herpes gE protein (manufactured by Yuanda Saiweixin and calibrated).

[0085] The kit also includes an ELISA plate and a plate sealing film.

[0086] Put the above antibodies, reagents, ELISA plates, and sealing films into the packaging box in order to complete the assembly of the kit.

[0087] 3. How to use the kit

[0088] 1) Coating: Dilute the herpes gE protein rabbit polyclonal antibody to 1 μg / mL using coating solution, coat the ELISA plate, 50 μL / well, at 4°C overnight;

[0089] 2) Blocking: Wash the coated ELISA plate in step 1) twice with washing solution, add blocking solution, 200 μL / well, and block at 37° C. for 1 hour.

[0090] 3) Dilution and loading: Take the herpes gE protein standard, and dilute it in series with the diluent according to the indicated protein content value. Take 500-7.81ng / ml concentration as the standard series, and take the diluent as the zero point of the standard series. Also dilute the sample to be tested to the range of the standard series. Add the standard of each concentration, the diluent and the diluted sample to be tested to the ELISA plate that has been closed in step 2) in turn. All loading wells are set up for parallel experiments. After loading, seal the plate with a sealing film and incubate at 37°C for 1 hour.

[0091] 4) Detection antibody incubation: After washing the ELISA plate in step 3) with a washing solution for 3 times, add 50 μL of mouse anti-herpes zoster virus monoclonal antibody 4G8 diluted with a diluent by volume ratio to each well. Seal the plate with a sealing film and incubate at 37° C. for 1 hour.

[0092] 5) Incubation with secondary antibody: Wash the ELISA plate incubated in step 4) with washing solution for 3 times, add goat anti-mouse-HRP diluted with diluent by volume, 50 μL per well, seal the plate with sealing film and incubate at 37°C for 1 hour.

[0093] 6) Color development: After the incubated ELISA plate in step 5) is washed three times with washing solution, 50 μL of TMB color development solution (purchased from Thermo) is added to each well, and the plate is sealed with a sealing film and placed at 37° C. for color development for 10 to 20 minutes.

[0094] 7) Stop plate reading: After color development is complete, add 50 μL of stop solution to all wells. Read the absorbance (OD value) at 450 nm on a microplate reader with 630 nm as the reference wavelength.

[0095] 4. Calculation of Antigen Content

[0096] The average OD detection value of each dilution sample of the standard sample minus the average OD detection value of the blank control is the OD detection value of the sample at that dilution. With the concentration value of each dilution of the standard sample (ng / mL) as the horizontal axis and the OD detection value as the vertical axis, ELISA calc software is used for four-parameter fitting. Subtract the blank from the sample OD detection value and substitute it into the fitting curve to calculate the antigen content (ng / mL) of the sample solution:

[0097] Antigen content (μg / mL) = Antigen content value (ng / mL) × dilution factor / 1000

[0098] Example 3 Neutralizing Activity of Monoclonal Antibodies to Herpes Zoster Virus

[0099] The antibody was diluted with a diluent containing complement at a ratio of 1:20, and the antibody dilution ratio was 1:50, 1:200, 1:800, and 1:3200. The virus diluted to 500-1000 PFU / ml was mixed with an equal volume of antibody, incubated at 37°C for 1 hour, and 200 μL of the virus and antibody mixture was inoculated into a monolayer of diploid cells in a 96-well plate. Each dilution of the antibody sample was repeated in 4 wells, and a cell control group and a virus control group were also prepared.

[0100] The cells were cultured in a 37°C, 5% CO2 incubator, and the results were observed and recorded daily. On the 7th day, the 50% neutralization titer was calculated according to the Karber method.

[0101] Test results: The monoclonal antibody 4G8 described in the present invention has a good neutralizing protective effect on varicella-zoster virus (VZV), and its neutralizing protective titer is 1:3162.

[0102] Example 4 Effect verification of herpes zoster virus detection kit

[0103] 1. Exclusiveness verification

[0104] The herpes gE protein sample and buffer solution A (2.11 mM Na2HPO4·12H2O+7.9 mM KH2PO4+0.15 MNaCl, pH 6.1) were diluted 10,000 times and 20,000 times, respectively, and the herpes gE protein standard was gradiently diluted to 7.81 ng / ml, and the herpes zoster virus detection kit prepared in Example 2 was used for detection.

[0105] The results showed that the OD average value corresponding to the concentration point of 7.81ng / ml of the standard curve was 0.182; the OD average values ​​of buffer solution A diluted 10000 and 20000 times were both 0.022, which was less than the OD value of 0.182 at the lowest concentration point (7.81ng / ml) in the standard curve; the OD average values ​​of herpes gE protein samples diluted 10000 and 20000 times were 1.969 and 1.442, respectively, which were greater than the OD value of 0.182 at the lowest concentration point (7.81ng / ml) in the standard curve, meeting the acceptable standard for specificity. It was proved that the kit of the present invention had good specificity when detecting the content of recombinant herpes zoster virus gE protein.

[0106] Table 4 Specificity verification results

[0107]

[0108] 2. Accuracy Verification

[0109] Take herpes gE protein samples, dilute to 250ng / ml, 62.5ng / ml, 15.63ng / ml according to their protein concentrations, do 6 parallels for each dilution, and load the samples in duplicate wells. Use the herpes zoster virus detection kit prepared in Example 2 to detect and obtain the antigen content (ng / ml) of the samples.

[0110] The recovery rate was calculated according to the following formula:

[0111]

[0112] The results showed that the sample recovery rate was between 86.80% and 123.20%, and the test results were within the acceptable standard range of 70% to 130%, which met the requirements. This proved that the kit of the present invention had good accuracy in detecting the content of recombinant herpes zoster virus gE protein.

[0113] Table 5 Accuracy verification results

[0114]

[0115] 3. Repeatability verification

[0116] Herpes gE protein samples were taken, and the same experimenter used the kit provided by the present invention (see Example 2) to measure the same batch of samples 6 times on the same day as a repeatability test verification, and the relative standard deviation (RSD) of the 6 results was calculated.

[0117] The results showed that the RSD of the six parallel tests on the herpes zoster gE protein samples was 4.91%, which met the requirement of the repeatability acceptance standard RSD≤20%, proving that the kit of the present invention has good repeatability when detecting the content of the recombinant herpes zoster virus gE protein.

[0118] Table 6 Repeatability verification results

[0119]

[0120] 4. Linearity and range verification

[0121] Herpes gE protein samples were taken and diluted to 500ng / mL, 250ng / mL, 125ng / mL, 62.5ng / mL, 31.25ng / mL, 15.63ng / mL, and 7.81ng / mL, respectively, and repeatability and accuracy verification were performed. The R2 of the standard curve was greater than 0.99, within the acceptable standard R2 ≥ 0.99 range; the recovery rate of each point of the standard curve was 95.56% to 104.21%, within the acceptable standard 80% to 120% range.

[0122] It has been verified that the kit of the present invention has good linearity when detecting the content of recombinant herpes zoster virus gE protein, and the detection range is 500 to 7.81 ng / ml.

[0123] Table 7 Linearity verification results

[0124]

[0125]

[0126] In summary, the monoclonal antibody or antigen-binding fragment thereof provided by the present invention has specificity for varicella-zoster virus (VZV) and can bind to it with high affinity, and the monoclonal antibody or antigen-binding fragment thereof can be used to prepare a kit for detecting herpes zoster virus. The detection method using the kit is simple to operate, highly sensitive, and the detection result is precise and accurate.

[0127] Although the present invention has been described in detail above, it is understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the spirit and scope of the present invention. The scope of the present invention is not limited to the detailed description above, but should be attributed to the claims.

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof against varicella-zoster virus, comprising: A light chain complementary determining region 1, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1; a light chain complementary determining region 2, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 2; and a light chain complementary determining region 3, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: NO:3 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and / or A heavy chain complementary determining region 1, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:4; a heavy chain complementary determining region 2, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:5; and a heavy chain complementary determining region 3, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:

5. The sequence shown in NO:6 has a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising: A light chain complementary determining region 1, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 1; a light chain complementary determining region 2, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: 2; and a light chain complementary determining region 3, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO: NO:3 has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and A heavy chain complementary determining region 1, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:4; a heavy chain complementary determining region 2, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:5; and a heavy chain complementary determining region 3, whose amino acid sequence is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:

5. The sequence shown in NO:6 has a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein: The antigen binding fragment is selected from one or more of Fab, Fab', (Fab')2, Fv, disulfide-bonded Fv, scFv, diabody or single domain antibody.

4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein The monoclonal antibody or antigen-binding fragment thereof is selected from one or more of a murine antibody, a chimeric antibody or a humanized antibody; Preferably, the monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody; more preferably, at least a portion of the constant region of the chimeric antibody is a human common constant region; Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 7, and / or the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 8; more preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:

8.

5. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The nucleotide sequence encoding the light chain variable region of the monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO.9, and / or the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO.

10.

6. A vector comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; Preferably, the nucleic acid comprises a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO.9, and / or a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO.10; Preferably, the vector is an expression vector.

7. A host cell comprising the vector according to claim 6; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; further preferably, the host cell is selected from one or more of yeast cells and mammalian cells, more preferably human embryonic kidney cells 293F.

8. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the nucleic acid according to claim 5, the vector according to claim 6 and / or the host cell according to claim 7 in the preparation of a product for detecting varicella-zoster virus; Preferably, the product is a detection kit.

9. A varicella-zoster virus detection kit comprising the monoclonal antibody or antigenic fragment thereof according to any one of claims 1 to 4.

10. The kit according to claim 9, further comprising one or more of a coating solution, a washing solution, a blocking solution, a diluent, a stop solution or a standard; Further preferably, the kit further comprises an ELISA plate and / or a sealing film.

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