Antibody capable of specifically recognizing rubella virus and application thereof

Abstract

The invention relates to an antibody or an antigen binding fragment capable of specifically recognizing rubella viruses, and a kit containing the antibody or the antigen binding fragment and used for detecting the rubella viruses. The antibody provided by the invention has relatively strong affinity to the rubella virus, and the kit provided by the invention is high in sensitivity and strong in specificity when being used for detecting the rubella virus.

Description

Technical Field

[0001] The present invention relates to the field of epidemic prevention and detection, and in particular to antibodies that specifically recognize rubella virus and applications thereof. Background Art

[0002] Rubella is an acute infectious disease caused by rubella virus (RV), with the main symptoms of fever, respiratory catarrh and rash. Early infection of rubella virus in pregnant women can easily lead to miscarriage or congenital rubella syndrome (CRS) in infants, which manifests as congenital defects such as blindness, congenital heart defect, deafness and intellectual disability, which is very harmful.

[0003] Rubella virus is the only member of the Rubivirus genus in the Togaviridae family, and has only one blood type. Humans are the only natural host of rubella virus. Rubella virus can be transmitted through droplets through the respiratory tract, or through close daily contact or vertical transmission from mother to child.

[0004] There are many diagnostic methods for rubella virus at present. The etiological method is the most accurate means of diagnosis, but this method has low sensitivity and is prone to missed detection. Molecular biological diagnostic methods have high requirements for laboratory conditions and operator skills and are not suitable for widespread application. Enzyme-linked immunosorbent assay (ELISA) has the advantages of being simple, rapid, with clear results and simple operation, and has become a development direction in the field of clinical and quarantine diagnosis.

[0005] Therefore, there is an urgent need to find monoclonal antibodies with high affinity to rubella virus to meet the needs of rubella virus detection. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art, provide a monoclonal antibody with high affinity for rubella virus, and on this basis provide a detection kit with high sensitivity and high specificity for rubella virus.

[0007] In a first aspect, the present invention provides an antibody or antigen-binding fragment that specifically recognizes Rubella virus, the antibody or antigen-binding fragment comprising:

[0008] a heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, wherein the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO:6, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:7; and

[0009] The light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 12, the amino acid sequence of the light chain CDR2 is shown in AAS, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 13.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises:

[0011] A heavy chain variable region comprising or consisting of an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having at least 85% homology, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to the amino acid sequence as shown in SEQ ID NO:8; and / or

[0012] A light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 85% homology, for example, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to the amino acid sequence as shown in SEQ ID NO: 14, or consisting of said amino acid sequence.

[0013] In some embodiments, the antibody subtype that specifically recognizes Rubella virus is IgG.

[0014] In a second aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid encoding the antibody or antigen-binding fragment of the first aspect of the present invention.

[0015] In some embodiments, the nucleic acid molecule comprises:

[0016] A nucleotide sequence encoding a heavy chain, which comprises a nucleotide sequence encoding a heavy chain variable region (VH) as shown in SEQ ID NO: 8, and comprises a nucleotide sequence encoding VH as shown in SEQ ID NO: 4, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99% homology thereto; and / or

[0017] A nucleotide sequence encoding a light chain, which comprises a nucleotide sequence encoding a light chain variable region (VL) as shown in SEQ ID NO: 14, and comprises a nucleotide sequence encoding VL as shown in SEQ ID NO: 11, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto.

[0018] In a third aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described in the second aspect of the present invention.

[0019] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect of the present invention, or the recombinant vector described in the third aspect.

[0020] In a fifth aspect, the present invention provides a protein conjugate comprising the antibody or antigen-binding fragment described in the first aspect of the present invention and a ligand.

[0021] In some embodiments, the ligand is selected from the group consisting of a radioisotope, a fluorescent group, and a delivery vehicle.

[0022] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment described in the first aspect of the present invention or the protein conjugate described in the fifth aspect.

[0023] In some embodiments, the pharmaceutical composition further contains other active ingredients.

[0024] In some embodiments, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0025] In a seventh aspect, the present invention provides use of the antibody or antigen-binding fragment, the nucleic acid molecule, the recombinant vector, the host cell, the protein conjugate or the pharmaceutical composition in the preparation of a drug for preventing or treating diseases caused by rubella virus infection.

[0026] In an eighth aspect, the present invention provides a kit for in vitro detection of Rubella virus, comprising the antibody or antigen-binding fragment described in the first aspect of the present invention or the protein conjugate described in the fifth aspect.

[0027] In some embodiments, the antibody, antigen-binding fragment or protein conjugate is labeled with a marker, wherein the marker can be selected from an enzyme, a chemiluminescent group and an isotope group.

[0028] In some embodiments, the kit is a double antibody sandwich ELISA detection kit.

[0029] In some embodiments, the kit further comprises polyclonal antibodies against rubella virus. The polyclonal antibodies can be obtained by immunizing mammals (such as rabbits, sheep, goats, etc.) with rubella virus and then purifying the serum.

[0030] In some embodiments, the kit further comprises a secondary antibody labeled with horseradish peroxidase (HRP) and a color developing solution containing 3,3',5,5'-tetramethylbenzidine (TMB). The color developing solution generally comprises solution A and solution B; one of which comprises H2O2 or carbamide peroxide, and the other comprises TMB as a color developing substrate.

[0031] The kit provided by the present invention adopts a high-affinity rubella virus monoclonal antibody, which can achieve high-sensitivity and high-specificity detection of rubella virus.

[0032] In a ninth aspect, the present invention provides use of the antibody or antigen-binding fragment, the protein conjugate or the kit in in vitro detection of rubella virus.

[0033] As a specific implementation, the application is specifically to detect Rubella virus using a method comprising the following steps:

[0034] (1) adding the monoclonal antibody against rubella virus provided in the first aspect to an ELISA plate, sealing it, and drying it;

[0035] (2) Add the sample to be tested and set up a negative control, incubate and then wash;

[0036] (3) adding polyclonal antibodies against rubella virus, incubating and then washing;

[0037] (4) adding horseradish peroxidase-labeled secondary antibody, incubating and then washing;

[0038] (5) Add a colorimetric solution containing 3,3',5,5'-tetramethylbenzidine, add sulfuric acid after incubation to terminate the reaction, and measure the absorbance at 630 nm using an enzyme reader.

[0039] The present invention provides a high-affinity monoclonal antibody against rubella virus. The kit provided on this basis has high sensitivity and specificity against rubella virus, can achieve high-specificity, rapid and simple detection and diagnosis, and has a strong application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram of cell culture and pathological changes; Figure 1 A is RK-13 ​​cells not inoculated with rubella virus. Figure 1 B RK-13 ​​cells not inoculated with rubella virus for 216 h.

[0041] Figure 2 The figure is a schematic diagram of the results of SDS-PAGE identification of purified Rubella virus monoclonal antibodies; wherein the first and second columns are purified antibodies, and the third column is a marker. DETAILED DESCRIPTION

[0042] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0043] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0044] Unless otherwise specified, the chemical reagents used in the examples are conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0045] definition

[0046] Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0047] As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their plural referents unless the context clearly dictates otherwise.

[0048] Unless the context clearly dictates otherwise, the term "or" is used to mean, and can be used interchangeably with, the term "and / or."

[0049] In the context of the present disclosure, unless otherwise stated, the word "comprising" and its variations such as "including" and "containing" should be understood to imply the inclusion of the stated elements, such as amino acid sequences, nucleotide sequences, properties, steps or groups thereof, but without excluding any other elements, such as amino acid sequences, nucleotide sequences, properties and steps. When used herein, the term "comprising" or any variation thereof may be replaced by the term "containing", "including" or sometimes by "having" or its equivalent variations. In certain embodiments, the word "comprising" also includes the situation of "consisting of".

[0050] Unless otherwise indicated, the term "antibody" used herein covers antibodies and antibody fragments in the broadest sense, as long as it recognizes and binds to the antigen of the present invention, that is, human CD44v containing the v5 exon. Specifically, the antibody of the present application binds to human CD44v5. The antibody of the present application generally refers to a monospecific antibody. However, the present application also considers antibodies with heterologous specificity (heterospecific) or multispecific antibodies. Antibodies bind to specific antigenic determinants or epitopes through specific binding sites.

[0051] "Antibody fragment" or "antigen-binding fragment" refers to a portion of a full-length antibody, typically comprising the antigen-binding or variable region. Examples of antibody fragments may include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0052] The most common basic structure of an antibody is a tetramer. Each tetramer includes two pairs of identical polypeptide chains, each pair having a smaller chain called a "light chain" (about 25 kDa) and a larger chain called a "heavy chain" (about 50-70 kDa). At the amino terminus of each chain, it includes a variable domain of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of the heavy chain can define a constant region that is primarily responsible for effector function. Typically, the light chains of human antibodies are divided into κ and λ light chains. In addition, the heavy chains of human antibodies are typically classified as α, δ, ε, γ or μ, and the isotypes of antibodies are defined as IgA, IgD, IgE, IgG and IgM and their subclasses, such as IgG1, IgG2, IgG3 and IgG4, respectively. The variable region / domain of each light chain / heavy chain (VL / VH) pair forms an antibody binding site. Therefore, a complete antibody typically has two binding sites.

[0053] As used herein, the term "human antibody" means an antibody that contains only human immunoglobulin protein sequences. If produced in mice, mouse cells, or hybridomas derived from mouse cells, human antibodies may contain mouse sugar chains. Similarly, "mouse antibody" or "rat antibody" means an antibody that contains only mouse or rat immunoglobulin sequences, respectively.

[0054] The term "humanized antibody" refers to an antibody obtained by replacing sequences in non-human antibodies (such as mouse antibodies) with human sequences. For example, humanized antibodies generally contain non-human CDR regions, but the remaining regions are all human sequences.

[0055] The antibody of the present invention may be an animal-derived antibody such as a mouse-derived antibody. The antibody of the present invention may be a chimeric antibody such as a human-mouse chimeric antibody. The antibody of the present invention may be a human antibody or a humanized antibody.

[0056] The antibodies of the present application may be subjected to a purification process to remove unwanted substances, thereby producing purified antibodies. Conventional methods for purifying antibodies include, but are not limited to, column chromatography methods known in the art.

[0057] The antibody or antigen binding fragment of the present invention can be an isolated antibody. The term "isolated" means that the antibody or antigen binding fragment is at least partially free of other biological materials or non-biological materials from the cells, cell cultures, growth media, expression systems that produce them. The materials may include nucleic acids, proteins, lipids, carbohydrates, buffers, salts or other materials, such as cell debris and growth media.

[0058] The present application also provides an isolated nucleic acid sequence comprising a nucleotide sequence encoding an antibody or fragment thereof of the present application. "Isolated nucleic acid" or "isolated polynucleotide" refers to a DNA or RNA removed from all or part of a polynucleotide in which the isolated polynucleotide is present in nature, or a DNA or RNA connected to a polynucleotide that is not connected to it in nature. An isolated nucleic acid molecule "comprising" a specific nucleotide sequence may also include, in addition to the specific sequence, an operably connected regulatory sequence that controls the expression of the coding region of the nucleic acid sequence. Due to codon degeneracy, those skilled in the art will appreciate that a specific amino acid sequence may be encoded by different nucleotide sequences.

[0059] "Percent homology" or "% homology" is used in the context of the present invention to describe the degree of similarity between two nucleotide sequences or two amino acid sequences, and has the same meaning as "percent identity". The percent homology of two sequences can be calculated as follows: after the two sequences are aligned, the number of positions where the residues are identical is divided by the total length of the aligned sequences and multiplied by 100%. Methods and tools for aligning two amino acid sequences or nucleotide sequences are well known in the art, such as the BLAST suite provided on the NCBI website (Altschul, SF et al. (1990) J. Mol. Biol. 215: 403-410).

[0060] "Nucleotide" in the context of the present invention includes DNA and RNA.

[0061] In the term "isolated nucleic acid molecule", "isolated" means that an operation has been performed to remove factors other than the desired component, and the isolated nucleic acid molecule no longer exists in its naturally occurring state, such as no longer existing in a chromosomal genome.

[0062] "Vector" refers to a carrier DNA molecule that can carry foreign genes into cells. Examples of vectors include, but are not limited to, plasmids, viral vectors, cosmids, and artificial chromosomes. A vector used to introduce foreign transgenes into cells and facilitate the expression of the transgenes is called an "expression vector."

[0063] "Host cell" herein refers to a cell containing exogenous recombinant DNA such as an expression vector, and also refers to a cell containing a polypeptide or protein expressed by the recombinant DNA. Such host cells can be used to produce the anti-CD44v5 antibody of the present invention. In one embodiment, the host cell is not particularly limited as long as it can be used for the expression of a polypeptide sequence such as the anti-CD44v5 antibody of the present invention.

[0064] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0065] As used herein, the term "treat" refers to, for example, a reduction in the severity of a disease or disorder; a shortening of the duration of a disease course; an improvement or elimination of one or more symptoms associated with a disease or disorder; providing a beneficial effect to a subject suffering from a disease or disorder, but not necessarily curing the disease or disorder. The term also includes the prevention or prophylaxis of a disease or disorder or its symptoms.

[0066] The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical preparation other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0067] According to the technical solution of the present invention, conservative substitutions can be made to certain amino acids in the amino acid sequence without changing the activity or function of the protein. "Conservative substitutions" of amino acids are well known in the art and generally refer to changing an amino acid residue to another amino acid residue with a similar side chain in structure or function. For example, an exemplary conservative substitution list is provided in Table 1 below.

[0068] Table 1

[0069]

[0070]

[0071] Example 1: Cultivation and purification of rubella virus

[0072] 1. Rubella virus culture

[0073] RK-13 ​​cells were cultured with MEM containing 3% to 5% newborn calf serum, and after the cells grew into a monolayer, rubella virus was inoculated at an MOI of 0.01 to 0.1, and MEM containing 0% to 2% newborn calf serum was used to culture rubella virus, and the virus was harvested after 8 to 11 days. The harvested rubella virus was inactivated with β-propiolactone at 4°C for 12 hours.

[0074] 2. Rubella virus purification

[0075] The harvested virus was centrifuged at 6000 rpm for 15-20 min to remove cell debris; after 10-fold ultrafiltration concentration, it was further purified by sucrose density gradient centrifugation: the sucrose concentrations used were 30% sucrose and 55% sucrose, respectively, and centrifuged at 30000 rpm for 16 h.

[0076] The pathological effects of RK-13 ​​cell culture and rubella virus inoculation are as follows Figure 1 shown.

[0077] Example 2: Animal Immunization

[0078] 1. The purified rubella virus protein is 100 μg / mL, and 1 mL of each of Freund's complete adjuvant is mixed and emulsified; after emulsification, immunization is injected subcutaneously at 4 points on the back of BALB / c mice, 0.2 mL / mouse.

[0079] 2. On the 14th and 28th days after the first immunization, 100 μg / mL of purified virus and 1 mL of Freund's incomplete adjuvant were mixed and emulsified, and injected subcutaneously at 4 points on the back of BALB / c mice, 0.2 mL / mouse.

[0080] 3. 35 days after the first immunization, blood was collected from the eye socket, serum was separated, and the antibody titer in the serum was tested by ELISA. If the antibody titer was not less than 1:10000, intraperitoneal impaction was performed and 100 μg / mL of purified rubella virus solution was injected into the peritoneal cavity of BALB / c mice, 0.1 mL / mouse.

[0081] Example 3: Cell fusion and screening

[0082] 1. Take the spleen of immunized BALB / c mice, mix the spleen cells with mouse myeloma cells SP2 / 0 at a ratio of 2:1 to 5:1, add 1 mL PEG1500 in a 37°C water bath; then slowly add 15 mL serum-free 1640 medium to terminate the fusion, centrifuge at 1500 r / min for 5 min, discard the supernatant, add 50 mL 10% fetal bovine serum 1640 medium to resuspend, add the cell suspension to a 96-well cell culture plate, 100 μL / well (each well has about 1*10 5 cells), and add HAT culture medium (100 μL / well) the next day. Change HAT culture medium every 2-3 days, and observe whether hybridomas appear for two consecutive weeks. After two weeks, switch to HT culture medium and observe the growth of fused cells.

[0083] 2. Screening of hybridoma cells: Observe the growth of hybridoma cells from the seventh day after cell fusion. When the cells cover more than 1 / 10 of the bottom area of ​​the well, aspirate the supernatant for antibody ELISA detection. Transfer the cells in the positive wells to 24-well plates for expansion and timely subcloning.

[0084] Example 4: Cloning of hybridoma cells (limiting dilution method)

[0085] Count the hybridoma cells and dilute the hybridoma cells with 1640 medium containing 10% fetal bovine serum. Add the diluted hybridoma cells to a 96-well plate for culture, 100 μl per well. Culture at 37°C and 5% CO2 for 7 to 10 days. When clones visible to the naked eye appear, detect the antibody activity in time. Observe under an inverted microscope, mark the wells where only a single clone grows, and take the supernatant for antibody detection. Move the cells in the positive wells to a 24-well plate for expansion culture, and then transfer to a 25 cm 2 or 175cm 2 Cell flask expansion. After expansion, freeze the cell lines as soon as possible, number them, and store them in liquid nitrogen.

[0086] Example 5: Preparation and detection of ascites of monoclonal antibody cell lines

[0087] 1. Resuscitate the frozen monoclonal cells according to the conventional method, culture them, and observe the cell growth under the microscope. 2 When the cells in the cell culture flask cover 70% to 80% of the bottom of the flask, the inoculation can be carried out; BALB / c female mice are intraperitoneally inoculated according to conventional methods, and ascites is collected regularly.

[0088] 2. Rubella virus monoclonal antibody ascites identification

[0089] After overnight coating with 1 μg / ml of rubella virus, the antibody titer of ascites was detected by indirect ELISA. The antibody titer test results are shown in Table 2 below. It can be seen that the antibody titer is 10 7 .

[0090] Table 2: Antibody titer test results

[0091] Dilution multiple <![CDATA[10 4 ]]> <![CDATA[10 5 ]]> <![CDATA[10 6 ]]> <![CDATA[10 7 ]]> <![CDATA[10 8 ]]> B5 3.954 3.851 1.819 0.244 0.062 Negative control 0.026 0.018 0.029 0.026 0.030

[0092] Example 6: Monoclonal antibody purification

[0093] The ascites was filtered through filter paper to preliminarily remove impurities, and the ascites was filtered through a 0.45 μm microporous filter to further remove impurities. The ascites was further purified through a Protein A chromatography column to obtain high-purity monoclonal antibodies.

[0094] The purified monoclonal antibody was identified by SDS-PAGE. Figure 2 shown.

[0095] The purified monoclonal antibody was sequenced and the results are shown in Table 3 below.

[0096] Table 3: Monoclonal antibody sequences

[0097]

[0098]

[0099] Note: The underlined characters in Table 3 are CDR regions, and the bold characters are CH1 end primers.

[0100] Example 7: Preparation and purification of rubella virus polyclonal antibodies

[0101] The following steps were used to prepare and purify the Rubella virus polyclonal antibody:

[0102] (1) Using the rubella virus obtained in Example 1 as an antigen, immunize one sheep, with an immunization dose of 0.2 mg of rubella virus per sheep. For the initial immunization, the antigen was emulsified with Freund's complete adjuvant and injected subcutaneously at multiple points on the back of the neck. Immunize five times, with an interval of 7 days between each immunization. Blood was collected from the jugular vein, placed at 37°C for 1 hour, and then at 4°C overnight to allow the serum to precipitate naturally.

[0103] (2) The serum to be extracted was centrifuged at 5000 rpm for 15 min at 4°C and the supernatant was collected;

[0104] (3) Take 20 mL of the supernatant and mix it with an equal volume of PBS solution. Slowly add 40 mL of saturated ammonium sulfate at 4°C overnight while stirring to fully precipitate the protein.

[0105] (4) Centrifuge at 13,000 rpm for 10 min at 4°C, discard the supernatant, dissolve the precipitate with 12 mL of PBS, and then slowly add 8 mL of saturated ammonium sulfate dropwise while stirring, and react at 4°C for 1 h;

[0106] (5) Centrifuge at 13,000 rpm for 10 min at 4°C, discard the supernatant, dissolve the precipitate with 13 mL of PBS, add 7 mL of saturated ammonium sulfate dropwise, and react at 4°C for 1 h;

[0107] (6) Centrifuge at 13,000 rpm for 10 min at 4°C, discard the supernatant, and dissolve the precipitate with a small amount of PBS (pH 7.4);

[0108] (7) The dissolved solution was ultrafiltered, desalted, concentrated, and stored at -20°C with glycerol for later use.

[0109] Example 8: Detection of Rubella Virus by Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay (ELISA)

[0110] Rubella virus testing is done as follows:

[0111] (1) Rubella virus monoclonal antibody (provided in Example 6) was diluted to a coating concentration of 1 μg / mL in 0.05 mol / L carbonate buffer at pH 9.6, added to 100 μL / well of the ELISA plate, incubated at 4°C overnight, dried, and washed three times with 0.01 mol / L PBST;

[0112] (2) Block with 10% calf serum in PBS (0.01 mol / L, pH 7.4), 200 μL / well, incubate at 37°C for 2 h, and spin dry;

[0113] (3) Add rubella virus samples, 100 μL / well of 5 μg / mL, 500 ng / mL, 50 ng / mL, and 5 ng / mL of the test samples, and set up a negative control, and incubate at 37°C for 1 h;

[0114] (4) Wash the plate three times with 0.01 mol / L PBST and spin dry; dilute the polyclonal antibody (provided in Example 7) 1000 times and add it to the ELISA plate (100 μL / well) at 37°C for 1 h;

[0115] (5) Wash the plate three times with 0.01 mol / L PBST, spin dry, add 10000-fold diluted commercial HRP-labeled rabbit anti-goat secondary antibody, 100 μL / well, and incubate at 37°C for 1 h;

[0116] (6) Wash the plate 5 times with 0.01 mol / L PBST, spin dry, and add TMB substrate solution A and solution B (solution A is H2O2 or carbamide peroxide, solution B is TMB, 50 μL / well each) at 37°C for 10 min;

[0117] (7) Add 50 μL / well of 2 mol / L sulfuric acid to terminate the reaction and measure the absorbance at 630 nm using an ELISA reader.

[0118] The endpoint method is used to read the value, and the negative sample is 0.01 mol / L PBST. The detection sensitivity of the method provided by the present invention is the sample corresponding to OD 630 The result shows that the sensitivity of the method provided by the present invention is 50 ng / mL, and the corresponding OD 630 The value is 0.2016.

[0119] Example 9: Specificity Verification

[0120] The method provided in Example 8 was used to detect multiple viruses, namely, in step (3), 100 μL / well of rubella virus (RV), yellow fever virus (YEV), Japanese encephalitis virus (JEV), human cytomegalovirus (HCMV), and human herpes simplex virus type 1 (HSV-1) were added respectively.

[0121] The OD of each group 630 The values ​​are shown in Table 4.

[0122] Table 4: OD of different viruses detected 630 value

[0123] sample RV YEV JEV HCMV HSV-1 Negative control OD value 2.178 0.060 0.027 0.065 0.039 0.022

[0124] From the above results, it can be seen that the method provided by the present invention has strong specificity and can only detect Rubella virus, without cross-detection with YEV, JEV, HCMV, and HSV-1.

[0125] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An antibody or antigen-binding fragment that specifically recognizes rubella virus, characterized in that: The antibody or antigen-binding fragment comprises: a heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, wherein the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO:6, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:7; and The light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3, wherein the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 12, the amino acid sequence of the light chain CDR2 is shown in AAS, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:

13.

2. The antibody or antigen-binding fragment according to claim 1, comprising: A heavy chain variable region comprising or consisting of an amino acid sequence as shown in SEQ ID NO:8, or an amino acid sequence having at least 85% homology, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to the amino acid sequence as shown in SEQ ID NO:8; and / or A light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having at least 85% homology, for example, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to the amino acid sequence as shown in SEQ ID NO: 14, or consisting of said amino acid sequence.

3. An isolated nucleic acid molecule comprising a nucleic acid encoding the antibody or antigen-binding fragment of claim 1 or 2. A recombinant vector comprising the nucleic acid molecule according to claim 3 . A host cell comprising the nucleic acid molecule according to claim 3 or the recombinant vector according to claim 4.

6. A protein conjugate comprising the antibody or antigen-binding fragment according to claim 1 or 2 and a ligand.

7. A pharmaceutical composition comprising the antibody or antigen-binding fragment of claim 1 or 2 or the protein conjugate of claim 6; Preferably, the pharmaceutical composition further contains other active ingredients and / or pharmaceutically acceptable carriers.

8. Use of the antibody or antigen-binding fragment of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant vector of claim 4, the host cell of claim 5, the protein conjugate of claim 6 or the pharmaceutical composition of claim 7 in the preparation of a drug for preventing or treating diseases caused by rubella virus infection.

9. A kit for in vitro detection of rubella virus, comprising the antibody or antigen-binding fragment of claim 1 or 2 or the protein conjugate of claim 6; Preferably, the antibody, antigen-binding fragment or protein conjugate is labeled with a marker, and the marker is preferably selected from an enzyme, a chemiluminescent group and an isotope group; preferably, the kit is a double antibody sandwich enzyme-linked immunosorbent assay kit; Preferably, the kit further comprises polyclonal antibodies to rubella virus, preferably obtained by purifying serum obtained from mammals after immunization with rubella virus; Preferably, the kit further comprises a secondary antibody labeled with horseradish peroxidase and a color developing solution containing 3,3',5,5'-tetramethylbenzidine.

10. Use of the antibody or antigen-binding fragment according to claim 1 or 2, the protein conjugate according to claim 6 or the kit according to claim 9 for in vitro detection of rubella virus.

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