Monoclonal antibody aiming at respiratory syncytial virus F protein, detection reagent based on monoclonal antibody and application of monoclonal antibody

Abstract

The invention relates to a monoclonal antibody aiming at respiratory syncytial virus F protein, a detection reagent based on the monoclonal antibody and application of the monoclonal antibody. According to the present invention, the monoclonal antibody or the antigen binding fragment thereof can specifically bind with the respiratory syncytial virus F protein with high affinity in vivo or in vitro, and has broad-spectrum effectiveness on various genotypes, such that the huge potential application value is provided in the clinical detection of respiratory syncytial virus infection.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology and molecular virology, and in particular to a monoclonal antibody or an antigen-binding fragment thereof against respiratory syncytial virus F protein, a detection reagent based thereon and an application thereof. Background Art

[0002] Respiratory Syncytial Virus (RSV) belongs to the Pneumoviridae family and the Orthopneumovirus genus. It is an enveloped, non-segmented, negative-sense RNA virus. RSV is an important pathogen that causes acute respiratory infections in humans.

[0003] In recent years, with the analysis of the pre-fusion (pre-F) conformation of RSV F protein and the identification of new neutralization-sensitive epitopes, the research and development of RSV immune preparations has made breakthrough progress.

[0004] The genome size of RSV is about 15-16kb, encoding 11 proteins, including 8 structural proteins and 3 non-structural proteins (NS1, NS2 and M2-2). Structural proteins include 3 transmembrane surface proteins (G, F, SH), 2 matrix proteins (M and M2-1) and 3 nucleocapsid proteins (L, N and P); among them, G protein is responsible for mediating the binding of the virus to the host cell, while F protein mediates the fusion of the virus and the host cell membrane, allowing the virus to enter the cell; F protein and G protein are essential for viral replication, and both contain B cell and T cell epitopes, and are important viral antigen proteins that stimulate the body to produce humoral and cellular immunity. The G protein coding region has a large variation, and can be divided into subtype A and subtype B according to its variation, and the neutralizing antibodies induced by G protein are subtype specific; the F protein coding region is highly conserved, and the amino acid sequences of F proteins of subtype A and subtype B are at least 90% identical, so the neutralizing antibodies induced by F protein can inhibit RSV infection of both subtypes A and B at the same time. The antigenic epitopes of F protein related to neutralization activity are mainly II, IV, V and Four, of which epitopes II and IV are present in the pre-fusion and post-fusion F proteins; V and These are the specific antigenic sites of the F protein before fusion. The F protein after fusion does not have these two epitopes. The neutralizing titer of epitope monoclonal antibody is 10 to 100 times that of epitope II monoclonal antibody, and the neutralizing activity of V epitope monoclonal antibody is also high. Therefore, most of the RSV neutralizing activity in the serum is mainly directed against the antigenic site of the pre-fusion F protein.

[0005] The detection method of RSV infection includes nucleic acid detection, antigen detection, serological specific antibody detection and virus isolation and culture, among which serum specific IgM antibody positive is of great reference significance for etiological diagnosis. At present, the domestic method for detecting RSV infection includes electron microscopy detection, culture identification method, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, agglutination method, fluorescent immunospecific antibody detection, etc. The colloidal gold detection method based on high-affinity monoclonal antibodies is of great significance for convenient and fast RSV etiological detection. Therefore, it is urgently needed to develop a monoclonal antibody with high affinity for RSV antigen for convenient and fast RSV etiological detection. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a specific monoclonal antibody with high affinity to the F protein of respiratory syncytial virus (RSV), its related products, and its preparation method and use.

[0007] The inventors of the present invention have obtained several monoclonal antibodies against RSV F protein after a large number of experimental screenings, which can specifically recognize and target RSV F protein and have a high affinity with RSV F protein. Therefore, the monoclonal antibodies of the present invention are particularly suitable for immunological detection of RSV.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof against respiratory syncytial virus F protein, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises:

[0010] (1) the three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:7, and the three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:8; or,

[0011] (2) three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO: 13, and three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO: 14; or,

[0012] (3) three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:23, and three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:24; or,

[0013] (4) three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:27, and three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:28; or,

[0014] (5) three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:37, and three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:38.

[0015] In a preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0016] (1) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; wherein the amino acid sequence of SEQ ID NO:5 is AAT; or,

[0017] (2) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:11, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:12, respectively; wherein the amino acid sequence of SEQ ID NO:5 is AAT; or,

[0018] (3) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, respectively; wherein the amino acid sequence of SEQ ID NO:21 is YAS; or,

[0019] (4) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:12, respectively; wherein the amino acid sequence of SEQ ID NO:5 is AAT; or,

[0020] (5) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively; wherein the amino acid sequence of SEQ ID NO:35 is KVS.

[0021] Further preferably, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0022] (1) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:7 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:7; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:8; or

[0023] (2) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 13 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 13; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 14; or

[0024] (3) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:23 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:23; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:24; or

[0025] (4) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:27 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:27; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:28; or

[0026] (5) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:37 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:37; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:38 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:38.

[0027] More preferably, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0028] (1) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:8; or

[0029] (2) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 13, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 14; or

[0030] (3) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 24; or

[0031] (4) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 27, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 28; or

[0032] (5) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:37, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:38.

[0033] In addition, the monoclonal antibody or antigen-binding fragment thereof further comprises a constant region; preferably, the constant region is any one selected from the following: a constant region of an IgG, IgA or IgM antibody.

[0034] In a preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0035] (1) a heavy chain having an amino acid sequence as set forth in SEQ ID NO:9 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:9; and a light chain having an amino acid sequence as set forth in SEQ ID NO:10 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:10; or

[0036] (2) a heavy chain having an amino acid sequence as set forth in SEQ ID NO:15, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:15; and a light chain having an amino acid sequence as set forth in SEQ ID NO:16, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:16; or

[0037] (3) a heavy chain having an amino acid sequence as set forth in SEQ ID NO:25 or an amino acid sequence that has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:25; and a light chain having an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence that has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26; or

[0038] (4) a heavy chain having an amino acid sequence as set forth in SEQ ID NO:29 or an amino acid sequence that has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:29; and a light chain having an amino acid sequence as set forth in SEQ ID NO:30 or an amino acid sequence that has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:30; or

[0039] (5) a heavy chain having an amino acid sequence as set forth in SEQ ID NO:39 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:39; and a light chain having an amino acid sequence as set forth in SEQ ID NO:40 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:40.

[0040] Most preferably, the monoclonal antibody or antigen-binding fragment thereof comprises:

[0041] (1) a heavy chain with an amino acid sequence as shown in SEQ ID NO:9, and a light chain with an amino acid sequence as shown in SEQ ID NO:10; or

[0042] (2) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain having an amino acid sequence as shown in SEQ ID NO: 16; or

[0043] (3) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 25, and a light chain having an amino acid sequence as shown in SEQ ID NO: 26; or

[0044] (4) a heavy chain having an amino acid sequence as shown in SEQ ID NO:29, and a light chain having an amino acid sequence as shown in SEQ ID NO:30; or

[0045] (5) a heavy chain having an amino acid sequence as shown in SEQ ID NO:39, and a light chain having an amino acid sequence as shown in SEQ ID NO:40.

[0046] In some specific embodiments, the monoclonal antibody or antigen-binding fragment thereof further has a leader sequence (eg, a signal peptide sequence) at the N-terminus of the heavy chain variable region and / or the light chain variable region.

[0047] In some feasible embodiments, the antigen-binding fragment of the monoclonal antibody is selected from Fab, Fab', F(ab') 2 , Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, human antibody, chimeric antibody or bispecific or multispecific antibody.

[0048] The monoclonal antibody or antigen-binding fragment thereof of the present invention can bind to RSV F protein with high affinity and specificity, and thus can be used for immunological detection of respiratory syncytial virus.

[0049] In a second aspect, the present invention provides a polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect. The polynucleotide is not limited by the method of its production and can be obtained by genetic engineering recombination technology or chemical synthesis method.

[0050] In a possible embodiment, the polynucleotide is a set of polynucleotides.

[0051] In some preferred embodiments, the polynucleotide set comprises:

[0052] (I) a first polynucleotide encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7, preferably, the first polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO:41 or its corresponding mRNA molecule; and a second polynucleotide encoding a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8, preferably, the second polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO:42 or its corresponding mRNA molecule; or

[0053] (II) a first polynucleotide encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 13, preferably, the first polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 43 or its corresponding mRNA molecule; and a second polynucleotide encoding a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 14, preferably, the second polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 44 or its corresponding mRNA molecule; or

[0054] (III) a first polynucleotide encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23, preferably, the first polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 45 or its corresponding mRNA molecule; and a second polynucleotide encoding a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 24, preferably, the second polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 46 or its corresponding mRNA molecule; or

[0055] (IV) a first polynucleotide encoding a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 27, preferably, the first polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 47 or its corresponding mRNA molecule; and a second polynucleotide encoding a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 28, preferably, the second polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO: 48 or its corresponding mRNA molecule; or

[0056] (V) a first polynucleotide encoding a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:37, preferably, the first polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO:49 or its corresponding mRNA molecule; and a second polynucleotide encoding a light chain variable region with an amino acid sequence as shown in SEQ ID NO:38, preferably, the second polynucleotide is a DNA molecule comprising a nucleotide sequence as shown in SEQ ID NO:50 or its corresponding mRNA molecule.

[0057] In a third aspect, the present invention provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above, and at least one expression control element operably linked to the polynucleotide.

[0058] In a fourth aspect, the present invention provides a vector comprising the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above.

[0059] The vector of the present invention may be a cloning vector or an expression vector, for example, it may be a plasmid, a cosmid, a phage, etc.

[0060] In some preferred embodiments, the vector is an expression vector, preferably a eukaryotic expression vector.

[0061] In a fifth aspect, the present invention provides a host cell transformed with the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the vector as described in the fourth aspect;

[0062] The host cell includes, but is not limited to, prokaryotic cells, such as Escherichia coli cells, eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cell can also be a cell line, such as a 293T cell line.

[0063] Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0064] In a sixth aspect, the present invention provides a detection reagent for detecting respiratory syncytial virus, which comprises the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above.

[0065] In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention further comprises a detectable label; in certain preferred embodiments, the kit further comprises a second antibody, which specifically recognizes the monoclonal antibody or antigen-binding fragment thereof of the present invention or an anti-idiotypic antibody; preferably, the second antibody further comprises a detectable label; such detectable labels are well known to those skilled in the art, and include, but are not limited to, radioactive isotopes, fluorescent substances, luminescent substances, colored substances and enzymes (e.g., horseradish peroxidase), etc.

[0066] In a preferred embodiment, the detection reagent is a double antibody sandwich immunoassay reagent, which comprises two monoclonal antibodies or antigen-binding fragments thereof selected from the monoclonal antibodies or antigen-binding fragments thereof described in the first aspect above, wherein one monoclonal antibody or antigen-binding fragment thereof is defined as item (1), (2) or (4) in the first aspect above, and the other monoclonal antibody or antigen-binding fragment thereof is defined as item (3) or (5) in the first aspect above.

[0067] In the seventh aspect, the present invention provides the use of the monoclonal antibody or its antigen-binding fragment as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the expression vector as described in the fourth aspect, the host cell as described in the fifth aspect and / or the detection reagent as described in the sixth aspect in the preparation of a product for detecting the presence or level of respiratory syncytial virus in a sample and / or for diagnosing respiratory syncytial virus infection.

[0068] Optionally, the sample is a biological sample from a subject.

[0069] In an eighth aspect, the present invention provides a method for preparing the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, the method comprising: allowing the host cell as described in the fifth aspect above to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for the expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from the culture of the host cell.

[0070] In the ninth aspect, the present invention provides a method for detecting the presence or level of respiratory syncytial virus in a sample, the method comprising using the monoclonal antibody or its antigen-binding fragment as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the host cell as described in the fifth aspect above and / or the detection reagent as described in the sixth aspect above.

[0071] In some preferred embodiments of this method, the monoclonal antibody or antigen-binding fragment thereof further comprises a detectable label.

[0072] In some other preferred embodiments of the method, the method further comprises: using a secondary antibody carrying a detectable label to detect the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0073] The method can be used for diagnostic purposes (eg, the sample is a sample from a patient), or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

[0074] Therefore, in some specific embodiments, the present invention provides a method for diagnosing whether a subject is infected with respiratory syncytial virus, comprising: using the monoclonal antibody or antigen-binding fragment thereof of the present invention as described in the first aspect above, detecting the presence of respiratory syncytial virus F protein in a sample from the subject. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention further comprises a detectable label; in other preferred embodiments, the method further comprises using a second antibody carrying a detectable label to detect the monoclonal antibody or antigen-binding fragment thereof or anti-idiotypic antibody of the present invention. In a feasible embodiment, the subject is a mammal, such as a human.

[0075] The general method of using monoclonal antibodies or their antigen-binding fragments to detect the presence of target viruses or antigens in samples or their levels is well known to those skilled in the art. In certain preferred embodiments, the detection method can use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competitive method and similar detection methods.

[0076] Beneficial Effects

[0077] The inventors have confirmed through experiments that the monoclonal antibody of the present invention can specifically bind to RSV F protein with high affinity both in vitro and in vivo, and it shows high affinity for both the F protein of type A RSV and the F protein of type B RSV, and therefore has broad-spectrum effectiveness and is suitable for the detection of respiratory syncytial virus infection of all genotypes; in view of this, the monoclonal antibody or its antigen-binding fragment of the present invention has extremely high potential to be developed as a specific detection antibody for respiratory syncytial virus, and it can provide a lower detection limit and a higher detection sensitivity, and has great clinical application prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0079] Figure 1 The figure shows the SDS-PAGE identification results of each monoclonal antibody.

[0080] Figure 2 The binding force curves of the monoclonal antibody of the present invention and RSV-A type F protein (Figure A) and RSV-B type F protein (Figure B) detected by ELISA method.

[0081] Figure 3 The graph shows the results of Western blot detection of the immunogen RSV sc-Dimer (30 ng) using various monoclonal antibodies.

[0082] Figure 4 The results of SPR determination of the affinity of the monoclonal antibody of the present invention to RSV-A type F protein and RSV-B type F protein are shown.

[0083] Figure 5 The figure shows the result of immunofluorescence staining of mouse lungs infected with RSV A virus using the monoclonal antibody of the present invention.

[0084] Figure 6 The figure shows the result of immunofluorescence staining of mouse lungs infected with RSV B virus using the monoclonal antibody of the present invention. DETAILED DESCRIPTION

[0085] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0086] Unless explicitly stated otherwise, 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.

[0087] The term "and / or" should be understood to mean that a single option can be selected, or a combination of two or more options.

[0088] The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of the amino acid residues in the candidate amino acid sequence and the identical amino acid residues in the reference amino acid sequence when the amino acid sequences are aligned (and gaps are introduced if necessary) to obtain the maximum percentage sequence identity (and any conservative substitutions are not considered as part of the sequence identity). Sequence alignment can be performed using various methods in the art to determine the percentage (%) amino acid sequence identity.

[0089] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analog, which generally includes at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. The antibody fragment retains at least some of the binding specificity of the parent antibody. Specifically, the antigen-binding fragment can be selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabodies, etc.

[0090] The "Fab" fragment consists of a light chain and the CH1 and variable regions of a heavy chain.

[0091] A "Fab'" fragment contains a light chain and a portion of a heavy chain including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, with an interchain disulfide bond formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0092] The "F(ab')2" fragment contains two light chains and portions of two heavy chains including a VH domain, a CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0093] The "Fv" region contains the variable regions from both the heavy and light chains, but lacks the constant regions.

[0094] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, which domains are contained in a single polypeptide chain. Generally speaking, the scFv polypeptide comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0095] "Bispecific antibodies" are small antigen-binding fragments with two antigen-binding sites. The fragments contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are allowed to pair with the complementary domains of another chain and form two antigen-binding sites.

[0096] "Affinity" or "binding affinity" refers to the binding force between an antibody and its antigen. The affinity of an antibody for its antigen is usually represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art.

[0097] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form.

[0098] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0099] Example 1: Preparation, screening, identification and sequencing of monoclonal antibodies

[0100] Step 1: Design and produce specific antigens

[0101] 1) Antigen design:

[0102] RSV sc-Dimer protein was prepared according to the method described in Example 1 of patent CN202311316283.6.

[0103] 2) Antigen production & purification:

[0104] RSV sc-Dimer protein was secreted and expressed using the 293F expression system, and the protein was newly purified by the His tag affinity layer. It was further purified into PBS buffer by molecular sieve chromatography according to the molecular weight. The protein concentration was determined by the BCA method, and the protein was packaged and frozen in a -80 degree refrigerator for later use.

[0105] Step 2: Immunize mice with antigen

[0106] The RSV sc-Dimer antigen protein produced and purified as described above was used to immunize female BALB / c mice aged 6-8 weeks, wherein 3 mice were immunized with the denatured antigen protein and 3 mice were immunized with the non-denatured antigen protein; the protocol was as follows: a single immunization dose of 50 μg was used for each mouse, and for the first immunization, the immunogen was prepared into an emulsion with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple points in the abdomen; for the second and third immunizations, the same dose of the immunogen was prepared into an emulsion with an equal volume of incomplete Freund's adjuvant and injected subcutaneously at multiple points in the abdomen 2 weeks after the previous immunization.

[0107] Step 3: Blood collection and titer testing

[0108] A. Serum Collection

[0109] One week after the last immunization, 50-60 μL of blood was collected from the mouse orbital venous plexus. After standing at 4°C overnight, the blood was centrifuged at 4000 rpm and 4°C for 15 minutes to separate the upper serum for testing.

[0110] B. ELISA method to detect the antibody titer of immunized mouse serum

[0111] The indirect ELISA method was used to determine the antibody titer of the immunized mouse serum, and the titer qualification standard was set as OD450-Blank greater than 1.0 when the mouse serum was diluted 1:8000.

[0112] (1) Coating: Dissolve an appropriate amount of RSV sc-Dimer protein in coating buffer and dilute to 5 μg / mL. Then add 100 μL to each well of a 96-well plate using a pipette. Tap the plate to mix the sample, seal with plastic wrap, and coat overnight at 4°C.

[0113] (2) Washing: Wash the plate once with washing solution at 300 μL / well and dry the ELISA plate;

[0114] (3) Blocking: Block the ELISA plate with blocking solution at 300 μL / well for 1 h at room temperature;

[0115] (4) Washing: Wash the plate twice with washing solution at 300 μL / well and dry the ELISA plate;

[0116] (5) Sample loading: Add the serially diluted serum sample and sample diluent at 100 μL / well;

[0117] (6) Adding secondary antibody: Add goat anti-mouse HRP secondary antibody to 96-well plate at 100 μL / well and incubate at room temperature for 2 h;

[0118] (7) Washing: Wash the plate five times with washing solution at 300 μL / well and dry the ELISA plate;

[0119] (8) Color development: Add 200 μL / well of color development solution and incubate at room temperature for 12 min;

[0120] (9) Termination and detection: 50 μL / well of stop solution was added to terminate the reaction, and then the reaction was detected using an ELISA reader at a wavelength of 450 nm.

[0121] The experimental results are shown in Tables 1, 2 and 3 below.

[0122] Table 1. Results of serum ELISA test after three immunizations with non-denatured protein

[0123]

[0124]

[0125] Table 2. Results of serum ELISA test after three immunizations with denatured protein

[0126]

[0127] Table 3. Results of serum ELISA test after four immunizations with denatured protein

[0128]

[0129]

[0130] The immunogen was coated at 5 μg / mL, and the serum of immune mice was diluted 8000 times. When OD450-Blank>1.0, it was considered as qualified. Among the qualified immune mice, the immune mice with high ELISA titer were preferred for shock immunization. The spleen of the mice was taken for hybridoma fusion 3-4 days after shock immunization. Unqualified animals were given additional immunization until the titer was qualified, and the maximum number of immunizations was 5 times.

[0131] Step 4: Cell fusion to produce hybridoma cells

[0132] The spleen cells of the immunized mice were taken and fused with SP2 / 0 cells by electrofusion method. The electrofusion was performed for two rounds to obtain hybridoma cells. The positive primary clone cells binding to RSV sc-Dimer were screened by indirect ELISA method.

[0133] Step 5: Limiting dilution and subclone screening

[0134] Using HT selection medium, each positive hybridoma cell obtained by screening was inoculated in half a 96-well cell culture plate at a seeding density of 0.75 cells per well, and cultured statically at 5% CO2 and 37°C. Take the supernatant for indirect ELISA detection about the 7th day after culture to confirm that the antibody to be tested binds positively to the antigen. For the specific detection process, refer to the main clone indirect ELISA detection process. Select ELISA specific binding positive monoclonal cells and perform 1-2 rounds of limiting dilution again until stable, positive monoclonal cells are obtained. Through the above experimental process, a total of 10 positive mouse hybridoma cell lines were obtained.

[0135] Step 6: Amplification of specific hybridomas and antibody purification

[0136] A. Specific hybridoma expansion

[0137] The large-scale preparation of monoclonal antibodies mainly adopts in vitro culture method. Hybridoma cells are placed in culture bottles for culture. During the culture process, hybridoma cells produce and secrete monoclonal antibodies. The culture supernatant is collected and centrifuged to remove the cells and their debris to obtain the required monoclonal antibodies. 5mL / 20mL hybridoma cells are transferred to 100mL / 500mL culture bottles, and a certain amount of culture medium is added regularly for cell expansion to expand the cells to the required volume. After 6-8 days of culture, the material is collected. The collected hybridoma cell supernatant is centrifuged using a desktop centrifuge, the centrifugation condition is 4000g, the centrifugation time is 30min, and the cell supernatant after centrifugation is collected; the cell supernatant after centrifugation is filtered using a 0.45μM filter membrane.

[0138] B. Antibody Purification

[0139] The hybridoma cell culture supernatant after centrifugation was purified using the Protein A affinity purification method to harvest mouse monoclonal antibodies. The operation steps are as follows.

[0140] (1) Select a protein A column of appropriate specifications according to the expression level.

[0141] (2) Water balance: Wash with ultrapure water for 2CV and replace the 25% ethanol storage solution;

[0142] (3) Equilibration of chromatography column: AC Binding + 1 / 5 stock buffer for 10 CV;

[0143] (4) Loading: Pour the sample;

[0144] (5) Elution: Elution with eluent for 5-10 CV until the protein column is in equilibrium;

[0145] (6) Elution: AC elution, discard the first 1CV and collect the last 1.5CV;

[0146] (7) Neutralization: Add 2 M Tris, pH 8.0 to neutralize the eluted antibody;

[0147] (8) Balance: AC Binding balance 5CV to neutral;

[0148] (9) CIP cleaning: CIP cleaning for more than 5CV;

[0149] (10) Alkali flushing: AC Binding flushing alkali until the pH at the outlet is neutral;

[0150] (11) Storage: Equilibrate the column with 25% ethanol for 2CV.

[0151] C. Mouse monoclonal antibody detection (antibody concentration and purity detection)

[0152] Start the micro-spectrophotometer and spot the sample with the corresponding buffer of the test sample. When the absorbance value is between ±0.015 at a wavelength of 280nm, it indicates that the instrument baseline is stable. Spot the purified antibody samples in turn and record the absorbance value. The test data is divided by the IgG extinction coefficient (1.414), and the resulting value is the concentration of the test sample (in mg / mL).

[0153] Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was used to verify the purity of the purified antibody. This method separates the proteins in the test sample in the electrophoresis gel according to their different molecular weights, thereby verifying the purity of the target antibody. Take 5μg of the test sample and add 5μL 4R / 4N, heat it in a 100℃ water bath, and then centrifuge it at 10000rpm. Add enough 1×SDS electrophoresis buffer to the electrophoresis tank, and use a micropipette to add the treated sample solution and protein molecular weight standards to the sample well. Connect the power supply, first electrophoresed at a constant voltage of 100V until the bromophenol blue dye enters the separation gel from the stacking gel, then adjust the current to 140V and continue electrophoresis to the bottom of the gel plate, and turn off the power supply. Soak the gel in the staining solution, heat it in a microwave for 60s, and stain it at room temperature on a gently shaking platform. Replace the destaining solution to cover the gel, heat it in a microwave for 60 seconds, destain it at room temperature on a gently shaking platform, and repeat the destaining operation until a blue band and a clean background are obtained.

[0154] The results of denaturing and non-denaturing SDS-PAGE detection of monoclonal antibodies produced by the 10 hybridoma cells obtained in step 5 are as follows: Figure 1 As shown; Figure 1 The top is a non-reducing gel (i.e., a sample without DTT added to the antibody), and its band molecular weight is about 150 KDa, corresponding to the intact antibody; Figure 1The lower part is the reducing gel (i.e., the sample with antibody plus DTT), and the molecular weights of the two bands thereof are respectively about 50 KDa (i.e., corresponding to the antibody heavy chain) and about 25 KDa (i.e., corresponding to the antibody light chain).

[0155] Figure 1 The results showed that 10 hybridoma mouse monoclonal antibodies of good quality were obtained through amplification and purification, which were named Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9 and Ab-10 respectively.

[0156] Example 2: ELISA method to detect the binding of monoclonal antibodies and immunogens

[0157] In this embodiment, the ELISA method was used to detect the binding of each purified monoclonal antibody to RSV-A type F protein and RSV-B type F protein. The specific detection process is as follows:

[0158] 1) Coating: Coating with RSV-A type F protein or RSV-B type F protein, the coating concentration is 3 μg / mL, 5-fold concentration gradient dilution, 7 concentration gradients are arranged in a 96-well plate, 100 μL / well, and coating is carried out at 4°C overnight;

[0159] 2) Blocking: discard the liquid in the plate and pat dry, add 2% BSA in PBST solution, 200 μL / well, seal and incubate at room temperature for 1 hour;

[0160] 3) Washing: 200 μL / well PBST washing solution, wash the plate twice, and pat dry for the last time;

[0161] 4) Antibody dilution and sample addition: dilute the antibody to be tested to 1 μg / mL, and add 100 μL of each of Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, and Ab-10 from left to right to the corresponding well plate, mix well, and react at room temperature for 2 hours;

[0162] 5) Washing: 200 μL / well washing solution, wash the plate 3 times, and pat dry for the last time;

[0163] 6) Secondary antibody incubation: Rabbit Anti-Mouse IgG F(ab')2 / HRP (1:2000) diluted secondary antibody, 100 μL / well, mixed evenly, incubated at room temperature for 1 hour;

[0164] 7) Washing: 200 μL / well washing solution, wash the plate 3 times, and pat dry for the last time;

[0165] 8) Color development: Add 60 μL to each well and incubate at room temperature in the dark for 20 min;

[0166] 9) Termination and detection: Add 60 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0167] The antibody-antigen binding curve was drawn with the Log value of the antigen protein concentration as the horizontal axis and the measured OD450 value as the vertical axis.

[0168] The binding force curves of monoclonal antibodies to RSV-A type F protein and RSV-B type F protein are shown in Figure 2 As shown in Figures A and B in the figure, it shows that Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, and Ab-10 can all effectively bind to the F proteins of RSV-A and RSV-B through spatial epitopes; among them, Ab-2, Ab-8, Ab-9, and Ab-10 antibodies have strong binding ability to the F proteins of RSV-A and RSV-B.

[0169] Example 3: Western blot detection of the binding of monoclonal antibodies to immunogens

[0170] In this example, Western blot was used to detect the binding of each purified monoclonal antibody to the immunogen RSV sc-Dimer. The specific detection process is as follows:

[0171] 1) Loading: Add the immunogen RSV sc-Dimer into the loading buffer at a loading volume of 30 ng / lane;

[0172] 2) Electrophoresis: Constant voltage 100V, until bromophenol blue runs to the bottom of the gel plate;

[0173] 3) Transfer: constant voltage 110V, 90min;

[0174] 4) Blocking: Block at 4°C overnight;

[0175] 5) Primary antibody incubation: Add hybridoma supernatant to the multichannel hybridization instrument and incubate at room temperature for 2 hours;

[0176] 6) Elution: Use a decolorizing shaker to elute the membrane 3 times, 5 min each time;

[0177] 7) Secondary antibody incubation: goat anti-mouse IgG (H+L) / HRP final concentration 1:10000, room temperature for 2 hours;

[0178] 8) Elution: Use a decolorizing shaker to elute the membrane 3 times, 5 min each time;

[0179] 9) Color development: Chemiluminescence imaging system is used for color development.

[0180] The results are as follows Figure 3 As shown; Figure 3The results show that Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, and Ab-10 can all effectively bind to RSV sc-Dimer protein.

[0181] Example 4: Surface Plasmon Resonance (SPR) Determination of Affinity of 10 Mouse Hybridoma Antibodies to RSV-A F Protein and RSV-B F Protein

[0182] First, the anti-mouse IgG antibody was fixed on the CM5 chip using the amino coupling method. After fixation, the mouse hybridoma monoclonal antibodies (numbered Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10) were bound by the capture method. RSV AF protein monomer and RSV BF protein monomer were used as mobile phases to carry out experiments to determine the binding affinity between the antibody and the F protein. The mobile buffer was a PBST solution containing 0.05% Tween 20. The affinity test results of each monoclonal antibody with RSV-A type F protein and RSV-B type F protein are shown in the following table. Figure 4 The affinity data statistical results are shown in Table 4 below.

[0183] Table 4. Affinity of monoclonal antibodies to RSV-A type F protein and RSV-B type F protein

[0184]

[0185] Depend on Figure 4 As can be seen from Table 4, monoclonal antibodies Ab-2, Ab-6, Ab-8, Ab-9, and Ab-10 have high affinity to RSV F protein (pM ​​level); among them, the affinity to RSV A type F protein can reach pM level, and the affinity to RSV B type F protein can reach ten pM level; in addition, monoclonal antibodies Ab-1 and Ab-5 have affinity to RSV A type and B type F protein to nM level, and Ab-3, Ab-4, and Ab-7 have weaker affinity to RSV A type and B type F protein, and their order of magnitude is between hundreds of nM and μM.

[0186] Therefore, monoclonal antibodies Ab-2, Ab-6, Ab-8, Ab-9, and Ab-10 with higher affinity to the antigen were selected for sequencing, and they are the antibodies to be protected by the present invention.

[0187] Example 5: Sequencing of Antibodies

[0188] Antibody sequencing experimental steps:

[0189] 1) Extract RNA after hybridoma cell lysis;

[0190] 2) Reverse transcription: Using the extracted RNA as a template, the Sino-Biotech Reverse Transcription Kit was used to obtain cDNA in two steps (by first opening the RNA secondary structure and then allowing the primers to bind to the RNA and polymerize the cDNA to achieve the purpose of obtaining cDNA);

[0191] 3) Using the cDNA obtained by reverse transcription as a template, amplify by PCR, insert the amplified fragment into an expression vector or commercial vector, and sequence to obtain a plasmid containing the correct sequence.

[0192] After sequencing and sequence identification, the relevant sequence information of monoclonal antibodies Ab-2, Ab-6, Ab-8, Ab-9, and Ab-10 is shown in the sequence table, wherein:

[0193] The HCDR1-3, LCDR1-3, VH, VL, H, and L sequences of Ab-2 are shown in SEQ ID NOs: 1-10, respectively;

[0194] The HCDR1-3, LCDR1-3, VH, VL, H, and L sequences of Ab-6 are shown in SEQ ID NOs: 11, 2, 3, 4, 5, 12, 13, 14, 15, and 16, respectively;

[0195] The HCDR1-3, LCDR1-3, VH, VL, H, and L sequences of Ab-8 are shown in SEQ ID NOs: 17-26, respectively;

[0196] The HCDR1-3, LCDR1-3, VH, VL, H, and L sequences of Ab-9 are shown in SEQ ID NOs: 1, 2, 3, 4, 5, 12, 27, 28, 29, and 30, respectively;

[0197] The HCDR1-3, LCDR1-3, VH, VL, H, and L sequences of Ab-10 are shown in SEQ ID NOs: 31-40, respectively.

[0198] Example 6: Octet assay to detect epitope competition of monoclonal antibodies

[0199] Experimental method: SA Sensor was used to capture biotinylated RSV sc-dimer protein. The first step of the mobile phase was used to determine the binding of three known antibodies (CR9501, Am22 and Palivizumab), and PBST was used as a control. The second step of the mobile phase was used to determine Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, and Ab-10. The analysis was used to confirm whether Ab 1-10 had epitope competition with the three known antibodies.

[0200] The results of the Octet experiment are shown in Table 5 below.

[0201] Table 5. Octet assay to determine competitive epitopes between monoclonal antibodies and three known antibodies

[0202]

[0203]

[0204] Table 5 shows that Ab-2, Ab-6, Ab-9 and Am22 antibodies have competitive binding ( Ab-8 had competitive binding with CR9501 antibody (epitope V), and other antibodies were not found to have obvious competitive binding with the three antibodies.

[0205] According to the above results, on the one hand, Ab-2, Ab-6, Ab-8, Ab-9 and Ab-10 all have high affinity binding ability with RSV sc-Dimer; on the other hand, because they bind to different antigenic epitopes, Ab-2, Ab-6, Ab-9 and Ab-8 or Ab10 have the potential to be paired antibodies for double antibody sandwich immunoassay (one as a capture antibody and the other as a detection antibody), for example, they can be used as candidate antibodies for RSV double antibody sandwich immunoassay test strips.

[0206] Example 7: Feasibility verification of using the monoclonal antibody of the present invention to detect mice infected with RSV

[0207] In this example, the feasibility of using the monoclonal antibody of the present invention to detect mice infected with RSV was verified by using the immunofluorescence staining method based on the monoclonal antibody of the present invention to detect the immunofluorescence staining of the lungs of mice infected with RSV virus.

[0208] The experimental steps of immunofluorescence staining are as follows:

[0209] 1) Dewaxing of paraffin sections: sequentially place the sections into environmentally friendly dewaxing solution I for 10 min - environmentally friendly dewaxing solution II for 10 min - environmentally friendly dewaxing solution III for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - and wash with distilled water.

[0210] 2) Antigen repair: Microwave citric acid (pH 6.0) at medium heat for 8 minutes, stop heating for 8 minutes, and switch to medium-low heat for 7 minutes. During the repair process, prevent excessive evaporation of the buffer and do not dry the slides. After repair, cool naturally. Place the slides in PBS (pH 7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time.

[0211] 3) Circle serum blocking: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue, add % BSA solution, and block for 30 minutes.

[0212] 4) Add primary antibody: Add diluted primary antibody (Ab-2, Ab-6, Ab-8, Ab-9, Ab-10 for detection, 1:500 dilution), and lay the slices flat in a humidified box and incubate overnight at 4°C.

[0213] 5) Add secondary antibody: Place the slide in PBS solution (pH 7.4) and wash on a shaker for 3 times, 5 minutes each time. Add Alexa Fluor 488-labeled goat anti-mouse IgG secondary antibody and incubate at room temperature for 50 minutes in the dark.

[0214] 6) DAPI counterstaining of cell nuclei: Place the slide in PBS (PH7.4) and wash on a decolorizing shaker for 3 times, 5 minutes each time. Add DAPI staining solution and incubate at room temperature for 10 minutes in the dark.

[0215] 7) Quench tissue autofluorescence: add autoluminescence quencher B solution for 5 minutes, rinse with running water for 10 minutes. Place the slide in PBS solution (PH7.4) and wash on a shaker for 3 times, 5 minutes each time.

[0216] 8) Sealing: Seal the slices with anti-fluorescence quenching sealing medium.

[0217] 9) Image acquisition: DAPI excitation wavelength 330-380nm, emission wavelength 420nm; 488 excitation wavelength 465-495nm, emission wavelength 515-555nm; CY3 excitation wavelength 510-560nm, emission wavelength 590nm; CY5 excitation wavelength 608-648nm, emission wavelength 672-712nm.

[0218] 10) Result analysis.

[0219] The cell nucleus of the DAPI channel is blue, and the positive cell nucleus of the 488 channel is green.

[0220] Immunofluorescence staining results of the lungs of mice infected with RSV A virus Figure 5 As shown; Figure 5 It shows that compared with the control, Ab-2, Ab-6, Ab-8, Ab-9, and Ab-10 can bind to infected tissue cells.

[0221] Immunofluorescence staining results of the lungs of mice infected with RSV B virus Figure 6 As shown; Figure 6 Show: Compared with the control, Ab-2, Ab-6, Ab-8, Ab-9, and Ab-10 can all bind to infected tissue cells.

[0222] The above results indicate that the monoclonal antibodies Ab-2, Ab-6, Ab-8, Ab-9 and Ab-10 of the present invention can effectively detect RSV virus infection, whether in tissues infected with RSV type A virus or in tissues infected with RSV type B virus.

[0223] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against respiratory syncytial virus F protein, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) the three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:7, and the three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:8; or, (2) three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO: 13, and three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO: 14; or, (3) three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:23, and three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:24; or, (4) three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:27, and three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:28; or, (5) three heavy chain complementarity determining regions in the heavy chain variable region as shown in SEQ ID NO:37, and three light chain complementarity determining regions in the light chain variable region as shown in SEQ ID NO:

38.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; wherein the amino acid sequence of SEQ ID NO:5 is AAT; or, (2) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:11, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:12, respectively; wherein the amino acid sequence of SEQ ID NO:5 is AAT; or, (3) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, respectively; wherein the amino acid sequence of SEQ ID NO:21 is YAS; or, (4) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:12, respectively; wherein the amino acid sequence of SEQ ID NO:5 is AAT; or, (5) a heavy chain variable region comprising heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, respectively; and a light chain variable region comprising light chain complementary determining regions LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively; wherein the amino acid sequence of SEQ ID NO:35 is KVS.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:7 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:7; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:8; or (2) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 13 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 13; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 14; or (3) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:23 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:23; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:24 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:24; or (4) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:27 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:27; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:28 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:28; or (5) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:37 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:37; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO:38 or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

38.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:8; or (2) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 13, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 14; or (3) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 23, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 24; or (4) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 27, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 28; or (5) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:37, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:

38.

5. The monoclonal antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain having an amino acid sequence as set forth in SEQ ID NO:9 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:9; and a light chain having an amino acid sequence as set forth in SEQ ID NO:10 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:10; or (2) a heavy chain having an amino acid sequence as set forth in SEQ ID NO:15, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:15; and a light chain having an amino acid sequence as set forth in SEQ ID NO:16, or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:16; or (3) a heavy chain having an amino acid sequence as set forth in SEQ ID NO:25 or an amino acid sequence that has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:25; and a light chain having an amino acid sequence as set forth in SEQ ID NO:26 or an amino acid sequence that has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26; or (4) a heavy chain having an amino acid sequence as set forth in SEQ ID NO:29 or an amino acid sequence that has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:29; and a light chain having an amino acid sequence as set forth in SEQ ID NO:30 or an amino acid sequence that has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:30; or (5) a heavy chain having an amino acid sequence as set forth in SEQ ID NO:39 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:39; and a light chain having an amino acid sequence as set forth in SEQ ID NO:40 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

40.

6. The monoclonal antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain with an amino acid sequence as shown in SEQ ID NO:9, and a light chain with an amino acid sequence as shown in SEQ ID NO:10; or (2) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 15, and a light chain having an amino acid sequence as shown in SEQ ID NO: 16; or (3) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 25, and a light chain having an amino acid sequence as shown in SEQ ID NO: 26; or (4) a heavy chain having an amino acid sequence as shown in SEQ ID NO:29, and a light chain having an amino acid sequence as shown in SEQ ID NO:30; or (5) a heavy chain having an amino acid sequence as shown in SEQ ID NO:39, and a light chain having an amino acid sequence as shown in SEQ ID NO:

40.

7. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein: The antigen binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, human antibody, chimeric antibody or bispecific or multispecific antibody.

8. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. The polynucleotide according to claim 8, wherein The polynucleotide is a polynucleotide group, and the polynucleotide group includes: (I) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:41 or its corresponding mRNA molecule, and a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:42 or its corresponding mRNA molecule; or (II) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:43 or its corresponding mRNA molecule, and a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:44 or its corresponding mRNA molecule; or (III) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:45 or its corresponding mRNA molecule, and a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:46 or its corresponding mRNA molecule; or (IV) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:47 or its corresponding mRNA molecule, and a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:48 or its corresponding mRNA molecule; or (V) a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:49 or its corresponding mRNA molecule, and a DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:50 or its corresponding mRNA molecule.

10. A nucleic acid construct comprising the polynucleotide according to any one of claims 8 to 9, and at least one expression control element operably linked to the polynucleotide.

11. A vector comprising the polynucleotide according to any one of claims 8 to 9, or the nucleic acid construct according to claim 10.

12. A host cell transformed with the polynucleotide according to any one of claims 8 to 9, the nucleic acid construct according to claim 10 or the vector according to claim 11.

13. A detection reagent for detecting respiratory syncytial virus, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

14. The detection reagent according to claim 13, characterized in that The detection reagent is a double antibody sandwich immunoassay reagent, which comprises two monoclonal antibodies or antigen-binding fragments thereof selected from the monoclonal antibodies or antigen-binding fragments thereof as described in any one of claims 1 to 7, wherein one monoclonal antibody or antigen-binding fragment thereof is defined by item (1), (2) or (4) of any one of claims 1 to 7, and the other monoclonal antibody or antigen-binding fragment thereof is defined by item (3) or (5) of any one of claims 1 to 7.

15. Use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 7, the polynucleotide as described in any one of claims 8 to 9, the nucleic acid construct as described in claim 10, the vector as described in claim 11, the host cell as described in claim 12 and / or the detection reagent as described in claim 13 or 14 in the preparation of a product for detecting the presence or level of respiratory syncytial virus in a sample and / or for diagnosing respiratory syncytial virus infection.

16. The use according to claim 15, characterized in that The sample is a biological sample from a subject.

17. A method for preparing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the method comprising: The host cell of claim 12 is allowed to express the monoclonal antibody or its antigen-binding fragment under conditions suitable for the expression of the monoclonal antibody or its antigen-binding fragment, and the expressed monoclonal antibody or its antigen-binding fragment is recovered from the culture of the host cell.

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