Monoclonal antibodies against respiratory syncytial virus f protein, assay reagents based thereon, and uses thereof

Abstract

The present application relates to monoclonal antibodies against respiratory syncytial virus F protein, detection reagents based thereon and applications thereof. The monoclonal antibodies or antigen-binding fragments thereof of the present application can specifically bind to respiratory syncytial virus F protein with high affinity both in vivo and in vitro, and have broad-spectrum effectiveness for each genotype, thus having great potential application value in clinical detection of respiratory syncytial virus infection.

Description

TECHNICAL FIELD

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

[0002] Respiratory syncytial virus (RSV) belongs to the family of pneumoviridae, orthopneumovirus, is a kind of enveloped, non-segmented, negative-sense RNA virus. RSV is an important pathogen causing acute respiratory infection 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 development of RSV immunological preparations has made a breakthrough.

[0004] The genome of RSV is about 15-16 kb in size, encoding 11 kinds of proteins, including 8 structural proteins and 3 non-structural proteins (NS1, NS2 and M2-2). The 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, the G protein is responsible for mediating the binding of the virus to the host cell, while the F protein mediates the fusion of the virus with the host cell membrane, allowing the virus to enter the cell; both F protein and G protein are essential for viral replication and contain B cell and T cell epitopes, which are important viral antigen proteins for stimulating the body to produce humoral and cellular immunity. The G protein coding region varies greatly and can be divided into A and B subtypes according to its variation, and the neutralizing antibodies induced by G protein have subtype specificity; the F protein coding region is highly conserved, and the F protein amino acid sequences of A and B subtypes are at least 90% identical, so the neutralizing antibodies induced by F protein can simultaneously inhibit A and B subtype RSV infection. The F protein has four antigenic epitopes related to neutralizing activity, among which epitopes II and IV exist in pre-fusion and post-fusion F proteins; V and VI are specific antigenic sites of pre-fusion F protein, and post-fusion F protein does not have these two epitopes. The neutralizing titer of epitope monoclonal antibody is 10-100 times that of No. II epitope monoclonal antibody, and the neutralizing activity of V epitope monoclonal antibody is also high, therefore, most of the RSV neutralizing activity in serum is mainly directed against the antigenic sites of pre-fusion F protein.

[0005] ​​The detection methods of RSV infection include nucleic acid detection, antigen detection, serological specific antibody detection and virus isolation culture, etc., wherein the positive serum specific IgM antibody has important reference significance for etiological diagnosis. At present, the detection methods of RSV infection at home include electron microscope detection, culture identification method, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, agglutination method, fluorescence immune specific antibody detection, etc. The colloidal gold detection method based on the high-affinity monoclonal antibody has important significance for the convenient and rapid RSV etiology detection. Therefore, at present, it is urgently needed to develop a monoclonal antibody with high affinity to RSV antigen for the convenient and rapid RSV etiology detection. SUMMARY

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

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

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

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

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

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

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

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

[0014] (5) 3 heavy chain complementarity determining regions in a heavy chain variable region as shown in SEQ ID NO: 37, and 3 light chain complementarity determining regions in a light chain variable region as shown in SEQ ID NO: 38.

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

[0016] (1) a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in 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 complementarity determining regions HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in 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 complementarity determining regions HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively; and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in 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 complementarity determining regions HCDR1, HCDR2 and HCDR3 of amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 of amino acid sequences set forth in 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 complementarity determining regions HCDR1, HCDR2 and HCDR3 of amino acid sequences set forth in SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, respectively; and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 of amino acid sequences set forth 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 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 set forth in SEQ ID NO: 7; and a light chain variable region having an amino acid sequence 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 set forth in SEQ ID NO: 8; or

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

[0024] (3) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:23 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence 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 an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:24; or

[0025] (4) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:27 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence 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 an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:28; or

[0026] (5) Heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:37 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:37; and light chain variable region having an amino acid sequence as shown in SEQ ID NO:38 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:38.

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

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

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

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

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

[0032] (5) a heavy chain variable region having an amino acid sequence of SEQ ID NO: 37, and a light chain variable region having an amino acid sequence of 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 group consisting of a constant region of an IgG, IgA or IgM antibody.

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

[0035] (1) a heavy chain having an amino acid sequence of 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 of SEQ ID NO: 9; and a light chain having an amino acid sequence of 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 of SEQ ID NO: 10; or

[0036] (2) a heavy chain having an amino acid sequence of 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 of SEQ ID NO: 15; and a light chain having an amino acid sequence of 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 of SEQ ID NO: 16; or

[0037] (3) a heavy chain having an amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 25; and a light chain having an amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of 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 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: 39; and a light chain having an amino acid sequence as set forth in SEQ ID NO: 40 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: 40.

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

[0041] (1) a heavy chain having an 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

[0042] (2) a heavy chain having an 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

[0043] (3) a heavy chain having an 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

[0044] (4) a heavy chain having an 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

[0045] (5) a heavy chain having an 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.

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

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

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

[0049] In a second aspect, the present application provides polynucleotides encoding the monoclonal antibody or the antigen-binding fragment thereof of the first aspect described above. The polynucleotides are not limited by the method for producing them, and can be obtained by genetic engineering recombination techniques or chemical synthesis methods.

[0050] In possible embodiments, the polynucleotides are a polynucleotide group.

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

[0052] (I) a first polynucleotide encoding a heavy chain variable region with 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 a corresponding mRNA molecule thereof; and a second polynucleotide encoding a light chain variable region with 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 a corresponding mRNA molecule thereof; or

[0053] (II) a first polynucleotide encoding a heavy chain variable region with 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 a corresponding mRNA molecule thereof; and a second polynucleotide encoding a light chain variable region with 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 a corresponding mRNA molecule thereof; or

[0054] (III) a first polynucleotide encoding a heavy chain variable region of 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 a corresponding mRNA molecule thereof; and a second polynucleotide encoding a light chain variable region of 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 a corresponding mRNA molecule thereof; or

[0055] (IV) a first polynucleotide encoding a heavy chain variable region of 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 a corresponding mRNA molecule thereof; and a second polynucleotide encoding a light chain variable region of 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 a corresponding mRNA molecule thereof; or

[0056] (V) a first polynucleotide encoding a heavy chain variable region of 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 a corresponding mRNA molecule thereof; and a second polynucleotide encoding a light chain variable region of 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 a corresponding mRNA molecule thereof.

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

[0058] In a fourth aspect, the present application provides a vector comprising a polynucleotide according to the second aspect described above, or a nucleic acid construct according to the third aspect described above.

[0059] The vector of the present application can be a cloning vector or an expression vector, for example, it can be a plasmid, a cosmid, a bacteriophage, etc.

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

[0061] In a fifth aspect, the present application provides a host cell transformed with a polynucleotide according to the second aspect described above, a nucleic acid construct according to the third aspect described above, or a vector according to the fourth aspect described above.

[0062] The host cell includes, but is not limited to, a prokaryotic cell, such as an E. coli cell; a eukaryotic cell, such as a yeast cell, an insect cell, a plant cell and an animal cell (such as a mammalian cell, for example, a mouse cell, a human cell, 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, further preferably a mammalian cell.

[0064] In a sixth aspect, the present application provides a detection reagent for detecting respiratory syncytial virus, comprising the monoclonal antibody or antigen-binding fragment thereof according to the first aspect described above.

[0065] In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof according to the present application is further comprised in 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 according to the present application or an anti-idiotypic antibody; preferably, the second antibody is further comprised in a detectable label; such detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent substances, luminescent substances, colored substances and enzymes (such as horseradish peroxidase), etc.

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

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

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

[0069] In an eighth aspect, the present application provides a method of producing the monoclonal antibody or antigen-binding fragment thereof of the first aspect described above, the method comprising: expressing the monoclonal antibody or antigen-binding fragment thereof of the fifth aspect described above from the host cell under conditions suitable for 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 a ninth aspect, the present application provides a method of detecting the presence or level of respiratory syncytial virus in a sample, the method comprising using the monoclonal antibody or antigen-binding fragment thereof of the first aspect described above, the polynucleotide of the second aspect described above, the nucleic acid construct of the third aspect described above, the expression vector of the fourth aspect described above, the host cell of the fifth aspect described above, and / or the detection reagent of the sixth aspect described above.

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

[0072] In other preferred embodiments of the method, the method further comprises: detecting the monoclonal antibody or antigen-binding fragment thereof of the present application using a second antibody carrying a detectable label.

[0073] The method can be used for diagnostic purposes (e.g., the sample is a sample from a patient) or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0074] Thus, in some embodiments, the present application provides a method of diagnosing whether a subject is infected with respiratory syncytial virus, the method comprising: detecting the presence of respiratory syncytial virus F protein in a sample from the subject using the monoclonal antibody or antigen-binding fragment thereof of the present application of the first aspect described above. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present application further comprises a detectable label; in other preferred embodiments, the method further comprises detecting the monoclonal antibody or antigen-binding fragment thereof of the present application or an anti-idiotypic antibody using a second antibody carrying a detectable label. In workable embodiments, the subject is a mammal, e.g., a human.

[0075] General methods of using monoclonal antibodies or antigen-binding fragments thereof to detect the presence or level of a target virus or antigen in a sample are 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, competition method, and the like.

[0076] Beneficial effects

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

[0078] One or more embodiments are illustrated by way of example in the figures that form a part of this patent document. This illustration is not to be considered limiting in relation to the embodiments will be limited to the embodiments illustrated here and any variations that occur to those skilled in the art upon reading this description. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0079] Figure 1 Figure for SDS-PAGE identification results of each monoclonal antibody.

[0080] Figure 2 Figure for binding force curve of the monoclonal antibody of the present application to RSV-A type F protein (A) and RSV-B type F protein (B) detected by ELISA method.

[0081] Figure 3 Figure for results of detecting immunogen RSV sc-Dimer (30 ng) with each monoclonal antibody by Western blot.

[0082] Figure 4 Figure showing the affinity results of the monoclonal antibody of the present application to RSV-A type F protein and RSV-B type F protein determined by SPR method.

[0083] Figure 5 Figure showing the results of immunofluorescence staining of the lung of a mouse infected with RSV A type virus using the monoclonal antibody of the present application.

[0084] Figure 6 Figure showing the results of immunofluorescence staining of the lung of a mouse infected with RSV B type virus using the monoclonal antibody of the present application. DETAILED DESCRIPTION

[0085] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of the present application.

[0086] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art to which this application belongs.

[0087] The term "and / or" should be understood to mean either a single option, 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 amino acid residues in a candidate amino acid sequence that have the same amino acid residue as the reference amino acid sequence when the amino acid sequences are aligned (and gaps introduced as necessary) to achieve maximum percent sequence identity (and no conserved substitutions are counted as part of the sequence identity). Sequence alignment can be performed using a variety of methods known in the art to determine percent (%) amino acid sequence identity.

[0089] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogs, which generally include at least a portion of an antigen-binding region or variable region of a parent antibody, such as one or more CDRs. Fragments of an antibody retain at least certain binding specificities of the parent antibody. In particular, an antigen-binding fragment can be selected from a Fab, Fab', Fab'-SH, Fv, scFv, F(ab')2, diabody, and the like.

[0090] A "Fab" fragment consists of one light chain and a portion of one heavy chain, including the CH1 and variable regions.

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

[0092] A "F(ab')2" fragment includes two light chains and two portions of a heavy chain that include the VH domain, the CH1 domain, and a portion of the constant region between the CH1 and CH2 domains, with an interchain disulfide bond between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0093] An "Fv" region comprises 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 are contained in a single polypeptide chain. Generally, the scFv polypeptide comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0095] "Bispecific antibody" is a small antigen-binding fragment having two antigen-binding sites. The fragment comprises 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 pair the two domains in the same chain, the domains pair with 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 typically represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdisand kon, respectively). Affinity can be measured by common methods known in the art.

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

[0098] The preferred embodiments of the present application will be described in detail below with reference to the examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and substitutions to the present application without departing from the spirit and principles of the present application.

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

[0100] Step 1: Design and production of specific antigens

[0101] 1) Antigen design:

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

[0103] 2) Antigen production & purification:

[0104] RSV sc-Dimer protein was expressed by 293F expression system, purified by His-tag protein affinity column, and further purified by molecular sieve chromatography according to the molecular weight to PBS buffer. The protein concentration was determined by BCA method, and the protein was frozen at -80°C for standby after aliquot.

[0105] Step 2: Immunization of mice with antigens

[0106] The 6-8 week old female BALB / c mice were immunized with the antigen protein RSV sc-Dimer produced and purified above, wherein 3 mice were immunized with denatured antigen protein and 3 mice were immunized with non-denatured antigen protein; the regimen was as follows: single immunization dose of 50 μg per mouse, for the first immunization, the immunogen was mixed with an equal volume of complete Freund's adjuvant to form an emulsifier, and was injected subcutaneously in multiple points on the abdomen; for the second and third immunizations, the same dose of immunogen was mixed with an equal volume of incomplete Freund's adjuvant to form an emulsifier, and was injected subcutaneously in multiple points on the abdomen at an interval of 2 weeks from the previous immunization.

[0107] Step 3: Bleeding and titering

[0108] A. Serum collection

[0109] One week after the last immunization, 50-60 μL of blood was taken from the orbital plexus of the mice. After standing at 4°C overnight, the upper serum was separated by centrifugation at 4000 rpm and 4°C for 15 min.

[0110] B. ELISA method for detecting the antibody titer of the serum of the immunized mice

[0111] The antibody titer of the serum of the immunized mice was determined by indirect ELISA, and the qualified standard was that the OD450-Blank of the mouse serum diluted 1:8000 times was greater than 1.0.

[0112] (1) Coating: an appropriate amount of RSV sc-Dimer protein was dissolved and diluted with coating buffer to 5 μg / mL, then 100 μL was added to each well of a 96-well plate using a pipette, the sample was mixed by tapping the plate, and the plate was sealed with plastic wrap and coated at 4°C overnight;

[0113] (2) Washing the plate: wash the plate once with 300 μL / well of washing solution, and dry the enzyme-labeled plate;

[0114] (3) Blocking: block the enzyme-labeled plate with 300 μL / well of blocking solution at room temperature for 1 h;

[0115] (4) Washing the plate: wash the plate twice with 300 μL / well of washing solution, and dry the enzyme-labeled plate;

[0116] (5) Sample addition: add the gradient-diluted serum sample and sample diluent to the plate at 100 μL / well;

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

[0118] (7) Washing: Then wash the plate with 300 μL / well of washing solution for 5 times, and dry the plate;

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

[0120] (9) Termination and detection: Add 50 μL / well of termination solution to terminate the reaction, and then use an enzyme label instrument to detect, with the detection wavelength being 450 nm.

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

[0122] Table 1: ELISA detection results of sera after three immunizations of non-denatured proteins

[0123]

[0124]

[0125] Table 2: ELISA detection results of sera after three immunizations of denatured proteins

[0126]

[0127] Table 3: ELISA detection results of sera after four immunizations of denatured proteins

[0128]

[0129]

[0130] The immunization was performed with 5 μg / mL of immunogen, and the serum of the immunized mouse was diluted 8000 times. When OD450-Blank>1.0, it was considered that the immunization was qualified. The immunized mouse with high ELISA titer was preferentially selected for impact immunization. The mouse spleen was taken for hybridoma fusion after 3-4 days of impact immunization. The unqualified animal was additionally immunized until the titer was qualified, and the immunization was performed at most 5 times.

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

[0132] The spleen cells of the immunized mouse were taken, and were fused with SP2 / 0 cells by using an electric fusion method. The electric fusion was performed for two rounds, and hybridoma cells were obtained. The positive master clone cells combined with RSV sc-Dimer were obtained by using an indirect ELISA method.

[0133] Step 5: Limited dilution and subcloning screening

[0134] Using HT selection medium, each positive hybridoma cell obtained by screening was inoculated in a half-block 96-well cell culture plate at a seeding density of 0.75 cells per well, and incubated at 37°C in a 5% CO2 incubator. The supernatant was taken about 7 days after incubation for indirect ELISA detection to confirm that the antibody to be tested was positively bound to the antigen. The specific detection process is referred to the indirect ELISA detection process of the master clone. The ELISA-specific positive monoclonal cells were selected for 1-2 rounds of limited dilution again until stable positive monoclonal cells were obtained. Through the above experimental process, a total of 10 positive mouse hybridoma cell lines were obtained.

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

[0136] A. Specific hybridoma amplification

[0137] A large amount of monoclonal antibody is prepared by in vitro culture. The hybridoma cells are cultured in a culture bottle. During the culture process, the hybridoma cells produce and secrete monoclonal antibodies. The culture supernatant is collected, centrifuged to remove cells and debris, and the desired monoclonal antibody is obtained. 5mL / 20mL hybridoma cells are transferred into a 100mL / 500mL culture bottle, and a certain amount of culture medium is added regularly for cell expansion. The cells are expanded to the desired volume. After 6-8 days of culture, the material is collected. The collected hybridoma cell supernatant is centrifuged using a table centrifuge at 4000g for 30min, and the centrifuged cell supernatant is collected. The centrifuged cell supernatant is filtered using a 0.45μM filter.

[0138] B. Antibody purification

[0139] The centrifuged hybridoma cell culture supernatant is purified using Protein A affinity purification method, and the mouse monoclonal antibody is harvested. The operation steps are as follows.

[0140] (1) Select the appropriate size of Protein A column according to the expression amount,

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

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

[0143] (4) Sample loading: pour the sample loading;

[0144] (5) Elution: elution liquid elution 5-10CV to protein column balance;

[0145] (6) Elution: AC Elution elution, the first CV is discarded, and the last 1.5CV is collected;

[0146] (7) Neutralization: Neutralize the eluted antibody with 2M Tris, pH 8.0;

[0147] (8) Equilibration: Equilibrate the column with AC Binding Buffer for 5 CV to neutral pH;

[0148] (9) CIP Cleaning: Clean the column with CIP for 5 CV or more;

[0149] (10) Rinse Alkali: Rinse the column with AC Binding Buffer until the pH at the outlet is neutral;

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

[0151] C. Mouse Monoclonal Antibody Detection (Antibody Concentration and Purity Detection)

[0152] Start the micro-spectrophotometer, and point the sample to be detected with the corresponding buffer. When the absorbance value is between ±0.015 at a wavelength of 280 nm, it indicates that the instrument baseline is stable. Then, point the purified antibody sample successively, and record the absorbance value. Divide the detection data by the IgG extinction coefficient (1.414), and the obtained value is the concentration (unit: mg / mL) of the detection sample.

[0153] Verify the purity of the purified antibody using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This method separates the target antibody according to the different molecular weights of the proteins in the sample, thereby verifying the purity of the target antibody. Add 5 μg of the detection sample to 5 μL of 4R / 4N, heat in a 100°C water bath, and then centrifuge at 10000 rpm. Add enough 1x SDS electrophoresis buffer to the electrophoresis tank, and use a micropipette to add the treated sample solution and the protein molecular weight marker to the sample well. Connect the power supply, and first electrophorese at a constant voltage of 100 V until the bromophenol blue dye enters the separation gel from the concentrated gel, and then continue to electrophorese at 140 V until the bottom of the gel plate, and turn off the power. Soak the gel in the staining solution, heat in a microwave oven for 60 s, and place on a gently shaking platform at room temperature for staining. Replace the decolorizing solution to cover the gel, heat in a microwave oven for 60 s, place on a gently shaking platform at room temperature for decolorization, and repeat the decolorization operation until a blue band and a clean background are obtained.

[0154] The denatured and non-denatured SDS-PAGE detection results of the monoclonal antibodies produced by the 10 hybridoma cells obtained in step five are shown in Figure 1 . Figure 1 The upper part is a non-reducing gel (i.e., the sample of the antibody without DTT), and the band has a molecular weight of about 150 KDa, which corresponds to the intact antibody. Figure 1The lower is the reduced gel (i.e. the sample of antibody plus DTT), and the molecular weight of the two bands is about 50 KDa (i.e. corresponding to the heavy chain of the antibody) and about 25 KDa (i.e. corresponding to the light chain of the antibody), respectively.

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

[0156] Example 2: Detection of the binding of monoclonal antibodies to immunogens by ELISA

[0157] In this example, the binding of each purified monoclonal antibody to RSV-A type F protein and RSV-B type F protein is detected by the method of ELISA, and the specific detection process is as follows:

[0158] 1) Coating: coat RSV-A type F protein or RSV-B type F protein, the coating concentration is 3 μg / mL, 5-fold concentration gradient dilution, 96-well plate according to column to pull 7 concentration gradients, 100 μL / well, 4°C coating overnight;

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

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

[0161] 4) Antibody dilution and sample addition: dilute the antibody to be tested to 1 μg / mL, and add 100 μL 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 h;

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

[0163] 6) Secondary antibody incubation: dilute the secondary antibody Rabbit Anti-Mouse IgG F(ab')2 / HRP (1:2000), 100 μL / well, mix well, and incubate at room temperature for 1 h;

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

[0165] 8) Color development: add 60 μL per well, and incubate at room temperature for 20 min in the dark;

[0166] 9) Termination with detection: 60 μL of termination solution was added to each well, and OD value was immediately measured at 450 nm wavelength.

[0167] The antibody-antigen binding force curve was plotted with the Log value of the antigen protein concentration as the abscissa and the measured OD450 value as the ordinate.

[0168] The binding force curves of the monoclonal antibodies to RSV-A type F protein and RSV-B type F protein are shown in A and B of FIG. 1, respectively, which shows that Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, and Ab-10 can effectively bind to the F proteins of RSV-A and RSV-B through spatial epitopes; wherein the binding abilities of Ab-2, Ab-8, Ab-9, and Ab-10 to the F proteins of RSV-A and RSV-B are relatively strong. Figure 2 Example 3: Western blot detection of the binding of monoclonal antibodies to immunogens

[0169] In this example, the binding of each purified monoclonal antibody to the immunogen RSV sc-Dimer was detected by Western blot, and the specific detection process was as follows:

[0170] 1) Loading: The immunogen RSV sc-Dimer was added to the loading buffer, and the loading amount was 30 ng / lane;

[0171] 2) Electrophoresis: constant voltage 100 V until the bromophenol blue runs to the bottom of the gel plate;

[0172] 3) Membrane transfer: constant voltage 110 V, 90 min;

[0173] 4) Blocking: blocking at 4°C overnight;

[0174] 5) Primary antibody incubation: adding hybridoma supernatant to a multi-channel hybridization instrument, incubating at room temperature for 2 h;

[0175] 6) Elution: eluting the membrane 3 times using a decolorizing shaker, 5 min each time;

[0176] 7) Secondary antibody incubation: goat anti-mouse IgG (H+L) / HRP final concentration 1:10000, room temperature for 2 hours;

[0177] 8) Elution: eluting the membrane 3 times using a decolorizing shaker, 5 min each time;

[0178] 9) Color development: chemiluminescence imaging system for color development.

[0179] The results are shown in FIG. 2.

[0180] Figure 3 Figure 3 ​​It is shown that Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10 can all effectively bind to the RSV sc-Dimer protein.

[0181] Example 4: Surface plasmon resonance (SPR) assay of the affinities of 10 mouse hybridoma antibodies to RSV-A type F protein and RSV-B type F protein

[0182] First, the anti-mouse IgG antibody was immobilized on a CM5 chip using an amino coupling method, and after immobilization, the mouse hybridoma monoclonal antibody (numbered Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10) was bound using a capture method, and experiments were carried out using RSV A-F protein monomer and RSV B-F protein monomer as the mobile phase, to determine the binding affinity of the antibody to the F protein, and the flow buffer was a PBST solution containing 0.05% Tween 20. The affinity detection results of each monoclonal antibody to RSV-A type F protein and RSV-B type F protein are shown in Figure 4 Table 4, and the affinity data statistics are shown in the following Table 4.

[0183] Table 4, affinity of monoclonal antibody to RSV-A type F protein and RSV-B type F protein

[0184]

[0185] From Figure 4 and Table 4, it can be seen that the affinities of monoclonal antibodies Ab-2, Ab-6, Ab-8, Ab-9, and Ab-10 to RSV F protein are relatively high (in the order of pM); among them, the affinity to RSV A type F protein can reach the order of pM, and the affinity to RSV B type F protein can reach the order of ten pM; in addition, the affinities of monoclonal antibodies Ab-1 and Ab-5 to RSV A type and B type F protein can reach the order of nM, and the affinities of Ab-3, Ab-4, and Ab-7 to RSV A type and B type F protein are relatively weak, and the order of magnitude is between hundred nM and μM.

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

[0187] Example 5: Sequencing of the antibody

[0188] Antibody sequencing experiment steps:

[0189] 1) After lysis of the hybridoma cells, RNA was extracted;

[0190] 2) Reverse transcription: using extracted RNA as a template, using the Yikui Technology reverse transcription kit, two steps are taken to obtain cDNA (by first opening the RNA secondary structure, then allowing the primer to bind to the RNA and polymerizing the cDNA to achieve the purpose of obtaining cDNA);

[0191] 3) Using the cDNA obtained by reverse transcription as a template, amplifying by PCR, inserting the amplified fragments into an expression vector or a commercial vector, and sequencing 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 NO: 1-10, respectively;

[0194] The HCDR1-3, LCDR1-3, VH, VL, H, and L sequences of Ab-6 are shown in SEQ ID NO: 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 NO: 17-26, respectively;

[0196] The HCDR1-3, LCDR1-3, VH, VL, H, and L sequences of Ab-9 are shown in SEQ ID NO: 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 NO: 31-40, respectively.

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

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

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

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

[0202]

[0203]

[0204] Table 5 shows that antibodies Ab-2, Ab-6, Ab-9, and Am22 have competitive binding ( Epitope), Ab-8 competitively binds to CR9501 antibody (epitaxe V), while no other antibody was found to have significant competitive binding with the three antibodies.

[0205] Based on the above results, we can conclude that: on the one hand, Ab-2, Ab-6, Ab-8, Ab-9, and Ab-10 all have high affinity binding to RSV sc-Dimer; on the other hand, because they bind to different antigenic epitopes, Ab-2, Ab-6, and Ab-9, along with Ab-8 or Ab-10, have the potential to serve as paired antibodies for double-antibody sandwich immunoassay (one as a capture antibody and the other as a detection antibody), for example, they could be 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 RSV-infected mice

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

[0208] The steps for immunofluorescence staining are as follows:

[0209] 1) Dewaxing paraffin sections to water: Place the sections in 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 then wash with distilled water.

[0210] 2) Antigen retrieval: Microwave citric acid (pH 6.0) on medium heat for 8 minutes, turn off the heat for 8 minutes, and then microwave on medium-low heat for 7 minutes. During the retrieval process, prevent excessive evaporation of the buffer solution and avoid drying the slide. After retrieval, allow it to cool naturally. Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker, 5 minutes each time.

[0211] 3) Circle blocking with serum: After slightly drying the section, draw a circle around the tissue with a histochemical pen, 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, 1:500 dilution) to the slice, and incubate the slice in a wet box at 4°C overnight.

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

[0214] 6) DAPI re-stain the nucleus: Place the slide in PBS (PH 7.4) and shake on a decolorizing shaker for 3 times, 5 min each time. Add DAPI staining solution, and incubate at room temperature for 10 min in the dark.

[0215] 7) Quench the tissue autofluorescence: Add autofluorescence quencher B solution for 5 min, and rinse with running water for 10 min. Place the slide in PBS solution (PH 7.4) and wash on a shaker for 3 times, 5 min each time.

[0216] 8) Mount the slide: Mount the slide with anti-fluorescence quenching mounting agent.

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

[0218] 10) Analyze the results.

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

[0220] The results of immunofluorescence staining of the lungs of mice infected with RSV type A virus are shown in Figure 5 . Figure 5 It is shown that: compared with the control, Ab-2, Ab-6, Ab-8, Ab-9, and Ab-10 can all bind to the infected tissue cells.

[0221] The results of immunofluorescence staining of the lungs of mice infected with RSV type B virus are shown in Figure 6 . Figure 6 It is shown that: compared with the control, Ab-2, Ab-6, Ab-8, Ab-9, and Ab-10 can all bind to the infected tissue cells.

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

[0223] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A monoclonal antibody or its antigen-binding fragment against the respiratory syncytial virus F protein, characterized in that, The monoclonal antibody or its antigen-binding fragment 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, (3) The three heavy chain complementarity determination regions in the heavy chain variable region as shown in SEQ ID NO:23, and the three light chain complementarity determination regions in the light chain variable region as shown in SEQ ID NO:

24.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises: (1) A heavy chain variable region comprising heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and a light chain variable region comprising light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 as shown in 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, (3) Heavy chain variable region, which includes heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19 respectively; and light chain variable region, which includes light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3 as shown in 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.

3. The monoclonal antibody or its antigen-binding fragment according to claim 2, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises: (1) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:7; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:8; or (3) Heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:23 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:23; and light chain variable region having an amino acid sequence as shown in SEQ ID NO:24 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

24.

4. The monoclonal antibody or its antigen-binding fragment according to claim 3, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises: (1) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:8; or (3) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:23, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:

24.

5. The monoclonal antibody or its antigen-binding fragment according to claim 4, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises: (1) A heavy chain having an amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:9; and a light chain having an amino acid sequence as shown in SEQ ID NO:10 or an amino acid sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:10; or (3) A heavy chain having an amino acid sequence as shown in SEQ ID NO:25 or having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:25; and a light chain having an amino acid sequence as shown in SEQ ID NO:26 or having at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

26.

6. The monoclonal antibody or its antigen-binding fragment according to claim 5, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises: (1) The heavy chain with the amino acid sequence shown in SEQ ID NO:9, and the light chain with the amino acid sequence shown in SEQ ID NO:10; or (3) The heavy chain with an amino acid sequence as shown in SEQ ID NO:25, and the light chain with an amino acid sequence as shown in SEQ ID NO:

26.

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

8. A polynucleotide encoding as claimed in claim 1 7. The monoclonal antibody or its antigen-binding fragment as described in any one of the above.

9. The polynucleotide of claim 8, wherein, The polynucleotide is a polynucleotide group, which includes: (I) A DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:41, and a DNA molecule or its corresponding mRNA molecule comprising the nucleotide sequence shown in SEQ ID NO:42; or (III) A DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:45, and a DNA molecule or its corresponding mRNA molecule containing the nucleotide sequence shown in SEQ ID NO:

46.

10. A nucleic acid construct comprising a polynucleotide as described in any one of claims 8-9, and at least one expression regulatory element operatively linked to said polynucleotide.

11. A vector comprising a polynucleotide as described in any one of claims 8-9, or a nucleic acid construct as described in claim 10.

12. A host cell wherein the polynucleotide of any one of claims 8-9, the nucleic acid construct of claim 10, or the vector of claim 11 is transformed.

13. A detection reagent for detecting respiratory syncytial virus, comprising a monoclonal antibody or an antigen-binding fragment thereof as described in 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 contains two monoclonal antibodies or antigen-binding fragments selected from the monoclonal antibodies or antigen-binding fragments thereof as described in any one of claims 1-7, wherein one monoclonal antibody or antigen-binding fragment is defined by claim (1) of any one of claims 1-7, and the other monoclonal antibody or antigen-binding fragment is defined by claim (3) of any one of claims 1-7.

15. The use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-7, the polynucleotide as described in any one of claims 8-9, 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 products for detecting the presence or level of respiratory syncytial virus in a sample and / or for diagnosing respiratory syncytial virus infection.

16. The application as described in claim 15, characterized in that, The sample was a biological sample from the subject.

17. A method for preparing a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-7, the method comprising: Under conditions suitable for the expression of the monoclonal antibody or its antigen-binding fragment, the host cell of claim 12 is made to express 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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