CN115838419A

CN115838419A - Anti-respiratory syncytial virus antibodies and uses related thereto

Info

Publication number: CN115838419A
Application number: CN202211196049.XA
Authority: CN
Inventors: 李燕飞; 吴学胜; 欧阳海桥; 全红花; 武云波; 游梅香; 龚春喜; 池朗山; 林育佳
Original assignee: Shenzhen Heavy Chain Biotechnology Co ltd
Current assignee: Shenzhen Heavy Chain Biotechnology Co ltd
Priority date: 2022-09-27
Filing date: 2022-09-27
Publication date: 2023-03-24
Anticipated expiration: 2042-09-27
Also published as: CN115838419B

Abstract

The present invention relates to novel monoclonal anti-Respiratory Syncytial Virus (RSV) antibodies, methods for their preparation, and their related uses for immunodetection. The monoclonal anti-RSV antibody of the invention exhibits high sensitivity and high specificity against RSV, and can be used for detecting RSV at nanogram concentration in a chromatographic platform, and can realize more sensitive detection of the concentration such as picogram in other platforms such as chemiluminescence method.

Description

Anti-respiratory syncytial virus antibodies and uses related thereto

Technical Field

The invention relates to the field of virus detection, and in particular relates to a monoclonal anti-Respiratory Syncytial Virus (RSV) antibody, a preparation method thereof and a related application thereof in immunodetection.

Background

Respiratory Syncytial Virus (RSV) belongs to the genus Pneumovirus of the family Paramyxoviridae and is an enveloped, single-stranded RNA virus. RSV virus causes around 6400 million infections annually, resulting in over 300 million hospitalizations for infants, leading to 16 million deaths. Therefore, sensitive and rapid detection of RSV infection is extremely important, and antibodies against RSV virus are an important part of immunodiagnosis of RSV infection.

RSV expresses two major surface glycoproteins: fusion proteins (F proteins) and attachment proteins (G proteins). The genetic variability of the F protein is less than that of the G protein, which binds to specific receptors on the surface of the host cell membrane, mediates fusion of the viral envelope with the host cell plasma membrane, and causes viral nucleic acid to enter the host cell and be replicated. Thus, RSV F protein is a very attractive target for immunodetection and antibody-based therapies.

The existing antibodies for resisting RSV F protein at present have defects in the aspects of sensitivity and specificity, can cause false negative results in clinic and still have great improvement space. Therefore, there remains a need in the art for an anti-RSV F protein antibody with high specificity and high sensitivity.

Disclosure of Invention

As previously mentioned, there is a need in the art for an anti-RSV antibody with high specificity and high sensitivity.

The invention induces mice to generate immune response by taking the purified RSV fusion protein as immunogen, obtains hybridoma cell strains capable of secreting anti-RSV monoclonal antibodies by using a hybridoma technology, collects supernatants by an in vitro roller bottle culture method to obtain a large amount of anti-RSV monoclonal antibodies, and obtains the anti-RSV antibodies with high sensitivity and high specificity by screening. Thus, the present invention has been achieved.

In a first aspect, the invention provides a monoclonal anti-Respiratory Syncytial Virus (RSV) antibody, the heavy chain of which comprises the heavy chain variable region V set forth in SEQ ID NOS: 13-15, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain of said antibody includes light chain variable region V represented by SEQ ID NO 16-18 respectively _L CDR1、V _L CDR2 and V _L CDR3。

In a second aspect, the invention provides a hybridoma cell strain, which is deposited at the China general microbiological culture Collection center (CGMCC) of the institute of microbiology, china institute of sciences, no. 3, west Lu 1, beijing, toyoho, 7.14 days 2022, with the collection number of CGMCC No.45240.

In a third aspect, the present invention provides an expression vector comprising genes encoding the heavy and/or light chain of a monoclonal anti-RSV antibody, wherein the heavy chain of said antibody comprises a heavy chain variable region V represented by SEQ ID NOS: 13-15, respectively _H CDR1、V _H CDR2 and V _H CDR3, saidThe light chain of the body comprises a light chain variable region V represented by SEQ ID NOS 16-18, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In a fourth aspect, the present invention provides an expression cell comprising the expression vector of the third aspect.

In a fifth aspect, the present invention provides a method of detecting Respiratory Syncytial Virus (RSV), for non-diagnostic or diagnostic purposes, comprising the step of using the monoclonal anti-RSV antibody of the first aspect.

In a sixth aspect, the invention provides the use of a monoclonal anti-RSV antibody according to the first aspect in the manufacture of a reagent for the detection of Respiratory Syncytial Virus (RSV).

In a seventh aspect, the invention provides a kit for detecting Respiratory Syncytial Virus (RSV), the kit comprising the monoclonal anti-RSV antibody of the first aspect and instructions for directing how to detect Respiratory Syncytial Virus (RSV).

In conclusion, the invention provides a novel anti-RSV monoclonal antibody, which shows high sensitivity and high specificity aiming at RSV, can be used for RSV detection of nanogram and even picogram concentration, and greatly improves the sensitivity of RSV detection and analysis. The anti-RSV antibody of the invention can be applicable to various immunochromatography detections such as fluorescent microsphere chromatography, colloidal gold chromatography and colored microsphere chromatography; ELISA assays such as direct, indirect, sandwich and competitive; a chemiluminescence method; electrochemical luminescence method, etc. The anti-RSV antibodies of the invention exhibit high sensitivity and high specificity for RSV, can be used for nanogram concentrations of RSV in the chromatographic platform, and can achieve more sensitive detection of picogram concentrations, for example, in other platforms such as chemiluminescence.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings used in the detailed description or the prior art description will be briefly described below.

FIG. 1 shows SDS-PAGE electrophorograms (loading 5. Mu.g per well) of antibodies RSV101, RSV200 and RSV300 of the invention.

FIG. 2 shows OD measured by microplate reader of RSV101, RSV200 and RSV300 antibodies of the invention _450nm Absorbance curve.

FIG. 3 shows OD measurements of antibodies RSV101 and RSV102 of the invention measured by a microplate reader _450nm Absorbance curve.

Fig. 4 shows a schematic diagram of a colloidal gold colorimetric card used in the present invention.

FIG. 5 shows sensitivity curves for exemplary fluorescent microsphere immunochromatographic test agents prepared from antibodies of the present invention to recombinant antigens of RSV fusion proteins.

Detailed Description

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It is to be understood that the following description is intended to illustrate the present invention by way of example only and is not intended to limit the scope of the invention, which is defined by the appended claims. Also, it is understood by those skilled in the art that modifications may be made to the technical aspects of the present invention without departing from the spirit and gist of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this invention belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

Where numerical ranges are provided, such as concentration ranges, percentage ranges, or ratio ranges, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the subject matter.

In the context of the present invention, many embodiments use the expression "comprising", "including" or "consisting essentially of … …". The expressions "comprising", "including" or "consisting essentially of … …" are generally to be understood as open-ended expressions that include not only the elements, components, assemblies, method steps, etc., specifically listed after the expression, but also other elements, components, assemblies, method steps. In addition, the expressions "comprising", "including" or "consisting essentially of … …" may in some cases also be understood as a closed expression, meaning that only the elements, components, assemblies, method steps specifically listed after the expression are included, but not any other elements, components, assemblies, method steps. At this time, the expression is equivalent to the expression "consisting of … …".

For a better understanding of the present teachings and not to limit the scope of the present teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions used in the specification and claims, as well as other numerical values, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

The term "antibody" as used herein refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of an antibody can be defined accordingly as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region (hinge region) of about 12 or more amino acids, and the heavy chain also contains about 3 or more amino acidsAnd (3) region "D". Each heavy chain is composed of a heavy chain variable region (V) _H ) And heavy chain constant region (C) _H ) And (4) forming. The heavy chain constant region consists of 3 domains (C) _H1 、C _H2 And C _H3 ) And (4) forming. Each light chain is composed of a light chain variable region (V) _L ) And light chain constant region (C) _L ) And (4) forming. The light chain constant region consists of a domain C _L And (4) forming. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). V _H And V _L Regions may also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). For each heavy or light chain, the variable regions each comprise three CDRs, namely CDR1, CDR2 and CDR3. Thus, each V _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. Variable region (V) of each heavy/light chain pair _H And V _L ) Respectively, forming an antigen binding site.

The assignment rules for assigning amino acids to regions or domains are defined in a number of documents: kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda M.d. (1987 and 1991)); chothia & Lesk j.mol.biol.1987;196 parts by weight; chothia et al, nature 1989; 342 from 878 to 883; ehrenmann, francois, quentin Kaas, and Marie-PauleLefranc, "IMGT/3Dstructure-DB and IMGT/DomainGapAlign," a database and a tool for immunoglobulin or antibodies, T cell receptors, MHC, igSF and MhcSF, "Nucleic acids research 2009;38 (Suppl _ 1) D301-D307.

The exact boundaries of the CDRs have been defined differently according to different systems, the Kabat system not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining 3 CDRs, which are referred to as Kabat CDRs; chothia found that certain sub-parts within the CDRs of the Kabat system, despite a large diversity at the amino acid sequence level, have nearly identical conformation of the peptide backbone, and these sub-parts are covered byCalled Chothia CDR, chothia CDR and Kabat CDR overlapping boundary. The boundaries of the above overlap are again described by Padlan and MacCallum, and CDR boundary definitions may not strictly adhere to the above system, such as the AbM definition. Herein, the CDRs may be defined according to any of these systems, although the preferred embodiment uses the antibody numbering system of Chothia et al to define the CDRs. V of antibodies according to the Chothia numbering system _H CDR1 is located at positions 26 to 32, V _H CDR2 is located at positions 52 to 57, V _H CDR3 is located at positions 99 to 108, and V _L CDR1 is in position 24 to 39, V _L CDR2 is located at positions 55 to 61 and V _L CDR3 is located at positions 94 to 102.

As used herein, the term "monoclonal antibody" or "monoclonal antibody" refers to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. The antibody molecule may be an immunoglobulin, whether it is a natural immunoglobulin or an immunoglobulin that is partially or wholly synthetically obtained. The antibody molecules may also include all polypeptides or proteins having binding domains of antibody domains, antibody fragments having antibody domains are molecules such as Fab, scFv, fv, dAb, fd, and diabodies. Monoclonal antibodies have high specificity for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using the hybridoma technique first reported by Kohler et al (A)

G,Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]Nature,1975;256 (5517): 495), but can also be obtained using recombinant DNA techniques (see, e.g., U.S. Pat. No.4,816,567).

As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As described above, the present invention aims to provide a monoclonal anti-RSV antibody having high sensitivity and high specificity.

In a first aspect, the present invention provides a monoclonal Respiratory Syncytial Virus (RSV) antibody, which is a monoclonal RSV antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NOS: 13-15, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprises a light chain complementarity determining region V having amino acid sequences represented by SEQ ID NOS 16-18, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In some embodiments, the antibody comprises at least six CDRs.

In some preferred embodiments, the antibody comprises:

1) A heavy chain variable region comprising or consisting of the sequence:

SEQ ID NO:23, or an amino acid sequence corresponding to SEQ ID NO:23 has a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more, preferably 95% or more.

2) A light chain variable region comprising or consisting of the sequence:

SEQ ID NO:24, or an amino acid sequence corresponding to SEQ ID NO:24 has a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more, preferably 95% or more.

In some embodiments, the antibody is a whole antibody comprising a variable region and a constant region. For the antibodies of the invention, any Framework Region (FR) as well as any constant region can be usedAnd (4) a zone. The amino acid sequence of the FR or constant region used in the antibody of the present invention may be the amino acid sequence of the original FR or constant region from which it is derived, or may be a different amino acid sequence obtained by substituting, deleting, adding and/or inserting 1 or more amino acids into the amino acid sequence of the original FR or constant region. The structures used to support the CDRs or sets of CDRs of the invention are generally of antibody heavy or light chain sequence or major portion thereof, wherein the CDRs or sets of CDRs are located in naturally occurring V encoded by rearranged immunoglobulin genes _H And V _L The CDR or set of CDRs of an antibody variable domain are in the corresponding position.

In some embodiments, the antibody can be one of a nanobody, a Fab, a dAb, a Fd, an Fv, a scFv, and a diabody.

In some embodiments, the antibody further comprises a constant region sequence.

In some embodiments, the constant region sequence may be a constant region sequence selected from any one of IgG, lgA, igM, igE, igD.

In some embodiments, the constant region sequence species is derived from a mouse.

In an exemplary embodiment, the constant region sequence may comprise or consist of: consists of the following sequences:

has an amino acid sequence shown as SEQ ID NO. 25; and

has the amino acid sequence shown in SEQ ID NO. 26.

In a preferred embodiment, the antibody is produced by a hybridoma cell line having a collection number of CGMCC No.45240 and deposited in CGMCC (CGMCC) of China Committee for culture Collection of microorganisms, china institute of sciences, china institute of microbiology, no. 3, west Lu 1, kyoho, beijing, 2022, 7 months and 14 days.

In a third aspect, the present invention provides an expression vector comprising a gene encoding a monoclonal anti-RSV antibody, wherein the monoclonal anti-RSV antibody is a monoclonal anti-RSV antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NOS: 13-15, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprising a light chain complementarity determining region V having amino acid sequences represented by SEQ ID NOS 16-18, respectively _L CDR1、V _L CDR2 and V _L CDR3。

In an exemplary embodiment, for the monoclonal anti-RSV antibody, the heavy chain variable region comprises SEQ ID NO:23 or an amino acid sequence substantially identical to SEQ ID NO: 23. a heavy chain variable region having a sequence with more than 80%, more than 85%, more than 90% or more than 95% identity in the amino acid sequence set forth, the light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:24 or an amino acid sequence substantially identical to SEQ ID NO:24 has a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more.

In a preferred embodiment, the expression vector may be a plasmid vector, such as pcdna3.1, pEE12, pCAGGS.

As described above, the present inventors induced immune response in mice by using purified RSV fusion protein (F protein, SEQ ID NO: 27) as an immunogen, then obtained hybridoma cell lines capable of secreting anti-RSV monoclonal antibodies by hybridoma technology, collected supernatants by in vitro roller bottle culture method to obtain a large amount of anti-RSV antibodies, and screened to obtain monoclonal anti-RSV antibodies with high sensitivity and high specificity, which was named RSV300.

Other antibodies or chimeric molecules that retain the specificity of the original antibody can be produced using monoclonal and other antibody and recombinant DNA techniques, which can include the introduction of DNA encoding the immunoglobulin variable regions or Complementarity Determining Regions (CDRs) of an antibody into the constant regions or constant regions of a different immunoglobulin plus framework regions.

In one embodiment, the expression cell can be a mammalian cell, such as a chinese hamster ovary cell, a small hamster kidney cell, a monkey kidney cell, a mouse thymoma cell, a human embryonic kidney cell. In a more specific embodiment, the expression cells may be, for example, SV 40-transformed monkey kidney cells (COS-7, ATCC CRL1651), human embryonic kidney cells (HEK 293 or HEK293 cells subcloned for growth in suspension culture, graham et al, 1977, J.Gen Virol.36: 59), baby hamster kidney cells (BHK, ATCC CCL 10), chinese hamster ovary cells/-DHFR 1 (CHO, urlaub et al, 1980, proc.Natl.Acad.Sci. USA77:4216; for example, but not limited to, DG 44), mouse thymoma cells (NSO), mouse testicular supporting cells (TM 4, mather,1980, biol. Reprod.23, 243-251), monkey kidney cells (CV-1, ATCC CCL 70), african green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo mouse liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (HepG 2, HB 8065), mouse mammary tumors (MMT 060562, ATCC CCL 51), TR1 cells (Mather et al, 1982, annals N.Y. Acad. Sci.383: 44-68), MRC5 cells, FS4 cells, and the like. In a preferred embodiment, the expression cell may be a HEK293 cell.

In a specific embodiment, the detection is performed by immunochromatography, enzyme-labeled antibody method (ELISA), chemiluminescence, electrochemiluminescence.

In a preferred embodiment, the enzyme-labeled antibody method may be a direct method, an indirect method, a sandwich method, and a competition method.

In a preferred embodiment, the immunochromatography includes, but is not limited to, fluorescent microsphere immunochromatography, colloidal gold immunochromatography, immunochromatography based on colored latex microspheres, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography, and quantum dot immunochromatography.

In the present invention, the monoclonal anti-RSV antibody can be used as a coating antibody. For example, monoclonal anti-RSV antibodies are bound to a solid phase, such as a solid support. The solid support used in the detection method of the present invention is not particularly limited, and may be a porous or non-porous material, such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, and the like.

The detection method of the present invention can be used for point-of-care testing (POCT) or electrochemical immunoassay systems. The detection method according to the invention or any exemplary form thereof may be used in automated and semi-automated systems and optimized on a case-by-case basis.

Without wishing to be bound by theory, the monoclonal anti-RSV antibodies may also be used as labeled antibodies. For example, the monoclonal anti-RSV antibody is bound to magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, or the like.

For example, when an immunochromatographic test based on colored latex microspheres is performed, an immunochromatographic rapid test card can be assembled in a conventional manner using latex microspheres labeled with an anti-RSV antibody of the present invention, a nitrocellulose membrane (NC membrane) coated with another anti-RSV antibody, a sample pad, absorbent paper, a polyester plate, or the like. During detection, the substance to be detected in the positive sample is combined with the latex microspheres marked with the anti-RSV antibody, and agglutination reaction occurs at room temperature, so that after the positive sample is placed for a period of time, the result can be observed and judged by naked eyes.

For another example, when a colloidal gold immunochromatographic test is performed, an anti-RSV antibody may be labeled with colloidal gold, a nitrocellulose membrane (NC membrane) may be coated with another anti-RSV antibody, and a test line (T line) may be obtained by scribing. And assembling according to the preparation method of the immune test strip to obtain the colloidal gold test strip. During detection, an object to be detected in a positive sample is combined with the colloidal gold-labeled anti-RSV antibody to form a compound, the compound is combined with the envelope antibody at the T line to form a sandwich compound, and the colloidal gold is aggregated and precipitated to show red, so that the sample is indicated to be positive.

For another example, when performing immunochromatography test using fluorescent microspheres, an immunochromatography rapid test card can be assembled by labeling time-resolved fluorescent microspheres with the monoclonal anti-RSV antibody of the present invention, coating a nitrocellulose membrane (NC membrane) with another anti-RSV antibody, and using a sample pad or the like. When the detection is carried out, an object to be detected in a sample is combined with a fluorescent microsphere labeled antibody in a combination pad and is subjected to forward chromatography through capillary action, and after the object reaches a detection area, the object is further combined with another anti-RSV antibody fixed on a T line of a detection line to form a sandwich type of double-antibody sandwich. And after the chromatography is finished, reading the fluorescence intensity of the T line and the C line by using an immunofluorometer, calculating a T/C value, and calculating the content of the object to be measured in the sample by using a standard curve arranged in the immunofluorometer.

Thus, in a specific embodiment, another monoclonal anti-RSV antibody is also used in the assay, and the monoclonal anti-RSV antibody is used as one of the coating antibody and the labeled antibody, and the other monoclonal anti-RSV antibody is used as the other of the coating antibody and the labeled antibody.

It is noted that in the context of the present invention, "another monoclonal anti-RSV antibody" refers to an antibody capable of binding to the same antigen, preferably to a different epitope of the same antigen, as the monoclonal anti-RSV antibody of the invention, which may or may not be the monoclonal anti-RSV antibody of the invention. For example, in the assay, when the antibody RSV300 is selected as the labeled antibody, RSV300 may still be selected as the coating antibody, but preferably another different monoclonal anti-RSV antibody is selected as the coating antibody, e.g., a heavy chain complementarity determining region V comprising amino acid sequences represented by SEQ ID NOS: 1-3, respectively, may be selected _H CDR1、V _H CDR2 and V _H CDR3 and light chain complementarity determining region V having amino acid sequences represented by SEQ ID NOS 4 to 6, respectively _L CDR1、V _L CDR2 and V _L Monoclonal anti-RSV antibodies to CDR3, such as RSV101 or RSV102, or comprising monoclonal antibodies having the sequences set forth in SEQ ID NOS: 7-9, respectivelyHeavy chain complementarity determining region V of the amino acid sequence of (1) _H CDR1、 V _H CDR2 and V _H CDR3 and light chain complementarity determining region V having amino acid sequences shown by SEQ ID NOS 10-12, respectively _L CDR1、V _L CDR2 and V _L Monoclonal anti-RSV antibodies to CDR3, e.g., RSV200.

Thus, in a preferred embodiment, the further monoclonal anti-RSV antibody may be a monoclonal anti-RSV antibody according to the first aspect of the invention, e.g. RSV300, or may be a monoclonal antibody other than a monoclonal anti-RSV antibody according to the invention.

In a preferred embodiment, in the detection method of the present invention, the labeled antibody is different from the coating antibody.

In a further preferred embodiment, the other monoclonal anti-RSV antibody is a monoclonal anti-RSV antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth respectively in SEQ ID NOS: 1-3 _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprises a light chain complementarity determining region V having amino acid sequences represented by SEQ ID NOS: 4-6, respectively _L CDR1、 V _L CDR2 and V _L CDR3, e.g., RSV101 or RSV102. In a more preferred embodiment, for another monoclonal anti-RSV antibody, the heavy chain variable region comprises SEQ ID NO:19 or an amino acid sequence substantially identical to SEQ ID NO:19, and the light chain variable region comprises a sequence with more than 80%, more than 85%, more than 90% or more than 95% identity to the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence substantially identical to SEQ ID NO:20 has a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more.

In yet another preferred embodiment, the other monoclonal anti-RSV antibody is a monoclonal anti-RSV antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NOS: 7-9, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprises a light chain complementarity determining region V having amino acid sequences represented by SEQ ID NOS 10-12, respectively _L CDR1、V _L CDR2 and V _L And (3) CDR3. In a more preferred embodiment, for the alternative monoclonal anti-RSV antibody, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:21 or a sequence identical to SEQ ID NO:21, and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO:22 or a sequence having more than 80%, more than 85%, more than 90% or more than 95% identity with the amino acid sequence shown in SEQ ID NO:22 has a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more. As a specific example, the monoclonal anti-RSV antibody can be RSV200.

In the detection method of the present invention, the labeled antibody and the coating antibody may be the same. That is, when the monoclonal anti-RSV antibody of the present invention is used for immunochromatography for detection of RSV virus, it is used as a labeled antibody for labeling magnetic beads, microspheres, enzymes, fluorochromes, biotin, colloidal gold, etc., and at the same time, it is also used as a coating antibody for coating a solid support such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, etc.

In a sixth aspect, the invention provides use of a monoclonal anti-RSV antibody of the first aspect in the manufacture of a reagent for detecting Respiratory Syncytial Virus (RSV).

In yet another preferred embodiment, the ELISA may be a direct method, an indirect method, a sandwich method and a competition method.

As described above, the monoclonal anti-RSV antibody can be used as a coating antibody in assays. For example, monoclonal anti-RSV antibodies are bound to a solid phase, such as a solid support. The solid support used in the detection method of the present invention is not particularly limited, and may be a porous or non-porous material, such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, and the like.

Similarly, the monoclonal anti-RSV antibody can be used as a labeled antibody. For example, the monoclonal anti-RSV antibody is bound to magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, or the like.

In a specific embodiment, another monoclonal anti-RSV antibody may also be used in the use, and the monoclonal anti-RSV antibody is used as one of the coating antibody and the labeled antibody, and the another monoclonal anti-RSV antibody is used as the other of the coating antibody and the labeled antibody.

As described above, the "another monoclonal anti-RSV antibody" refers to an antibody that binds to the same antigen as the monoclonal anti-RSV antibody of the invention, preferably to a different epitope of the same antigen, and may or may not be the monoclonal anti-RSV antibody of the invention. For example, in this use, when the antibody RSV300 is selected as the marker antibody, RSV300 may still be selected as the coating antibody, but preferably another different monoclonal anti-RSV antibody is selected as the coating antibody, e.g., antibodies RSV101, RSV102 or RSV200 may be selected as described previously.

In a preferred embodiment, the other monoclonal anti-RSV antibody is different from the monoclonal anti-RSV antibody of the invention.

In a preferred embodiment, the other monoclonal anti-RSV antibody is a monoclonal anti-RSV antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NOS: 1-3, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprisesA light chain complementarity determining region V having amino acid sequences shown in SEQ ID NOS 4-6 _L CDR1、V _L CDR2 and V _L And (3) CDR3. As a specific example, the monoclonal anti-RSV antibody can be RSV101 or RSV102.

In yet another preferred embodiment, for the other monoclonal anti-RSV antibody, the heavy chain variable region comprises SEQ ID NO:19 or an amino acid sequence substantially identical to SEQ ID NO:19, and the light chain variable region comprises a sequence having more than 80%, more than 85%, more than 90% or more than 95% identity to the amino acid sequence set forth in SEQ ID NO:20 or an amino acid sequence substantially identical to SEQ ID NO:20 has a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more. As a specific example, the monoclonal anti-RSV antibody can be RSV101.

The inventors have found that when a monoclonal anti-RSV antibody of the invention, such as RSV300, is used in combination with a monoclonal anti-RSV antibody, such as RSV101 or RSV102, comprising a heavy chain complementarity determining region represented by SEQ ID NOS: 1-3 and a light chain complementarity determining region represented by SEQ ID NOS: 4-6, respectively, either as a labeled antibody or a coated antibody, it exhibits significant sensitivity in use in immunochromatographic reactions, such as color latex microsphere-based and colloidal gold-based immunochromatographic reactions.

Accordingly, the present invention also provides a monoclonal anti-RSV antibody combination for immunodetection comprising a first monoclonal anti-RSV antibody as a marker antibody and a second monoclonal anti-RSV antibody as a coating antibody, wherein the first monoclonal anti-RSV antibody and the second monoclonal anti-RSV antibody may be specifically as follows:

as a first antibody combination, the first monoclonal anti-RSV antibody is a monoclonal anti-RSV antibody of the first aspect of the invention, e.g., RSV300, and the second monoclonal anti-RSV antibody is a monoclonal anti-RSV antibody comprising a heavy chain variable region comprising a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NOS: 1-3, respectively, and a light chain variable region _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprises a light chain variable regionLight chain complementarity determining regions V of amino acid sequences represented by SEQ ID NOS 4 to 6, respectively _L CDR1、 V _L CDR2 and V _L CDR3。

In an exemplary embodiment, for the second monoclonal anti-RSV antibody, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID No. 19 or a sequence identical to SEQ ID NO:19 has a sequence with more than 80%, more than 85%, more than 90% or more than 95% identity, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:20 or a sequence which is identical to the amino acid sequence shown in SEQ ID NO:20 has a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more.

As a second antibody combination, the first monoclonal anti-RSV antibody is a monoclonal anti-RSV antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences represented by SEQ ID NOs 1 to 3, respectively _H CDR1、 V _H CDR2 and V _H CDR3, the light chain variable region comprises a light chain complementarity determining region V having amino acid sequences represented by SEQ ID NOS: 4-6, respectively _L CDR1、V _L CDR2 and V _L CDR3, e.g., RSV101 or RSV102.

In an exemplary embodiment, for the first monoclonal anti-RSV antibody, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID No. 19 or a sequence identical to SEQ ID NO:19 has a sequence with more than 80%, more than 85%, more than 90% or more than 95% identity, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:20 or a sequence which is identical to the amino acid sequence shown in SEQ ID NO:20 has a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more.

The second monoclonal anti-RSV antibody is a monoclonal anti-RSV antibody of the first aspect of the invention, e.g., RSV300.

In a preferred embodiment, the kit may further comprise: another monoclonal anti-RSV antibody comprising a heavy chain variable region and a light chain variable region, wherein:

the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences represented by SEQ ID NOS: 1-3, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprises a light chain complementarity determining region V having amino acid sequences represented by SEQ ID NOS: 4-6, respectively _L CDR1、V _L CDR2 and V _L CDR3, e.g., RSV101 or RSV102.

The kit of the present invention can be used for point-of-care testing (POCT) or electrochemical immunoassay systems. The kit according to the invention and any exemplary form thereof may be used and optimized in automated and semi-automated systems.

Examples

In the following examples, the methods of preparation of the antibodies of the invention and characterization of relevant properties are shown. Unless otherwise specified, the test methods employed therein were all conventional methods, and, unless otherwise specified, the test materials used in the following examples were all purchased from a conventional reagent store. Unless defined otherwise, all 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.

It should be noted that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The foregoing summary, as well as the following detailed description, is intended merely to be illustrative of the invention and is not intended to be in any way limiting. The scope of the invention is to be determined by the appended claims without departing from the spirit and scope of the invention.

Example 1: preparation of monoclonal antibody against RSV F protein

Antigen coupling and immunization: the purified RSV F protein (SEQ ID NO: 27) was used as an immunogen to immunize mice. The mice are female BALB/c mice with 6-8 weeks of age. Mice were immunized 4 times in total, each time at 2 weeks intervals, at an immunization dose of 100. Mu.g/mouse. First immunization RSV F protein was mixed in equal volumes with Freund's complete adjuvant (Sigma-Aldrich) and injected subcutaneously in multiple dorsal injections, and the last three immunizations were mixed in equal volumes with RSV F protein and Freund's incomplete adjuvant (Sigma-Aldrich) and injected intraperitoneally. And 7 days after the fourth immunization, blood is collected after the tail of the mouse is cut off, serum is separated, and the antibody titer level of antiserum of the immunized mouse is detected by adopting an indirect ELISA method so as to observe the immune response effect. Selecting a serum antibody titer higher than 1:10000 mice were subjected to cell fusion experiments, which were boosted 3 days before the cell fusion experiments by intraperitoneal injection with the unadjuvanted RSV F protein (100 μ g/mouse).

Establishment of hybridoma cells: on the day of fusion, spleens from immunized mice were removed under sterile conditions and the organs were made into single cell suspensions. Taking mouse myeloma cells (SP 2/0) and the spleen cells of the immunized BALB/c mice according to the ratio of 1:5, mixed well and washed twice before being fused with PEG. Add pre-warmed PEG1500, gently shake, wash cells with pre-warmed serum-free RPMI-1640 medium, and resuspend cells in HAT selective medium. Plating the cell suspension into 96-well culture plates at 200. Mu.L/well, and at 37 ℃ 5% CO ₂ The cells are cultured under conditions. After 4 to 7 days of culture, the cells were cultured in HT medium, and when the fused cells grew to 1/10-1/5 of the bottom area of the wells of the 96-well plate, the supernatant was subjected to antibody detection.

Screening of positive hybridoma cells: RSV F protein was diluted with coating buffer (0.05 mol/L, pH9.6, PBS) to a final concentration of 1. Mu.g/ml, added to a 96-well plate at 100. Mu.L/well and coated overnight at 4 ℃; discarding the coating solution, washing with Phosphate Buffer Solution (PBST) for 3 times, and patting to dry; blocking with PBST containing 2% BSA, 150. Mu.L/well, incubating at 37 ℃ for 2h, washing 3 times with PBST, patting dry; the fusion cell supernatant, 1 dilution of immune mouse positive serum (as positive control) and 1 dilution of mouse negative serum (as negative control) at 100 μ L/well were added to the corresponding wells, incubated at 37 ℃ for 1h, washed 3 times with PBST, patted dry; adding 1: goat anti-mouse IgG labeled with horseradish peroxidase (HRP) at a dilution of 4000 (purchased from Sigma), 100. Mu.L/well, incubated at 37 ℃ for 1h, washed 3 times with PBST, patted dry; adding tetramethyl benzidine (3,3 ',5,5' -tetramethyllbenzidine, TMB) substrate, 100 μ L/hole, and developing in dark at room temperature for 10min; the reaction was stopped by adding 50. Mu.L of 2mol/L sulfuric acid per well.

Detecting the OD of all holes in the microplate at the wavelength of 450nm of the microplate reader _450nm The value is obtained. OD of negative serum _450nm Absorbance OD of ≦ 0.1 for determining absorbance OD of well (RSV F protein) _450nm Value is negative well OD _450nm Positive at least 2.1 times of the total amount of the composition was determined as a criterion. Positive hybridoma cells were selected for further cloning.

Cloning of positive cell lines: sampling and counting antibody-secreting positive cell wells, diluting to 100 cells/10 mL of culture medium, plating the diluted cell suspension at 100. Mu.L/well to a 96 well cell culture plate, setting at 37 ℃ and 5% CO ₂ Culturing in a cell culture box. After 6-7 days, the formation of clonal cells was observed microscopically, and a single long hole of Long Sheng was marked, and the cell supernatant was removed and subjected to ELISA test (the same as the fusion test described above) to select positive monoclonal cells. Limiting dilution is carried out on positive hole cells, ELISA values are measured 5-6 days after each limiting dilution, and OD obtained through ELISA detection is selected _450nm And (4) carrying out limited dilution on the monoclonal wells with higher positive values until the whole plate result of the 96-well plate is positive by ELISA (enzyme-Linked immunosorbent assay). And (4) selecting a monoclonal fixed strain with a high positive value. Finally, three cell strains which stably secrete the anti-RSV antibody are obtained and named as hybridoma cell strains F2H11, B8H12 and C5B7 respectively.

The hybridoma cell strain F2H11 is deposited in the China general microbiological culture Collection center (CGMCC) of the microbiological research institute of China academy of sciences No. 3, beijing, chaoyang, beijing, 7 months and 14 days in 2022, with the collection number of CGMCC No.45238.

The hybridoma cell strain B8H12 is deposited in the China general microbiological culture Collection center (CGMCC) of the microbiological research institute of China academy of sciences No. 3, beijing, chaoyang, beijing, 7 months and 14 days in 2022, with the collection number of CGMCC No.45239.

The hybridoma cell strain C5B7 is preserved in China general microbiological culture Collection center (CGMCC) of the microbiological research institute of China academy of sciences No. 3, west Lu 1, beijing, the rising area, 7 months and 14 days in 2022, and the preservation number is CGMCC No.45240.

Preparing and purifying cell supernatant monoclonal antibody: the three cell lines were cultured in RPMI-1640 medium containing 15% serum on a 10cm dish, and expanded to about 4X 10 ⁷ At cell/dish, centrifuge at 800rpm for 5min, discard supernatant and transfer cells to 2L spinner flask, add serum-free medium to make cell density about 3X 10 ⁵ And (4) culturing each cell per ml in a spinner flask. After further culturing for 1-2 weeks, the cell death rate reaches 80% -90% (at the time, the cell density is about 1X 10) ⁶ -2×10 ⁶ Pieces/ml), collecting cell suspension, centrifuging at 6000rpm for 20min, collecting supernatant, and purifying the supernatant by Protein A immunochromatography.

The monoclonal antibody prepared from the F2H11 hybridoma cell line was designated RSV101; the monoclonal antibody prepared from the B8H12 hybridoma cell line is marked as RSV200; the monoclonal antibody produced by the C5B7 hybridoma cell line was designated RSV300.

The concentrations of RSV101, RSV200 and RSV300 monoclonal antibodies were approximately 2-4mg/mL as determined by microspectrophotometry. The purified monoclonal antibody was assayed for concentration and dispensed (100. Mu.L/tube, 1 mg/ml) and stored at 4 ℃ to 8 ℃.

Three antibodies (5. Mu.g per well) were identified by SDS-PAGE electrophoresis and the results are shown in FIG. 1. As can be seen from the figure, all three antibodies exhibited an antibody heavy chain band of about 51kD and an antibody light chain band of about 26 kD.

And (3) purity detection: analyzing the three monoclonal antibodies by using size exclusion chromatography (SEC-HPLC), and calculating the purity percentage of the main peak by using a peak area normalization method under the condition of ensuring that all components in a sample to be detected have peaks, wherein the purity is more than 98%.

Example 2: binding ability of antibody to RSV F protein

RSV F protein was diluted to a concentration of 1. Mu.g/ml with 0.05mol/L carbonate buffer pH9.6, added to a 96-well microplate at 100. Mu.L/well, coated overnight at 4 ℃, washed 3 times with PBST on an automatic plate washer, and patted dry. Blocked with PBST containing 2% BSA, 150. Mu.L/well, incubated at 37 ℃ for 2h, washed 3 times with PBST, and patted dry. The antibodies of the anti-RSV F protein monoclonal antibodies RSV101, RSV200 and RSV300 are subjected to gradient dilution by PBS buffer solution with pH7.4 and 0.02M, the initial concentration of the antibody is 1.5ug/ml, and the gradient dilution is sequentially carried out according to three times to obtain a series of monoclonal antibody samples with different concentrations. The diluted monoclonal antibody sample is added to the enzyme label plate at a rate of 100 mu L/well, incubated at 37 ℃ for 1h, washed 3 times and patted dry. Adding 1: horse Radish Peroxidase (HRP) -labeled goat anti-mouse IgG (purchased from sigma) at a dilution of 4000, 100. Mu.L/well, incubated at 37 ℃ for 1h, washed 3 times with PBST, and patted dry. TMB substrate was added at 100. Mu.L/well and developed in the dark at room temperature for 10min. The reaction was terminated by adding 2mol/L sulfuric acid at 50. Mu.L/well. OD determination by enzyme-linked immunosorbent assay _450nm The results of the ELISA described above were analyzed by software to obtain EC50 values of the RSV101, 200 and 300 antibodies, and the results are shown in fig. 2.

As can be seen from FIG. 2, the EC50 values for binding of RSV101, 200, 300 antibodies to RSV-F (strain A) were 8.514ng/ml, 6.459ng/ml and 6.035ng/ml, respectively.

Example 3: cloning and sequencing of anti-RSV antibody variable region sequences

Total RNA was isolated from the above three hybridoma cell lines, cDNA was prepared by reverse transcription to clone an immunoglobulin sequence from the hybridoma cell line, and the antibody variable region sequence of the hybridoma cell line was determined as follows.

Extraction of RNA: total RNA extraction and immediate reverse transcription were performed on the hybridoma cell lines described above with reference to the instructions of the Total RNA M5 extraction kit (from Peking Polymermai Biotech Co., ltd.).

Reverse transcription of RNA into cDNA: the total RNA extracted in the previous step was reverse transcribed with reference to M5 First Strand cDNA Synthesis Kit (available from Biotech Co., ltd., beijing) to prepare cDNA, which was frozen at-20 ℃ for use.

c. PCR amplification and recovery of variable region sequences: the cDNA obtained in the above step is used as a template, and a universal heavy chain primer Mu Ig V is used _H 5' -A and Mu IgG V _H 3' -2, amplification of immunoglobulin heavy chain (IgH) cDNA by PCR; similarly, the light chain primer Mu Ig kappa V was used _L 5' -A and Mu Ig kappa V _L 3' -1, amplifying immunoglobulin light chain (IgK) cDNA by PCR, and recovering PCR products; the PCR reaction was carried out using thermostable PfuDNA polymerase throughout.

d. Cloning and sequencing of variable region sequences: according to the instructions of the Cloning vector pTOPO-Blunt Cloning kit (available from Beijing Convergence technologies, inc.), the heavy chain and light chain variable region genes were ligated to pTOPO vector, respectively, to transform E.coli DH 5. Alpha. And positive clones were picked up and subjected to sequencing by Beijing Rui Boxing, biotech, inc.

The sequences of the heavy chain variable region gene and the light chain variable region gene of the antibody of the hybridoma cell strain obtained by sequencing are analyzed, and the sequences of the complementarity determining regions of the heavy chain and the complementarity determining regions of the light chain are shown in the following table 1.

Table 1: complementarity determining regions of heavy and light chains of antibodies RSV101, RSV200 and RSV300 (according to the Chothia numbering system)

In addition, the sequences of the variable regions of the heavy and light chains of each anti-RSV antibody were sequenced as follows:

the heavy chain variable region sequence of the antibody RSV101 is shown as SEQ ID NO. 19, and the light chain variable region sequence is shown as SEQ ID NO. 20; the heavy chain variable region sequence of the antibody RSV200 is shown as SEQ ID NO:21, and the light chain variable region sequence is shown as SEQ ID NO: 22; the heavy chain variable region sequence of antibody RSV300 is shown in SEQ ID NO:23 and the light chain variable region sequence is shown in SEQ ID NO: 24.

Example 4: preparation and purification of recombinant antibodies

Constructing recombinant antibody, preparing cell strain for stably expressing anti-blocking agent antibody through eukaryotic expression, and carrying out large-scale culture and purification on the cell strain.

Amplifying the mouse monoclonal antibody heavy and light chain variable region gene (V) by adopting a PCR method and taking cDNA obtained by reverse transcription as a template _H And V _L ) And sequencing to determine the sequence. V for antibody _L And V _H Gene, and the plasmid pCDNA3.1 is used in constructing recombinant antibody eukaryotic expression vector by means of molecular cloning. The heavy chain and light chain gene expression plasmids of the antibody are electrically transduced into CHO host cells, after the electric transduction, the CHO host cells are added into a pressure screening culture medium (50 mu M MSX) to be cultured for 20 days, and then the supernatant is taken to be subjected to ELISA detection (horse radish peroxidase (HRP) marked goat anti-mouse IgG is used as a secondary antibody for screening, the method is the same as the above), and the stably expressed recombinant antibody cell strains are screened.

Furthermore, the light chain CDR3 of RSV101 was mutated to QQYSNFPCMutation of T to QQYSNFPFT, the remaining sequence was kept unchanged, and a recombinant antibody eukaryotic expression vector (also using plasmid pCDNA3.1) was constructed in the same manner. The heavy chain and light chain gene expression plasmids of the antibody are electrically transduced into CHO host cells, and recombinant antibody cell strains with stable expression are screened out, the obtained recombinant antibody is marked as RSV102, and CDRs of the recombinant antibody are shown in the following table 2:

table 2: CDRs of the heavy and light chains of antibody RSV102 (according to the Chothia numbering system)

And (3) carrying out large-scale cell culture on the screened stable transgenic cell strain by adopting a cell roller bottle culture technology, and preparing a recombinant antibody. Cells were plated at (0.2-0.3). Times.10 using medium (Vega CHO) ⁶ cells/ml were inoculated into roller bottles containing 300ml of medium (Vega CHO) in 1L roller bottles, the number of which was determined according to the production requirements, and the roller bottles inoculated with cells were placed in a cell spinner and cultured in a cell incubator. The culture conditions were 900 rpm, the temperature was 37 ℃ and the carbon dioxide was 5%. And (4) after culturing for 7-9 days, sampling under a microscope, observing, and centrifuging to collect samples when the cell viability is less than 50%. And (3) carrying out affinity purification on the sample by using a protein A affinity chromatographic column to obtain an antibody, namely the recombinant monoclonal antibody.

The binding ability of the recombinant antibody RSV102 to the RSV F protein was tested according to the method described in example 2 and the results are shown in FIG. 3. It can be seen that the EC50 value for RSV102 antibody binding to RSV-F (strain A) is 7.9ng/ml.

Example 5: immunochromatography test based on colored latex microspheres

The monoclonal anti-RSV antibody of the invention is evaluated by an immunochromatography method based on colored latex microspheres.

The recombinant antibody labeled latex microsphere comprises the following steps:

1. cleaning microspheres: adding a certain amount of 0.1M 4-morpholine ethanesulfonic acid (MES) buffer solution with pH of 6.0 into 1mg microsphere, mixing, centrifuging at 20000rpm for 20min, and removing supernatant;

2. and (3) activation: adding a certain volume of MES buffer solution with pH of 6.0, ultrasonically mixing uniformly, adding 12 mu g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and 12 mu g N-hydroxysuccinimide (NHS), reacting for 15min at 37 ℃, mixing uniformly once every 5min, centrifuging at 20000rpm, and discarding the supernatant after 20 min;

3. coupling: adding a certain volume of 50mM boric acid buffer solution with pH =8.0, ultrasonically mixing, adding 0.2mg of anti-RSV F monoclonal antibody a listed in the following table 3, and mixing and reacting at 37 ℃ overnight;

4. and (3) sealing: blocking was performed for 1h by adding 1ml BSA (5%), followed by centrifugation at 20000rpm for 20min and discarding the supernatant.

5. Cleaning: washing with 0.1M Tris-HCl (pH8.5) for 2 times, centrifuging at 20000rpm for 20min, and removing supernatant;

6. and (3) storage: 0.5ml of 25mM MES (pH = 7.4) was added for storage, and 1% of BSA was determined by diluting with 25mM MES (pH = 7.4), 0.1% Tween20 by 50-fold, followed by lyophilization and sealing for use.

Recombinant antibody-coated nitrocellulose membrane (NC membrane): anti-RSV monoclonal antibody b (listed in Table 3 below) was coated on NC membranes of Sidoris 140 as a test line (T line) diluted to a final concentration of 1.0mg/ml with 10mM pH 7.4PBS and 2% sucrose, and sealed overnight at 37 ℃ for use.

Preparing a sample pad: the sample pad was treated with 1 mM PBS, pH7.4, and lyophilized for use.

Assembling an immunochromatography system: and assembling the red latex microspheres marked with the antibodies, the NC membrane coated with the antibodies, the sample pad, the absorbent paper and the polyester plate into the immunochromatography rapid test card in a conventional manner.

Table 3: anti-RSV monoclonal antibodies a and b used in this example

Antibody combination numbering	Labeled antibody a	Coating antibody b
			1	RSV101	RSV101
2	RSV101	RSV102
			3	RSV101	RSV200
4	RSV101	RSV300
			5	RSV102	RSV101
6	RSV102	RSV102
			7	RSV102	RSV200
8	RSV102	RSV300
			9	RSV200	RSV101
10	RSV200	RSV102
			11	RSV200	RSV200
12	RSV200	RSV300
			13	RSV300	RSV101
14	RSV300	RSV102
			15	RSV300	RSV200
16	RSV300	RSV300

And (3) immune test: samples of virus cultures and samples containing different concentrations (1 ng/ml, 10ng/ml and 100 ng/ml) of recombinant antigen were tested at different dilutions (1. Specifically, 80 μ l of a sample to be tested is dripped into the rapid test card, and after the rapid test card is placed at room temperature for 15 minutes, the result is observed and judged by naked eyes. The antigen-antibody binding activity in the sample is indicated by the light and dark color of the developed bands, and the results are shown in Table 4, using the manufacturer A RSV latex chromatographic reagent as a control. And comparing the color of the developed T line with a standard color card, selecting the closest color, and marking the activity of the product by the grade number of the color number corresponding to the color. As shown in FIG. 4, the color chart is divided into nine grades C1-C9, wherein C1 is strong positive and represents the highest unit activity, the middle gradient is decreased, and C9 is weakest positive and represents the lowest unit activity. In the figure, B indicates a blank.

Table 4: immunochromatography reaction result based on colored latex microspheres

The result shows that when the antibody is used in immunochromatography detection based on the colored latex microspheres, the sensitivity of the antibody is one to two levels higher than that of similar products of a manufacturer A no matter against virus cultures or against recombinant antigens when different labeled antibodies and coating antibodies are adopted, and the test sensitivity can reach nanogram level. Of these, antibody combinations 4, 10, 13 and 14 showed the highest sensitivity.

Example 6: colloidal gold immunochromatographic assay

Preparing colloidal gold: 200ml of ultrapure water was added to a Erlenmeyer flask, the flask was heated to boiling, and then 1ml of a 2% chloroauric acid (Sigma-Aldrich Co., ltd., product No. 16961-25-4) solution was added thereto, and immediately after boiling, 1ml of a 2% trisodium citrate (Sigma-Aldrich Co., product No. 6132-04-3) aqueous solution was added thereto, followed by boiling under stirring for 10 minutes, followed by natural cooling.

Labeling the colloidal gold conjugate: 10ml of the above colloidal gold was put into a beaker, and 120. Mu.l of 0.2M K was added while stirring ₂ CO ₃ Adjusting the pH value to 7.0, and continuing stirring for 10 seconds; 100 μ g of anti-RSV monoclonal antibody a (labeled antibody) as listed in Table 3 above was added and stirring was continued for 5 minutes; adding 0.1ml 10% BSA, and stirring for 5 minutes; 12000g was centrifuged for 10 minutes, the supernatant was discarded, and the precipitate was volume-adjusted to 1ml with a colloid Jin Xishi liquid (10mM PB,150mM NaCl,0.2% BSA,0.1% TritonX-100,3% Sucrose,0.01% Proclin300) as an anti-RSV monoclonal antibody colloidal gold complex.

Preparing a colloidal gold pad: diluting the colloidal gold compound with 10 times of the solution Jin Xishi, soaking glass fiber (Shanghai gold mark company), and lyophilizing to obtain gold mark pad.

Nitrocellulose membrane (NC membrane) coating: the anti-RSV monoclonal antibody b (coating antibody) as listed in Table 3 above was diluted to 1.5mg/ml to prepare a working solution for test line, which was streaked onto the corresponding site of nitrocellulose membrane (Millipore, cat. No.: HF 135002) by a dot-streaked instrument and dried at 50 ℃ for 1 hour for use.

Assembling the colloidal gold immunochromatography test reagent strip: and assembling the gold label pad, the nitrocellulose membrane coated with the antibody, absorbent paper, a polyester plate and the sample pad into the colloidal gold immunoassay reagent strip.

And (3) sensitivity detection: samples of virus cultures at different dilutions and recombinant antigens at different concentrations were tested separately. Specifically, 80 μ l of the sample to be tested is dropped on the sample pad, and the sample is placed at room temperature for 15-30 minutes, and the result is judged. The activity of binding of the antigen to the antibody in the sample can be indicated by the shade of the color of the band. The color of the T-line strip of the colloidal gold test paper reaction coloration is compared with a standard color card, the closest color is selected, the grade number of the color number corresponding to the color is used for marking the activity of the product, and the result is shown in the following table 5, and the RSV colloidal gold chromatography reagent of manufacturer A is used as a contrast. The results show that the antibody combinations of the invention exhibit comparable sensitivity, or one to two orders of sensitivity, to the manufacturer a RSV colloidal gold chromatographic reagents when different labeled and coated antibodies are used in the invention, as compared to the manufacturer a RSV colloidal gold chromatographic reagents. Of these, antibody combinations 4, 10 and 13 showed the highest sensitivity.

Table 5: colloidal gold-based immunochromatographic reaction results (recombinant antigen unit ng/ml)

And (3) specific detection: with the RSV colloidal gold chromatography reagent of manufacturer A as a control, 30 nasopharyngeal swab samples of healthy people were tested using the reagent strips prepared with the four antibodies of the invention. The results show that, like the reagent of manufacturer A, the reagent strips prepared by the four antibodies of the invention have no false positive results, and the specificity is 100%.

Example 7: fluorescent microsphere immunochromatographic assay

Respectively coating an NC membrane (coated antibody) and a time-resolved fluorescent microsphere (labeled antibody) with the antibody to prepare a chromatography product, comprising the following steps of:

1. labeling time-resolved fluorescent microspheres: adding a certain amount of 0.1M MES buffer solution with pH 6.5 into 1mg microsphere, mixing uniformly, centrifuging at 20000rpm for 20min, and removing supernatant; adding MES buffer solution with a certain volume and pH 6.5, ultrasonically mixing, adding 20 μ g EDC and 40 μ g NHS, reacting at room temperature for 30min, mixing once every 5min, centrifuging at 20000rpm,20min, and discarding supernatant; adding a certain volume of 50mM boric acid buffer solution with pH =8.0, ultrasonically mixing, adding 0.05-0.1mg of the anti-RSV monoclonal antibody a listed in the above table 3, and mixing and reacting at room temperature for 2 hours; adding 1mL 100mM Tris-HCl pH8.5 containing 1% BSA for blocking for 2 hr, centrifuging at 20000rpm for 20min, and discarding the supernatant; washing with 0.1M Tris-HCl (pH8.5) for 2 times, centrifuging at 20000rpm for 20min, and removing supernatant; and (3) storage: 0.5ml of 25mM MES (pH = 7.4) was added for storage, and the mixture was diluted 50-fold with 25mM MES (pH = 7.4), 1% BSA, and 0.1% Tween20, and lyophilized and sealed for use.

2. Coating an NC film: the anti-RSV monoclonal antibody b listed in the above Table 3 was diluted with 10mM pH7.4 PBS,2% sucrose to a final concentration of 1.0mg/ml, coated on NC membrane of Celtolite 140, and sealed overnight at 37 ℃ for use.

3. Sample pad preparation: the sample pad was treated with 1 mM PBS, pH7.4, and lyophilized for use.

4. Assembling and testing: and assembling the time-resolved fluorescent microspheres marked with the antibodies, the NC membrane coated with the antibodies and the sample pad into an immunochromatography rapid test card, adding 80 mu l of random clinical samples, standing at room temperature for 15 minutes, and detecting by using an immunofluorometer.

Sensitivity of antibody to F protein recombinant antigen:

first, the detection of the F protein recombinant antigen at different concentrations (0.1-500 ng/ml) by the antibody combinations 4 and 10 was examined, and the results are shown in Table 6 below and FIG. 5. As shown in the following Table 6, the detection results of the antibody combination 4 on the F protein recombinant antigen show that when the antigen concentration is 0.1ng/ml, the T/C value is more than twice of the T/C value (0.52) of a detection group without the F protein, and by taking the T/C value as the standard, the sensitivity of the recombinant antibody on the F protein recombinant antigen can reach 0.1ng/ml in a fluorescent microsphere immunochromatographic system. The same detection is carried out by taking the RSV N protein recombinant antigen as a negative control, and the T/C value is not obviously changed along with the increase of the N protein concentration.

Table 6: sensitivity results of antibody combination 4 to F protein recombinant antigen

Next, the results of detection of the F protein recombinant antigen at different concentrations by the antibody combinations 2, 4,8, 10, and 15 of the present example and the RSV latex chromatographic reagent of manufacturer a were compared, and are shown in table 7. Compared with the RSV latex chromatographic reagent of manufacturer A, the antibody of the invention can detect the F protein recombinant antigen with the concentration of 0.1ng/ml in the fluorescence chromatographic reagent, and the latex reagent of manufacturer A has a negative detection result of the concentration, which shows that the monoclonal antibody of the invention has high sensitivity.

Table 7: sensitivity results of antibodies to viral cultures and recombinant antigens of F protein

Sensitivity of antibodies to virus culture: the antibody pair dilutions were tested as 1:100 to 1: sensitivity of virus cultures in the range 20000, the results are shown in Table 8. In contrast to the RSV latex chromatographic reagent from manufacturer A, the antibody of the invention was compared to the antibody of the present invention in a fluorescent chromatographic reagent to detect a viral culture 1:20000, and in the 1: at 20000 dilution, the latex reagent of manufacturer A showed negative results, indicating that the monoclonal antibody of the invention has higher sensitivity.

Table 8: results of sensitivity of the antibodies of the invention to viral cultures

Sequence listing

SEQ ID NO:1(V _H CDR1)：GYSITSDY

SEQ ID NO:2(V _H CDR2)：SYIGS

SEQ ID NO:3(V _H CDR3)：TIRNYFDY

SEQ ID NO:4(V _L CDR1)：SASQGISNYLN

SEQ ID NO:5(V _L CDR2)：DTSSLHS

SEQ ID NO:6(V _L CDR 3): QQYSNFPXT (wherein, X is C or F)

SEQ ID NO:7(V _H CDR1)：GFNIKDY

SEQ ID NO:8(V _H CDR2)：DPENGN

SEQ ID NO:9(V _H CDR3)：YGTSYWFPY

SEQ ID NO:10(V _L CDR1)：KASQDINSYLS

SEQ ID NO:11(V _L CDR2)：RANRLVD

SEQ ID NO:12(V _L CDR3)：LQFDEFPYT

SEQ ID NO:13(V _H CDR1)：GYTFSSN

SEQ ID NO:14(V _H CDR2)：LPGSGS

SEQ ID NO:15(V _H CDR3)：EELGYFDY

SEQ ID NO:16(V _L CDR1)：RASQDINNYLN

SEQ ID NO:17(V _L CDR2)：YTSRLHS

SEQ ID NO:18(V _L CDR3)：QQGSTLPPT

SEQ ID NO:19(V _H )：EVQLQQPGAELVKPGGSVKLSCKASGYSITSDYWVKQRPG RQLEWIGSYIGSKISLTVDKPSSTAYMQLSSLTSEDSAVYYCARTIRNYFDYWGQGTT VTVSS

SEQ ID NO:20(V _L )：DIVMTQTHKFMSTSVGDRVSLTCSASQGISNYLNWYQEKPG QSPKLLIYSDTSSLHSGVPARFTGTQSGTDFTFTISSVQAEDEALYFCQQYSNFPCTFGA GTKLELK

SEQ ID NO:21(V _H )：EVQLQQPGAELVKPGGSVKLSCKASGFNIKDYWVKQRPGR QLEWIGDPENGNKISLTVDKPSSTAYMQLSSLTSEDSAVYYCARYGTSYWFPYWGQG TTVTVSS

SEQ ID NO:22(V _L )：DIVMTQTHKFMSTSVGDRVSLTCKASQDINSYLSWYQEKPG QSPKLLIYSRANRLVDGVPARFTGTQSGTDFTFTISSVQAEDEALYFCLQFDEFPYTFG AGTKLELK

SEQ ID NO:23(V _H )：EVQLQQPGAELVKPGGSVKLSCKASGYTFSSNWVKQRPGR QLEWIGLPGSGSKISLTVDKPSSTAYMQLSSLTSEDSAVYYCAREELGYFDYWGQGTT VTVSS

SEQ ID NO:24(V _L )：DIVMTQTHKFMSTSVGDRVSLTCRASQDINNYLNWYQEKP GQSPKLLIYSYTSRLHSGVPARFTGTQSGTDFTFTISSVQAEDEALYFCQQGSTLPPTFG AGTKLELK

SEQ ID NO:25(C)：AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTW NSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRG PTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFV NNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKN TEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

SEQ ID NO:26(C)：ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKID GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSF NRNEC

27 (RSV fusion protein): MELPILKANAITTILAAVTFCFASSQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLMKQELDKYKNAVTE LQLLMQSTPAANNRARRELPRFMNYTLNNTKKTNVTLSKKRKRRFLGFLLGVGSAIA SGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVN KRSCRISNIETVIEFQHKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITND QKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKE GSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDI FNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNK GVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLA FIRKSDELLHNVNAGKSTTNIMITTIIIEIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGI NNIAFSN

Claims

1. A monoclonal anti-Respiratory Syncytial Virus (RSV) antibody, which is a monoclonal anti-RSV antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NOs: 13-15, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprises amino acid sequences shown by SEQ ID NO 16-18 respectivelyLight chain complementarity determining region V of (4) _L CDR1、V _L CDR2 and V _L CDR3；

Preferably, the antibody comprises the heavy chain variable region of SEQ ID NO. 23 or a heavy chain variable region substantially identical to SEQ ID NO:23, and a light chain variable region represented by SEQ ID No. 24 or a light chain variable region having a sequence with 80% or more, 85% or more, 90% or more, or 95% or more identity to the amino acid sequence represented by SEQ ID NO:24 has a sequence identity of 80% or more, 85% or more, 90% or more, or 95% or more;

preferably, the antibody is produced by hybridoma cell strains which are deposited in 2022 years, 7 months and 14 days in China general microbiological culture Collection center (CGMCC) of the microbiological research institute of China academy of sciences No. 3 of West Lu No. 1 of the facing-Yang district, beijing, and have the preservation number of CGMCC No.45240.

2. A hybridoma cell strain which is preserved in China general microbiological culture Collection center (CGMCC) of the microbiological research institute of China academy of sciences No. 3 of Beijing, chaoyang, beijing, 7 months and 14 days 2022, wherein the preservation number is CGMCC No.45240.

3. An expression vector comprising a gene encoding a monoclonal anti-RSV antibody, wherein the monoclonal anti-RSV antibody is a monoclonal anti-RSV antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain complementarity determining region V having amino acid sequences set forth in SEQ ID NOS: 13-15, respectively _H CDR1、V _H CDR2 and V _H CDR3, the light chain variable region comprises a light chain complementarity determining region V having amino acid sequences represented by SEQ ID NOS 16-18, respectively _L CDR1、V _L CDR2 and V _L CDR3；

Preferably, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:23 or a sequence identical to SEQ ID NO:23, and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO:24 or a sequence having more than 80%, more than 85%, more than 90% or more than 95% identity with the amino acid sequence shown in SEQ ID NO:24 has a sequence with 80% or more, 85% or more, 90% or more, or 95% or more identity;

preferably, the expression vector is a plasmid vector such as pcdna3.1, pEE12, pCAGGS.

4. An expression cell comprising the expression vector of claim 3; preferably, the expression cell is a mammalian cell, such as a chinese hamster ovary cell, a small hamster kidney cell, a monkey kidney cell, a mouse thymoma cell, a human embryonic kidney cell.

5. A method of detecting Respiratory Syncytial Virus (RSV), for non-diagnostic purposes, comprising the step of using the monoclonal anti-RSV antibody of claim 1.

6. The method of claim 5, wherein the detection is performed by immunochromatography, enzyme-labeled antibody method (ELISA), chemiluminescence, electrochemiluminescence; preferably, the immunochromatography comprises a fluorescent microsphere immunochromatography, a colloidal gold immunochromatography, an immunochromatography based on colored latex microspheres, a time-resolved fluorescent microsphere immunochromatography, a magnetic microsphere immunochromatography and a quantum dot immunochromatography; ELISA such as direct method, indirect method, sandwich method and competition method.

7. Use of the monoclonal anti-RSV antibody of claim 1 in the manufacture of a reagent for detecting Respiratory Syncytial Virus (RSV).

8. The use according to claim 6, wherein the detection is performed by immunochromatography, enzyme-labeled antibody method (ELISA), chemiluminescence, electrochemiluminescence; preferably, the immunochromatography comprises a fluorescent microsphere immunochromatography, a colloidal gold immunochromatography, an immunochromatography based on colored latex microspheres, a time-resolved fluorescent microsphere immunochromatography, a magnetic microsphere immunochromatography and a quantum dot immunochromatography; ELISA such as direct method, indirect method, sandwich method and competition method.

9. A kit for detecting Respiratory Syncytial Virus (RSV), the kit comprising the monoclonal anti-RSV antibody of claim 1 and instructions for directing how to detect Respiratory Syncytial Virus (RSV).