Anti-respiratory syncytial virus F protein antibody or antigen binding fragment thereof, biological product and application thereof

Abstract

The invention discloses an antibody for resisting respiratory syncytial virus F protein or an antigen binding fragment of the antibody, a biological product and application of the biological product, and relates to the technical field of immunodetection. The antibody comprises a light chain complementarity determining region in a light chain as shown in SEQ ID NO: 1 and a heavy chain complementarity determining region in a heavy chain as shown in SEQ ID NO: 2. The monoclonal antibody has the characteristics of high binding specificity and high affinity with the F protein of the respiratory syncytial virus, can be effectively used for quantitative and / or qualitative detection of the respiratory syncytial virus, can also be used for various detection methods such as ELISA (enzyme-linked immuno sorbent assay) detection, immunochemiluminescence detection and immunofluorescence detection, and is wide in application range and high in applicability. In addition, the respiratory syncytial virus detection product or diagnosis product provided by the invention has the technical advantages of lower detection limit and higher sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of immunoassay technology, and in particular to an antibody or an antigen-binding fragment thereof against respiratory syncytial virus F protein, a biological product and applications thereof. Background Art

[0002] Human respiratory syncytial virus (RSV) is one of the main causes of severe respiratory diseases in infants, young children and the elderly. According to statistics, the economic burden of RSV infection worldwide exceeds 80 billion US dollars each year. Since the discovery of RSV in the 1950s, domestic and foreign researchers have conducted a lot of experimental explorations on RSV vaccines. Compared with other respiratory viruses, natural infection with RSV cannot produce lasting immunity and is prone to repeated infection with the same subtype of RSV.

[0003] RSV virus belongs to the family Paramyxoviridae and is a member of the genus Pneumovirus. The RSV genome is a single-stranded negative-sense, non-segmented RNA with a total length of 15.2kb, containing 10 genes and encoding 11 proteins. The RSV virus contains an envelope formed by a phospholipid bilayer, with fusion protein (F protein), adsorption protein (G protein) and small hydrophobic protein (SH protein) embedded on the surface. G and F proteins are the two main glycoproteins on the surface of the virus, playing an important role in mediating the entry of the virus into cells. F protein induces a natural immune response by binding to pattern recognition receptors CD14 and TLR4, while G protein and nucleocapsid protein N have a weaker ability to induce a natural immune response in the body.

[0004] F protein belongs to type I transmembrane protein. F protein is first synthesized into 574 amino acid precursor protein F0. There are 5-6 post-translationally modified N-linked glycosylation modifications on F0 protein. The protein-mediated membrane fusion process is also the process of its structure changing from pre-fusion state to fusion state. When G protein binds to the receptor on the target cell membrane, the pre-fusion F protein begins to trigger conformational change to a stable post-fusion form. Due to its key role in RSV invasion and high conservation, the induced antibodies can inhibit the infection of RSV subtypes A and B at the same time.

[0005] RSV F protein is the target of neutralizing antibodies and the main antigen for vaccine development. A large number of studies have confirmed that the recognition site of neutralizing antibodies against F protein is mainly on the pre-fusion F protein, and the immunogenicity and protective effect of recombinant proteins designed based on pre-fusion F are significantly better than vaccines designed based on post-fusion F protein.

[0006] In summary, RSV antigen detection has the advantages of being portable, rapid, and capable of bedside testing. Antibody raw materials are the main biologically active components of immunological detection reagents, and the selection of target proteins directly affects the test results. F protein is an important protein for the fusion of the virus and host cell membranes. Based on its immunogenicity and high conservatism, it can ensure the sensitivity and specificity of the test results as a target for RSV virus detection. The development of monoclonal antibodies targeting the syncytial virus F protein is of great significance for the diagnosis of syncytial virus and the discovery of antibody drugs with strong efficacy against syncytial virus.

[0007] The lateral flow immunochromatography rapid diagnostic kit is a test paper-based diagnostic product suitable for instant testing. The lateral flow immunochromatography rapid diagnostic kit also has some shortcomings: first, the detection method can only provide qualitative results, which is not very applicable for pathogens (specific bacteria, mycoplasma, viruses, etc.) that require precise quantitative results to diagnose the infection; second, the detection method requires that the test sample must be in a liquid state and have a certain viscosity to flow through the porous nitrocellulose membrane.

[0008] Nevertheless, the lateral flow immunochromatography rapid diagnostic kit is still a detection technology with important application value, which can achieve the general standards required for rapid diagnosis, such as sensitivity, stability, economy, user-friendliness and no equipment required. These advantages of the lateral flow immunochromatography rapid diagnostic kit are of great significance, especially for units or regions that lack professional equipment and poorly trained technicians.

[0009] In view of this, the present invention is proposed. Summary of the invention

[0010] The object of the present invention is to provide an antibody or antigen-binding fragment thereof against respiratory syncytial virus F protein, a biological product and application thereof to solve the above technical problems.

[0011] Another object of the present invention is to provide.

[0012] The present invention is achieved in that:

[0013] In the first aspect, the present invention provides an antibody or an antigen-binding fragment thereof against respiratory syncytial virus F protein, which comprises a light chain complementary determining region in the light chain as shown in SEQ ID NO:1 and a heavy chain complementary determining region in the heavy chain as shown in SEQ ID NO:2.

[0014] In a second aspect, the present invention also provides a biological product, which includes the above-mentioned anti-respiratory syncytial virus F protein antibody or its antigen-binding fragment, and the biological product is selected from a reagent, a kit, a test strip, an antibody chip, an antibody probe, and a detector.

[0015] In a third aspect, the present invention also provides the use of antibodies or antigen-binding fragments thereof against respiratory syncytial virus F protein in the preparation of respiratory syncytial virus detection products, diagnostic products or respiratory syncytial virus enrichment products.

[0016] In a fourth aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned anti-respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof.

[0017] In a fifth aspect, the present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0018] In a sixth aspect, the present invention further provides a recombinant cell, comprising: the above-mentioned recombinant vector.

[0019] The present invention has the following beneficial effects:

[0020] The present invention provides an antibody against respiratory syncytial virus F protein, which has high binding specificity and high affinity to the respiratory syncytial virus F protein, and can be effectively used not only for quantitative and / or qualitative detection of respiratory syncytial virus (RSV), but also for various detection methods such as ELISA detection, immunochemiluminescence detection and immunofluorescence detection, and has a wide range of applications and strong applicability.

[0021] The respiratory syncytial virus detection product or diagnostic product provided by the present invention has the technical advantages of low detection limit and high sensitivity; and the selected antibodies specifically target antigenic epitopes with highly conserved amino acid sequences, are not affected by common antigenic protein mutations of viral mutant strains, can reduce detection costs, shorten detection time, and improve detection efficiency. In particular, when the detection product or diagnostic product is a lateral flow immunochromatography rapid diagnostic kit, it can achieve the general standards required for rapid diagnosis, such as sensitivity, stability, economy, user-friendliness, and no need for equipment. These advantages of the lateral flow immunochromatography rapid diagnostic kit are of great significance, especially for units or regions that lack professional equipment and well-trained technicians.

[0022] In addition, the antibodies against respiratory syncytial virus F protein provided by the present invention can be used to design therapeutic targets for respiratory syncytial virus (RSV), and therapeutic preparations for respiratory syncytial virus (RSV) can be designed. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 It is a schematic diagram of the detection principle of the lateral flow immunochromatographic test strip;

[0025] Figure 2 The protein electrophoresis result of the respiratory syncytial virus (RSV) recombinant protein (antigen) in the embodiment of the present invention;

[0026] Figure 3 The results of detecting inactivated syncytial virus culture (different dilutions) using a lateral flow immunochromatography rapid test reagent developed using antibodies in the embodiments of the present invention;

[0027] Figure 4 The results of using the respiratory syncytial virus (RSV) F protein solution standard substance in the embodiments of the present invention to detect the lateral flow immunochromatography rapid test reagent developed based on the antibody of the present invention (different dilutions);

[0028] Figure 5 The results of testing nasal swab samples of normal persons using the lateral flow immunochromatography rapid test reagent developed using the antibody of the present invention;

[0029] Figure 6 The graphs are the binding curves of the antibody of the present invention with the recombinant respiratory syncytial virus (RSV) F protein antigen at gradient concentrations; wherein 0.5 μg is the binding curve when each well is coated with 50 ng of antigen (100 μL per well), and 1 μg is the binding curve when each well is coated with 100 ng of antigen (100 μL per well). DETAILED DESCRIPTION

[0030] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0031] Definition

[0032] The term "antigen binding fragment" refers to all proteins / protein fragments containing CDR regions, especially antibodies or antibody functional fragments. "Antigen binding fragments" include antigen compound binding fragments of the above-mentioned antibodies, including Fab, F(ab')2, Fd, Fv, scFv, bispecific antibodies, multispecific antibodies and antibody minimum recognition units, as well as single-chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, etc. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, bifunctional and humanized antibodies, and related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0033] The term "antibody" herein is used in the broadest sense and may include full-length monoclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, so long as they exhibit the desired biological activity, such as specific binding to respiratory syncytial virus F protein or a fragment thereof.

[0034] In the present invention, the term "complementarity determining region or complementarity determining region", "CDR" refers to the highly variable region of the heavy and light chains of immunoglobulins, and refers to the region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of the antibody or antigen-binding fragment with its recognized antigen or epitope. In a specific embodiment of the present invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0035] In the present invention, the heavy chain complementary determining region is represented by HCDR, which includes HCDR1, HCDR2 and HCDR3; the light chain complementary determining region is represented by LCDR, which includes LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. for all protein sequences of the immunoglobulin superfamily in 1997. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. Over the past few decades, the accumulation of sequences has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered to be a widely used standard for numbering antibody residues. The present invention uses the Kabat annotation standard to mark the CDR region, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0036] Typically, the variable region VH of the heavy chain of an antibody can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0037] The variable region VL of the light chain of an antibody can be obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0038] In the first aspect, the present invention provides an antibody or an antigen-binding fragment thereof against respiratory syncytial virus F protein, which comprises a light chain complementary determining region in the light chain as shown in SEQ ID NO:1 and a heavy chain complementary determining region in the heavy chain as shown in SEQ ID NO:2.

[0039] SEQ ID NO:1

[0040] DIQMTQTTSSLSASLGDRVTISCSASWDISKYLNWYQQKPDGTVKLLIYYDSILVSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQHYSKLGR TFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYT CEATHKTSTSPIVKSFNRNEC.

[0041] SEQ ID NO:2

[0042] QIQLVQSGPELKKPGETVKISCKASGYTFTDIKIHWVKQAPGKGLKWMGWMIFETGEPPCADDFKGRFAFSLETSATTAYLQINILKNEDTAIYFCASERORGSDVFRTFPY WGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD CGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0043] By inputting the above SEQ ID NO: 1 and SEQ ID NO: 2 sequences into the CDR labeling system, the corresponding CDR sequences can be obtained.

[0044] The amino acid sequence of the complementary determining region is discovered and disclosed for the first time by the present invention, and is a new sequence that can give the antibody or its antigen-binding fragment the ability to specifically recognize and bind to the F protein of the respiratory syncytial virus. Therefore, the antibody or its antigen-binding fragment provided by the present invention can be used for the detection of respiratory syncytial virus, and the corresponding respiratory syncytial virus detection products, diagnostic products, and RSV treatment products are developed. The proposal of the present invention provides core materials for the detection method of RSV.

[0045] In a preferred embodiment of the present invention, the light chain complementary determining region includes CDR-L1, CDR-L2 and CDR-L3, and their amino acid sequences are shown in SEQ ID NOs: 10-12, respectively; the heavy chain complementary determining region includes CDR-H1, CDR-H2 and CDR-H3, and their amino acid sequences are shown in SEQ ID NOs: 13-15, respectively.

[0046] CDR-L1 SASWDISKYLN SEQ ID NO:10 CDR-L2 YDSILVS SEQ ID NO:11 CDR-L3 QHYSKLGRT SEQ ID NO:12 CDR-H1 DIKIH SEQ ID NO:13 CDR-H2 WMIFETGEPPCADDFKG SEQ ID NO:14 CDR-H3 ERORGSDVFRTFPY SEQ ID NO:15

[0047] In a preferred embodiment of the present invention, the antibody or its functional fragment further comprises a heavy chain framework region, and / or a light chain framework region;

[0048] In a preferred embodiment of the present invention, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 that are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 16-19, for example, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown in SEQ ID NOs: 16-19.

[0049] The heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 that are at least 80% homologous to the amino acid sequences shown in SEQ ID NOs: 20 to 23, in sequence. For example, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to the amino acid sequences shown in SEQ ID NOs: 20 to 23, in sequence.

[0050] LFR1 DIQMTQTTSSLSASLGDRVTISC SEQ ID NO:16 LFR2 WYQQKPDGTVKLLIY SEQ ID NO:17 LFR3 GVPSRFSGSGSGTDYSLTISNLEPEDIATYYC SEQ ID NO:18 LFR4 FGGGTKLEIKRADAAPTVSIF SEQ ID NO:19 HFR1 QIQLVQSGPELKKPGETVKISCKASGYTFT SEQ ID NO:20 HFR2 WVKQAPGKGLKWMG SEQ ID NO:21 HFR3 RFAFSLETSATTAYLQINILKNEDTAIYFCAS SEQ ID NO:22 HFR4 WGQGTLVTVSAAKTTPPSVYP SEQ ID NO:23

[0051] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof further comprises a constant region, and the constant region comprises a heavy chain constant region and / or a light chain constant region;

[0052] In a preferred embodiment of the present invention, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region.

[0053] In a preferred embodiment of the present invention, the heavy chain constant region is selected from IgG1, and the light chain constant region is selected from κ-type light chain constant region;

[0054] In a preferred embodiment of the present invention, the species of origin of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, rabbit, chicken or human.

[0055] In a preferred embodiment of the present invention, the species of the constant region is mouse or human;

[0056] In a preferred embodiment of the present invention, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of an antibody. The antigen-binding fragment of the above-mentioned antibody usually has the same binding specificity as the antibody from which it is derived. It is easy for a person skilled in the art to understand based on the contents described in the present invention that the functional fragment of the above-mentioned antibody can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or by chemical reduction to split disulfide bonds.

[0057] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0058] In a second aspect, the present invention also provides a biological product, which includes the above-mentioned anti-respiratory syncytial virus F protein antibody or its antigen-binding fragment, and the biological product is selected from a reagent, a kit, a test strip, an antibody chip, an antibody probe, and a detector.

[0059] In one embodiment, the above-mentioned reagent is a product of an antibody against respiratory syncytial virus F protein or an antigen-binding fragment thereof, and may also include functional components such as a protein stabilizer and a protective agent.

[0060] In a preferred embodiment of the present invention, the reagent includes: an antibody or an antigen-binding fragment thereof against respiratory syncytial virus F protein, and the antibody or the antigen-binding fragment thereof is labeled with a detectable marker. The form of the reagent includes but is not limited to solid, liquid, and semi-solid.

[0061] The antibody chip refers to a chip formed by fixing the above-mentioned anti-RSV F protein antibody or antigen-binding fragment thereof on a carrier.

[0062] Detectable markers refer to a class of substances that have properties that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0063] In a preferred embodiment of the present invention, the detectable marker is selected from fluorescent dyes, enzymes that catalyze the color development of substrates, radioactive isotopes, chemiluminescent agents and nanoparticle markers.

[0064] In actual use, those skilled in the art can select a suitable marker according to the detection conditions or actual needs. No matter which marker is used, it belongs to the protection scope of the present invention.

[0065] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5 .5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), prephycocyanin-chlorophyll protein (preCP), etc.).

[0066] In an alternative embodiment, the enzyme that catalyzes the color development of the substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxidase.

[0067] In an alternative embodiment, the radioactive isotopes include but are not limited to 212 Bi, 131 I. 111 In, 90 Y. 186 Re, 211 At 125 I. 188 Re, 153 Sm, 213 Bi, 32 P. 94 mTc, 99 mTc, 203 Pb, 67 Ga, 68 Ga, 43 Sc, 47 Sc, 110 mIn、 97 Such as 62 Cu, 64 Cu, 67 Cu, 68 Cu, 86 Y. 88 Y. 121 Sn, 161 Tb, 166 Ho, 105 Rh,177 Lu, 172 Lu and 18 F.

[0068] In an optional embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0069] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles and colloids; the nanoparticles include but are not limited to organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0070] In alternative embodiments, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

[0071] In alternative embodiments, colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0072] In a preferred embodiment of the present invention, the kit includes a solid phase, and the antibody or antigen-binding fragment thereof is coated on the solid phase; for example, the antibody or antigen-binding fragment thereof is connected to the solid phase by chemical coupling.

[0073] In a preferred embodiment of the present invention, the solid phase is selected from microspheres, plates and membranes.

[0074] In a preferred embodiment of the present invention, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0075] In a preferred embodiment of the present invention, the test strip includes a sample pad, a conjugate pad, an NC membrane, a water-absorbing pad and a bottom plate; a detection line and a quality control line are provided on the NC membrane, the detection line is coated with a detection antibody, the quality control line is coated with a polyclonal antibody, the conjugate pad is coated with a capture antibody labeled with a marker, and the epitopes of the detection antibody and the capture antibody are different; the detection antibody is the above-mentioned anti-respiratory syncytial virus F protein antibody or its antigen-binding fragment or another anti-respiratory syncytial virus F protein antibody or its antigen-binding fragment; the capture antibody is the above-mentioned anti-respiratory syncytial virus F protein antibody or its antigen-binding fragment or another anti-respiratory syncytial virus F protein antibody or its antigen-binding fragment.

[0076] In another embodiment, the test strip may also include a sample pad, a red blood cell filter pad or a red blood cell removal pad, a binding pad, an NC membrane and a water absorbent pad which are sequentially arranged on the bottom plate. The pore size of the red blood cell filter pad or the red blood cell removal pad is smaller than the diameter of the red blood cells, thereby filtering out the red blood cells in the blood sample and avoiding the interference of the red blood cells on the test results during the test.

[0077] The concentration of the capture antibody coated on the conjugate pad is 10-15 μg / mL, and the concentration of the detection antibody coated on the detection line is 1.5-2 mg / mL. For example, the concentration of the capture antibody coated on the conjugate pad is 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL or 15 μg / mL. The concentration of the detection antibody coated on the detection line is 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL and 2 mg / mL.

[0078] In a preferred embodiment of the present invention, the marker on the conjugate pad is a nanoparticle marker.

[0079] In a preferred embodiment of the present invention, the nanoparticle marker is selected from nanoparticles or colloids.

[0080] Colloidal gold is one of the earliest labeling materials used in immunochromatographic test kits. The absorption wavelength is in the visible light region, which is easy to observe directly with the naked eye, and the color changes to red, blue or purple as the size and shape of the colloidal gold particles change. The colloidal gold raw material is usually prepared by reducing chloroauric acid with trisodium citrate, which is simple and reliable. The colloidal gold surface prepared by this method carries a considerable amount of negative charge, making its properties very stable in a dry state and even in solution. In addition, the large amount of negatively charged surface aggregation enables colloidal gold to be mildly coupled to biological macromolecules such as nucleotides, polypeptides or proteins that are positively charged under specific pH conditions in a simple way of electrostatic adsorption, and this coupling can retain the activity of biological macromolecules to the maximum extent. The above advantages make colloidal gold the labeling material in most commercial immunochromatographic test strips on the market.

[0081] The lateral flow immunochromatography rapid diagnostic kit is a test paper-based method for reporting results suitable for immediate testing. Its core component is the lateral flow immunochromatography test strip ( Figure 1 ), generally in the form of narrow strips, usually 4-6 mm wide and no longer than 6-7 cm. A standard lateral flow immunochromatographic test strip should consist of four main parts: sample pad, conjugate pad, detection area and absorbent pad.

[0082] The main material of the sample pad area is made of cellulose. When in use, the sample pad area is fully immersed in the object to be tested;

[0083] The main material of the binding pad area is made of glass fiber, and the binding pad area generally contains colloidal gold (coupled) antigen or colloidal gold (coupled) antibody;

[0084] The main material of the detection area is made of nitrocellulose membrane (NC membrane), and there are mainly detection lines and quality control lines in the detection area, and the detection lines and quality control lines contain corresponding antibodies respectively;

[0085] The main material of the absorbent pad area is made of cellulose.

[0086] When the solution to be tested is loaded onto the sample pad area of ​​the lateral flow test paper, the solution will flow from the sample pad area into the binding pad area under the action of chromatography. When the solution contains the substance to be tested, the substance to be tested will bind to the colloidal gold (coupled) antigen or colloidal gold (coupled) antibody in the binding pad area. The colloidal gold (coupled) antibody or colloidal gold (coupled) antigen rehydrated by the solution or the complex formed with the sample to be tested will pass through the detection area (NC membrane) and the absorbent pad area in turn under the action of capillary force. At this time, the lateral flow test paper will have two result display models, namely the standard model ( Figure 1 ) and competition models.

[0087] The detection principle is as follows: When the lateral flow immunochromatographic test paper adopts the standard model (detecting the F protein antigen of syncytial virus), the detection line (containing the specific antibody targeting the F protein antigen of syncytial virus) can only capture the complex of the antigen to be tested and the colloidal gold (coupled) primary antibody: If the antigen to be tested exists in the solution, the detection line can capture the complex formed by the antigen to be tested and the colloidal gold (coupled) primary antibody. Since the colloidal gold (coupled) primary antibody itself will always be captured by the quality control line (containing the corresponding secondary antibody), at this time, both the detection line and the quality control line will change color, and the result is judged as a positive reaction. If the antigen to be tested does not exist in the solution, only the quality control line (containing the corresponding secondary antibody) can capture the colloidal gold (coupled) primary antibody and change color. At this time, the quality control line is colored but the detection line is not colored, and the result is judged as a negative reaction.

[0088] In a preferred embodiment of the present invention, the amino acid sequences of the light chain and heavy chain of another anti-respiratory syncytial virus F protein antibody or its antigen-binding fragment are shown in SEQ ID NOs: 3-4, respectively;

[0089] In a preferred embodiment of the present invention, the detection antibody is the above-mentioned anti-respiratory syncytial virus F protein antibody or its antigen-binding fragment, and the capture antibody is another anti-respiratory syncytial virus F protein antibody or its antigen-binding fragment.

[0090] The antibody on the quality control line is a polyclonal antibody against the species of the capture antibody. For example, if the capture antibody is a mouse antibody, the polyclonal antibody coated on the quality control line is an anti-mouse polyclonal antibody, such as a goat anti-mouse polyclonal antibody.

[0091] In a third aspect, the present invention also provides the use of antibodies or antigen-binding fragments thereof against respiratory syncytial virus F protein in the preparation of respiratory syncytial virus detection products, diagnostic products or respiratory syncytial virus enrichment products.

[0092] In a preferred embodiment of the present invention, the detection product or diagnostic product is a reagent, a test kit, a test strip, an antibody chip, an antibody probe or a detector;

[0093] For example, the respiratory syncytial virus enrichment product is: the antibody against the respiratory syncytial virus F protein is coupled with magnetic beads, and then the magnetic beads are reacted with the sample to enrich the respiratory syncytial virus, and then eluted. In other embodiments, the antibody against the respiratory syncytial virus F protein can also be fixed on an affinity column to achieve the separation and enrichment of the respiratory syncytial virus.

[0094] In a fourth aspect, the present invention also provides a nucleic acid molecule encoding the above-mentioned anti-respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof.

[0095] Taking into account the degeneracy of codons, the gene sequence encoding the above-mentioned antibody can be modified in its coding region without changing the amino acid sequence to obtain a gene encoding the same antibody amino acid sequence; the gene can also be artificially synthesized and modified according to the codon preference of the host expressing the antibody to improve the expression efficiency of the antibody.

[0096] For example, the nucleotide sequences encoding the antibody light chain and heavy chain are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0097] In a fifth aspect, the present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0098] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, which can refer to any recombinant polynucleotide construct that can directly or indirectly (such as packaging into a virus) introduce the target DNA fragment into the host cell through transformation, transfection or transduction to express the target gene.

[0099] One type of vector is a plasmid, which is a circular double-stranded DNA molecule that can connect the target DNA fragment to the plasmid ring. Another type of vector is a viral vector, which can connect and package the target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0100] A person skilled in the art can also transcribe the nucleic acid sequence of the present invention into RNA by in vitro transcription, and further transfect, transduce or transform the RNA into a host cell to express the antibody of the present invention or its functional fragment to exert the biological efficacy of the present invention.

[0101] In a sixth aspect, the present invention further provides a recombinant cell, comprising: the above-mentioned recombinant vector.

[0102] In a preferred embodiment of the present invention, the recombinant cell is a bacterium, a fungus or a 293 cell, a 293T cell, a 293FT cell, a CHO cell, a COS cell, or a Per6 cell. 293 series cells, Per6 cells and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins, and are well known to those skilled in the art.

[0103] In a preferred embodiment of the present invention, the bacteria is Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli or Bacillus subtilis.

[0104] In a preferred embodiment of the present invention, the fungus is Trichoderma reesei or yeast.

[0105] The host cells mentioned above include transformants and transformed cells, which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parental cells in nucleic acid content, but may contain mutations.

[0106] In a preferred embodiment of the present invention, the yeast is selected from at least one of the following genera: Dekkera, Brettanomyces, Hansenula, Kluyveromyces, Pichia, Candida, Kluyveromyces, Debaryomyces, Kazachstania, Wickerhamomyces, Lindnera, Zygotorulaspora, Zygosaccharomyces, Rhodosporidium and Schizosaccharomyces.

[0107] The above-mentioned recombinant cells are prepared by transforming the recombinant expression vector into a host cell (such as a microorganism) by conventional methods in the art; the host microorganism can be any conventional host microorganism in the art, as long as the recombinant expression vector can stably replicate itself and the foreign gene carried can be effectively expressed. The host microorganism is a bacterium or a fungus.

[0108] In a preferred embodiment of the present invention, the recombinant cell refers to at least one of a resting cell of a recombinant bacterium, a living cell of a recombinant bacterium, a dead cell of a recombinant bacterium, and a cell fragment of a recombinant bacterium.

[0109] Dormant cells, also known as resting cells, are a special cell state. In this state, cells do not grow and reproduce, but still contain various enzyme systems and have oxidation and fermentation capabilities. Under suitable conditions, dormant cells can resume growth. The characteristics of dormant cells include: a. Cells maintain growth potential: Although in a dormant state, these cells can re-enter the cell cycle and restore their proliferation ability when given appropriate stimulation. b. Strong specificity: Dormant cells have strong specificity in the reaction and can improve the substrate conversion rate. c. Not easy to be contaminated by foreign bacteria: Due to its characteristics, dormant cells can reduce the inhibition of bacterial growth and enzyme synthesis by products during use.

[0110] Dead bacteria of recombinant bacteria include but are not limited to bacteria obtained by inactivation by heat, pressure, radiation, etc.

[0111] Cell disruption products refer to: including but not limited to, cell disruption products obtained by changing the permeability of cell membranes through ultrasonic, mechanical, chemical, biological and other means, resulting in leakage of cell contents.

[0112] In a preferred embodiment of the present invention, the dead bacteria are selected from at least one of a precipitate of dead bacteria and a cell-free supernatant of dead bacteria. The cell-free supernatant of dead bacteria refers to the "exudate contents" remaining after removing the outer shell of the dead bacteria.

[0113] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0114] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0115] Example 1

[0116] This example provides a method for preparing hybridomas producing monoclonal antibodies.

[0117] 1. Design of respiratory syncytial virus (RSV) F protein antigen

[0118] According to the amino acid sequence obtained by translating the respiratory syncytial virus (RSV) F protein gene (GenBank: M11486.1) (SEQ ID NO: 5; the optimized gene sequence is shown in SEQ ID NO: 6) on GenBank, the amino acid sequence of the F protein of the respiratory syncytial virus (RSV) (A2) was selected for sequence comparison, and it was found that the sequence had a high homology with other respiratory syncytial virus (RSV) F proteins. After immunogenicity, hydrophilicity and surface accessibility analysis, the full-length 574 amino acids of the CDS region of the F protein of the respiratory syncytial virus (RSV) (A2) were finally selected as the subsequent recombinant protein, i.e., the sequence of the antigen (SEQ ID NO: 7).

[0119] The relevant information on the antigenic epitopes and / or functional localization of the respiratory syncytial virus (RSV) F protein (IEDB analysis software) is shown in Table 1.

[0120] Table 1 Relevant information on antigenic epitopes and / or functional localization of respiratory syncytial virus (RSV) F protein

[0121]

[0122]

[0123] 2. Preparation of recombinant respiratory syncytial virus (RSV) F protein

[0124] After gene synthesis and molecular cloning design, the recombinant respiratory syncytial virus F protein was finally expressed in the E. coli system, and the protein was the antigen. The corresponding expressed protein recombinant protein was identified by SDS-PAGE ( Figure 2 ).

[0125] 3. Immunization of mice

[0126] 1mg / mL recombinant respiratory syncytial virus (RSV) F protein was mixed with Freund's complete adjuvant at a ratio of 1:1 to 500μL for emulsification, and then subcutaneously injected into 6-8 week old female Balb / c mice at multiple points, with each mouse inoculated with 100μg of recombinant respiratory syncytial virus (RSV) F protein (antigen). Three weeks later, 1mg / mL antigen was mixed with Freund's incomplete adjuvant at a ratio of 1:1 to 500μL for emulsification, and then subcutaneously injected at multiple points, with each mouse receiving an antigen vaccination dose of 50μg, as a booster immunization. After intervals of three weeks, six weeks, and nine weeks, immunization was repeated according to the aforementioned booster immunization process, for a total of four booster immunizations.

[0127] 4. Immune serum titer determination

[0128] 10 days after the fourth booster immunization, blood was collected from the tail vein of mice, and the titer of immune serum was determined by indirect ELISA. 50 μg of synthetic antigen recombinant respiratory syncytial virus (RSV) F protein was dissolved in 10 mL 0.05 M pH 9.6 phosphate buffer, coated on a polystyrene 96-well plate, 100 μL / well, and incubated at 4°C overnight. The plate was washed three times with PBST (0.02 M PBS containing 0.05% v / v Tween-20), blocked with 100 μl / well of 10 mM PBS containing 1% BSA blocking solution at 37°C for 2 h, washed three times with PBST (0.02 M PBS containing 0.05% v / v Tween-20), and prepared for use. Mouse immune serum was incubated with 10 mM PBS containing 1% BSA for 10 2 ~10 6 The plate was diluted 1:10000 and added to a 96-well plate, 100 μL / well, and then incubated at 37°C for 1 hour. After that, the plate was washed three times with PBST (0.02MPBS containing 0.05% v / v Tween-20), and then 100 μL / well of 1:10000 diluted horseradish peroxidase labeled goat anti-mouse IgG (Sigma, INC.) was added, and then incubated at 37°C for 30 minutes, and the plate was washed as above. Then, 100 μL / well of TMB was added to each well for color development, and the reaction was stopped at 37°C at room temperature and protected from light for 10 minutes, and then 2M H2SO4 was added in an amount of 50 μL / well to terminate the reaction. The absorbance at 450 nm was measured, and the mouse serum before immunization was used as a negative control. The ratio of the measured value to the control value ≥ 2.1 was used as a positive judgment value to determine the immune serum titer (Table 2).

[0129] Table 2 Absorption values ​​at 450nm of mouse immune serum after immunization of mice at different dilution multiples

[0130]

[0131] 5. Preparation of hybridomas

[0132] Take serum with a titer greater than 1:10 5For mice, 3 days before fusion, take the synthesized antigen respiratory syncytial virus (RSV) F protein and mix it with an equal volume of PBS, and then inject it intraperitoneally into BALB / c mice to be fused at a volume of 50μg / 500μL per mouse for booster immunization. Feeder layer cell preparation process: Balb / c mice (about 4 weeks old) were bled out of the orbital vein and soaked in 75% ethanol for 3 minutes, with the ventral side facing up; the chest cavity was opened to separate the thymus, grinded with a cell sieve, and the cells were suspended in preheated basal medium. Preparation of peritoneal macrophages: On the day of fusion, select one healthy mouse (one spleen), remove its eyeball and bleed until the blood stops dripping. Kill by cervical dislocation, soak and disinfect in 75% alcohol for 5 minutes, move to the clean bench, and fix on the dissection board with the abdomen facing up. Lift the mouse abdominal skin with tweezers, cut a small incision with scissors (be careful not to damage the peritoneum to prevent the outflow of peritoneal fluid), fully expose the peritoneum by blunt dissection, and wipe with alcohol for disinfection. Use a disposable sterile syringe to draw 5-10mL of basal culture medium and inject it into the mouse peritoneal cavity. Hold the syringe in the right hand and keep it still. Use tweezers in the left hand to pick up an alcohol cotton ball and gently rub the mouse abdomen for 1-2 minutes to encourage macrophages to migrate out. Use a syringe to draw out the culture medium in the peritoneal cavity and transfer it to a 15mL centrifuge tube. After mixing the peritoneal macrophages and thymocytes, centrifuge at 1200r / min for 10 minutes and discard the supernatant. Resuspend in preheated 1×HAT medium containing 20% ​​FBS (hypoxanthine (H), aminopterin (A) and thymidine (T) (HAT, Sigma)) and store at 37°C for use.

[0133] On the morning of the day of cell fusion, the preparation process of spleen cells is as follows: take mice that have been boosted for 3 to 4 days, remove the eyeballs to collect blood, and collect the separated serum as the positive control serum for anti-detection. At the same time, kill the mice by cervical dislocation, soak them in 75% alcohol for 5 minutes for disinfection, and immediately put them in the clean bench. Fix the mice on the dissection table, open the abdomen aseptically, and then lift the skin on the right side of the abdomen to see the spleen. Change the ophthalmic scissors, cut the peritoneum with sterile surgical scissors, remove the spleen with forceps, rinse the spleen with saline, use scissors to cut the spleen, place it in a disposable cell sieve, gently squeeze the spleen with the inner core of the syringe, and repeatedly rinse the cell sieve with saline until only connective tissue is left in the cell sieve, and then use the disposable cell sieve to filter the cell suspension again. Harvest the spleen cell suspension, centrifuge at 1200r / min for 10 minutes, and wash once with 30-40mL of resuspension centrifugation (try to remove red blood cell clusters). The spleen cells were resuspended and added to the basic culture medium containing 10% FBS, and then placed in a T75 cell flask and cultured in a 37° C., 5% CO 2 incubator for 2-3 hours to allow the macrophages in the cell suspension to adhere to the wall.

[0134] In the afternoon of the day of cell fusion, the preparation process of myeloma cells is as follows: discard the supernatant of 3 bottles of T75 myeloma cells, blow down with 50mL preheated saline, centrifuge together with spleen cells, and centrifuge at 1200r / min for 10 minutes. Mix the spleen cells and 3 bottles of T75 myeloma cells thoroughly, discard the supernatant, resuspend with 40ml preheated saline, and centrifuge at 1200r / min for 10 minutes.

[0135] Cell fusion process: discard the supernatant, discard the supernatant as much as possible, use a dropper to suck out the remaining liquid to avoid affecting the concentration of the cell fusion agent, and try to remove red blood cells. Use your fingers to gently tap the bottom of the centrifuge tube to mix and make the precipitated cells loose and even into a paste. Fusion at room temperature: The cell fusion agent and the culture medium containing the feeding layer need to be placed in an incubator for warmth when preparing spleen cells. Use a Pasteur pipette to absorb the 1mL cell fusion agent solution (try to add it to the centrifuge tube wall as close to the cells as possible). Gently mix within 60s-90s. After the timing is completed, add 30mL of preheated 37℃ basal culture medium at one time to dilute the cell fusion agent and lose its fusion-promoting effect, and let it stand at 37℃ for 5min. Centrifuge at 800r / min for 6 minutes and discard the supernatant. Add preheated 20% FBS1×HAT culture medium and gently blow and suck the precipitated cells to suspend and mix them (mix thoroughly to reduce cell clusters), and move gently. In this experiment, 10 96-well cell culture plates were prepared, with 200 μL per well, requiring 200 mL of 20% FBS 1×HAT medium.

[0136] The cells were cultured in an incubator at 37°C and 5% CO2. The cells were observed under a microscope 7 days after cell fusion. The culture wells in the 96-well cell culture plate with obvious cell clones were counted and the fusion rate was calculated. Fusion rate = (number of fused cells / total number of cells) × 100%.

[0137] 6. Screening of hybridoma cells secreting monoclonal antibodies against respiratory syncytial virus (RSV) F protein

[0138] The cell culture supernatant was screened by indirect ELISA, and the positive cloned hybridoma cells with higher titers were selected for subcloning, and the limiting dilution method was used for continuous cloning 2-3 times until the cell positive rate reached 100%. The culture supernatants of the 10 hybridoma cell lines with higher titers were tested by indirect ELISA, and 0.01M PBS was used for dilution testing. The results are shown in Table 3:

[0139] Table 3 Absorbance values ​​of culture supernatants of hybridoma cell lines with different clone numbers at different dilution multiples

[0140]

[0141] As can be seen from Table 3, by comparing the detection data of the ELISA method performed on the hybridoma cell line culture supernatant, a cell line that stably secretes anti-RSV F protein monoclonal antibodies and has a high antibody titer can be further selected, and the OD value of the cell supernatant after 1:100 dilution is greater than 0.5, marked as 9F5. The cells with a positive rate of 100% after cloning were expanded and cultured and then frozen in liquid nitrogen.

[0142] 7. Preparation and purification of ascites

[0143] Hybridoma cell line 9F5 was cultured at 1×10 6 / mouse was injected into the abdominal cavity of 8-10 week old BALB / c female mice pretreated with liquid paraffin, and the abdominal fluid was extracted when the abdomen of the mice was swollen after 10-14 days of feeding and observation. Monoclonal antibodies were purified by affinity chromatography Protein A Sepharose, and the purity of monoclonal antibodies was determined by SDS-PAGE, and the purity reached more than 90%.

[0144] Example 2

[0145] This example is used to characterize the monoclonal antibody.

[0146] 1. Determination of antibody concentration: The monoclonal antibody against respiratory syncytial virus (RSV) F protein was obtained by purifying the ascites prepared by hybridoma cell 9F5. The concentration was determined by Nanodrop nucleic acid protein analyzer produced by Thermofisher, and the concentration was >1 mg / ml.

[0147] 2. Antibody subtype identification: The Thermofisher mouse monoclonal antibody subtype identification kit was used to identify the subtype of the hybridoma cell line. The subtype of the antibody secreted by 9F5 was IgG1, and the light chain was κ chain.

[0148] 3. Identification of the potency of purified antibodies: 10 μg of synthetic respiratory syncytial virus (RSV) F protein was dissolved in 10 mL of 0.05 M carbonate coating buffer at pH 9.6, added to a 96-well plate, 100 μL per well, and incubated at 4°C overnight. The plate was washed three times with PBS (containing 0.05% v / v Tween-20), blocked with 10 mM PBS containing 1% BSA blocking solution 150 μL / well at 37°C for 2 h, washed three times with PBS (containing 0.05% v / v Tween-20), 100 μL of purified antibody S1 was added to each well, diluted from 1 ug / ml 1:3 to S7, incubated at 37°C for 1 h, washed three times with PBS (containing 0.05% v / v Tween-20), horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody was added as secondary antibody, incubated at 37°C for 30 min, washed three times with PBS (containing 0.05% v / v Tween-20), 100 μL was added to each well, TMB was used for color development, incubated at 37°C for 15 min, and 2M The reaction was terminated with H2SO4 solution and the absorbance was detected by ELISA at 450nm. For 1mg / mL antibody, the titer of 9F5 antibody in positive wells (P / N>2.0) could reach 81000.

[0149] Table 4 9F5 antibody titer absorbance value

[0150]

[0151] 4. Antibody binding test:

[0152] The respiratory syncytial virus (RSV) F protein was diluted to 0.5 μg / mL and 1 μg / mL with 1×CB, and added to the wells of the ELISA plate at 100 μL / well, and duplicate wells were made, and placed at 4°C overnight or 37°C for adsorption for 2 hours. The coated microplate was dried, washed once according to the operating procedure (AFP procedure) set by the plate washer, and the blocking solution was added at 200 μL / well, placed in a 37°C incubator for 2 hours, and then placed at 4°C overnight. Before use, the blocked microplate was taken out from 4°C, dried, and the cleaning solution (1×PBS-T) was added to wet the ELISA plate; the monoclonal antibody of the present invention was pre-diluted to 30 μg / mL with 1×PBS, and the pre-dilution multiple m was recorded, and the highest concentration (S1) was 3 μg / mL after diluting 10 times, and then diluted in a 1:3 gradient (diluted in a 96-deep well plate), with a total of 8 dilution gradients (S1-S8).

[0153] Add 100 μL of diluted antibody to a 96-well microplate that has been patted clean on absorbent paper and incubate at 37°C for 30 min. After incubation, spin dry the plate and pat dry on absorbent paper. Wash the plate three times with a plate washer. Add 100 μL of 1× PBS to each well in columns 1-4. Add 200 μL of urea treatment solution to each well in columns 5 and 6 and incubate at 37°C for 30 min. After incubation, spin dry the plate and pat dry on absorbent paper. Dry, wash the ELISA plate 3 times with a plate washer, add 100 μL of GAM-HRP enzyme-labeled secondary antibody diluted 10,000 times with secondary antibody diluent to each well, and incubate at 37°C for 30 minutes; after incubation, spin dry the ELISA plate, pat dry on absorbent paper, wash the ELISA plate 3 times with a plate washer, take TMB color development solution, add 100 μL color development solution to each well, and incubate at 37°C for 10 minutes; after color development, add 50 μL of stop solution to each well. Set the ELISA reader to read at 450nm / 630nm.

[0154] The ELISA antigen-antibody binding experiment was tested under the conditions of 0.5μg / mL and 1μg / mL antigen coating, and the data were obtained by testing the absorbance value OD ( Figure 6 ), and the corresponding polynomial curve was fitted. The corresponding binding showed obvious gradient with the change of antibody dilution ratio. At the same time, the affinity binding reflected by the two curves under the conditions of 0.5μg / mL and 1μg / mL antigen coating reflected the specificity of binding and reflected that the concentration corresponding to the antibody binding force reached 6.85E-10mol / L (under the condition of 0.5μg / mL antigen coating, the highest OD reading was 50%, that is, the concentration of antigen-antibody complex accounted for half of the total concentration, and the antibody concentration value K 0.5 ) and 6.0E-10mol / L (under 1μg / mL antigen coating conditions, 50% of the highest OD reading, that is, when the concentration of the antigen-antibody complex accounts for half of the total concentration, the antibody concentration value K1). .40562E-10

[0155] Binding strength calculation: Calculate the average OD readings of the duplicate wells of S1-S8 antibody concentrations before and after urea treatment under 0.5μg / mL and 1μg / mL antigen coating conditions. Substitute the readings into the binding strength calculation formula:

[0156]

[0157] 5. Specificity detection:

[0158] Biotinylated Antibodies

[0159] The antibody was dialyzed with 20mM PB (pH8.5) solution, placed at 4°C and dialyzed for three times, biotin was dissolved to 2mg / ml with DMF (dimethylformamide), and the antibody:biotin molar ratio was 1:20. The volume of biotin was measured and added to the dialyzed antibody, vortexed and mixed, and allowed to react for 2 hours in the dark. The ligation product was dialyzed with 0.01M PBS solution, placed at 4°C and dialyzed for three times.

[0160] Double antibody sandwich ELISA screening

[0161] Use 1×CB to dilute the coated RSV antibody to 1μg / mL, add 100μL / well to the ELISA plate wells, and place at 4℃ overnight or 37℃ for adsorption for 2 hours. Shake dry the coated microplate, wash it once according to the operating program set by the plate washer (AFP program), add blocking solution at 200μL / well, place it in a 37℃ incubator for 2h, and then place it at 4℃ overnight.

[0162] Before use, take out the sealed microplate from 4°C and spin dry, and dilute the respiratory syncytial virus (RSV) F protein to 0.1μg / mL, 0.2μg / mL, 0.5μg / mL, and 1μg / mL respectively with the secondary antibody diluent. Incubate at 37℃ for 30min, wash the plate with a plate washer 3 times after incubation, pat dry on absorbent paper, add 100μL of RSV biotin-labeled antibody diluted 0.2ug / ml with secondary antibody diluent to each well, incubate at 37℃ for 30min, wash the plate with a plate washer 3 times after incubation, spin dry the plate, pat dry on absorbent paper, add 100μL of SA-HRP (1mg / ml) diluted 30000 times with secondary antibody diluent to each well, incubate at 37℃ for 30min, wash the plate with a plate washer 3 times after incubation, spin dry the plate, pat dry on absorbent paper, take TMB colorimetric solution, add 100μL colorimetric solution to each well, incubate at 37℃ for 10min; add 50μL stop solution to each well after color development. Set the ELISA reader to read at 450nm.

[0163] The orthogonal design of the F protein antibody of syncytial virus was used for coating and labeling, indicating that the antibody of the present invention has good specificity (Table 5A to Table 5J).

[0164] Table 5A

[0165]

[0166] Table 5B

[0167]

[0168]

[0169] Table 5C

[0170]

[0171] Table 5D

[0172]

[0173] Table 5E

[0174]

[0175]

[0176] Table 5F

[0177]

[0178] Table 5G

[0179]

[0180] Table 5H

[0181]

[0182]

[0183] Table 5I

[0184]

[0185] Table 5J

[0186]

[0187] As can be seen from Table 5A to Table 5J, by comparing the experimental data corresponding to the antibody pairing experiment, it is shown that except for the detection hole corresponding to the enzyme-labeled antibody that is the same as the coated antibody, which will be shielded, other enzyme-labeled antibodies can still bind to the respiratory syncytial virus (RSV) F protein antigen in the presence of the coated antibody, and the binding effect increases with the increase in the concentration of the added antigen, thus reflecting the specificity of 9F5 as a coated antibody. And when it is paired with 5A5 as a labeled antibody to form an immune sandwich reaction, it will show the best sensitivity to the respiratory syncytial virus (RSV) antigen.

[0188] 6. Monoclonal antibody sequencing

[0189] Take about 44 mg (batch number V20220526; 3.22 mg / mL) of the monoclonal antibody of respiratory syncytial virus (RSV) F protein prepared by the purified hybridoma cell 9F5. The sequencing of the light chain variable region and the heavy chain variable region shows that the amino acid sequences of the light chain and heavy chain of the antibody are shown in SEQ ID NO: 1 and 2, respectively. The light chain variable region sequences of the above-mentioned F protein monoclonal antibody are CDR-L1 (SASWDISKYLN), CDR-L2 (YDSILVS) and CDR-L3 (QHYSKLGRT), and the heavy chain variable region sequences are CDR-H1 (DIKIH), CDR-H2 (WMIFETGEPPCADDFKG) and CDR-H3 (ERORGSDVFRTFPY). Antibodies with corresponding variable region sequences have high affinity and good specificity; therefore, the corresponding antibody variable regions can be used for the development of recombinant antibodies, single-chain antibodies, bispecific antibodies, and for the development of related products for diagnostic or therapeutic purposes.

[0190] Example 3

[0191] In this example, a lateral flow immunochromatographic detection reagent was developed using a monoclonal antibody against the syncytial virus F protein.

[0192] The antibody of the present invention can be used for the development of immunodiagnostic reagents such as enzyme-linked immunosorbent assay, chemiluminescence, lateral flow immunochromatography and immunofluorescence detection.

[0193] 1. Preparation of colloidal gold:

[0194] The gold was prepared by reduction method, and the gold preparation conditions were 5 mL of 2% chloroauric acid + 10 mL of 1% trisodium citrate.

[0195] 2. Colloidal gold labeled mouse anti-respiratory syncytial virus (RSV) F protein monoclonal antibody

[0196] Physical adsorption method, by adjusting the pH value, colloidal gold and antibody (antibody 5A5 targeting respiratory syncytial virus (RSV) F protein, the amino acid sequences of the light chain and heavy chain are shown in SEQ ID NO: 3 and 4 respectively).

[0197] The specific labeling conditions are as follows: at pH 7.5, 10 μL of 0.1 M potassium carbonate solution is added to each milliliter of colloidal gold, and the labeling concentration of mouse anti-respiratory syncytial virus (RSV) F protein monoclonal antibody is 10 μg / mL.

[0198] 3. Testing line and quality control line

[0199] Test line: Take appropriate concentration (1.5 mg / mL) of mouse anti-RSV F protein monoclonal antibody 9F5 and coat it on nitrocellulose membrane to prepare the test line. Dry at 37°C. Spray volume is 0.1 μL / mm.

[0200] Quality control line: 0.5 mg / mL goat anti-mouse IgG polyclonal antibody was taken to prepare the quality control line on the fiber membrane and dried at 37°C. The spray volume was 0.1 μL / mm.

[0201] The colloidal gold-labeled mouse anti-respiratory syncytial virus (RSV) F protein monoclonal antibody in step 2 is sprayed onto a polyester film and dried to prepare a colloidal gold conjugate pad. The sample pad, the colloidal gold conjugate pad, the nitrocellulose membrane sprayed with a detection line and a quality control line, and the absorbent paper are then laid on the PVC base plate in the designed order.

[0202] 4. Inspection methods

[0203] Return the test card (test strip), sample diluent and sample to 18-30°C. The test method for the test card or test strip is as follows:

[0204] a. Take out the test card or test strip from the aluminum foil bag, mark the sample, and place it flat on a horizontal work surface;

[0205] b. Take 20 μL of nasopharyngeal or oropharyngeal swab sample extract and add it directly into the sample well (test card) or the sample point at the lower end of the arrow (test strip);

[0206] c. Add 100 μL (2 to 3 drops) of sample diluent;

[0207] d. Read the results within 15 to 20 minutes. Test results after 20 minutes will be invalid.

[0208] 5. Interpretation of test results

[0209] a. Positive test line: The test line and the quality control line are colored. This indicates that the sample has detected the F protein antigen of respiratory syncytial virus (RSV), which may be an early infection or an active infection, and the final confirmation needs to be combined with clinical symptoms.

[0210] b. Negative: Only one red quality control line appears in the test window, indicating that the F protein antigen of respiratory syncytial virus (RSV) is not detected in the sample.

[0211] c. Invalid: No red quality control line appears in the detection window.

[0212] Example 4

[0213] This example provides a performance test of the respiratory syncytial virus antigen lateral flow immunochromatography reagent provided in Example 3.

[0214] 1. Sensitivity test

[0215] a. Inactivated syncytial virus culture test

[0216] The lateral flow immunochromatographic rapid test reagent developed using the antibody of the present invention detects inactivated syncytial virus culture, and the minimum detection level can be as low as 10TCID 50 / mL of virus culture.

[0217] TCID 50 / mL refers to the amount of virus required to cause cytopathic effect or death (CPE) in half of the cells in the culture plate or test tube, which is used to characterize the titer of the virus. Note: The amount of virus here is not the specific concentration, but the multiple of the original sample dilution. For example, 1mL of culture medium, after dilution 1000 times, just causes 50% of the cells to be infected, then TCID 50 1000 / mL. It indicates the dilution factor required to infect 50% of cells with the virus contained in each mL of sample.

[0218] Therefore, TCID 50 The larger the value, the more virus copies in the inactivated supernatant, and the larger the dilution multiple is needed (to achieve the effect of infecting 50% of cells). Then, the corresponding inactivated supernatant is used to detect the test strips of the kit, and the corresponding gradient results are as follows: Figure 3 100TCID 50 / mL represents the sample with the lowest number of original virus copies. That is, the lowest test can be as low as 100TCID 50 / mL of virus culture.

[0219] b. Purified RSV F protein test

[0220] The respiratory syncytial virus (RSV) F protein solution standard substance produced by Hangzhou Huakui Jinpei Biotechnology Co., Ltd. was used to test the lateral flow immunochromatography rapid test reagent developed by the antibody of the present invention. Figure 4 As shown, standard recombinant F protein of respiratory syncytial virus (RSV) can be detected as low as 50 ng / mL.

[0221] 2. Specificity test (clinical sample test)

[0222] The lateral flow immunochromatography rapid test reagent developed using the antibody of the present invention was used to test nasal swab samples from 8 normal subjects. The test results are as follows: Figure 5 As shown. Figure 5 It can be seen that the test results are clear and the background is clean, indicating that the product has good specificity.

[0223] Example 5

[0224] In this example, a double antibody sandwich ELISA platform detection reagent was developed using a monoclonal antibody against the syncytial virus F protein.

[0225] 1. Biotinylated Antibodies

[0226] The 5A5 antibody was dialyzed with a 20mM PB (pH8.5) solution, and the dialyzed solution was changed three times at 4°C. Biotin was dissolved to 2mg / ml with DMF (dimethylformamide), and the antibody:biotin molar ratio was 1:20. The volume of biotin was measured and added to the dialyzed antibody, vortexed and mixed, and the reaction was allowed to stand in the dark for 2h. The ligation product was dialyzed with a 0.01M PBS solution, and the solution was changed three times at 4°C. After the dialysis was completed, the concentration of 5A5-Biotin was determined using the Nanodrop nucleic acid protein analyzer produced by Thermofisher, and an equal volume of glycerol was added and stored at -20°C.

[0227] 2. Double Antibody Sandwich ELISA for Detection of RSV F Protein

[0228] Dilute the coated 9F5 antibody to 1 μg / mL with 1×CB, add 100 μL / well to the ELISA plate, and place at 4°C overnight or 37°C for 2 hours. Shake the coated microplate dry, wash it once according to the operating program set by the plate washer (AFP program), add blocking solution at 200 μL / well, place it in a 37°C incubator for 2 hours, and then place it at 4°C overnight.

[0229] Before use, take out the sealed microplate from 4°C and spin dry, and dilute the respiratory syncytial virus (RSV) F protein to 1.37ng / ml, 4.12ng / ml, μg / mL, 12.35ng / ml, 37.04ng / ml, 0.11ug / ml, 0.33ug / ml, and 1μg / mL respectively with secondary antibody diluent. Incubate at 37℃ for 30min, wash the plate with a plate washer 3 times after incubation, pat dry on absorbent paper, add 100μL of RSV biotin-labeled antibody diluted 0.2ug / ml with secondary antibody diluent to each well, incubate at 37℃ for 30min, wash the plate with a plate washer 3 times after incubation, spin dry the plate, pat dry on absorbent paper, add 100μL of SA-HRP (1mg / ml) diluted 30000 times with secondary antibody diluent to each well, incubate at 37℃ for 30min, wash the plate with a plate washer 3 times after incubation, spin dry the plate, pat dry on absorbent paper, take TMB colorimetric solution, add 100μL colorimetric solution to each well, incubate at 37℃ for 10min; add 50μL stop solution to each well after color development. Set the ELISA reader to read at 450nm.

[0230] The double-antibody sandwich enzyme-free platform detection reagent developed based on the antibody of the present invention was used to detect the respiratory syncytial virus (RSV) F protein solution standard material produced by Hangzhou Huakui Jinpei Biotechnology Co., Ltd. As shown in Table 6, the standard recombinant F protein of respiratory syncytial virus (RSV) can be detected at a minimum of 4.12 ng / mL.

[0231] Table 6 Results of double-antibody sandwich ELISA platform detection of RSV F protein solution standard substances at different concentrations

[0232]

[0233]

[0234] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against respiratory syncytial virus F protein, characterized in that: It includes a light chain complementarity determining region in the light chain as shown in SEQ ID NO:1 and a heavy chain complementarity determining region in the heavy chain as shown in SEQ ID NO:

2.

2. The anti-respiratory syncytial virus F protein antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The light chain complementary determining region includes CDR-L1, CDR-L2 and CDR-L3, and their amino acid sequences are shown in SEQ ID NOs: 10-12, respectively; the heavy chain complementary determining region includes CDR-H1, CDR-H2 and CDR-H3, and their amino acid sequences are shown in SEQ ID NOs: 13-15, respectively.

3. The antibody or antigen-binding fragment thereof against respiratory syncytial virus F protein according to claim 2, characterized in that: The antibody or its functional fragment further comprises a heavy chain framework region, and / or a light chain framework region; Preferably, the light chain framework region comprises LFR1, LFR2, LFR3 and LFR4 which have at least 80% homology to the amino acid sequences shown in SEQ ID NOs: 16-19, in sequence; The heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4, which have at least 80% homology to the amino acid sequences shown in SEQ ID NOs: 20-23, in sequence; Preferably, the antibody or antigen-binding fragment thereof further comprises a constant region, and the constant region comprises a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ type or λ type light chain constant region; Preferably, the heavy chain constant region is selected from IgG1, and the light chain constant region is selected from κ-type light chain constant region; Preferably, the species of origin of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, rabbit, chicken or human; Preferably, the species origin of the constant region is mouse or human; Preferably, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

4. A biological product, characterized in that It comprises the antibody or antigen-binding fragment thereof against respiratory syncytial virus F protein according to any one of claims 1 to 3, and the biological product is selected from the group consisting of reagents, kits, test strips, antibody chips, antibody probes, and detectors.

5. The biological product according to claim 4, characterized in that The reagent comprises: the antibody or antigen-binding fragment thereof against respiratory syncytial virus F protein, and the antibody or antigen-binding fragment thereof is labeled with a detectable marker; Preferably, the detectable marker is selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent agents and nanoparticle markers.

6. The biological product according to claim 4, characterized in that The kit comprises a solid phase, and the antibody or antigen-binding fragment thereof is coated on the solid phase; Preferably, the solid phase is selected from microspheres, plates and membranes; Preferably, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microplates, glass, capillaries, nylon and nitrocellulose membranes; Preferably, the test strip comprises a sample pad, a conjugate pad, an NC membrane, a water-absorbing pad and a bottom plate; a detection line and a quality control line are provided on the NC membrane, the detection line is coated with a detection antibody, the quality control line is coated with a polyclonal antibody, the conjugate pad is coated with a capture antibody labeled with a marker, and the detection antibody and the capture antibody have different epitopes; the detection antibody is an antibody against respiratory syncytial virus F protein or an antigen-binding fragment thereof according to any one of claims 1 to 3, or another antibody against respiratory syncytial virus F protein or an antigen-binding fragment thereof; the capture antibody is an antibody against respiratory syncytial virus F protein or an antigen-binding fragment thereof according to any one of claims 1 to 3, or another antibody against respiratory syncytial virus F protein or an antigen-binding fragment thereof; Preferably, the marker on the conjugate pad is a nanoparticle marker; Preferably, the nanoparticle-based marker is selected from nanoparticles or colloids; Preferably, the amino acid sequences of the light chain and heavy chain of the other anti-respiratory syncytial virus F protein antibody or its antigen-binding fragment are shown in SEQ ID NO: 3-4, respectively; Preferably, the detection antibody is an antibody against respiratory syncytial virus F protein or an antigen-binding fragment thereof according to any one of claims 1 to 3, and the capture antibody is another antibody against respiratory syncytial virus F protein or an antigen-binding fragment thereof.

7. Use of an antibody or antigen-binding fragment thereof against respiratory syncytial virus F protein according to any one of claims 1 to 3 in the preparation of a respiratory syncytial virus detection product, a diagnostic product or a respiratory syncytial virus enrichment product; Preferably, the detection product or diagnostic product is a reagent, a kit, a test strip, an antibody chip, an antibody probe or a detector.

8. A nucleic acid molecule, characterized in that It encodes the antibody or antigen-binding fragment thereof against respiratory syncytial virus F protein according to any one of claims 1 to 3.

9. A recombinant vector, characterized in that: It comprises the nucleic acid molecule according to claim 8.

10. A recombinant cell, characterized in that It includes: The recombinant vector according to claim 9.

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