Antibody, biological product, nucleic acid molecule, vector and application thereof against RSV F protein

Abstract

The present invention discloses an antibody, biological product, nucleic acid molecule, vector, and application thereof against RSV F protein, and relates to the field of immunoassay technology. The antibody against human respiratory syncytial virus F protein provided by the present invention has high specificity and high affinity characteristics and can be used to develop corresponding biological products, such as reagents, kits, test strips, antibody chips, antibody probes, etc., in particular products for respiratory syncytial virus detection. The antibody provided by the present invention can be used in various detection methods such as ELISA detection, immunochemiluminescence detection, and immunofluorescence detection, and has the advantages of a wide range of applications and strong applicability.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassays, and in particular to an antibody, biological product, nucleic acid molecule, vector and application thereof against RSV F protein. Background Art

[0002] Human respiratory syncytial virus (RSV) is a leading cause of severe respiratory illness in infants, young children, and the elderly. According to statistics, RSV infection results in an annual global economic burden exceeding $80 billion. Since its discovery in the 1950s, researchers both domestically and internationally have conducted extensive experimental research into RSV vaccines. Compared to other respiratory viruses, natural RSV infection does not produce long-lasting immunity, making repeated infection with the same RSV subtype more likely.

[0003] RSV virus belongs to the family Paramyxoviridae and the genus Pneumovirus. The RSV genome is a single-stranded, negative-sense, non-segmented RNA with a total length of 15.2 kb, containing 10 genes and encoding 11 proteins. 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 and play an important role in mediating the entry of the virus into cells. F protein induces an innate immune response by binding to pattern recognition receptors CD14 and TLR4, while G protein and nucleocapsid protein N have a weaker ability to induce the body's innate immune response.

[0004] The F protein is a type I transmembrane protein. It is synthesized as a 574-amino acid precursor protein, F0. The F0 protein is modified by five to six post-translational N-linked glycosylation modifications. The protein-mediated membrane fusion process is also a structural change from a pre-fusion state to a fusion state. Upon binding of the G protein to a receptor on the target cell membrane, the pre-fusion F protein initiates conformational changes to a stable post-fusion form. Due to its critical role in RSV invasion and its high degree of conservation, the induced antibodies can inhibit infection with both RSV subtypes A and B.

[0005] RSV F protein is the target of neutralizing antibodies and the primary antigen for vaccine development. Numerous studies have confirmed that the primary recognition site for neutralizing antibodies against the F protein is located on the pre-fusion F protein. Recombinant vaccines designed based on the pre-fusion F protein exhibit significantly superior immunogenicity and protective efficacy compared to vaccines designed based on the post-fusion F protein.

[0006] In summary, RSV antigen testing has the advantages of being portable, rapid, and capable of bedside testing. Antibody raw materials are the main bioactive components of immunological detection reagents, and the choice of target protein directly affects the test results. F protein is an important protein for the fusion of the virus and the host cell membrane. Based on its immunogenicity and high conservation, 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 detection technology with important application value. 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 poorly trained technicians. However, the lateral flow immunochromatography rapid diagnostic kit also has some shortcomings: first, the detection method can only provide qualitative results, and is not very applicable for pathogens (specific bacteria, mycoplasmas, viruses, etc.) that require precise quantitative results to diagnose infections; 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] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of the present invention is to provide an antibody, biological product, nucleic acid molecule, vector and application thereof against RSV F protein to solve the above technical problems.

[0010] The present invention is achieved in that:

[0011] In the first aspect, the present invention provides an antibody or an antigen-binding fragment thereof against human 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.

[0012] In a second aspect, the present invention also provides a biological product, which includes the above-mentioned anti-human respiratory syncytial virus F protein antibody or its antigen-binding fragment, and the biological product is selected from reagents, kits, test strips, antibody chips, antibody probes, and detectors.

[0013] In a third aspect, the present invention also provides an application of an anti-human respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof in the preparation of respiratory syncytial virus detection products, diagnostic products, and RSV enrichment products.

[0014] In a fourth aspect, the present invention further provides a nucleic acid molecule encoding the above-mentioned anti-RSV F protein antibody or an antigen-binding fragment thereof.

[0015] In a fifth aspect, the present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0016] In a sixth aspect, the present invention further provides a recombinant cell comprising the above-mentioned recombinant vector.

[0017] The present invention has the following beneficial effects:

[0018] The antibodies against human respiratory syncytial virus F protein provided by the present invention have high specificity and high affinity and can be used to develop corresponding biological products, such as reagents, kits, test strips, antibody chips, antibody probes, etc., particularly products and diagnostic products for respiratory syncytial virus detection. The antibodies provided by the present invention can be used in various detection methods such as ELISA, immunochemiluminescence, and immunofluorescence, offering the advantages of wide application and strong applicability.

[0019] The respiratory syncytial virus detection 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 and are not affected by common antigenic protein mutations of viral mutant strains, which can reduce detection costs, shorten detection time, and improve detection efficiency. In particular, when the detection 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Schematic diagram of the detection principle of lateral flow immunochromatographic test strip;

[0022] Figure 2 1 is a graph showing the protein electrophoresis results of the respiratory syncytial virus (RSV) recombinant protein (antigen) in an embodiment of the present invention;

[0023] Figure 3This is a graph showing the results of detecting inactivated syncytial virus culture (different dilutions) using a lateral flow immunochromatographic rapid test reagent developed using the antibody in a preferred embodiment of the present invention;

[0024] Figure 4 This is a graph showing the results of testing (at different dilutions) a lateral flow immunochromatographic rapid test reagent developed based on the antibody of the present invention using a respiratory syncytial virus (RSV) F protein solution standard substance in a preferred embodiment of the present invention;

[0025] Figure 5 This is a graph showing the results of testing nasal swab samples from eight normal subjects using a lateral flow immunochromatographic rapid test reagent developed using the antibody of the present invention in a preferred embodiment of the present invention;

[0026] Figure 6 The figures are the binding curves of the antibody of the present invention with 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

[0027] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, 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 invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0028] Definition of noun

[0029] 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, as well as related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin".

[0030] 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.

[0031] In the present invention, the terms "complementarity determining region" or "CDR" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, and refer to the regions containing one or more, or all, of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to its recognized antigen or epitope. In specific embodiments of the present invention, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0032] 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 this field 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 CDR regions, but CDR regions marked by other methods also fall within the scope of protection of the present invention.

[0033] Typically, the variable region VH of the heavy chain of an antibody can be obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0034] The variable region VL of the antibody light chain can be obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0035] In the first aspect, the present invention provides an antibody or an antigen-binding fragment thereof against human 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.

[0036] SEQ ID NO: 1

[0037] DVQITQSPSYLAASPGETITINCRSSDFGSKYLAWYQEKPGKTNKLLIYSGSRAQSGIPSRFSGSGSGTDFTLTISSLEPEDFAIYYCQSHNEYLWTF GGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC.

[0038] SEQ ID NO:2

[0039] DVQLVESGGGLVQPGGSRKLSCAASGLTFSSIGMEWVRQAPEKGLEWVAYISHYFITIYYANVVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARSGIHNAADYWGQGT SVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP CPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERT ISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG.

[0040] 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.

[0041] The amino acid sequence of the complementary determining region described above is discovered and disclosed for the first time by the present invention. It is a novel sequence that can confer upon the antibody or its antigen-binding fragment the ability to specifically recognize and bind to the respiratory syncytial virus F protein. Therefore, the antibody or its antigen-binding fragment provided by the present invention can be used to detect respiratory syncytial virus and develop corresponding respiratory syncytial virus detection products, diagnostic products, and RSV treatment products. The present invention provides core materials for RSV detection methods.

[0042] 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.

[0043] CDR-L1 RSSDFGSKYLA SEQ ID NO: 10 CDR-L2 SGSRAQS SEQ ID NO:11 CDR-L3 QSHNEYLWT SEQ ID NO:12 CDR-H1 SIGME SEQ ID NO:13 CDR-H2 YISHYFITIYYANVVKG SEQ ID NO:14 CDR-H3 SGIHNAADY SEQ ID NO:15

[0044] 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.

[0045] In a preferred embodiment of the present invention, the light chain framework region includes LFR1, LFR2, LFR3 and LFR4 that are at least 85% 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 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.

[0046] The heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 that are at least 85% homologous to the amino acid sequences shown in SEQ ID NOs: 20-23, for example, the heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4 that are at least 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: 20-23, for example.

[0047]

[0048]

[0049] 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;

[0050] 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;

[0051] 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.

[0052] 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 or human;

[0053] In a preferred embodiment of the present invention, the species of origin of the constant region is mouse or human.

[0054] In preferred embodiments of the present invention, the antigen-binding fragment is selected from any one of antibody F(ab')2, Fab', Fab, Fv, and scFv. Antigen-binding fragments of such antibodies generally have the same binding specificity as the antibody from which they are derived. Those skilled in the art will readily appreciate, based on the disclosure herein, that functional fragments of such antibodies can be obtained by, for example, enzymatic digestion (including pepsin or papain) and / or chemical reduction to cleave disulfide bonds.

[0055] The antigen-binding fragments of the above antibodies can also be synthesized by recombinant genetic techniques known to those skilled in the art or by an automatic peptide synthesizer, such as those sold by Applied BioSystems.

[0056] In a second aspect, the present invention also provides a biological product, which includes the above-mentioned anti-human respiratory syncytial virus F protein antibody or its antigen-binding fragment, and the biological product is selected from reagents, kits, test strips, antibody chips, antibody probes, and detectors.

[0057] In one embodiment, the reagent is a preparation of an antibody or antigen-binding fragment thereof against respiratory syncytial virus F protein, and may also include functional components such as protein stabilizers and protective agents. Protein stabilizers are selected from sucrose, trehalose, BSA, glycerol, mannitol, Triton X-100, and Tween-20. Protective agents are selected from cryoprotectants, such as polyols and sugars. Polyols, for example, are selected from sorbitol, mannitol, or mixtures thereof.

[0058] In a preferred embodiment of the present invention, the reagent comprises an antibody or antigen-binding fragment thereof against human respiratory syncytial virus F protein, wherein the antibody or antigen-binding fragment is labeled with a detectable marker. The reagent may be in a solid, liquid, or semi-solid form.

[0059] The antibody chip refers to a chip formed by immobilizing the above-mentioned anti-RSV F protein antibody or antigen-binding fragment thereof on a carrier.

[0060] A detectable marker is a substance that has properties that can be observed directly by the naked eye or detected or detected by an instrument, such as luminescence, color development, radioactivity, etc., through which qualitative or quantitative detection of the corresponding target can be achieved.

[0061] In a preferred embodiment of the present invention, the detectable marker is selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents and nanoparticle markers.

[0062] 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 falls within the scope of protection of the present invention.

[0063] 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), peridinin-chlorophyll protein (preCP), etc.).

[0064] 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-phosphate glucose deoxidase.

[0065] In an alternative embodiment, 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.

[0066] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxalates and their derivatives.

[0067] In an optional embodiment, the nanoparticle markers include but are not limited to nanoparticles and colloids; nanoparticles include but are not limited to organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

[0068] 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.

[0069] In a preferred embodiment of the present invention, the solid phase is selected from microspheres, plates and membranes;

[0070] 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.

[0071] In a preferred embodiment of the present invention, the test strip includes a sample pad, a conjugate pad, an NC membrane, an absorbent pad, and a base 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, and the conjugate pad is coated with a capture antibody labeled with a marker, and the epitopes of the antigens recognized by the detection antibody and the capture antibody are different; the detection antibody is the above-mentioned anti-human respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof, or another anti-human respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof; the capture antibody is the above-mentioned anti-human respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof, or another anti-human respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof;

[0072] In a preferred embodiment of the present invention, the concentration of the capture antibody coated on the conjugate pad is 10-15 μg / mL; the concentration of the detection antibody coated on the test 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 test 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. At these coating concentrations, good detection sensitivity and specificity are achieved.

[0073] In a preferred embodiment of the present invention, the marker on the conjugate pad is a nanoparticle marker.

[0074] In an alternative embodiment, the colloid includes, but is not limited to, colloidal metals, including, but not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0075] Colloidal gold was one of the earliest labeling materials used in immunochromatographic test kits. Its absorption wavelength is in the visible light region, making it easily observable with the naked eye. Colloidal gold particles change color, such as red, blue, or purple, depending on their size and shape.

[0076] The lateral flow immunochromatographic rapid diagnostic kit is a test paper-based method for reporting results suitable for immediate testing. Its core component is the lateral flow immunochromatographic test strip ( Figure 1 ), which includes: a sample pad, a conjugate pad, a detection area and a water absorbent pad.

[0077] The main material of the sample pad is made of cellulose, and when in use, the sample pad is fully immersed in the object to be tested;

[0078] The main material of the binding pad area is made of glass fiber, and the binding pad area contains colloidal gold (coupled) antibodies;

[0079] 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 respectively contain corresponding antibodies;

[0080] The main material of the absorbent pad area is made of cellulose.

[0081] After the test solution is loaded onto the sample pad of a lateral flow test strip, the solution flows from the sample pad into the conjugate pad under the action of chromatography. If the solution contains a substance to be tested, it will bind to the colloidal gold (conjugated) antigen or colloidal gold (conjugated) antibody in the conjugate pad. The colloidal gold (conjugated) antibody or colloidal gold (conjugated) antigen, rehydrated by the solution, or the complex formed with the test sample, will pass through the detection zone (NC membrane) and the absorbent pad in sequence under the action of capillary forces.

[0082] Figure 1 The detection principle is as follows: When the lateral flow immunochromatographic test strip uses the standard model (detecting the F protein antigen of syncytial virus), the test line (containing a specific antibody targeting the F protein antigen of syncytial virus) can only capture the complex formed by the test antigen and the colloidal gold (conjugated) primary antibody. If the test antigen is present in the solution, the test line will capture the complex formed by the test antigen and the colloidal gold (conjugated) primary antibody. Since the colloidal gold (conjugated) primary antibody itself is always captured by the quality control line (containing the corresponding secondary antibody), both the test line and the quality control line will change color, and the result is determined to be a positive reaction. If the test antigen is not present in the solution, only the quality control line (containing the corresponding secondary antibody) will capture the colloidal gold (conjugated) primary antibody and change color. In this case, the quality control line will develop color while the test line will not, and the result is determined to be a negative reaction.

[0083] In a preferred embodiment of the present invention, the amino acid sequences of the light chain and heavy chain of another anti-human respiratory syncytial virus F protein antibody or its antigen-binding fragment are shown in SEQ ID NOs: 3-4, respectively.

[0084] The amino acid sequence of SEQ ID NO: 3 is as follows:

[0085] DIQMTQTTSSLSASLGDRVTISCSASWDISKYLNWYQQKPDGTVKLLIYYDSILVSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQHYSKLGR TFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYT CEATHKTSTSPIVKSFNRNEC.

[0086] The amino acid sequence of SEQ ID NO:4 is as follows:

[0087] QIQLVQSGPELKKPGETVKISCKASGYTFTDIKIHWVKQAPGKGLKWMGWMIFETGEPPCADDFKGRFAFSLETSATTAYLQINILKNEDTAIYF

[0088] CASERORGSDVFRTFPYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVT

[0089] VPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQP

[0090] REEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAE

[0091] NYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0092] In a preferred embodiment of the present invention, the detection antibody is the above-mentioned anti-RSV F protein antibody or its antigen-binding fragment, and the capture antibody is another anti-HRSV F protein antibody or its antigen-binding fragment.

[0093] 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 mouse, the polyclonal antibody coated on the quality control line should be an anti-mouse polyclonal antibody, such as a goat anti-mouse polyclonal antibody.

[0094] In a third aspect, the present invention also provides an anti-human respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof for use in preparing respiratory syncytial virus detection products, diagnostic products, and RSV enrichment products;

[0095] For example, a product for RSV enrichment involves coupling magnetic beads with antibodies against RSV F protein, which are then reacted with the sample to enrich RSV, followed by elution. In other embodiments, antibodies against RSV F protein can also be immobilized on an affinity column to achieve RSV isolation and enrichment.

[0096] 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.

[0097] In a fourth aspect, the present invention further provides an isolated nucleic acid molecule encoding the above-mentioned anti-RSV F protein antibody or antigen-binding fragment thereof.

[0098] 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.

[0099] 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.

[0100] In a fifth aspect, the present invention also provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0101] A recombinant vector is an expression vector or a cloning vector, preferably an expression vector, and can refer to any recombinant polynucleotide construct that can introduce the target DNA fragment directly or indirectly (such as packaging into a virus) into the host cell by transformation, transfection or transduction to express the target gene.

[0102] One type of vector is a plasmid, a circular double-stranded DNA molecule that can be used to connect a target DNA fragment to the plasmid loop. Another type of vector is a viral vector, which can be used to connect and package a target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, or oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0103] Those skilled in the art can also use the nucleic acid sequence of the present invention as a template to transcribe 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.

[0104] In a sixth aspect, the present invention further provides a recombinant cell, comprising: the above-mentioned recombinant vector.

[0105] In preferred embodiments of the present invention, the recombinant cells are bacteria, fungi, or 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, or Per6 cells. 293 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.

[0106] In a preferred embodiment of the present invention, the bacteria is Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli or Bacillus subtilis.

[0107] In a preferred embodiment of the present invention, the fungus is Trichoderma reesei or yeast.

[0108] The host cells include transformants and transformed cells, which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations.

[0109] 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.

[0110] The recombinant cells are prepared by transforming a recombinant expression vector into a host cell (e.g., a microorganism) using conventional methods in the art. The host microorganism can be any of a variety of conventional host microorganisms in the art, as long as the recombinant expression vector can stably replicate and the exogenous gene carried by it can be effectively expressed. The host microorganism is a bacterium or a fungus.

[0111] In a preferred embodiment of the present invention, the recombinant cell refers to at least one of resting cells of recombinant bacteria, living cells of recombinant bacteria, dead cells of recombinant bacteria, and cell fragments of recombinant bacteria.

[0112] Resting cells, also known as quiescent cells, are a special cell state. In this state, cells do not grow or reproduce, but still contain various enzymes and possess oxidation and fermentation capabilities. Under appropriate conditions, resting cells can resume growth. Characteristics of resting cells include: a. Cells maintain growth potential: Despite being dormant, these cells can re-enter the cell cycle and resume proliferation when given appropriate stimulation. b. High specificity: Resting cells are highly specific in their reactions, which can improve substrate conversion rates. c. Resistant to contamination by foreign bacteria: Due to their characteristics, resting cells can reduce the inhibition of bacterial growth and enzyme synthesis by products during use.

[0113] Dead bacteria of recombinant bacteria include but are not limited to bacteria obtained by inactivation by heat, pressure, radiation, etc.

[0114] Cell disruptors refer to products obtained by changing the permeability of cell membranes through, but not limited to, ultrasonic, mechanical, chemical, biological, or other methods, resulting in leakage of cell contents.

[0115] In a preferred embodiment of the present invention, the dead bacteria are selected from at least one of a sediment 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.

[0116] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0117] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0118] Example 1

[0119] This example provides a method for preparing hybridomas that produce monoclonal antibodies.

[0120] 1. Design of respiratory syncytial virus (RSV) F protein antigen

[0121] The amino acid sequence obtained by translating the respiratory syncytial virus (RSV) F protein gene (GenBank: M11486.1) on GenBank (SEQ ID NO: 5; the optimized gene sequence is shown in SEQ ID NO: 6) was analyzed, and the amino acid sequence of the F protein of respiratory syncytial virus (RSV) (A2) was selected for sequence alignment. It was found that this sequence had 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 respiratory syncytial virus (RSV) (A2) were finally selected as the sequence of the subsequent recombinant protein, i.e., the antigen (SEQ ID NO: 7).

[0122] Relevant information on the antigenic epitopes and functional localization of respiratory syncytial virus (RSV) F protein (IEDB analysis software) is shown in Table 1:

[0123] Table 1 Relevant information on the antigenic epitopes and functional localization of respiratory syncytial virus (RSV) F protein

[0124]

[0125]

[0126] 2. Preparation of recombinant respiratory syncytial virus (RSV) F protein

[0127] 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 obtained. The corresponding expressed protein recombinant protein was identified by SDS-PAGE ( Figure 2 ).

[0128] 3. Immunization of mice

[0129] 1mg / mL recombinant respiratory syncytial virus (RSV) F protein was mixed with Freund's complete adjuvant in a 1:1 ratio to form 500μL of emulsified solution. Six- to eight-week-old female Balb / c mice were then subcutaneously injected at multiple points, with each mouse receiving 100μg of recombinant respiratory syncytial virus (RSV) F protein (antigen). Three weeks later, 1mg / mL antigen was mixed with Freund's incomplete adjuvant in a 1:1 ratio to form 500μL of emulsified solution. Each mouse received a 50μg antigen dose as a booster immunization. Three, six, and nine weeks later, the mice were re-immunized according to the aforementioned booster immunization procedure, for a total of four booster immunizations.

[0130] 4. Immune serum titer determination

[0131] Ten days after the fourth booster immunization, blood was collected from the tail vein of the mice, and the immune serum titer was determined by indirect ELISA. 50 μg of synthetic antigen recombinant respiratory syncytial virus (RSV) F protein was dissolved in 10 mL of 0.05 M pH 9.6 phosphate buffer and coated on a polystyrene 96-well plate at 100 μL / well 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 10 mM PBS containing 1% BSA blocking solution at 100 μl / well at 37°C for 2 h, and washed three times with PBST (0.02 M PBS containing 0.05% v / v Tween-20) and prepared for use. Mouse immune serum was diluted 102-106 times in 10 mM PBS containing 1% BSA and added to a 96-well plate at 100 μL / well. The plate was then incubated at 37°C for 1 hour. The plate was then washed three times with PBST (0.02 M PBS containing 0.05% v / v Tween-20). A 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma, Inc.) was added at 100 μL / well. The plate was then incubated at 37°C for 30 minutes and washed as above. TMB (100 μL / well) was then added to each well for color development. The reaction was incubated at 37°C in the dark for 10 minutes. The reaction was terminated by adding 2 M H2SO4 (50 μL / well). Absorbance at 450 nm was measured, and pre-immune mouse serum was used as a negative control. A positive titer was determined when the ratio of the assay value to the control value was ≥2.1 (Table 2).

[0132] Table 2 Absorption values ​​at 450 nm after immunization of mice with mouse immune serum at different dilution multiples

[0133] Dilution multiple Immunization of mice 1 Immunization of mice 2 Immunization of mice 3 Immunization of mice 4 10000 1.75 1.71 1.96 1.92 50000 0.65 0.77 0.91 0.85 100000 0.24 0.25 0.33 0.29 500000 0.09 0.10 0.10 0.10 Preimmune mouse serum 0.05 0.05 0.05 0.05

[0134] 5. Preparation of Hybridomas

[0135] Take serum with titer greater than 1:10 5Three days before fusion, a synthetic antigen, respiratory syncytial virus (RSV) F protein, was mixed with an equal volume of PBS and injected intraperitoneally into BALB / c mice to be fused for booster immunization at 50 μg / 500 μL per mouse. Feeder layer cell preparation process: Balb / c mice (approximately 4 weeks old) were sacrificed by orbital venous exsanguination and immersed in 75% ethanol for 3 minutes, with the ventral side facing up. The thoracic cavity was opened and the thymus isolated, triturated using a cell strainer, and the cells were suspended in prewarmed basal medium. Preparation of peritoneal macrophages: On the day of fusion, one healthy mouse (one spleen per mouse) was selected, and the eyeball was removed and exsanguinated until no blood could be seen. The mice were sacrificed by cervical dislocation, and the mice were disinfected by immersion in 75% ethanol for 5 minutes. The mice were then transferred to a laminar flow hood and mounted on a dissecting board with the abdomen facing up. The mouse's abdominal skin was lifted with forceps and a small incision was made with scissors (be careful not to damage the peritoneum to prevent loss of peritoneal fluid). The peritoneum was fully exposed by blunt dissection and wiped with alcohol for disinfection. Use a disposable sterile syringe to draw 5-10 mL of basal culture medium and inject it into the mouse's peritoneal cavity. Hold the syringe still with your right hand, and use forceps with your left hand to gently massage the mouse's abdomen for 1-2 minutes to encourage the macrophages to migrate out. Then, use a syringe to aspirate the culture medium in the peritoneal cavity and transfer it to a 15 mL centrifuge tube. Mix the peritoneal macrophages and thymocytes, centrifuge at 1200 rpm for 10 minutes, and discard the supernatant. Resuspend the cells in preheated 1× HAT medium (hypoxanthine (H), aminopterin (A), and thymidine (T) (HAT, Sigma)) containing 20% ​​FBS and store at 37°C until ready to use.

[0136] On the morning of the day of cell fusion, the spleen cell preparation process 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 in 75% alcohol for 5 minutes for disinfection, and immediately place 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 to ophthalmic scissors, use sterile surgical scissors to cut the peritoneum, and use tweezers to remove the spleen. After rinsing the spleen with saline, use scissors to cut the spleen into pieces and place it in a disposable cell sieve. Use the inner core of the syringe to gently squeeze the spleen and repeatedly rinse the cell sieve with saline until only connective tissue remains in the cell sieve. 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 a basal 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.

[0137] On the afternoon of the day of cell fusion, the following myeloma cell preparation process was performed: 3 bottles of T75 myeloma cells were washed, the supernatant was discarded, 50 mL of preheated saline was added, and the spleen cells were centrifuged together at 1200 rpm for 10 minutes. The spleen cells and 3 bottles of T75 myeloma cells were thoroughly mixed, the supernatant was discarded, and the cells were resuspended in 40 mL of preheated saline and centrifuged at 1200 rpm for 10 minutes.

[0138] Cell fusion process: Discard the supernatant and discard as much of it as possible. Use a pipette to remove any remaining liquid to avoid affecting the concentration of the cell fusion agent and to minimize the removal of red blood cells. Gently tap the bottom of the centrifuge tube with your fingers to mix thoroughly until the precipitated cells form a paste. Fusion at room temperature: The cell fusion agent and culture medium containing the feeder layer should be kept warm in an incubator while preparing spleen cells. Use a Pasteur pipette to draw up 1 mL of the aliquoted cell fusion agent solution (swirl it around the tube as close to the cells as possible). Gently mix within 60-90 seconds. After the timer expires, add 30 mL of preheated 37°C basal culture medium all at once to dilute the cell fusion agent and eliminate its fusogenic effect. Incubate at 37°C for 5 minutes. Centrifuge at 800 rpm for 6 minutes and discard the supernatant. Add preheated 20% FBS 1× HAT culture medium and gently pipette the precipitated cells to suspend and mix thoroughly (mix thoroughly to reduce cell clumping). This experiment was conducted by preparing 10 96-well cell culture plates with 200 μL per well, which required 200 mL of 20% FBS 1× HAT medium.

[0139] The cells were cultured in an incubator at 37°C and 5% CO2. Cell fusion was observed under a microscope 7 days after the cells were fused. 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%.

[0140] 6. Screening of hybridoma cells secreting monoclonal antibodies against respiratory syncytial virus (RSV) F protein

[0141] The cell culture supernatants were screened by indirect ELISA. Hybridoma cells with high titers were selected for subcloning and cloned 2-3 times using limiting dilution until a 100% cell positivity rate was achieved. The culture supernatants of the ten high-titer hybridoma cell lines obtained were tested by indirect ELISA, diluted with 0.01M PBS, and the results are shown in Table 3:

[0142] Table 3 Absorbance values ​​of culture supernatants of hybridoma cell lines with different clone numbers at different dilution multiples

[0143]

[0144] As shown in Table 3, by comparing the ELISA data from hybridoma cell line culture supernatants, we were able to further select cell lines that stably secreted anti-RSV F protein monoclonal antibodies with high antibody titers. These cell lines exhibited an OD value greater than 0.5 after a 1:100 dilution of the supernatant and were designated 5A5. Cloned cells with a 100% positive rate were expanded and cultured before being frozen in liquid nitrogen.

[0145] 7. Preparation and purification of ascites

[0146] Hybridoma cell line 5A5 was cultured at 1×10 6 The antibody was injected into the peritoneal cavity of 8-10 week-old female BALB / c mice pretreated with liquid paraffin. After 10-14 days of observation, ascites was extracted when the abdomen became distended. The monoclonal antibody was purified using affinity chromatography on Protein G Sepharose Fast Flow. The purity of the monoclonal antibody was determined by SDS-PAGE, which showed a purity exceeding 90%.

[0147] Example 2

[0148] Ascites prepared from hybridoma cell line 5A5 was purified to obtain monoclonal antibodies against respiratory syncytial virus (RSV) F protein. Antibody concentration determination, antibody subtype identification, antibody potency identification, antibody binding capacity testing, double antibody sandwich ELISA screening and monoclonal antibody sequencing were then performed.

[0149] 1. Determination of antibody concentration: The concentration was >1 mg / ml using the Nanodrop nucleic acid protein analyzer produced by Thermofisher.

[0150] 2. Antibody subtype identification: The subtype of the hybridoma cell line was identified using the Thermofisher mouse monoclonal antibody subtype identification kit. The subtype of the antibody secreted by 5A5 was IgG1, and the light chain was a kappa chain.

[0151] 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 (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 150 μL / well of 10mM PBS containing 1% BSA blocking solution at 37°C for 2h, washed three times with PBS (containing 0.05% v / v Tween-20), and 100 μL of purified antibody S1 was added to each well, diluted from 1ug / ml 1:3 to S7, and incubated at 37°C for 1h. The plate was washed three times with PBS (containing 0.05% v / v Tween-20), and horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody was added as the secondary antibody, incubated at 37°C for 30min, washed three times with PBS (containing 0.05% v / v Tween-20), and 100 μL of TMB was added to each well for color development. After incubation at 37°C for 15min, 2M The reaction was terminated with H2SO4 solution and detected by microplate reader at absorbance of 450nm. At 1mg / mL antibody, the titer of 5A5 antibody in positive wells (P / N>2.0) could reach 81,000.

[0152] Table 4 5A5 antibody titer absorbance values

[0153]

[0154] 4. Antibody binding test:

[0155] Respiratory syncytial virus (RSV) F protein was diluted to 0.5 μg / mL and 1 μg / mL using 1×CB, and added to the wells of the ELISA plate at a volume of 100 μL / well. The wells were then placed at 4°C overnight or at 37°C for adsorption for 2 hours. The coated microplate was dried and washed once according to the operating program set by the plate washer (AFP program). Blocking solution was added at a volume of 200 μL / well, and the plate was placed in a 37°C incubator for 2 hours, and then placed at 4°C overnight. Before use, the blocked microplate was removed from 4°C, dried, and a cleaning solution (1×PBS-T) was added to wet the ELISA plate. The monoclonal antibody described in the present invention was pre-diluted to 30 μg / mL using 1×PBS, and the pre-dilution multiple m was recorded. After a 10-fold dilution, i.e., 3 μg / mL, the highest concentration (S1) was obtained, and then a 1:3 gradient dilution (in a 96-deep-well plate) was performed, for a total of 8 dilution gradients (S1-S8).

[0156] 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. 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 GAM-HRP enzyme-labeled secondary antibody, pre-diluted 10,000-fold in secondary antibody diluent, to each well 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 TMB colorimetric solution to each well and incubate at 37°C for 5-10 min. After color development, add 50 μL of stop solution to each well. Set the plate reader to read at 450 nm / 630 nm.

[0157] 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 measuring the absorbance OD ( Figure 6 ), and the corresponding polynomial curve was fitted. The corresponding binding showed an obvious gradient with the change of the 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 the binding and reflected that the concentrations corresponding to the antibody binding force reached 8.22E-11mol / L (under the condition of 0.5μg / mL antigen coating, 50% of the highest OD reading, that is, when the concentration of the antigen-antibody complex accounts for half of the total concentration, the corresponding antibody concentration value K0.5) and 3.09E-10mol / L (under the condition of 1μg / mL antigen coating, 50% of the highest OD reading, that is, when the concentration of the antigen-antibody complex accounts for half of the total concentration, the corresponding antibody concentration value K1).

[0158] Binding affinity calculation: Calculate the average OD readings of the duplicate wells for S1-S8 antibody concentrations before and after urea treatment under 0.5 μg / mL and 1 μg / mL antigen coating conditions. Substitute the readings into the binding affinity calculation formula:

[0159] 5. Specificity detection:

[0160] Biotinylated antibodies

[0161] Dialyze the antibody against 20mM PB (pH 8.5) at 4°C, exchanging the dialyzer three times. Dissolve biotin in DMF (dimethylformamide) to 2mg / ml at a molar ratio of 1:20. Add the biotin to the dialyzed antibody, vortex to mix, and incubate in the dark for 2 hours. Dialyze the ligated product against 0.01M PBS at 4°C, exchanging the dialyzer three times.

[0162] Double antibody sandwich ELISA screening

[0163] Dilute the coated RSV antibody to 1 μg / mL with 1×CB and add 100 μL / well to the ELISA plate. Incubate at 4°C overnight or at 37°C for 2 hours. Spin dry the coated microplate and wash once using the AFP program set on the plate washer. Add blocking solution at 200 μL / well and incubate at 37°C for 2 hours, then at 4°C overnight.

[0164] Before use, the sealed microplate was taken out from 4°C and dried, and the respiratory syncytial virus (RSV) F protein was diluted to 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 1 μg / mL respectively with the secondary antibody diluent. Incubate at 37°C for 30 min. After incubation, wash the plate three times with a plate washer and pat dry on absorbent paper. Add 100 μL of RSV biotin-labeled antibody pre-diluted to 0.2 μg / ml in secondary antibody diluent to each well and incubate at 37°C for 30 min. Wash the plate three times with a plate washer, spin dry, and pat dry on absorbent paper. Add 100 μL of SA-HRP (1 mg / ml) pre-diluted 30,000-fold in secondary antibody diluent to each well and incubate at 37°C for 30 min. Wash the plate three times with a plate washer, spin dry, and pat dry on absorbent paper. Add 100 μL of TMB colorimetric solution to each well and incubate at 37°C for 10 min. After color development, add 50 μL of stop solution to each well. Set the microplate reader to read at 450 nm.

[0165] 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)

[0166] Table 5A

[0167]

[0168] Table 5B

[0169]

[0170]

[0171] Table 5C

[0172]

[0173] Table 5D

[0174]

[0175] Table 5E

[0176]

[0177] Table 5F

[0178]

[0179]

[0180] Table 5G

[0181]

[0182] Table 5H

[0183]

[0184] Table 5I

[0185]

[0186] Table 5J

[0187]

[0188]

[0189] As shown in Tables 5A to 5J, a comparison of the experimental data from the antibody pairing experiments indicates that, with the exception of the detection wells corresponding to the enzyme-labeled antibody, which is the same as the coating antibody, all other enzyme-labeled antibodies can still bind to the RSV F protein antigen in the presence of the coating antibody. The binding effect increases with increasing antigen concentration, demonstrating the specificity of 5A5 as a labeled antibody. Furthermore, when paired with 9F5 as the coating antibody to form an immune sandwich reaction, the sensitivity to RSV antigen is optimal.

[0190] 6. Monoclonal antibody sequencing

[0191] Approximately 38 mg (batch number V20220526; 3.58 mg / mL) of a monoclonal antibody against the respiratory syncytial virus (RSV) F protein prepared from purified hybridoma cell 5A5 was obtained. Sequencing of the light and heavy chains revealed that the amino acid sequences of the light and heavy chains of the antibody are shown in SEQ ID NOs: 1 and 2, respectively. The light chain complementary determining region sequences of the F protein monoclonal antibody are CDR-L1 (RSSDFGSKYLA), CDR-L2 (SGSRAQS), and CDR-L3 (QSHNEYLWT), and the heavy chain complementary determining region sequences are CDR-H1 (SIGME), CDR-H2 (YISHYFITIYYANVVKG), and CDR-H3 (SGIHNAADY). Antibodies with corresponding variable region sequences have high affinity and good specificity.

[0192] Therefore, the antibodies provided by the present invention can be used for the development of recombinant antibodies, single-chain antibodies, bispecific antibodies, and related products for diagnostic or therapeutic purposes.

[0193] Example 3

[0194] Development of a lateral flow immunochromatographic detection kit using monoclonal antibodies against syncytial virus F protein.

[0195] 1. Preparation of colloidal gold:

[0196] The gold was prepared by the reduction method, and the gold preparation conditions were 5 mL of 2% chloroauric acid + 10 mL of 1% trisodium citrate.

[0197] 2. Colloidal gold labeled mouse anti-respiratory syncytial virus (RSV) F protein monoclonal antibody

[0198] 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: 1 and 2 respectively) are combined.

[0199] The specific labeling conditions are as follows: 10 μL of 0.1 M potassium carbonate solution was added to each mL of colloidal gold at pH 7.5, and the labeling concentration of mouse anti-respiratory syncytial virus (RSV) F protein monoclonal antibody was 10 μg / mL.

[0200] 3. Test line and quality control line

[0201] Test line: Prepare a test line by coating a nitrocellulose membrane with mouse anti-RSV F protein monoclonal antibody 9F5 at an appropriate concentration (1.5 mg / mL). Dry at 37°C. Spray volume: 0.1 μL / mm.

[0202] Quality control line: Prepare a quality control line on a fiber membrane using 0.5 mg / mL goat anti-mouse IgG polyclonal antibody and dry at 37°C. Spray volume: 0.1 μL / mm.

[0203] The colloidal gold-labeled mouse anti-respiratory syncytial virus (RSV) F protein monoclonal antibody in step 2 was sprayed onto a polyester film and dried to prepare a colloidal gold conjugate pad. The sample pad, colloidal gold conjugate pad, nitrocellulose membrane sprayed with test and quality control lines, and absorbent paper were then laid on a PVC base plate in the designed order.

[0204] 4. Inspection methods

[0205] Return the test card (test strip), sample diluent, and sample to 18-30°C. The test card or test strip testing method is as follows:

[0206] a. Remove the test card or test strip from the aluminum foil bag, mark the sample, and place it flat on a level work surface;

[0207] b. Take 20 μL of nasopharyngeal or oropharyngeal swab sample extract and add it directly to the sample well (test card) or the sample injection area below the arrow (test strip);

[0208] c. Add 100 μL (2 to 3 drops) of sample diluent;

[0209] d. Read the results within 15 to 20 minutes. Test results after 20 minutes will be invalid.

[0210] 5. Interpretation of test results

[0211] a. Positive test line: The test line and the quality control line show color. This indicates that the sample has detected the F protein antigen of respiratory syncytial virus (RSV), which may indicate early infection or current infection. Final confirmation must be combined with clinical symptoms.

[0212] b. Negative: Only one red control line appears in the test window, indicating that the F protein antigen of respiratory syncytial virus (RSV) is not detected in the sample.

[0213] c. Invalid: No red quality control line appears in the detection window.

[0214] Example 4

[0215] The sensitivity, minimum detection limit and specificity of the lateral flow immunochromatographic detection kit prepared in Example 3 were tested.

[0216] 1. Sensitivity test

[0217] a. Inactivated syncytial virus culture test

[0218] The lateral flow immunochromatographic rapid test reagent developed using the antibody of the present invention can detect inactivated syncytial virus culture, and the minimum detection level can be as low as 10TCID 50 / mL of virus culture.

[0219] 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 dilution of the original sample. For example, 1mL of culture medium, after dilution 1000 times, just causes 50% of the cells to be infected, then TCID 50 1000 / mL. This indicates the dilution factor required to infect 50% of cells with the virus in each mL of sample.

[0220] Therefore, TCID 50The larger the value, the more virus copies in the inactivated supernatant stock solution, and the larger the dilution multiple is required (to achieve the effect of infecting 50% of cells). Then, the corresponding inactivated supernatant stock solution is used to detect the test strips of the kit, and the corresponding gradient results are as follows: Figure 3 As shown. 10TCID 50 / mL represents the sample with the lowest viral copy number in the original solution. That is, the lowest testable virus copy number is as low as 100TCID 50 / mL of virus culture.

[0221] b. Purified respiratory syncytial virus (RSV) F protein test

[0222] The respiratory syncytial virus (RSV) F protein solution standard substance produced by Hangzhou Huakui Jinpei Biotechnology Co., Ltd. was used to detect the lateral flow immunochromatographic rapid test reagent developed by the antibody of the present invention. Figure 4 As shown, the standard recombinant F protein of respiratory syncytial virus (RSV) can be detected as low as 50 ng / mL.

[0223] 2. Specificity test (clinical sample test)

[0224] The lateral flow immunochromatographic 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 were 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.

[0225] Example 5

[0226] In this example, a double-antibody sandwich ELISA platform detection reagent was developed using a monoclonal antibody against the syncytial virus F protein.

[0227] Biotinylated antibodies

[0228] The 5A5 antibody was dialyzed against 20mM PB (pH 8.5) at 4°C with three changes of the dialyzed solution. Biotin was dissolved in DMF (dimethylformamide) to 2mg / ml at a molar ratio of 1:20. Biotin was added to the dialyzed antibody, vortexed, and allowed to react for 2 hours in the dark. The ligation product was dialyzed against 0.01M PBS at 4°C with three changes of the dialyzed solution. After dialysis, the concentration of 5A5-Biotin was determined using a Thermofisher Nanodrop nucleic acid protein analyzer. An equal volume of glycerol was added and stored at -20°C.

[0229] Detection of Respiratory Syncytial Virus (RSV) F Protein by Double Antibody Sandwich ELISA

[0230] Dilute the coated 9F5 antibody to 1 μg / mL with 1×CB and add 100 μL / well to the ELISA plate. Incubate at 4°C overnight or at 37°C for 2 hours. Shake dry the coated microplate and wash once using the AFP program set on the plate washer. Add blocking solution at 200 μL / well and incubate at 37°C for 2 hours, then at 4°C overnight.

[0231] Before use, the sealed microplate was taken out from 4 ° C and dried, and the respiratory syncytial virus (RSV) F protein was diluted to 1.37 ng / ml, 4.12 ng / ml, μg / mL, 12.35 ng / ml, 37.04 ng / ml, 0.11 ug / ml, 0.33 ug / ml, and 1 μg / mL respectively with secondary antibody diluent. Incubate at 37°C for 30 min. After incubation, wash the plate three times with a plate washer and pat dry on absorbent paper. Add 100 μL of RSV biotin-labeled antibody pre-diluted to 0.2 μg / ml in secondary antibody diluent to each well and incubate at 37°C for 30 min. Wash the plate three times with a plate washer, spin dry, and pat dry on absorbent paper. Add 100 μL of SA-HRP (1 mg / ml) pre-diluted 30,000-fold in secondary antibody diluent to each well and incubate at 37°C for 30 min. Wash the plate three times with a plate washer, spin dry, and pat dry on absorbent paper. Add 100 μL of TMB colorimetric solution to each well and incubate at 37°C for 10 min. After color development, add 50 μL of stop solution to each well. Set the microplate reader to read at 450 nm.

[0232] The dual-antibody sandwich ELISA platform detection reagent developed with the antibodies described herein was tested using a respiratory syncytial virus (RSV) F protein solution standard substance produced by Hangzhou Huakui Jinpei Biotechnology Co., Ltd. As shown in Table 6, the standard recombinant RSV F protein could be detected at a minimum concentration of 4.12 ng / mL.

[0233] Table 6 Detection results of double antibody sandwich ELISA method for detecting respiratory syncytial virus (RSV) F protein

[0234]

[0235] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against human respiratory syncytial virus F protein, characterized in that: It includes a heavy chain and a light chain, the heavy chain includes a heavy chain complementary determining region, and the light chain includes a light chain complementary determining region; 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, and 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.

2. The anti-human respiratory syncytial virus F protein antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof further comprises a heavy chain framework region, and a light chain framework region.

3. The anti-human respiratory syncytial virus F protein antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The light chain framework region includes LFR1, LFR2, LFR3 and LFR4, which are at least 85% homologous to the amino acid sequences shown in SEQ ID NOs: 16-19; The heavy chain framework region includes HFR1, HFR2, HFR3 and HFR4, which have at least 85% homology to the amino acid sequences shown in SEQ ID NOs: 20-23.

4. The anti-human respiratory syncytial virus F protein antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The antibody or antigen-binding fragment thereof further comprises a constant region, which comprises a heavy chain constant region and / or a light chain constant region.

5. The anti-human respiratory syncytial virus F protein antibody or antigen-binding fragment thereof according to claim 4, characterized in that: 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.

6. The anti-human respiratory syncytial virus F protein antibody or antigen-binding fragment thereof according to claim 5, characterized in that: The heavy chain constant region is selected from IgG1, and the light chain constant region is selected from κ-type light chain constant regions.

7. The anti-human respiratory syncytial virus F protein antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

8. The anti-human respiratory syncytial virus F protein antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The light chain is shown in SEQ ID NO: 1, and the heavy chain is shown in SEQ ID NO:

2.

9. A biological product, characterized in that It comprises the antibody against human respiratory syncytial virus F protein or its antigen-binding fragment according to any one of claims 1 to 8, and the biological product is selected from the group consisting of reagents, kits, test strips, antibody chips, antibody probes, and detectors.

10. The biological product according to claim 9, characterized in that The reagent comprises: the anti-human respiratory syncytial virus F protein antibody or its antigen-binding fragment, and the antibody or its antigen-binding fragment is labeled with a detectable marker; The detectable marker is selected from fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents and nanoparticle markers.

11. The biological product according to claim 9, characterized in that The kit comprises a solid phase, and the antibody or antigen-binding fragment thereof is coated on the solid phase.

12. The biological product according to claim 11, characterized in that The solid phase is selected from the group consisting of microspheres, plates, and membranes.

13. The biological product according to claim 11, characterized in that The solid phase is selected from the group consisting of magnetic microspheres, plastic microspheres, plastic microparticles, latex microspheres, microporous plates, glass, capillaries, nylon and nitrocellulose membranes.

14. The biological product according to claim 9, characterized in that 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 antigens recognized by the detection antibody and the capture antibody are different; when the detection antibody is an anti-human respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, the capture antibody is another anti-human respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof; when the detection antibody is another anti-human respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof; the capture antibody is an anti-human respiratory syncytial virus F protein antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

15. The biological product according to claim 14, characterized in that The concentration of the capture antibody coated on the conjugate pad is 10-15 μg / mL; The concentration of the detection antibody coated on the detection line is 1.5-2 mg / mL.

16. The biological product according to claim 14, characterized in that The marker on the conjugate pad is a nanoparticle marker.

17. The biological product according to claim 16, characterized in that The nanoparticle marker is selected from nanoparticles or colloids.

18. The biological product according to claim 14, characterized in that The amino acid sequences of the light chain and heavy chain of the other anti-human respiratory syncytial virus F protein antibody or its antigen-binding fragment are shown in SEQ ID NOs: 3-4, respectively.

19. The biological product according to claim 14, characterized in that The detection antibody is the antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to any one of claims 1 to 8, and the capture antibody is another antibody against human respiratory syncytial virus F protein or its antigen-binding fragment.

20. Use of the anti-human respiratory syncytial virus F protein antibody or the antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a respiratory syncytial virus detection product or a respiratory syncytial virus enrichment product.

21. The use according to claim 20, characterized in that The detection product is a reagent, a test kit, a test strip, an antibody chip, an antibody probe or a detector.

22. An isolated 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 8.

23. A recombinant vector, characterized in that It includes the nucleic acid molecule according to claim 22.

24. A recombinant cell, characterized in that It comprises the recombinant vector according to claim 23.

Images