An antibody against respiratory syncytial virus F protein or its antigen-binding fragment, biological product and its application

Abstract

The present invention discloses an antibody against respiratory syncytial virus F protein or its antigen-binding fragment, a biological product and its application, which relates to the technical field of immunoassay. The antibody comprises a light chain complementary determining region in the light chain shown in SEQ ID NO: 1 and a heavy chain complementary determining region in the heavy chain shown in SEQ ID NO: 2. It has high binding specificity and high affinity with respiratory syncytial virus F protein, and can not only be effectively used for quantitative and / or qualitative detection of respiratory syncytial virus, but also be used in various detection methods such as ELISA detection, immunochemiluminescence detection and immunofluorescence detection, etc. It has a wide application range and strong applicability. In addition, the respiratory syncytial virus detection product or diagnostic product provided by the present invention has the technical advantages of lower detection limit and higher sensitivity.

Description

Technical Field

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

[0002] Human respiratory syncytial virus (RSV) is one of the main causes of severe respiratory diseases in infants and the elderly. It is estimated that the economic burden caused by RSV infection exceeds 80 billion US dollars globally every year. Since RSV was discovered in the 1950s, domestic and foreign researchers have conducted a large number 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 Paramyxoviridae family and is a member of the Pneumovirus genus. The RSV genome is a single-stranded negative-stranded, non-segmented RNA, with a full length of 15.2 kb, containing 10 genes and encoding 11 proteins. The RSV virus contains an envelope formed by a phospholipid bilayer, on which the fusion protein (F protein), the attachment protein (G protein) and the small hydrophobic protein (SH protein) are embedded. The G and F proteins are the two main glycoproteins on the virus surface and play an important role in mediating the process of virus entry into cells. The F protein causes a natural immune response by binding to the pattern recognition receptors CD14 and TLR4, while the G protein and the nucleocapsid protein N have a weaker ability to cause a natural immune response in the body.

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

[0005] The 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 neutralizing antibody recognition sites against the F protein are mainly on the pre-fusion F protein. The recombinant protein immunogenicity and protective effect designed based on pre-fusion F are significantly better than those of vaccines designed based on post-fusion F protein.

[0006] In summary, RSV antigen detection has advantages such as portability, rapidity, and the ability to perform point-of-care testing. Antibody raw materials are the main bioactive components of immunological detection reagents, and the selection of target proteins directly affects the test results. The F protein is an important protein for the fusion of the virus with the host cell membrane. Based on its immunogenicity and high conservation, using it as a target for RSV virus detection can ensure the sensitivity and specificity of the test results. The development of monoclonal antibodies targeting the F protein of syncytial virus is of great significance for diagnosing syncytial virus and discovering antibody drugs with strong efficacy against syncytial virus.

[0007] The lateral flow immunoassay rapid diagnostic kit is a test strip-based diagnostic product suitable for point-of-care testing. The lateral flow immunoassay rapid diagnostic kit also has some deficiencies: Firstly, this detection method can only provide qualitative results and is not very applicable to pathogens (specific bacteria, mycoplasmas, viruses, etc.) that require diagnosis of the infection situation based on precise quantitative results. Secondly, this detection method requires the test sample to be in a liquid state and have a certain viscosity to flow through the porous nitrocellulose membrane.

[0008] Nevertheless, the lateral flow immunoassay rapid diagnostic kit is still a detection technology with important application value, capable of meeting the general standards required for rapid diagnosis, such as sensitivity, stability, economy, user-friendliness, and equipment-free. These advantages of the lateral flow immunoassay rapid diagnostic kit are particularly significant for units or regions lacking professional equipment and technicians who have not received good training.

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

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

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

[0012] The present invention is implemented as follows:

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

[0014] In the second aspect, the present invention further provides a biological product, which comprises the above-mentioned antibody against the F protein of respiratory syncytial virus or its antigen-binding fragment, and the biological product is selected from reagents, kits, test strips, antibody chips, antibody probes, and detectors.

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

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

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

[0018] In a sixth aspect, the present invention also provides a recombinant cell comprising: the recombinant vector as described above.

[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 for respiratory syncytial virus F protein. It can not only be effectively used for the quantitative and / or qualitative detection of respiratory syncytial virus (RSV), but also for various detection methods such as ELISA detection, immunochemical luminescence detection, and immunofluorescence detection. It has a wide application range and strong applicability.

[0021] The respiratory syncytial virus detection product or diagnostic product provided by the present invention has the technical advantages of a lower detection limit and higher sensitivity; and the selected antibody specifically targets an antigenic epitope with a highly conserved amino acid sequence, is not affected by the common antigen protein mutations of virus mutants, can reduce the detection cost, shorten the detection time, and improve the detection efficiency. In particular, when the detection product or diagnostic product is a lateral flow immunochromatographic rapid diagnostic kit, it can meet the general standards required for rapid diagnosis, such as sensitivity, stability, economy, user-friendliness, and equipment-free. These advantages of the lateral flow immunochromatographic rapid diagnostic kit are particularly significant for units or regions lacking professional equipment and well-trained technical personnel.

[0022] In addition, the antibody against respiratory syncytial virus F protein provided by the present invention can be used to design a therapeutic target for respiratory syncytial virus (RSV), and a therapeutic agent against 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 following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

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

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

[0026] Figure 3 It is the result of detecting inactivated syncytial virus cultures (at different dilutions) using the lateral flow immunochromatographic rapid test reagent developed with the antibody in the embodiment of the present invention;

[0027] Figure 4 It is the result of detecting the lateral flow immunochromatographic rapid test reagent developed based on the antibody of the present invention (at different dilutions) using the respiratory syncytial virus (RSV) F protein solution reference material in the embodiment of the present invention;

[0028] Figure 5 It is the result of detecting nasal swab samples of normal people using the lateral flow immunochromatographic rapid test reagent developed with the antibody of the present invention;

[0029] Figure 6 It is the binding curve of the antibody of the present invention with the recombinant respiratory syncytial virus (RSV) F protein antigen at gradient concentrations; among them, 0.5 μg is the binding curve of coating 50 ng of antigen per well (100 μL per well), and 1 μg is the binding curve of coating 100 ng of antigen per well (100 μL per well). Detailed Embodiments

[0030] Reference to the embodiments of the present invention will now be provided in detail, with one or more examples described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0031] Definition of Terms

[0032] The term "antigen-binding fragment" generally refers to any protein / protein fragment 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 the minimum antibody recognition unit, 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".

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

[0034] In the present invention, the term "complementary determining region or complementarity-determining region", "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins, which refer to regions containing one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody or antigen-binding fragment to the antigen or epitope it recognizes. In the specific embodiments of the present invention, the CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0035] In the present invention, the heavy chain complementary determining regions are denoted as HCDR and include HCDR1, HCDR2 and HCDR3; the light chain complementary determining regions are denoted as LCDR and include LCDR1, LCDR2 and LCDR3. Commonly used CDR labeling methods in the art include: the Kabat numbering scheme, the IMGT numbering scheme, the Chothia and Lesk numbering scheme, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. 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 the widely adopted standard for numbering antibody residues. The present invention uses the Kabat annotation standard to label the CDR regions, but CDR regions labeled by other methods also fall within the scope of protection of the present invention.

[0036] Under normal circumstances, the variable region VH of the heavy chain of an antibody can be obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0037] The variable region VL of the light chain of an antibody can be obtained by connecting the CDRs and FRs numbered as follows in the following combined arrangement: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

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

[0043] Inputting the above SEQ ID NO:1 and SEQ ID NO:2 sequences into the CDR labeling system can obtain the corresponding CDR sequences.

[0044] The amino acid sequences of the above complementarity-determining regions are first discovered and revealed in the present invention, and are a new sequence, which can endow the antibody or its antigen-binding fragment with 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 for the detection of respiratory syncytial virus, and to develop corresponding respiratory syncytial virus detection products, diagnostic products, and RSV treatment products. The present invention provides the core material for the detection method of RSV.

[0045] In a preferred embodiment of the application of the present invention, the light-chain complementarity-determining regions include CDR-L1, CDR-L2, and CDR-L3, and their amino acid sequences are shown as SEQ ID NO: 10-12 in sequence; the heavy-chain complementarity-determining regions include CDR-H1, CDR-H2, and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 13-15 in sequence.

[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 application of the present invention, the antibody or its antigen-binding fragment further includes a heavy-chain framework region, and / or, a light-chain framework region;

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

[0049] The heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4 which have at least 80% homology with the amino acid sequences shown in SEQ ID NOs: 20-23 in sequence. For example, the heavy chain framework region comprises HFR1, HFR2, HFR3 and HFR4 which have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequences shown in SEQ ID NOs: 20-23 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 application of the present invention, the antibody or its antigen-binding fragment 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 application of the present invention, the heavy chain constant region is selected from the heavy chain constant regions 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 regions.

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

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

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

[0056] In a preferred embodiment of the application 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 antibody generally has the same binding specificity as its source antibody. Those skilled in the art can easily understand from the content recorded in the present invention that the functional fragment of the above antibody can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemically reducing and cleaving disulfide bonds.

[0057] The antigen-binding fragment of the above antibody can also be obtained by recombinant genetic techniques known to those skilled in the art or by synthesis using, for example, an automated peptide synthesizer, such as those sold by Applied BioSystems.

[0058] In a second aspect, the present invention also provides a biological product, which includes the above-mentioned antibody against respiratory syncytial virus F protein or its antigen-binding fragment, and the biological product is selected from reagents, reagent kits, test strips, antibody chips, antibody probes, and detectors.

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

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

[0061] An antibody chip refers to a chip formed by immobilizing the above-mentioned antibody against respiratory syncytial virus F protein or its antigen-binding fragment on a carrier.

[0062] A detectable label refers to a class of substances with characteristics such as luminescence, color development, radioactivity, etc. that can be directly observed by the naked eye or detected or detected by an instrument, and qualitative or quantitative detection of the corresponding target can be achieved through these characteristics.

[0063] In a preferred embodiment of the application of the present invention, the detectable label is selected from fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

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

[0065] Fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, including but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, including but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, including but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, including but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, including but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0066] In alternative embodiments, the enzymes that catalyze the substrate to develop color include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0067] In alternative embodiments, the radioisotopes 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 Ru 62 Cu 64 Cu 67 Cu 68 Cu 86 Y 88 Y 121 Sn 161 Tb 166 Ho 105 Rh177 Lu, 172 Lu and 18 F.

[0068] In an alternative embodiment, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, ostracod luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rhodamine alkaloid and its derivatives, and peroxyoxalate and its derivatives.

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

[0070] In an alternative embodiment, the colloids include, but are not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latex.

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

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

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

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

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

[0076] In another embodiment, the test strip may further include a sample pad, a red blood cell filtration pad or a red blood cell removal pad, a conjugate pad, an NC membrane, and a water absorption pad, which are sequentially arranged on the bottom plate. The pore size of the red blood cell filtration pad or the red blood cell removal pad is smaller than the diameter of red blood cells, so as to filter out red blood cells in the blood sample and avoid interference of red blood cells on the test result during testing.

[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 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.

[0078] In a preferred embodiment of the application of the present invention, the label on the conjugate pad is a nanoparticle - type label.

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

[0080] Colloidal gold is one of the earliest label materials applied to immunochromatographic test kits. Its absorption wavelength is in the visible light region, which is easy to be directly observed by the naked eye, and the color shows red, blue or purple, etc. with the change of the size and shape of colloidal gold particles. The raw material of colloidal gold is usually prepared by reducing chloroauric acid with trisodium citrate in a simple and reliable way. The colloidal gold prepared by this method carries a considerable amount of negative charges on its surface, making its properties very stable in both the dry state and even in solution. In addition, the aggregation of a large number of negatively charged surfaces enables colloidal gold to be gently coupled with biopolymers such as nucleotides, polypeptides or proteins with positive charges under specific pH and other conditions through the simple way of electrostatic adsorption, and this coupling can retain the activity of biopolymers to the greatest extent. The above advantages make colloidal gold become the label material in most commercial immunochromatographic test strips on the market at present.

[0081] The lateral flow immunochromatographic rapid diagnostic kit is a result reporting method based on a test strip and suitable for point - of - care testing. Its core component is the lateral flow immunochromatographic test strip ( Figure 1 ), generally narrow - strip - shaped, usually with a width of 4 - 6 mm, and the length does not exceed 6 - 7 cm. A standard lateral flow immunochromatographic test strip should include four main parts, namely: a sample pad, a conjugate pad, a detection area and a water absorption 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 analyte.

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

[0084] The main material of the test area is made of nitrocellulose membrane (NC membrane). In the test area, there are mainly a test line and a control line, and the test line and the control line respectively contain corresponding antibodies;

[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 strip, the solution will flow from the sample pad area into the conjugate pad area under the action of chromatography. When the substance to be detected is contained in the solution, the substance to be detected will combine with the colloidal gold (coupled) antigen or colloidal gold (coupled) antibody in the conjugate pad area. The colloidal gold (coupled) antibody or colloidal gold (coupled) antigen or the complex formed with the sample to be tested rehydrated by the solution will pass through the test area (NC membrane) and the absorbent pad area in turn under the action of capillary force. At this time, the lateral flow test strip will have two result display models, namely the standard model ( Figure 1 ) and the competitive model.

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

[0088] In a preferred embodiment of the application of the present invention, the amino acid sequences of the light chain and heavy chain of another antibody against the F protein of respiratory syncytial virus or its antigen-binding fragment are respectively shown in SEQ ID NO: 3-4;

[0089] In a preferred embodiment of the application of the present invention, the detection antibody is the above-mentioned antibody against the F protein of respiratory syncytial virus or its antigen-binding fragment, and the capture antibody is another antibody against the F protein of respiratory syncytial virus 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 murine antibody, the polyclonal antibody coated on the quality control line is a polyclonal antibody against mouse, such as a polyclonal antibody of goat anti-mouse.

[0091] In a third aspect, the present invention also provides the use of an antibody against respiratory syncytial virus F protein or its antigen-binding fragment in the preparation of a respiratory syncytial virus detection product, a diagnostic product or a respiratory syncytial virus enrichment product.

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

[0093] Examples of respiratory syncytial virus enrichment products: Coupling an antibody against respiratory syncytial virus F protein with magnetic beads, then reacting the magnetic beads with a sample for enrichment of respiratory syncytial virus, and then eluting. In other embodiments, the antibody against respiratory syncytial virus F protein can also be immobilized on an affinity column to achieve separation and enrichment of respiratory syncytial virus.

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

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

[0096] For example, the nucleotide sequences encoding the light chain and heavy chain of the antibody are shown as 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 packaged into a virus) introduce a target DNA fragment into a host cell by transformation, transfection or transduction for expression of the target gene.

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

[0100] Those skilled in the art can also use in vitro transcription to transcribe the nucleic acid sequences of the present invention into RNA templates, and further transfect, transduce or transform the RNA into host cells, which can also express the antibodies or antigen-binding fragments of the present invention and exert the biological effects of the present invention.

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

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

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

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

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

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

[0107] The above recombinant cells are prepared by transforming a recombinant expression vector into a host cell (such as a microorganism) by conventional methods in the art; the host microorganism can be various conventional host microorganisms in the art as long as it can satisfy that 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 resting cells of recombinant bacteria, living cells of recombinant bacteria, dead cells of recombinant bacteria, and cell fragments of recombinant bacteria.

[0109] 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.

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

[0111] 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.

[0112] 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.

[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 that produce monoclonal antibodies.

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

[0118] Analysis was performed on the amino acid sequence translated from 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 respiratory syncytial virus (RSV) (A2) was selected for sequence alignment, and it was found that the homology of this sequence with the F proteins of other respiratory syncytial viruses (RSV) is very high. After immunogenicity, hydrophilicity, and surface accessibility analysis, the full-length 574-amino acid CDS region of the F protein of respiratory syncytial virus (RSV) (A2) was finally selected as the subsequent recombinant protein, that is, the sequence of the antigen (SEQ ID NO: 7).

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

[0120] Table 1 Table of relevant information on the antigenic epitopes and / or functional localization of the 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, it was finally expressed in the E. coli system to obtain recombinant respiratory syncytial virus F protein, which is the antigen. The corresponding expressed protein recombinant protein was identified by SDS-PAGE( Figure 2 ).

[0125] 3. Immunization of mice

[0126] 1 mg / mL of recombinant respiratory syncytial virus (RSV) F protein was mixed with Freund's complete adjuvant at a ratio of 1:1 and emulsified with 500 μL, then subcutaneously injected into 6-8-week-old female Balb / c mice at multiple points, and each mouse was inoculated with 100 μg of recombinant respiratory syncytial virus (RSV) F protein (antigen). Three weeks later, 1 mg / mL of antigen was mixed with Freund's incomplete adjuvant at a ratio of 1:1 and emulsified with 500 μL, and then subcutaneously injected at multiple points. The antigen inoculation dose for each mouse was 50 μg, and this was used as a booster immunization. At intervals of three weeks, six weeks, and nine weeks, immunization was repeated according to the aforementioned booster immunization procedure, and a total of 4 booster immunizations were performed.

[0127] 4. Determination of immune serum titer

[0128] Ten days after the fourth booster immunization, blood was collected from the tail veins of mice, and the titer of immune serum was determined by indirect ELISA. 50 μg of the synthetic antigen recombinant respiratory syncytial virus (RSV) F protein was dissolved in 10 mL of 0.05 M pH 9.6 phosphate buffer, and a polystyrene 96-well plate was coated, 100 μL per well, overnight at 4°C. The plate was washed three times with PBST (0.02 M PBS containing 0.05% v / v Tween-20), blocked with 100 μL per well of 10 mM PBS containing 1% BSA 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. The mouse immune serum was diluted 10 2 to 10 6 times with 10 mM PBS containing 1% BSA, added to the 96-well plate, 100 μL per well, and then incubated at 37°C for 1 h. After that, the plate was washed three times with PBST (0.02 M PBS containing 0.05% v / v Tween-20), and then 100 μL per well of a 1:10,000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG (Sigma, INC.) was added and incubated at 37°C for 30 min, and washed as above. Then 100 μL per well of TMB was added to each well for color development, and it was incubated at 37°C in the dark for 10 min at room temperature. Then 2 M H2SO4 was added at a volume of 50 μL per well to terminate the reaction. The absorbance at 450 nm was measured. The serum of mice before immunization was used as a negative control, and the immune serum titer was determined with a ratio of the measured value to the control value ≥ 2.1 as the positive judgment value (Table 2).

[0129] Table 2 Absorbance at 450 nm of mouse immune serum at different dilution multiples after immunizing mice

[0130]

[0131] 5. Preparation of hybridomas

[0132] Take the serum titer greater than 1:10 5For the mice, 3 days before fusion, the synthesized antigen respiratory syncytial virus (RSV) F protein was mixed with an equal volume of PBS, and BALB / c mice to be fused were intraperitoneally injected with 50 μg / 500 μL per mouse for booster immunization. Preparation process of feeder layer cells: Balb / c mice (about 4 weeks old) were sacrificed by orbital venous bleeding, soaked in 75% ethanol for 3 minutes, with the ventral side facing up; the chest cavity was opened to isolate the thymus, ground with a cell sieve, and the cells were resuspended with pre-warmed basal medium. Preparation of peritoneal macrophages: On the day of fusion, 1 healthy mouse (one mouse for one spleen) was selected, its eyeball was bled until no more blood dripped out. It was sacrificed by cervical dislocation, soaked in 75% alcohol for disinfection for 5 minutes, transferred into a laminar flow hood, and fixed on an anatomical board with the abdomen facing up. The abdominal skin of the mouse was lifted with forceps, and a small incision was made with scissors (note that the peritoneum should not be damaged to avoid the outflow of peritoneal fluid), and the peritoneum was fully exposed by blunt dissection and wiped with alcohol for disinfection. 5 - 10 mL of basal culture medium was aspirated with a disposable sterile syringe and injected into the mouse peritoneal cavity. The syringe was fixed with the right hand and remained stationary, and the left hand gently kneaded the mouse abdomen with an alcohol cotton ball for 1 - 2 minutes to promote the migration of macrophages. Then the culture fluid in the peritoneal cavity was aspirated with a syringe and transferred into a 15 mL centrifuge tube. After mixing the peritoneal macrophages and thymocytes, they were centrifuged at 1200 r / min for 10 minutes, and the supernatant was discarded. They were resuspended with pre-warmed 1×HAT medium containing 20% FBS (hypoxanthine (H), aminopterin (A) and thymidine (T) (HAT, Sigma)) and stored at 37℃ for use.

[0133] On the morning of the day of cell fusion, preparation process of spleen cells: Mice that had been boosted immunized 3 - 4 days ago were sacrificed by orbital bleeding, and the serum was collected and separated as the positive control serum for detection. At the same time, the mice were sacrificed by cervical dislocation, soaked in 75% alcohol for 5 minutes for disinfection, and immediately placed in a laminar flow hood. The mice were fixed on the anatomical table, the abdomen was opened aseptically, and then the skin of the right abdomen was lifted, and the spleen could be seen. Replace with ophthalmic scissors, cut the peritoneum with a sterile surgical scissors, take out the spleen with forceps, wash the spleen with physiological saline, then cut the spleen into pieces with scissors, place it in a disposable cell sieve, gently squeeze the spleen with the inner core of the syringe, and repeatedly wash the cell sieve with physiological saline until only connective tissue remained in the cell sieve. Then the cell suspension was filtered again with a disposable cell sieve. The spleen cell suspension was harvested, centrifuged at 1200 r / min for 10 minutes, and resuspended and centrifuged and washed once with 30 - 40 mL (try to remove the red blood cell mass). After resuspending the spleen cells and adding them to the basal culture medium containing 10% FBS, they were placed in a T75 cell flask and cultured in an incubator at 37℃ and 5% CO2 for 2 - 3 hours to allow the macrophages in the cell suspension to adhere to the wall.

[0134] On the afternoon of the day of the fused cells, the preparation process of myeloma cells: Discard the supernatant from 3 flasks of T75 myeloma cells. Use 50 mL of pre-warmed physiological saline to blow down the cells, and centrifuge together with splenocytes at 1200 r / min for 10 minutes. Mix the splenocytes and 3 flasks of T75 myeloma cells thoroughly, discard the supernatant, resuspend with 40 ml of pre-warmed physiological saline, and centrifuge at 1200 r / min for 10 minutes.

[0135] Cell fusion process: Discard the supernatant, try to discard it as completely as possible. Use a dropper to suck out the residual liquid to avoid affecting the concentration of the cell fusion agent, and try to remove red blood cells. Gently tap the bottom of the centrifuge tube with your finger to mix well and make the precipitated cells loose and evenly into a paste. Perform fusion at room temperature: The cell fusion agent and the medium containing the feeder layer need to be placed in an incubator for warming when preparing spleen cells. Use a Pasteur pipette to aspirate 1 mL of the dispensed cell fusion agent solution (add it in a circle close to the cell at the centrifuge tube wall as much as possible). Gently mix within 60 s - 90 s. After timing is completed, add 30 mL of pre-warmed basic medium at 37°C at one time to dilute the cell fusion agent and lose its cell fusion-promoting effect, and let it stand at 37°C for 5 min. Centrifuge at 800 r / min for 6 minutes and discard the supernatant. Add pre-warmed 20% FBS 1×HAT medium and gently pipette the precipitated cells to suspend and mix them well (mix thoroughly to reduce cell clumps), with gentle movements. For this experiment, according to the preparation of 10 96-well cell culture plates, 200 μL per well, 200 mL of 20% FBS 1×HAT medium is required.

[0136] Culture in an incubator at 37°C and 5% CO2. Observe under a microscope 7 days after cell fusion, count the culture wells with obvious cell clones in the 96-well cell culture plate, and calculate the fusion rate. Fusion rate = (number of fused cells / total number of cells) × 100%.

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

[0138] Screen the cell culture supernatant by indirect ELISA method, select the positive clone hybridoma cells with higher titers for subcloning, and continuously clone 2 - 3 times by the limiting dilution method until a 100% cell positive rate is achieved. Detect the culture supernatants of the finally obtained 10 hybridoma cell lines with higher titers by the indirect ELISA method, and at the same time dilute and detect them with 0.01M PBS. The measured results are shown in Table 3:

[0139] Table 3 Absorbance values of the 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 using the culture supernatant of the hybridoma cell line, cell lines that stably secrete monoclonal antibodies against respiratory syncytial virus (RSV) F protein with relatively high antibody titers can be further selected. When the cell supernatant was diluted 1:100, the OD value was greater than 0.5, and it was labeled as 9F5. The cells with a 100% positive rate after cloning were amplified and cryopreserved in liquid nitrogen.

[0142] 7. Preparation and purification of ascites

[0143] The hybridoma cell line 9F5 was injected into the abdominal cavity of 8 - 10-week-old female BALB / c mice pretreated with liquid paraffin at a dose of 1×10 6 / mouse. After 10 - 14 days of feeding and observation, ascites was collected when the mouse's abdomen became enlarged. The monoclonal antibody was purified by affinity chromatography using Protein A Sepharose, and its purity was determined by SDS-PAGE, with a purity of over 90%.

[0144] Example 2

[0145] In this example, the characteristics of the monoclonal antibody were identified.

[0146] 1. Determination of antibody concentration: The ascites prepared from the hybridoma cell 9F5 was purified to obtain a monoclonal antibody against respiratory syncytial virus (RSV) F protein, and its concentration was measured using a Nanodrop nucleic acid and protein analyzer produced by Thermofisher, and the concentration was >1 mg / ml.

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

[0148] 3. Titer determination of purified antibody: 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 overnight at 4°C. The plate was washed three times with PBS (containing 0.05% v / v Tween-20), blocked with 150 μL / well of 10 mM PBS containing 1% BSA at 37°C for 2 h, and then washed three times with PBS (containing 0.05% v / v Tween-20). 100 μL of purified antibody S1 was added to each well and serially diluted from 1 μg / ml to S7 in a 1:3 gradient, incubated at 37°C for 1 h, washed three times with PBS (containing 0.05% v / v Tween-20), and then added with horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody as the 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, developed with TMB, incubated at 37°C for 15 min, and then the reaction was terminated by adding 2 M H2SO4 solution. The absorbance was measured at 450 nm with an enzyme-linked immunosorbent assay reader. For the 1 mg / mL antibody, the titer of 9F5 antibody could reach 81000 in positive wells (P / N > 2.0).

[0149] Table 4 Absorbance values of 9F5 antibody titer

[0150]

[0151] 4. Antibody binding capacity test:

[0152] Respiratory syncytial virus (RSV) F protein was diluted to 0.5 μg / mL and 1 μg / mL respectively with 1×CB, added to the wells of an enzyme-linked immunosorbent assay plate at a volume of 100 μL / well, and duplicate wells were set up, and incubated overnight at 4°C or adsorbed at 37°C for 2 h. The coated microplate was drained, washed once according to the operation program (AFP program) set by the plate washer, blocked with the blocking solution at a volume of 200 μL / well, incubated in a 37°C incubator for 2 h, and then incubated overnight at 4°C. Before use, the blocked microplate was taken out from 4°C, drained, and the enzyme-linked immunosorbent assay plate was moistened with the washing solution (1×PBS-T); 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. After dilution by 10 times to 3 μg / mL as the highest concentration (S1), it was then serially diluted in a 1:3 gradient (diluted in a 96-well deep plate), with a total of 8 dilution gradients (S1 - S8).

[0153] Add 100 μL of the diluted antibody to a 96-well microplate that has been patted dry on absorbent paper, and incubate at 37 °C for 30 min. After incubation, centrifuge the microplate to discard the liquid, pat it dry on absorbent paper, and wash the microplate 3 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, centrifuge the microplate to discard the liquid, pat it dry on absorbent paper, and wash the microplate 3 times with a plate washer. Add 100 μL of GAM-HRP enzyme-labeled secondary antibody diluted 10,000-fold with secondary antibody diluent to each well, and incubate at 37 °C for 30 min. After incubation, centrifuge the microplate to discard the liquid, pat it dry on absorbent paper, and wash the microplate 3 times with a plate washer. Take the TMB chromogenic solution and add 100 μL of the chromogenic 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 / 630 nm.

[0154] Under the conditions of antigen coating at 0.5 μg / mL and 1 μg / mL respectively, the ELISA antigen-antibody binding force experiment was tested, and data ( Figure 6 ) were obtained by measuring the absorbance OD, and the corresponding polynomial curves were 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 antigen coating at 0.5 μg / mL and 1 μg / mL reflected the specificity of the binding, and the concentrations corresponding to the antibody binding force reached 6.85E-10 mol / L (under the condition of 0.5 μg / mL antigen coating, 50% of the highest OD reading, that is, when the concentration of the antigen-antibody complex accounted for half of the total concentration, the corresponding concentration value of the antibody K 0.5 ) and 6.0E-10 mol / L (under the condition of 1 μg / mL antigen coating, 50% of the highest OD reading, that is, when the concentration of the antigen-antibody complex accounted for half of the total concentration, the corresponding concentration value of the antibody K1). 40562E-10

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

[0156]

[0157] 5. Specificity detection:

[0158] Biotin-labeled antibody

[0159] The antibody was dialyzed using a 20 mM PB (pH 8.5) solution, changed the dialysis solution three times at 4 °C. Dissolve biotin in DMF (dimethylformamide) to 2 mg / ml, measure the volume of biotin according to the antibody:biotin molar ratio of 1:20, add it to the dialyzed antibody, vortex and mix well, and let it react in the dark for 2 h. Dialyze the conjugation product using a 0.01 M PBS solution, place it at 4 °C, and change the dialysis solution three times.

[0160] Screening by double antibody sandwich ELISA

[0161] Dilute the coated RSV antibody to 1 μg / mL with 1×CB, add it to the wells of the enzyme-linked immunosorbent assay (ELISA) plate at a volume of 100 μL / well, and place it at 4 °C overnight or adsorb it at 37 °C for 2 h. Discard the liquid in the coated microplate by shaking, wash it once according to the operating procedure set by the plate washer (AFP program), add the blocking solution at a volume of 200 μL / well, place it in an incubator at 37 °C for 2 h, and then place it at 4 °C overnight.

[0162] Before use, take out the blocked microplate from 4 °C and discard the liquid by shaking. Dilute the respiratory syncytial virus (RSV) F protein to 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 1 μg / mL with the secondary antibody diluent and incubate it at 37 °C for 30 min. After incubation, wash the ELISA plate 3 times with a plate washer and pat dry each well on the absorbent paper. Add 100 μL of RSV biotin-labeled antibody diluted to 0.2 μg / ml with the secondary antibody diluent in advance to each well, incubate it at 37 °C for 30 min. After incubation, wash the ELISA plate 3 times with a plate washer, discard the liquid by shaking, and pat dry each well on the absorbent paper. Add 100 μL of SA-HRP (1 mg / ml) diluted 30,000 times with the secondary antibody diluent in advance to each well, incubate it at 37 °C for 30 min. After incubation, wash the ELISA plate 3 times with a plate washer, discard the liquid by shaking, and pat dry each well on the absorbent paper. Take the TMB chromogenic solution and add 100 μL of the chromogenic solution to each well, incubate it at 37 °C for 10 min; after chromogenesis, add 50 μL of the termination solution to each well. Set the ELISA reader to read the value at 450 nm.

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

[0164] Table 5A

[0165]

[0166] Table 5B

[0167]

[0168]

[0169] Table 5C Table 5D

[0170]

[0171] Table 5E

[0172]

[0173]

[0174] Table 5F

[0175]

[0176] Table 5G

[0177]

[0178] Table 5H

[0179]

[0180]

[0181] Table 5I

[0182]

[0183] Table 5J

[0184]

[0185] It can be seen from Table 5A to Table 5J that by comparing the experimental data corresponding to the antibody pairing experiment, it shows that except for the detection wells corresponding to the enzyme-labeled antibody that is the same as the coated antibody being blocked, 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 shows an increase in the reading value with the increase in the concentration of the added antigen. Therefore, it reflects the specificity of 9F5 binding as the coated antibody. And when paired with 5A5 as the labeled antibody to form an immunocapture assay, it shows the best sensitivity to the respiratory syncytial virus (RSV) antigen.

[0186] 6. Monoclonal antibody sequencing

[0187] Approximately 44 mg of the monoclonal antibody against respiratory syncytial virus (RSV) F protein prepared from the purified hybridoma cell 9F5 (batch number V20220526; 3.22 mg / mL). 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 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, and bispecific antibodies, and for the development of related products for diagnostic or therapeutic use.

[0188] Example 3

[0189] In this example, a lateral flow immunochromatographic detection reagent was developed using a monoclonal antibody against syncytial virus F protein.

[0190] The antibody described in 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.

[0191] 1. Preparation of colloidal gold:

[0192] Prepared by the reduction method, and the gold preparation conditions are 5 mL of 2% chloroauric acid + 10 mL of 1% trisodium citrate.

[0193] 2. Labeling of colloidal gold with mouse monoclonal antibody against respiratory syncytial virus (RSV) F protein

[0194] By the physical adsorption method, the colloidal gold is combined with the antibody (the antibody 5A5 targeting the respiratory syncytial virus (RSV) F protein, and the amino acid sequences of the light chain and heavy chain are shown in SEQ ID NO: 3 and 4 respectively) by adjusting the pH value.

[0195] The specific labeling conditions are: under the condition of 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 the mouse monoclonal antibody against respiratory syncytial virus (RSV) F protein is 10 μg / mL.

[0196] 3. Test line and control line

[0197] Test line: The mouse monoclonal antibody 9F5 against respiratory syncytial virus (RSV) F protein at an appropriate concentration (1.5 mg / mL) is coated on the nitrocellulose membrane to prepare the test line. Dried at 37 °C. The spraying amount is 0.1 μL / mm.

[0198] Quality control line: Take 0.5 mg / mL goat anti-mouse IgG polyclonal antibody and prepare a quality control line on the fiber membrane, then dry it at 37 °C. The spraying volume is 0.1 μL / mm.

[0199] Spray the colloidal gold-labeled mouse anti-respiratory syncytial virus (RSV) F protein monoclonal antibody in Step 2 onto the polyester membrane, dry it, and prepare a colloidal gold conjugate pad. Then lay the sample pad, the colloidal gold conjugate pad, the nitrocellulose membrane sprayed with the test line and the quality control line, and the absorbent paper on the PVC bottom plate in the designed order.

[0200] 4. Detection method

[0201] Bring the test card (test strip), sample diluent, and sample to 18 - 30 °C. The detection method of the test card or test strip is as follows:

[0202] a. Take out the test card or test strip from the aluminum foil bag, mark the sample, and place it flat on the horizontal workbench.

[0203] b. Take 20 μL of the nasopharyngeal or oropharyngeal swab sample extract and directly add it to the sample addition hole (test card) or the sample addition place at the lower end of the indicator arrow (test strip).

[0204] c. Then add 100 μL (2 - 3 drops) of the sample diluent.

[0205] d. Interpret the result within 15 - 20 minutes. The test result is invalid after 20 minutes.

[0206] 5. Interpretation of test results

[0207] a. Positive test line: The test line and the quality control line are colored. It indicates that the F protein antigen of respiratory syncytial virus (RSV) is detected in the sample, and it may be in the early stage of infection or current infection. Final confirmation is required by combining with clinical symptoms.

[0208] b. Negative: Only one red quality control line appears in the test window. It means that the F protein antigen of respiratory syncytial virus (RSV) is not detected in the sample.

[0209] c. Invalid: No red quality control line appears in the test window.

[0210] Example 4

[0211] This example provides a performance test on the respiratory syncytial virus antigen lateral flow immunoassay reagent provided in Example 3.

[0212] 1. Sensitivity test

[0213] a. Inactivated syncytial virus culture test

[0214] The lateral flow immunoassay rapid test reagent developed using the antibody of the present invention was used to detect the inactivated syncytial virus culture, and the virus culture as low as 10 TCID 50 / mL could be detected at the lowest.

[0215] TCID 50 / mL refers to the amount of virus required to cause cytopathic effect (CPE) in half of the cells in a culture plate well or test tube, which is used to characterize the virus titer. Note: The amount of virus here is not the specific concentration, but the dilution factor of the original sample. For example, if 1 mL of the culture medium is diluted 1000 times and exactly causes 50% of the cells to be infected, then the TCID 50 is 1000 / mL. It represents the dilution factor required for the virus contained in each mL of the sample to cause 50% cell infection.

[0216] Therefore, the larger the TCID 50 value, the more virus copies in the original solution of the inactivated supernatant, and the greater the dilution factor required (to achieve the effect of infecting 50% of the cells). Then, the test strips of the kit were respectively detected with the corresponding original solution of the inactivated supernatant, and the corresponding gradient results were as Figure 3 shown. 100 TCID 50 / mL represents the sample with the least virus copies in the original solution. That is, the virus culture as low as 100 TCID 50 / mL could be detected at the lowest.

[0217] b. Test of purified respiratory syncytial virus (RSV) F protein

[0218] The lateral flow immunoassay rapid test reagent developed using the antibody of the present invention was detected with the standard substance of the respiratory syncytial virus (RSV) F protein solution produced by Hangzhou Huakui Jinpei Biotechnology Co., Ltd. As Figure 4 shown, the standard recombinant F protein of respiratory syncytial virus (RSV) as low as 50 ng / mL could be detected.

[0219] 2. Specificity test (clinical sample test)

[0220] The nasal swab samples of 8 normal people were detected with the lateral flow immunoassay rapid test reagent developed using the antibody of the present invention, and the detection results were as Figure 5 shown. It can be seen from Figure 5 this that the detection results are clearly visible and the background is clean, indicating that the product has good specificity.

[0221] Example 5

[0222] In this example, a double-antibody sandwich enzyme-linked immunosorbent assay platform detection reagent was developed using the monoclonal antibody of syncytial virus F protein.

[0223] 1. Biotin-labeled antibody

[0224] Dialyze the 5A5 antibody with 20 mM PB (pH 8.5) solution, place it at 4 °C and change the solution three times. Dissolve biotin in DMF (dimethylformamide) to 2 mg / ml. Measure the volume of biotin according to the antibody:biotin molar ratio of 1:20, add it to the dialyzed antibody, vortex and mix well, and react in the dark for 2 h. Dialyze the conjugation product with 0.01 M PBS solution, place it at 4 °C, and change the solution three times. After dialysis, measure the concentration of 5A5-Biotin using a Nanodrop nucleic acid and protein analyzer produced by Thermo Fisher, add an equal volume of glycerol and store at -20 °C.

[0225] 2. Detection of respiratory syncytial virus (RSV) F protein by double antibody sandwich ELISA

[0226] Dilute the coated 9F5 antibody to 1 μg / mL with 1×CB, add it to the wells of the enzyme-linked immunosorbent assay (ELISA) plate at a volume of 100 μL / well, and place it at 4 °C overnight or adsorb at 37 °C for 2 h. Discard the liquid in the coated microplate by shaking, wash it once according to the operating procedure (AFP program) set by the plate washer, add the blocking solution at a volume of 200 μL / well, place it in a 37 °C incubator for 2 h, and then place it at 4 °C overnight.

[0227] Before use, take out the blocked microplate from 4 °C and discard the liquid by shaking. Dilute the respiratory syncytial virus (RSV) F protein to 1.37 ng / ml, 4.12 ng / ml, μg / mL, 12.35 ng / ml, 37.04 ng / ml, 0.11 μg / ml, 0.33 μg / ml, 1 μg / mL with the secondary antibody diluent and incubate at 37 °C for 30 min. After incubation, wash the ELISA plate 3 times with the plate washer, pat dry each well on the absorbent paper, add 100 μL of RSV biotin-labeled antibody diluted to 0.2 μg / ml with the secondary antibody diluent to each well, incubate at 37 °C for 30 min. After incubation, wash the ELISA plate 3 times with the plate washer, discard the liquid by shaking, pat dry each well on the absorbent paper, add 100 μL of SA-HRP (1 mg / ml) diluted 30,000 times with the secondary antibody diluent to each well, incubate at 37 °C for 30 min. After incubation, wash the ELISA plate 3 times with the plate washer, discard the liquid by shaking, pat dry each well on the absorbent paper, take the TMB chromogenic solution, add 100 μL of the chromogenic solution to each well, and incubate at 37 °C for 10 min; after color development, add 50 μL of the termination solution to each well. Set the ELISA reader to read the value at 450 nm.

[0228] The detection reagent of the double-antibody sandwich enzyme immunoassay platform developed based on the antibody of the present invention was used to detect the standard substance of the respiratory syncytial virus (RSV) F protein solution produced by Hangzhou Huakuijinpei Biotechnology Co., Ltd. As shown in Table 6, the standard recombinant F protein of the respiratory syncytial virus (RSV) with a minimum detectable level of 4.12 ng / mL could be detected.

[0229] Table 6 Detection results of the double-antibody sandwich enzyme immunoassay platform for standard substances of RSV F protein solutions with different concentrations

[0230]

[0231]

[0232] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An antibody against respiratory syncytial virus F protein or an antigen-binding fragment thereof, characterized in that, It includes a light chain complementary determining region and a heavy chain complementary determining region. The light chain complementary determining region includes CDR-L1, CDR-L2, and CDR-L3, and their amino acid sequences are shown as SEQ ID NO: 10-12 in sequence; the heavy chain complementary determining region includes CDR-H1, CDR-H2, and CDR-H3, and their amino acid sequences are shown as SEQ ID NO: 13-15 in sequence.

2. The antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain framework region and a light chain framework region.

3. The antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to claim 2, characterized in that, The light chain framework region includes LFR1, LFR2, LFR3, and LFR4 which have at least 80% homology with the amino acid sequences shown as SEQ ID NO: 16-19 in sequence; The heavy chain framework region includes HFR1, HFR2, HFR3, and HFR4 which have at least 80% homology with the amino acid sequences shown as SEQ ID NO: 20-23 in sequence.

4. The antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to claim 2, characterized in that, The antibody or its antigen-binding fragment further includes a constant region, and the constant region includes a heavy chain constant region and / or a light chain constant region.

5. The antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to claim 4, wherein The heavy chain constant region is selected from the heavy chain constant regions 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 regions.

6. The antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to claim 5, wherein, The heavy chain constant region is selected from IgG1, and the light chain constant region is selected from the κ-type light chain constant region.

7. The antibody or antigen-binding fragment thereof against respiratory syncytial virus F protein according to claim 4, characterized in that, The species origin of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, rabbit, chicken, or human.

8. The antibody or antigen-binding fragment thereof against respiratory syncytial virus F protein according to claim 7, wherein The species origin of the constant region is mouse or human.

9. The antibody against respiratory syncytial virus F protein or an antigen-binding fragment thereof according to claim 4, wherein The amino acid of the light chain of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 1, and the amino acid of the heavy chain of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:

2.

10. The antibody against respiratory syncytial virus F protein or its antigen-binding fragment 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.

11. A biological product, characterized in that, It includes the antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to any one of claims 1-10, and the biological product is selected from reagents, reagent kits, test strips, antibody chips, antibody probes, and detectors.

12. The biological product according to claim 11, wherein The reagent includes: the antibody against respiratory syncytial virus F protein or its antigen-binding fragment, and the antibody or its antigen-binding fragment is labeled with a detectable label.

13. The biological product according to claim 12, wherein The detectable label is selected from fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

14. The biological product according to claim 11, wherein, The reagent kit includes a solid phase, and the antibody or its antigen-binding fragment is coated on the solid phase.

15. The biological product according to claim 14, wherein, The solid phase is selected from microspheres, plates, and membranes.

16. The biological product according to claim 14, wherein, The solid phase is selected from magnetic microspheres, plastic microspheres, plastic particles, latex microspheres, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

17. The biological product according to claim 11, wherein The test strip includes a sample pad, a conjugate pad, an NC membrane, an absorbent pad, and a base plate; on the NC membrane, there are a test line and a control line, the test line is coated with a test antibody, the control line is coated with a polyclonal antibody, the conjugate pad is coated with a capture antibody labeled with a label, and the epitopes of the test antibody and the capture antibody are different; the test antibody is the antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to any one of claims 1-10, or another antibody against respiratory syncytial virus F protein or its antigen-binding fragment; the capture antibody is the antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to any one of claims 1-10, or another antibody against respiratory syncytial virus F protein or its antigen-binding fragment.

18. The biological product according to claim 17, characterized in that, The label on the conjugate pad is a nanoparticle-based label.

19. The biological product according to claim 18, characterized in that, The nanoparticle-based label is selected from nanoparticles or colloids.

20. The biological product according to claim 17, characterized in that, The amino acid sequences of the light chain and heavy chain of the another antibody against respiratory syncytial virus F protein or its antigen-binding fragment are respectively as shown in SEQ ID NO: 3-4.

21. The biological product according to claim 17, wherein The test antibody is the antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to any one of claims 1-10, and the capture antibody is another antibody against respiratory syncytial virus F protein or its antigen-binding fragment.

22. Use of the antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to any one of claims 1-10 in the preparation of a respiratory syncytial virus detection product, a diagnostic product, or a respiratory syncytial virus enrichment product.

23. The application according to claim 22, wherein, The detection product or diagnostic product is a reagent, a kit, a test strip, an antibody chip, an antibody probe, or a detector.

24. A nucleic acid molecule, characterized in that, It encodes the antibody against respiratory syncytial virus F protein or its antigen-binding fragment according to any one of claims 1-10.

25. A recombinant vector, characterized in that, It includes the nucleic acid molecule according to claim 24.

26. A recombinant cell, characterized in that, It includes: The recombinant vector according to claim 25.

Images