Antigen detection kit developed based on human metapneumovirus F protein monoclonal antibody and application thereof

Abstract

The invention discloses an antigen detection kit developed based on a human metapneumovirus F protein monoclonal antibody and application of the antigen detection kit, and belongs to the technical field of antibodies. The monoclonal antibody is a monoclonal antibody 1 and / or a monoclonal antibody 2; the amino acid sequence of a light chain variable region of the monoclonal antibody 1 is as shown in SEQ ID NO.6, and the amino acid sequence of a heavy chain variable region of the monoclonal antibody 1 is as shown in SEQ ID NO.8; the amino acid sequence of the light chain variable region of the monoclonal antibody 2 is as shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2 is as shown in SEQ ID NO.4. The kit containing the two monoclonal antibodies is used for detecting the metapneumovirus, has the characteristics of high sensitivity and high specificity, and can realize rapid detection and auxiliary diagnosis of HMPV infection. Therefore, the monoclonal antibody for resisting the F protein of the HMPV and the detection kit of the monoclonal antibody have wide application prospects and auxiliary diagnosis values.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibodies, and particularly relates to an antigen detection kit based on a human metapneumovirus F protein monoclonal antibody and an application thereof. BACKGROUND

[0002] Respiratory tract infection is a worldwide health problem, which occupies a large amount of medical resources and causes heavy economic burden to families and society. Respiratory tract infection is a common disease in children, which ranks first in the incidence and mortality of children. Human metapneumovirus (HMPV), one of the main pathogens of respiratory tract infection, was first isolated in 2001. HMPV is the second important virus causing bronchiolitis in infants and young children after RSV. HMPV-positive patients show a wide range of clinical features, including cough, abnormal lung breath sounds, fever, sputum, rhinitis and wheezing, from mild upper respiratory tract infection to life-threatening lower respiratory tract infection. HMPV infection causes severe bronchiolitis and pneumonia in children, and the symptoms are difficult to distinguish from those caused by RSV. Children and infants, the elderly, people with underlying heart and lung diseases, and people with low immune function are more susceptible to the virus and more likely to develop severe illness. It is reported that about 5-15% of hospitalized children with respiratory diseases are caused by HMPV infection. Children with complex chronic diseases are more common due to HMPV infection. In addition, HMPV infection increases the susceptibility to bacterial superinfection, leading to increased morbidity and mortality. HMPV infection requires our timely attention, especially for children, people with underlying diseases and other people with low immune function.

[0003] Early detection and accurate treatment are the main ways to reduce HMPV infection. The main methods for identifying viral infection in clinical practice include in vitro culture, nucleic acid detection, antigen or antibody detection, etc. Because viruses grow slowly in cell culture, nucleic acid detection methods are the preferred diagnostic methods for identifying viruses. The nucleic acid detection methods currently used in clinical practice include ordinary PCR, fluorescent quantitative PCR, isothermal amplification, high-throughput sequencing, etc. Molecular diagnostic methods have the characteristics of accuracy and reliability, but such detection methods require special and expensive instruments, require high professional skills, have long detection time, and are expensive, which limits their widespread application. Antigen or antibody detection is a method for detecting antigens of pathogens or antibodies induced by pathogens in the body. Known antibodies are used to detect unknown antigens or known antigens are used to detect unknown antibodies, and the antigens or antibodies in the sample are quantitatively or qualitatively detected. The antibody detection method has a long window period, is lagging, and often has false positive results due to cross-reactions in the detection process. Although the antigen detection has lower sensitivity than nucleic acid detection, it has the advantages of low cost, short time, simple operation, and no need for professional equipment.

[0004] HMPV is an RNA virus belonging to the Paramyxoviridae family. The genome of HMPV encodes 9 proteins. F, G and SH are HMPV surface transmembrane glycoproteins, and the sequence of F protein is relatively conserved among different HMPV genotypes compared with G and SH. In addition, it has been found that the F protein of HMPV can induce neutralizing antibodies after infecting hamsters, suggesting that the F protein as an immunogen can stimulate animals to produce better antibodies. These research results show that the F protein of HMPV has high antigenicity and conservation. The antigen detection based on HMPV F protein has specificity for HMPV, which can effectively reduce the problems of misdiagnosis and missed diagnosis of HMPV. However, there is no antigen detection reagent based on HMPV F protein at present. SUMMARY

[0005] The present application provides a monoclonal antibody against human metapneumovirus F protein, the monoclonal antibody is monoclonal antibody 1 and / or monoclonal antibody 2; the amino acid sequence of the light chain variable region of the monoclonal antibody 1 is shown in SEQ ID NO. 6, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 8; the amino acid sequence of the light chain variable region of the monoclonal antibody 2 is shown in SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 4.

[0006] In an embodiment of the present application, the nucleotide sequence of the light chain variable region of the monoclonal antibody 1 is shown in SEQ ID NO. 5, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 7.

[0007] In an embodiment of the present application, the nucleotide sequence of the light chain variable region of the monoclonal antibody 2 is shown in SEQ ID NO. 1, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 3.

[0008] SEQ ID NO. 1:

[0009] CAGGTTCAGCTGAAGCAGTCTGAAACTGACCTGGCGAGGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGTCTTCTGGCTATATCTTCACAAGGTTTGGTATAAGTTGGGTGAGGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAAATTTATCCTACCAGTGGTAAGACTGACTACAATGAGAATTTCAAGGGCAAGGCCACACTGACTGCAGACACGTCCTCCAGCGCAGCGTTCATGGAATTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCGGGATATACTATGAGTACGACGGGCTATGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0010] SEQ ID NO. 2:

[0011] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGINNRPPGVPARFSGSVIGDKAALTITGAQTEDEAIYFCALWYNNHSVFGGGTKLTVL

[0012] SEQ ID NO. 3:

[0013] CAGGTTCAGCTGAAGCAGTCTGAAACTGACCTGGCGAGGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGTCTTCTGGCTATATCTTCACAAGGTTTGGTATAAGTTGGGTGAGGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGAAATTTATCCTACCAGTGGTAAGACTGACTACAATGAGAATTTCAAGGGCAAGGCCACACTGACTGCAGACACGTCCTCCAGCGCAGCGTTCATGGAATTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCGGGATATACTATGAGTACGACGGGCTATGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0014] SEQ ID NO. 4:

[0015] QVQLKQSETDLARPGASVKMSCKSSGYIFTRFGISWVRQRTGQGLEWIGEIYPTSGKTDYNENFKGKATLTADTSSSAAFMEFRSLTSEDSAVYFCARSGYTMSTTGYDYWGQGTTLTVSS

[0016] SEQ ID NO. 5:

[0017] GACATTGTGATGAGCCAGTCTCAAAAATTCATGTCCACATCAGTCGGAGACAGAGTCAGCATCAGCTGCAAGGCCAGTCAGAATGTGGGTAATCTTATAGCCTGGTATCAACAGATACCAGGACAATCTCCTAAAGCATTGATTTACTTGTCATCCTACCGCTACAGTGGAGTCCCTGATAGCTTCGCAGGCAGTGGATCTGGGACAGATTTCACTCTCATCATTAGTAATGTGCAGTCTGAAGACTTGGCAGACTATTTCTGTCACCAATATGGCAGCTCTCCGAATACATTCGGATCGGGGACCAGGTTGGAAATAGAA

[0018] SEQ ID NO. 6:

[0019] DIVMSQSQKFMSTSVGDRVSISCKASQNVGNLIAWYQQIPGQSPKALIYLSSYRYSGVPDSFAGSGSGTDFTLIISNVQSEDLADYFCHQYGSSPNTFGSGTRLEIE

[0020] SEQ ID NO. 7:

[0021] GAGTTCCAGTCGCAGCAATCTGGACCTGAGCTGGTGAAGCCTGTCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTATTCATTCACTGACCACAACATGAACTGGGTGAAGCAACACAATGGAAAGAGCCTTGAGTGGATTGGAGTAATTAATCCTCACTATGGGTCTTCTACCTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGTGGACCAATCGTCCAGCACAGCCTACATGCAGCTCATCAGCCTGACATCTGAAGATTCTGCAGTCTATTACTGTGCAAGAGGGGGATTACGACGGGACAATGCTATGGACTACTGCGGTCAAGGAACTTCAGTCACCGTCTCCTCA

[0022] SEQ ID NO. 8:

[0023] EFQSQQSGPELVKPVASVKISCKASGYSFTDHNMNWVKQHNGKSLEWIGVINPHYGSSTYNEKFKGKATLTVDQSSSTAYMQLISLTSEDSAVYYCARGGLRRDNAMDYCGQGTSVTVSS

[0024] The present application also provides a nucleic acid molecule carrying a gene encoding the monoclonal antibody.

[0025] The present application also provides a host cell carrying the nucleic acid molecule and capable of autonomously expressing the monoclonal antibody.

[0026] The present application also provides a kit for detecting human metapneumovirus F protein, which contains the monoclonal antibody 1 and the monoclonal antibody 2.

[0027] In one embodiment of the present application, the monoclonal antibody 1 is used as a detection antibody and the monoclonal antibody 2 is used as a capture antibody.

[0028] In one embodiment of the present application, the kit contains a test strip, which comprises a sample pad, a conjugate pad, a PVDF membrane and an absorbent paper, and the preparation method of the test strip comprises the following steps:

[0029] (1) The monoclonal antibody 1 is labeled with red latex microspheres;

[0030] (2) The detection line of the PVDF membrane is coated with monoclonal antibody 2, and the coating concentration of monoclonal antibody 2 is 1.5 mg / mL;

[0031] (3) Monoclonal antibody 1 labeled with red latex microspheres at a concentration of 50 ug / mL adsorbed onto the conjugate pad.

[0032] In one embodiment of the present invention, the preparation method of the test strip further includes: coating the control line of the PVDF membrane with goat anti-mouse IgG antibody.

[0033] The present invention also provides the application of the above-mentioned monoclonal antibody in the preparation of reagents or kits for detecting human metapneumovirus F protein.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) Based on hybridoma cell technology, this invention obtained two monoclonal antibodies, 4-1B and 2-11G, against the F protein of HMPV, and sequenced the light chain variable region and heavy chain variable region of 4-1B (monoclonal antibody 2) and 2-11G (monoclonal antibody 1). The titers of the 4-1B and 2-11G monoclonal antibodies were analyzed by ELISA. The OD450 values ​​of the 4-1B and 2-11G monoclonal antibodies for binding to the F protein were 3.063 and 3.137, respectively, indicating high binding capacity to the F protein.

[0036] (2) This invention uses immunochromatography to pair self-developed 4-1B and 2-11G monoclonal antibodies, and uses a kit containing 4-1B and 2-11G monoclonal antibodies to detect HMPV, which has the characteristics of high sensitivity and high specificity. This kit can realize rapid detection and auxiliary diagnosis of HMPV infection, and is simple and fast to operate, suitable for large-scale rapid clinical examinations, as well as primary medical units, epidemiological screening, home self-testing, etc., to realize rapid monitoring and screening of HMPV. Therefore, the anti-HMPV F protein monoclonal antibody and its detection kit described in this invention have broad application prospects and auxiliary diagnostic value. Attached Figure Description

[0037] Figure 1 The purification status of F protein was analyzed by SDS-PAGE in Example 1, where M: Markers: 1: Recombinant F protein (without DTT); 2: Recombinant F protein (with DTT).

[0038] Figure 2 This is the screening result of different coating concentrations of the capture antibody in Example 4.

[0039] Figure 3 This is the screening result of detecting different antibody labeling concentrations in Example 4.

[0040] Figure 4 The results of the screening of different coating concentrations of the blocking agent and C-line sheep anti-mouse IGG in Example 4.

[0041] Figure 5 The results of the screening of different concentrations of YGBP03 blocking agent in Example 4.

[0042] Figure 6 The results of the determination of the minimum detection limit of the method of the application in Example 4.

[0043] Figure 7 The cross-reaction of the method of the application with other respiratory viruses in Example 4, wherein 1: Mycoplasma pneumoniae and rhinovirus positive nasopharyngeal swab samples; 2: bocavirus, rhinovirus and parainfluenza virus positive nasopharyngeal swab samples; 3: adenovirus positive nasopharyngeal swab samples; 4: coronavirus positive nasopharyngeal swab samples; 5: respiratory syncytial virus and rhinovirus positive nasopharyngeal swab samples; 6: influenza A virus and H1N1 positive nasopharyngeal swab samples; 7: Mycoplasma pneumoniae positive nasopharyngeal swab samples; 8: respiratory syncytial virus positive nasopharyngeal swab samples; 9: Mycoplasma pneumoniae, influenza A virus and H1N1 positive nasopharyngeal swab samples. DETAILED DESCRIPTION

[0044] The instruments used in the embodiments of the application are as follows:

[0045] (1) Frozen high-speed centrifuge (Eppendorf 5424R); (2) Vortex shaker (Vortex-Genie 2); (3) XYZ three-dimensional membrane drawing and gold spraying instrument (Shanghai Jinbiao Biological Technology Co., Ltd., HM3030); (4) Numerical control strip cutting machine (Shanghai Jinbiao Biological Technology Co., Ltd., CTS300); (5) Microcomputer automatic cutting machine (Shanghai Jinbiao Biological Technology Co., Ltd., ZQ2002); (6) Ultrasonic cleaner (Jieliang, JP-020S); (7) PH meter (Sartorius, PB10).

[0046] The reagents and consumables used in the embodiments of the application are as follows:

[0047] (1) Nitrocellulose membrane (Sartorius, CN95); (2) Glass fiber membrane (Shanghai Jeten Biotech Co., Ltd., G-4); (3) Absorbent paper (Shanghai Jeten Biotech Co., Ltd., H-2); (4) Bottom plate (Shanghai Jeten Biotech Co., Ltd., J-B6); (5) Desiccant (Shanghai Jeten Biotech Co., Ltd., jn8709); (6) Aluminum foil bag (Shanghai Jeten Biotech Co., Ltd., jn8920); (7) Red latex microspheres (Duoer Material Technology Co., Ltd., DR0400CA); (8) Triangular culture bottle (CORNING, 431143); (9) lipo3000 (Invitrogen, L3000015); (10) HRP-labeled mouse anti-human IgG (Genview Biotech, CA-001); (11) Goat anti-mouse IgG (Genview Biotech, AB-055A); (12) Tween-20 (sigma, P9416-100mL); (13) MES (aladdin, M16014); (14) NaH2PO4 (BBI, A600878-0500); (15) Na2HPO4 (BBI, A610404-0500); (16) BSA (Hangzhou Tianlai Biotechnology; GHB034); (17) Sucrose (Diamond, A100335-0250); (18) PVP (K30) (BBI, A620435-0250); (19) YGBP03 blocking agent (Wuhan Genview Biotech Co., Ltd., PAB202403002); (20) YGBP02 blocking agent (Wuhan Genview Biotech Co., Ltd., PAB202403001); (21) R-040 blocking agent (Feifeng Biotechnology Co., Ltd., HIER-R-040); (22) N-Hydroxysuccinimide (NHS) (sigma, 130672); (23) N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochonide (EDC) (sigma, E7750).

[0048] Example 1 HMPV F protein expression and purification

[0049] The nucleic acid sequence of F protein (PDB: 7TJQ_C) was constructed into pCAGGS plasmid, 293F cells were inoculated in 100 mL medium, and the cells were grown to 80% density for transfection. The expression plasmid was mixed with the transfection reagent lipo3000 in the culture medium, and incubated at room temperature for 15-20 minutes. The expression plasmid-transfection reagent complex was added dropwise to the 293T cell culture dish, shaken gently, and returned to the incubator for further culture. After 48 hours, the cells were collected and the recombinant F protein was purified.Figure 1 As shown in Figure 2, the F protein was successfully expressed in the E. coli. Figure 1 As shown in Figure 2, the F protein was successfully expressed in the E. coli.

[0050] Example 2 Analysis of the effectiveness of the recombinant HMPV F protein

[0051] Serum samples from two patients positive for HMPV and one patient negative for HMPV were collected. The binding of the recombinant F protein to the serum of the HMPV-positive patients was detected by ELISA to further verify whether the recombinant F protein has natural activity.

[0052] The recombinant F protein was diluted with coating solution (containing 0.015 mol / L Na2CO3 + 0.035 mol / L NaHCO3) to a concentration of 0.5 ug / mL, 1.0 ug / mL, and 2.0 ug / mL. 100 uL of the F protein at different concentrations was added to each well of the enzyme-labeled plate and placed in a refrigerator at 4°C overnight. The enzyme-labeled plate was removed after adsorption was completed, the liquid in the wells was shaken off and dried on a special towel, and 300 uL of washing solution (containing PBS + 0.05% Tween-20 + 1% PC-300, pH 7.2) was added to each well of the enzyme-labeled plate. The plate was washed immediately, and the added washing solution was shaken off. The plate was washed once and then dried on a special towel. 150 uL of blocking solution (containing PBS + 1% BSA + 0.5% PC-300, pH 7.2) was added to each well of the enzyme-labeled plate, which was placed at room temperature for 4 hours. The positive / negative sample serum was diluted with PBST buffer (containing PBS + 0.05% Tween-20, pH 7.2) at a dilution ratio of 1:5, 1:25, 1:100, and 1:500. 100 uL of each was added to the reaction wells of the enzyme-labeled plate, mixed gently, sealed with a sealing film, and incubated in a 37°C oven for 30 minutes. The liquid in the wells was shaken off, 250 uL of washing solution was added per well, soaked for 30 seconds, and then the liquid in the wells was shaken off. The plate was washed and dried four times. 100 uL of enzyme-labeled secondary antibody (mouse anti-human IgG) was added to each well, the secondary antibody was used at a concentration of 1:5000, mixed gently, sealed with a sealing film, and incubated in a 37°C oven for 30 minutes. The liquid in the wells was shaken off, 250 uL of washing solution was added per well, soaked for 30 seconds, and then the liquid in the wells was shaken off. The plate was washed and dried four times. 100 uL of substrate (TMB) was added to each well, which was placed in a 37°C oven for color development for 15 minutes. 50 uL of stop solution (2M H2SO4) was added per well, mixed gently, and the OD value was measured at 450 nm on an enzyme-labeled instrument.

[0053] The results are shown in Table 1 below. The OD450 values ​​of the negative controls were all less than 0.25, while the OD values ​​of the two positive controls were both >0.6. Furthermore, the S / P value increased with increasing F protein coating concentration, indicating that the F antigen is an effective antigen.

[0054] Table 1. ELISA analysis of the binding of recombinant F protein to serum from HMPV-positive patients.

[0055]

[0056] Note: S / P = (Positive serum OD value - Blank OD) / (Negative control OD value - Blank OD). The optimal experimental conditions are a higher S / P value, with negative control OD value < 0.25 and positive control OD value > 0.6. If the above data can be obtained, it indicates that the antigen is an effective antigen.

[0057] Example 3: Preparation of HMPV F protein monoclonal antibody

[0058] (1) Immunized animals

[0059] Mice were injected with purified recombinant HMPV F protein. The initial immunization dose was 50 μg protein per mouse, with Freund's complete adjuvant, administered via multiple subcutaneous injections. A second immunization was administered 2 weeks later, with a dose of 50 μg metapneumovirus F protein per mouse, using Freund's incomplete adjuvant, administered intraperitoneally. A third immunization was administered 2 weeks later. The dosage, adjuvant, and injection method used for the third and subsequent immunizations were the same as for the second immunization, with a 2-week interval between each immunization, up to the fourth immunization.

[0060] (2) Cell fusion

[0061] Cell fusion was performed on immunized mice. Blood was collected from the tail vein 3 days before fusion, and antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). Once the titer reached 1:8000 or higher, mice were boosted with a dose of 50 μg metapneumovirus F protein per mouse, without adjuvant. After collecting blood from the mouse eyeballs, spleen cells from successfully immunized mice were mixed with myeloma SP2 / 0 cells at a ratio of 10:1 (fusion was performed in a 37°C water bath). The mixture was centrifuged, the supernatant discarded, and 50% PEG was added to the fusion mixture within 1 min. The mixture was incubated at 37°C with shaking for 1 min, allowed to stand for 1 min, and then 10 mL of serum-free 1640 medium was added within 2 min. The mixture was centrifuged at 800 rpm for 6 min, the supernatant discarded, and the cells were resuspended in 1640 medium containing HAT and transferred to 96-well plates. Cells were cultured at 37°C and 5% CO2. The medium was partially changed on the third day after fusion and completely changed on the seventh day.

[0062] (3) Screening and culture of positive clones

[0063] When the clones in the fusion plate are large enough, 100 μL of supernatant is taken from each well for detection, and the detection method is the same as that described above for detecting the antibody titer. Wells with OD values more than twice that of negative wells are regarded as positive wells, and the cells in the positive wells are subjected to further cloning and culture. The hybridoma clones that are screened positive are expanded from 96-well plates to 24-well plates and cultured for 3-5 days, and then subjected to further supernatant screening and detection. Positive clones are subjected to further subcloning and culture, and the remaining cells are cryopreserved. The hybridoma cells in the 24-well plates are collected, counted, and the cell density is adjusted to 10 cells / mL. The cells are plated in 96-well plates at 100 μL per well and cultured at 37°C in a 5% CO2incubator. After about 10 days of culture, clones are formed, and wells with only a single clone are selected, and the culture supernatant is removed for detection. Positive clones are selected, expanded to 24-well plates, and subjected to further supernatant detection. Positive clones are selected for a second round of subcloning and culture, and generally, multiple rounds of subcloning and culture are performed until all the detection wells are positive, i.e., a hybridoma cell line that can stably secrete mouse anti-metapneumovirus F protein monoclonal antibody is obtained.

[0064] (4) Detection of the titer of the supernatant of the hybridoma cell line

[0065] Screening positive hybridoma cell clones by ELISA (Table 2). Dilute F protein with coating solution (containing 0.015 mol / L Na2CO3 + 0.035 mol / L NaHCO3) to a concentration of 1.0 ug / mL, and add 100 uL F protein to each well of the enzyme-labeled plate. Place the enzyme-labeled plate in the refrigerator at 4°C overnight, remove the adsorbed enzyme-labeled plate, shake off the liquid in the wells and pat dry on a special towel, and add 300 uL washing solution (containing PBS + 0.05% Tween-20 + 1% PC-300, pH 7.2) to each well of the enzyme-labeled plate to start washing the plate. Immediately shake off the added washing solution, wash the plate once, and then pat dry with a special towel. Add 150 uL blocking solution (containing PBS + 1% BSA + 0.5% PC-300, pH 7.2) to each well of the enzyme-labeled plate, and place it at room temperature for 4 h. Add 100 uL volume of hybridoma cell supernatant to the reaction well, mix gently, seal the plate with a sealing film, and incubate in a 37°C oven for 30 min; shake off the liquid in the wells, add 250 uL / well of washing solution, soak for 30 s, and then shake off the liquid in the wells. Repeat the washing process 4 times and then pat dry; add 100 uL of enzyme-labeled secondary antibody (goat anti-mouse IgG) to each well, the concentration of the secondary antibody is 1:5000, mix gently, seal the plate with a sealing film, and incubate in a 37°C oven for 30 min; shake off the liquid in the wells, add 250 uL / well of washing solution, soak for 30 s, and then shake off the liquid in the wells. Repeat the washing process 4 times and then pat dry; add 100 uL of substrate (TMB) to each well, and develop color in a 37°C oven for 15 min; add 50 uL / well of stop solution (2M H2SO4), mix gently, and measure the OD value at 450 nm on an enzyme-labeled instrument.

[0066] Table 2 ELISA detection of positive hybridoma cell supernatant titer

[0067]

[0068] (5) Purification of monoclonal antibodies

[0069] Wash the hybridoma cells with sterile PBS solution, and inject 5x10 6 / 0.5 ml / mouse intraperitoneally into liquid paraffin pre-sensitized mice, collect the ascites after 7 to 10 days, centrifuge at room temperature at 3000 rpm for 10 min, and collect the supernatant. Coarsely purify the antibodies in the ascites by the caprylic acid-ammonium sulfate method, and purify the antibodies twice by 1 ml Protein G purification pre-packed column according to the purification manual provided by GE company, to obtain purified monoclonal antibodies.

[0070] Preparation of HMPV antigen detection kit (latex method)

[0071] (1) Red latex microspheres labeled 2-11G monoclonal antibody

[0072] Take 12.5 μL red latex microspheres in 487.5 μL labeling buffer (50 mM methanesulfonic acid MES, pH 6.0), mix well; take 5 μL NHS (20 mg / mL), 2.5 μL EDC (20 mg / mL) respectively into the microspheres, mix quickly, incubate at room temperature, 20 min; wash the microspheres twice with 500 μL labeling buffer by ultrasonic resuspension; take 25 ug, 50 ug, 75 ug monoclonal antibody 2-11G into 500 μL activated microspheres, mix quickly, then incubate at room temperature for 2 hours; block with 50 μL 20 mg / mL BSA (dissolved in 50 mM Tris-HCl, pH 8.0) for 1 hour at room temperature; wash 3 times with 500 μL 50 mM Tris-HCl (pH 8.0); resuspend the microspheres with 1000 μL 0.2% BSA (dissolved in 50 mM Tris-HCl, pH 8.0) by ultrasonic, which is the 2-11G monoclonal antibody-latex microsphere labeling complex.

[0073] (2) Preparation of test strip

[0074] A. Cut the PVDF film to a length of 30 cm and paste it on the PVC plate.

[0075] B. Dilute the monoclonal antibody 4-1B with the coating solution (containing 0.02 M PB + 1% BSA + 2% sucrose, pH 7.5) to 0.5 mg / mL, 0.7 mg / mL, 1 mg / mL, 1.5 mg / mL, and dilute the goat anti-mouse (GAM) IgG antibody to 1 mg / mL. Use the membrane marker to draw GAM and 4-1B on the PVDF, corresponding to C line and T line. C line is close to the absorbent paper and T line is close to the binding pad. Dry at 37°C overnight.

[0076] C. Cut the sample pad to 1.5 cm wide and 30 cm long; cut the binding pad to 1.5 cm wide and 30 cm long; treat the sample pad with 3 mL of sample pad treatment solution (containing PBS + 1% PVP (K30) + 2% sucrose + 75 ug / mL blocker, pH 7.2); treat the binding pad with 2 mL of binding pad treatment solution (containing PBS + 5% sucrose + 0.5% BSA + 0.1% Tween-20, pH 7.2). Dry at 37°C overnight.

[0077] D. Paste the treated sample pad, conjugate pad and absorbent paper on the PVC plate respectively, and cut into 3mm wide test strips with a cutting device.

[0078] E. Spot 1 μL of red latex microspheres-2-11G monoclonal antibody complex on the conjugate pad of each test strip, and assemble the test strip into the card shell according to the direction of the detection card.

[0079] (3) Result analysis of test strip

[0080] Drop 80-100 μL of the sample to be tested into the sample well of the test strip, and place it at room temperature for 5-10 minutes, and then judge the results as follows: if the C line does not appear red band, the test is invalid; if the C line and the T line appear red band, it indicates that the sample to be tested contains F protein of metapneumovirus, thereby judging that the subject is a metapneumovirus infected person; if the C line appears red band and the T line does not appear red band, it indicates that the sample to be tested does not contain F protein of metapneumovirus, thereby judging that the subject is a non-metapneumovirus infected person.

[0081] (4) Screening of 4-1B monoclonal antibody coating concentration of capture antibody (T line)

[0082] Coat 0.5 mg / mL, 0.7 mg / mL, 1 mg / mL, 1.5 mg / mL 4-1B monoclonal antibody on the T line position of the PVDF membrane, coat 1 mg / mL goat anti-mouse IgG on the C line position of the PVDF membrane, and adsorb 1 uL of 2-11G-red latex microsphere complex with a labeled concentration of 50 ug / mL on the conjugate pad of each test strip, and use 1 ng / mL F protein antigen to screen the best T line coating concentration by T line brightness. As shown in Figure 2 , when the T line is coated with 1.5 mg / mL 4-1B monoclonal antibody, the brightness of the T line is the strongest under the same antigen concentration.

[0083] (5) Screening of 2-11G monoclonal antibody labeled concentration of detection antibody

[0084] Coat 1.5 mg / mL 4-1B monoclonal antibody on the T line position of the PVDF membrane, coat 1 mg / mL goat anti-mouse IgG on the C line position of the PVDF membrane, and adsorb 1 uL of 2-11G-red latex microsphere complex with a labeled concentration of 50 ug / mL, 100 ug / mL, 150 ug / mL on the conjugate pad of the test strip, and use 1 ng / mL F protein antigen to screen the best labeled concentration of detection antibody by T line brightness. As shown in Figure 3 , when the F protein antigen concentration is consistent, the brightness of the T line is the strongest when the 2-11G labeled concentration is 50 ug / mL.

[0085] (6) Screening and quality control line (C line) of blocking agent

[0086] Coat 1.5 mg / mL 4-1B monoclonal antibody on the T line position of the PVDF membrane, coat 1 mg / mL and 1.5 mg / mL goat anti-mouse IgG on the C line position of the PVDF membrane, respectively, and adsorb 1 uL of 2-11G-red latex microsphere complex with a labeled concentration of 50 ug / mL on the conjugate pad of the test strip. Add different blocking agents in the sample diluent, and screen the optimal blocking agent and the coating concentration of the C line by the brightness of the T line and the C line. As shown in Figure 4 shown, when the antigen concentration is 1 ng / mL, the brightness of the T line and the C line does not change after adding the blocking agent YGBP03 compared with the control group (without adding blocking agent), and the brightness of the C line does not change when the coating concentration of the C line is 1 mg / mL and 1.5 mg / mL. Therefore, the blocking agent YGBP03 is selected in the subsequent experiment, and the coating concentration of the C line is 1 mg / mL.

[0087] (7) Screening of different concentrations of blocking agent YGBP03

[0088] Coat 1.5 mg / mL 4-1B monoclonal antibody on the T line position of the PVDF membrane, coat 1 mg / mL goat anti-mouse IgG on the C line position of the PVDF membrane, and adsorb 1 uL of 2-11G-red latex microsphere complex with a labeled concentration of 50 ug / mL on the conjugate pad of the test strip. Add different concentrations of 50 ug / mL, 75 ug / mL, and 100 ug / mL YGBP03 blocking agent in the sample diluent, and screen the optimal blocking agent concentration by the brightness of the T line and the C line. Figure 5 shown, when the antigen concentration is 1 ng / mL, the brightness of the T line and the C line is equivalent when the concentration of YGBP03 blocking agent is 75 ug / mL compared with the concentration of 0 ug / mL and 50 ug / mL, and therefore the concentration of 75 ug / mL YGBP03 blocking agent is selected as the optimal concentration.

[0089] (8) Detection performance of test strip

[0090] A. Minimum detection limit

[0091] Detect F protein with concentrations of 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, and 0 pg / mL, respectively, analyze the brightness of the T line band at different F protein detection concentrations, and the results are shown in Figure 6As shown, when the antigen concentration is 62.5 pg / mL, the T-band is very weak, and when the antigen concentration is 31.25 pg / mL, the T-band shows no color development. Therefore, the detection limit of the F protein antigen in this method is 62.5 pg / mL.

[0092] B. Cross-reactivity

[0093] The antigen detection kit established in this invention was evaluated for cross-reactivity using nasopharyngeal swab samples that tested positive for clinical influenza A virus, H1N1, parainfluenza virus, rhinovirus, adenovirus, mycoplasma pneumoniae, chlamydia pneumoniae, respiratory syncytial virus, coronavirus, and bocavirus nucleic acid. The results are shown in [reference needed]. Figure 7 .Depend on Figure 7 It is known that the method of the present invention has no cross-reaction with influenza A virus, H1N1, parainfluenza virus, rhinovirus, adenovirus, mycoplasma pneumoniae, chlamydia pneumoniae, respiratory syncytial virus, coronavirus, and bocavirus.

[0094] C. Sensitivity, specificity, and consistency

[0095] 134 clinical samples were collected. The detection reaction system established in this invention was used as the experimental method. Clinical test results (commercial nucleic acid detection kit, manufacturer: Ningbo Haier Gene Technology Co., Ltd., catalog number: 1060071) were used as the detection comparison method. The sensitivity, specificity and consistency of the HMPV antigen detection kit of this project were calculated by using a quadrant table (shown in Table 3).

[0096] Table 3 Comparison of the method of the present invention with commercial nucleic acid detection kits

[0097]

[0098] Of the 134 nasopharyngeal swabs: the detection method of this invention detected 60 positive HMPV antigens and 74 negative HMPV antigens; the comparative method detected 71 positive HMPV nucleic acid and 63 negative HMPV nucleic acid. As shown in the table above, the sensitivity of this kit is 80.3%, the specificity is 95.2%, and the concordance is 87.3%, demonstrating that this method has extremely high sensitivity and specificity.

[0099] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody targeting the F protein of human metapneumovirus, characterized in that, The monoclonal antibody is monoclonal antibody 1 and / or monoclonal antibody 2; the amino acid sequence of the light chain variable region of monoclonal antibody 1 is shown in SEQ ID NO. 6, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 8; the amino acid sequence of the light chain variable region of monoclonal antibody 2 is shown in SEQ ID NO. 2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

4.

2. The monoclonal antibody according to claim 1, characterized in that, The nucleotide sequence of the light chain variable region of the monoclonal antibody 1 is shown in SEQ ID NO.5, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.

7.

3. The monoclonal antibody according to claim 2, characterized in that, The nucleotide sequence of the light chain variable region of the monoclonal antibody 2 is shown in SEQ ID NO.1, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.

3.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule carries a gene encoding the monoclonal antibody according to any one of claims 1-3.

5. A host cell, characterized in that, The host cell carries the nucleic acid molecule of claim 4 and is capable of autonomously expressing the monoclonal antibody of any one of claims 1-3.

6. A kit for detecting human metapneumovirus F protein, characterized in that, The kit contains monoclonal antibody 1 and monoclonal antibody 2 as described in claim 1.

7. The reagent kit according to claim 6, characterized in that, In the kit, monoclonal antibody 1 is used as the detection antibody, and monoclonal antibody 2 is used as the capture antibody.

8. The reagent kit according to claim 7, characterized in that, The kit contains test strips, each test strip comprising a sample pad, a conjugate pad, a PVDF membrane, and absorbent paper. The preparation method of the test strip includes the following steps: (1) Monoclonal antibody 1 was labeled using red latex microspheres; (2) The detection line of the PVDF membrane is coated with monoclonal antibody 2, and the coating concentration of monoclonal antibody 2 is 1.5 mg / mL; (3) Monoclonal antibody 1 labeled with red latex microspheres at a concentration of 50 ug / mL adsorbed onto the conjugate pad.

9. The reagent kit according to claim 8, characterized in that, The preparation method of the test strip further includes: coating the control line of the PVDF membrane with goat anti-mouse IgG antibody.

10. The use of the monoclonal antibody according to any one of claims 1-3 in the preparation of reagents or kits for detecting human metapneumovirus F protein.

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