Enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen and quantitative detection method

Abstract

The present invention relates to the biological field, and discloses an enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen and a quantitative detection method. The enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen includes: a capture antibody: a human metapneumovirus-specific antibody HMPV01 or an antigen-binding fragment thereof, including the amino acid sequence SEQ ID NO:1 of the light chain of the HMPV01 antibody and the amino acid sequence SEQ ID NO:2 of the heavy chain of the HMPV01 antibody; a recognition antibody: an enzyme-labeled antibody HMPV02-HRP specific for human metapneumovirus or an antigen-binding fragment thereof. By screening the capture antibody HMPV01 and the recognition antibody HMPV02-HRP with specific amino acid sequences, efficiently preparing the antibodies by using a mammalian cell expression system, and combining with an optimized detection method, the binding efficiency of the antibody and the antigen is significantly improved, enabling the kit to detect low concentrations of human metapneumovirus antigen.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to an enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen and a quantitative detection method. Background Art

[0002] Human metapneumovirus (hMPV) belongs to the Paramyxoviridae family, the Pneumovirinae subfamily, and the Metapneumovirus genus, and is a single-stranded negative-sense RNA virus. hMPV is divided into two genotypes, A and B, and these two genotypes are further divided into four subtypes, A1, A2, B1, and B2. Its genome is 13 kb long and encodes nine proteins. Among them, the F protein is essential for membrane fusion and can mediate cell fusion without the participation of an adsorption protein, that is, the F protein of hMPV can adsorb cell receptors and can also activate the membrane fusion process it mediates, having the dual functions of adsorption and fusion. In addition, the F protein plays an important role in inducing immune protection, high levels of serum and antibodies. Due to the highly conserved nucleotide sequence of the F protein, the homology between strains A and B can reach 95%. The protective antibodies induced by the F protein can resist infections of different subtypes. The high immunogenicity and high conservation of the F protein determine the potential of the F protein as a vaccine target antigen.

[0003] When traditional enzyme-linked immunosorbent assay kits detect human metapneumovirus antigen, due to the low binding efficiency between antibodies and antigens, the detection sensitivity is low. In the early stage of virus infection, the content of virus antigen in the human body is often low, resulting in missed detections and delaying the early diagnosis and treatment of diseases. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides an enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen and a quantitative detection method, which solve the problems that when traditional enzyme-linked immunosorbent assay kits detect human metapneumovirus antigen, due to the low binding efficiency between antibodies and antigens, the detection sensitivity is low. In the early stage of virus infection, the content of virus antigen in the human body is often low, resulting in missed detections and delaying the early diagnosis and treatment of diseases.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: An enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen, comprising:

[0006] Capture antibody: a human metapneumovirus-specific antibody HMPV01 or its antigen-binding fragment, including the HMPV01 antibody light chain amino acid sequence SEQ ID NO:1, nucleotide sequence SEQ ID NO:5, the HMPV01 antibody heavy chain amino acid sequence SEQ ID NO:2, nucleotide sequence SEQ ID NO:6;

[0007] Detection antibody: an enzyme-labeled antibody HMPV02-HRP specific for human metapneumovirus or an antigen-binding fragment thereof, including the amino acid sequence SEQ ID NO:3 and nucleotide sequence SEQ ID NO:7 of the light chain of the HMPV02 antibody, and the amino acid sequence SEQ ID NO:4 and nucleotide sequence SEQ ID NO:8 of the heavy chain of the HMPV02 antibody.

[0008] Preferably, the capture antibody and the detection antibody are prepared by a mammalian cell expression system, and the specific steps are as follows:

[0009] A1. Insert the nucleotide sequence SEQ ID NO:5 of the light chain of the HMPV01 antibody and the nucleotide sequence SEQ ID NO:6 of the heavy chain of the HMPV01 antibody into the eukaryotic expression vector pcDNA3.4 respectively, insert the nucleotide sequence SEQ ID NO:7 of the light chain of the HMPV02 antibody and the nucleotide sequence SEQ ID NO:8 of the heavy chain of the HMPV02 antibody into the eukaryotic expression vector pGS, perform clone transformation and plasmid extraction to construct a recombinant expression vector;

[0010] A2. Transfect the recombinant expression vector into mammalian cells, including Expi293 cells, ExpiCHO cells, and CHO cells, and then perform transfection using a serum-free transfection reagent. After transfection, place the cells in an incubator for culture; A3. Then collect the cell culture supernatant, perform preliminary purification by affinity chromatography, including Protein A and Protein G affinity chromatography columns, then collect the eluate containing the target antibody, and perform refined purification by ion exchange chromatography or gel filtration chromatography to remove impurities and impure proteins to obtain the capture antibody and the detection antibody.

[0011] Preferably, the enzyme-labeled antibody HMPV02-HRP specific for human metapneumovirus is obtained by labeling the purified HMPV02 antibody using an HRP labeling kit.

[0012] The quantitative detection method of an enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen includes the following steps: S1. Solid-phase antibody coating: Dilute the capture antibody with coating buffer, add it to the ELISA plate wells, and incubate overnight to form a solid-phase antibody;

[0013] S2. Blocking: Then discard the coating solution, add the blocking solution for incubation, and then wash to remove unbound substances;

[0014] S3. Antigen binding: Then add the standard antigen or the sample to be tested diluted in gradients in sequence, incubate and then wash for specific detection;

[0015] S4. Enzyme-labeled antibody incubation: Then add the detection antibody, dilute it with PBS solution and incubate, and then wash;

[0016] S5, Color development termination: Then add the substrate chromogenic solution, develop color in the dark at room temperature, and then add the termination solution to terminate the reaction;

[0017] S6, Detection and calculation: Then measure the absorbance value at OD450nm, draw a standard curve with the standard antigen concentration as the abscissa and the absorbance value as the ordinate, and calculate the antigen concentration according to the standard curve equation and the absorbance value of the antigen sample to be detected to complete the detection.

[0018] Preferably, the coating buffer in S1 includes carbonate buffer, and the blocking solution in S2 includes PBS solution containing 2% BSA.

[0019] Preferably, the washing in S2 - S4 is carried out with a washing solution, and the washing solution includes PBS solution containing 0.05% Tween - 20.

[0020] Preferably, the standard antigen in S3 is recombinantly expressed and purified through a eukaryotic expression system, including the recombinantly expressed preF - conformation F glycoprotein A - 115 of human metapneumovirus, whose amino acid sequence is SEQ ID NO:9 and nucleotide sequence is SEQ ID NO:10.

[0021] Preferably, the substrate chromogenic solution in S5 includes TMB chromogenic solution, and the termination solution includes H2SO4 solution.

[0022] Preferably, the specific detection in S3 specifically includes the following steps:

[0023] B1, Dilute the capture antibody and coat the ELISA plate overnight, discard the capture antibody the next day, and add the blocking solution for blocking;

[0024] B2, Then serially dilute each antigen, including the standard antigen, SARS Cov - 2 RBD, RSV F, MERS S, Ebola GP proteins, then discard the blocking solution, add the diluted antigens, incubate at room temperature and then discard;

[0025] B3, Then dilute the detection antibody and add it to the ELISA plate, incubate at room temperature and then discard simultaneously, add the chromogenic solution for color development, then add the termination solution to terminate and place it in the ELISA reader to read the value at a wavelength of 450nm;

[0026] B4, Then use the logarithm of each antigen concentration as the abscissa and the OD450nm value corresponding to the concentration as the ordinate to fit the curve.

[0027] Preferably, the quantitative detection method of the ELISA kit for detecting human metapneumovirus antigen is applied to the quantitative detection of antigens in samples containing preF - conformation F glycoprotein during the vaccine production process.

[0028] The present invention provides an enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen and a quantitative detection method, which have the following beneficial effects:

[0029] 1. By screening the capture antibody HMPV01 and the recognition antibody HMPV02-HRP with specific amino acid sequences, efficiently preparing the antibodies using a mammalian cell expression system, and combining with an optimized detection method, the present invention significantly improves the binding efficiency of the antibodies to the antigen, enabling the kit to detect low concentrations of human metapneumovirus antigen, effectively solving the problem of low detection sensitivity of traditional kits, detecting the virus antigen in the human body at the initial stage of virus infection, avoiding missed detections, and providing strong support for the early diagnosis of diseases.

[0030] 2. The capture antibody and recognition antibody of the present invention have high specificity for the preF conformation F glycoprotein A-115 of human metapneumovirus. Through specific detection steps, there is no cross-reaction with a variety of other virus antigens, accurately identifying the target antigen, greatly reducing the probability of false positives in the detection results, and ensuring the accuracy and reliability of the detection results.

[0031] 3. The quantitative detection method of the present invention is simple to operate and has good repeatability, suitable for the detection of a variety of samples. Especially in the process of vaccine production, it can accurately quantitatively detect the antigen in samples containing the preF conformation F glycoprotein. By drawing a standard curve and calculating the antigen concentration, it can effectively monitor the antigen content in the vaccine production process, ensuring the stability and consistency of vaccine quality, and having important significance for the quality control of vaccine research and development and production.

[0032] 4. By using a mammalian cell expression system to prepare the capture antibody and recognition antibody, the present invention constructs a recombinant expression vector, and then through a series of operations such as transfection, culture, affinity chromatography, and refined purification, high-purity antibodies are obtained, ensuring the quality and yield of the antibodies, and providing a guarantee for the stable production and performance optimization of the kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the plasmid map of the eukaryotic expression vector pcDNA3.4 of the present invention;

[0034] Figure 2 It is a schematic diagram of the purification result of the hMPV preF protein A-115 protein of the present invention;

[0035] Figure 3 It is a schematic diagram of the purification results of the HMPV01 and HMPV02 antibodies of the present invention;

[0036] Figure 4 It is a schematic diagram of the determination results of the binding activities of the HMPV01 and HMPV02 antibodies of the present invention;

[0037] Figure 5 Schematic diagram of the results of the binding activity assay of HMPV01-HRP and HMPV02-HRP of the present invention;

[0038] Figure 6 Schematic diagram of the results of the paired detection of HMPV0 and HMPV02-HRP and HMPV02 and HMPV01-HRP of the present invention;

[0039] Figure 7 Schematic diagram of the results of the specific detection of the present invention;

[0040] Figure 8 Schematic diagram of the standard curve of the present invention. Detailed implementation manners

[0041] Next, in combination with the accompanying drawings of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0042] Please refer to the attached Figure 1 - attached Figure 8 , the embodiment of the present invention provides an enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen, including:

[0043] Capture antibody: a human metapneumovirus-specific antibody HMPV01 or its antigen-binding fragment, including the amino acid sequence SEQ ID NO:1 and nucleotide sequence SEQ ID NO:5 of the light chain of the HMPV01 antibody, and the amino acid sequence SEQ ID NO:2 and nucleotide sequence SEQ ID NO:6 of the heavy chain of the HMPV01 antibody;

[0044] Detection antibody: an enzyme-labeled antibody HMPV02-HRP specific for human metapneumovirus or its antigen-binding fragment, including the amino acid sequence SEQ ID NO:3 and nucleotide sequence SEQ ID NO:7 of the light chain of the HMPV02 antibody, and the amino acid sequence SEQ ID NO:4 and nucleotide sequence SEQ ID NO:8 of the heavy chain of the HMPV02 antibody.

[0045] Specifically, the capture antibody HMPV01: A monoclonal antibody was obtained by immunizing rabbits, and through affinity maturation optimization and humanization modification, the amino acid sequences of its light chain (SEQ ID NO: 1) and heavy chain (SEQ ID NO: 2) were obtained. X-ray crystal diffraction analysis showed that its CDR region could accurately recognize the conserved epitope (amino acid residues 115 - 132) of the hMPVpreF conformation F glycoprotein A-115, and the dissociation constant was significantly improved compared with that of traditional polyclonal antibodies.

[0046] The recognition antibody HMPV02-HRP: A monoclonal antibody was obtained by immunizing rabbits, and through affinity maturation optimization and humanization modification, the amino acid sequences of its light chain (SEQ ID NO: 3) and heavy chain (SEQ ID NO: 4) were obtained. After labeling with HRP, more than 95% of the binding activity was retained, and the enzyme activity recovery rate reached 82%, achieving a signal amplification effect.

[0047] The capture antibody and the recognition antibody were prepared by a mammalian cell expression system, and the specific steps are as follows:

[0048] A1. The nucleotide sequences of the light chain of the HMPV01 antibody SEQ ID NO: 5 and the heavy chain of the HMPV01 antibody SEQ ID NO: 6 were respectively inserted into the eukaryotic expression vector pcDNA3.4, and the nucleotide sequences of the light chain of the HMPV02 antibody SEQ ID NO: 7 and the heavy chain of the HMPV02 antibody SEQ ID NO: 8 were inserted into the eukaryotic expression vector pGS. Cloning transformation and plasmid extraction were carried out to construct recombinant expression vectors;

[0049] A2. The recombinant expression vectors were transfected into mammalian cells, including Expi293 cells, ExpiCHO cells, and CHO cells, and then a serum-free transfection reagent was used for transfection. After transfection, the cells were placed in an incubator for culture; A3. Then, the cell culture supernatant was collected and preliminarily purified by affinity chromatography, including Protein A and Protein G affinity chromatography columns. Then, the eluate containing the target antibody was collected, and ion exchange chromatography or gel filtration chromatography was used for refined purification to remove impurities and impure proteins, obtaining the capture antibody and the recognition antibody.

[0050] Specifically, the HMPV01 antibody gene was inserted into the pcDNA3.4 vector ( Figure 1 ), and the CMV enhancer was used to drive antibody expression. The transient transfection expression level in Expi293 cells reached 220 mg / L, which was 60% higher than that of the pCI vector.

[0051] The light chain and heavy chain of the HMPV02 antibody were simultaneously cloned into the pGS vector, and a high-expression cell line was screened through the glutamine synthetase (GS) pressure system. The stable expression level in CHO cells reached 1.8 g / L.

[0052] Purification process:

[0053] The antibody was captured by Protein A affinity chromatography ( Figure 3 ), and the purity reached 90%; endotoxin was further removed to <0.1 EU / mg by Q Sepharose ion exchange chromatography (pH 7.0), and the final purity was >98% (verified by SDS-PAGE).

[0054] Please refer to the attached Figure 5 , and the enzyme-labeled antibody HMPV02-HRP specific for human metapneumovirus was obtained by labeling the purified HMPV02 antibody using an HRP labeling kit.

[0055] Specifically, the coupling of HRP and HMPV02 antibody was mediated by using the Sulfo-SMCC cross-linker. The labeling rate was determined to be 4.2:1 (HRP: antibody) by ultraviolet spectrophotometry, and its detection sensitivity for A-115 antigen was verified by ELISA to reach 6.1×10 -5 μg / mL.

[0056] The quantitative detection method of the enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen includes the following steps: S1. Solid-phase antibody coating: Dilute the capture antibody with coating buffer, add it into the ELISA plate wells, and incubate overnight to form a solid-phase antibody;

[0057] S2. Blocking: Then discard the coating solution, add the blocking solution for incubation, and then wash to remove unbound substances;

[0058] S3. Antigen binding: Then add the serially diluted standard antigen or test sample in sequence, incubate and then wash for specific detection;

[0059] S4. Enzyme-labeled antibody incubation: Then add the recognition antibody, dilute it with PBS solution and incubate, and then wash;

[0060] S5. Color development termination: Then add the substrate chromogenic solution, develop color at room temperature in the dark, and then add the termination solution to terminate the reaction;

[0061] S6. Detection and calculation: Then measure the absorbance value at OD450nm, draw a standard curve with the standard antigen concentration as the abscissa and the absorbance value as the ordinate, and calculate the antigen concentration according to the standard curve equation and the absorbance value of the antigen sample to be detected to complete the detection.

[0062] The coating buffer in S1 includes carbonate buffer, and the blocking solution in S2 includes PBS solution containing 2% BSA.

[0063] The washing in S2 - S4 is carried out with a washing solution, and the washing solution includes a PBS solution containing 0.05% Tween - 20.

[0064] The standard antigen in S3 is recombinantly expressed and purified through a eukaryotic expression system, and includes the recombinantly expressed preF - conformation F glycoprotein A - 115 of human metapneumovirus, and its amino acid sequence is SEQ ID NO:9.

[0065] The substrate chromogenic solution in S5 includes a TMB chromogenic solution, and the stop solution includes an H2SO4 solution.

[0066] The specific detection in S3 specifically includes the following steps:

[0067] B1. Dilute the capture antibody and coat the enzyme - linked immunosorbent assay (ELISA) plate overnight, discard the capture antibody the next day, and add a blocking solution for blocking;

[0068] B2. Then serially dilute each antigen, including the standard antigen, SARS CoV - 2 RBD, RSV F, MERS S, and Ebola GP proteins. Then discard the blocking solution, add the serially - diluted antigens, incubate at room temperature and then discard;

[0069] B3. Then dilute the detection antibody and add it to the ELISA plate, incubate at room temperature and then discard simultaneously. Add the chromogenic solution for color development, then add the stop solution to terminate and place it in an ELISA reader to read the absorbance at a wavelength of 450 nm;

[0070] B4. Then use the logarithm of each antigen concentration as the abscissa and the OD450nm value corresponding to the concentration as the ordinate to fit a curve.

[0071] The quantitative detection method of the enzyme - linked immunosorbent assay (ELISA) kit for detecting human metapneumovirus antigen is applied to the quantitative detection of antigens in samples containing preF - conformation F glycoprotein during the vaccine production process.

[0072] Specifically,

[0073] Screen for capture antibody HMPV01 and recognition antibody HMPV02-HRP with specific amino acid sequences, and use mammalian cell expression systems (Expi293 cells, ExpiCHO cells, CHO cells) combined with specific eukaryotic expression vectors (pcDNA3.4, pGS) for efficient expression. After multiple purification processes such as affinity chromatography (Protein A, Protein G affinity chromatography columns) and ion exchange chromatography or gel filtration chromatography, high-purity antibodies are obtained. And the purified HMPV02 antibody is labeled using an HRP labeling kit, thereby obtaining antibodies with high affinity, high specificity, and good labeling effects, significantly improving the binding efficiency between the antibody and the antigen, enhancing the signal intensity during detection, achieving high-sensitivity detection of human metapneumovirus antigen, and being able to accurately detect low concentrations of virus antigen in the human body at the initial stage of virus infection. Thus, it solves the problem that in the traditional enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen, due to the low binding efficiency between the antibody and the antigen, the detection sensitivity is low. At the initial stage of virus infection, the virus antigen content in the human body is often low, resulting in missed detections and delaying the early diagnosis and treatment of the disease.

[0074] A kit for quantitatively detecting human metapneumovirus antigen, which contains capture antibody HMPV01 or its antigen-binding fragment, selected from the following sequences:

[0075] The amino acid sequence of the light chain of HMPV01 antibody SEQ ID NO:1

[0076] SYELTQPPSVSVSPGQTATITCSGTNLGAKYSCWYQQKPGQSPVLVIFQDNKRASGIPARFSAST

[0077] SGDTATLTISGTQVMDEADYFCQAWDSRTAVFGGGTTLTVLGQPKAAPSVTLFPPSSEELQAN

[0078] KATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS*

[0079] The amino acid sequence of the heavy chain of HMPV01 antibody SEQ ID NO:2

[0080] QVQLQESGPGLVKPSQTLSLTCTVSGGSVRGGGDYWSWIRRPPGKGLEWIGHIYNSGTTFYNP

[0081] SLKSRVTISIDTSKNQFSLKLRSVTAADTAVYYCGRVGSRATGLPDWIDPWGQGTLVTVSSASTK

[0082] GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT

[0083] VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT

[0084] PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE

[0085] YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES

[0086] NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0087] The recognition antibody HMPV02 or antigen-binding fragment thereof included is selected from the following sequences:

[0088] Amino acid sequence of the light chain of HMPV02 antibody, SEQ ID NO:3

[0089] SYELIQLPSVSVSPGQTARIPCSGDGLPKKYAHWYQQKAGQAPILLMYKDSERPSGIPERFSAFS

[0090] SGTTVTMTISGVQEEDEADYYCQSGDSTDTSVIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQAN

[0091] KATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS*

[0092] Amino acid sequence of the heavy chain of HMPV02 antibody, SEQ ID NO:4

[0093] QVQLVQSGPALVKSTQTLTLTCTFSGFALTTSGMCVSWVRQPPGKALEWLARIDWEDNTYYS

[0094] TSLKTRVTISKDPSKNQVVLTMTNMDPVDTATYYCARSYITDWKKDWFFDLWGRGTLVTVSSA

[0095] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

[0096] VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT

[0097] LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0098] WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI

[0099] AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0100] A method for quantitatively detecting human metapneumovirus antigen: Coating an ELISA plate with the human metapneumovirus-specific antibody HMPV01 to prepare a solid-phase antibody, adding standard antigens or antigen samples to be detected diluted with different concentration gradients at one time, using the human metapneumovirus-specific enzyme-labeled antibody HMPV02-HRP as the recognition antibody, adding a substrate chromogenic solution, adding a 2M H2SO4 solution to terminate the color development, measuring the OD450nm value, plotting a standard curve equation based on the measured absorbance value and the standard antigen concentration, and calculating the concentration of the sample to be detected according to the standard curve equation and the absorbance value of the antigen sample to be detected.

[0101] Example 1: Recombinant expression and purification of the hMPV preF protein A-115 protein. Through genetic engineering techniques, the antigen nucleotide sequence SEQ ID NO:10 was inserted into the eukaryotic expression vector pcDNA3.4 (the plasmid map is shown in Figure 1.), Clone, transform the recombinant expression vector and extract the plasmid for transfection of ExpiCHO cells for recombinant protein expression. One day before transfection, the ExpiCHO cells in the logarithmic growth phase were subcultured at high density, and the viable cell density was 3×10 6 / mL. After the cells grew overnight, the cell density was diluted to 6×10 6 / mL. Take 200 mL of the cell suspension for transfection. Dilute 200 μl of DNA and 1.4 mL of PEI40K transfection reagent with MetaCellCHO-320 medium respectively, and mix gently. Then gently drip the mixed transfection reagent into the diluted plasmid and let it stand at room temperature for 10 min to prepare the DNA complex. After the incubation, drop the DNA complex into the cell suspension to be transfected drop by drop, and transfer it to a shaker at 37 °C with 8% CO2 and culture at a rotation speed of 225 rpm. 24 h after transfection, add feeding and Enhancer, and cool down to 32 °C for continued culture.

[0102] On the 5th day after transfection, centrifuge the cell culture supernatant at 4 °C and 4700 rpm for 30 min. After removing impurities with a 0.22 μm filter membrane, concentrate it to about 300 mL by TFF and purify the protein by Strep II affinity chromatography. The specific implementation steps are as follows: (1) Column packing: Take a 20 mL empty column, press the lower gasket to the bottom of the column and compact it, rinse the gasket with PBS buffer (pH 7.4), and immediately close the lower outlet after PBS flows out from the lower outlet; Suspend the resin, take an appropriate amount of slurry with a pipette and add it to the column (the ratio of the preservation solution to the packing is 1:1), open the lower outlet to drain the protective liquid; Add an appropriate amount of PBS buffer to rinse the column packing, and close the lower outlet after the column packing drains. (2) Equilibration: Equilibrate with 5 column volumes of equilibration buffer (PBS buffer, pH 7.4) to make the packing in the same buffer system as the target protein. (3) Binding and loading: Resuspend the packing with an appropriate amount of sample and transfer it to the sample, and bind overnight on a shaker at 4 °C; The next day, add the sample and the packing to the equilibrated column, and multiple loadings can be carried out to increase the binding efficiency and collect the flow-through for subsequent SDS-PAGE to detect the binding of the protein. (4) Equilibration and impurity washing: Wash impurities with 10 column volumes of equilibration / washing buffer to remove non-specifically bound impurity proteins, and collect the washing solution. (5) Elution: Finally, elute the target protein with 5 column volumes of elution buffer (D-Biotin PBS buffer, pH 7.4), collect in fractions and concentrate and change the solution. (6) Quantification: Mix the concentrated protein and quantify it by ΜV280nm. Take a small amount of protein for SDS-PAGE detection, and the results are shown in Figure 2 . The rest is aliquoted and stored at -80 °C.

[0103] Example 2: Recombinant Expression and Purification of HMPV01 and HMPV02 Antibodies By genetic engineering techniques, the nucleotide light chain sequence SEQ ID NO:5 and heavy chain sequence SEQ ID NO:6 of the HMPV01 antibody were respectively inserted into the eukaryotic expression vector pcDNA3.4, and the nucleotide light chain sequence SEQ ID NO:7 and heavy chain sequence SEQ ID NO:8 of the HMPV02 antibody were inserted into the eukaryotic expression vector pGS. Then, the recombinant expression vectors were subjected to cloning transformation and plasmid extraction for transfection of Expi293 cells for recombinant protein expression. High-density passage was performed one day before transfection of Expi293 cells in the logarithmic growth phase, and the viable cell density was 2.5×10 6 / mL. After the cells grew overnight, the cell density was diluted to 3×10 6 / mL, and 200 μL of the cell suspension was taken for transfection respectively. 200 μL of DNA and 1.4 mL of PEI40K transfection reagent were respectively diluted with Opti-MEM medium and gently mixed. Then, the mixed transfection reagent was gently added dropwise to the diluted plasmid, and left standing at room temperature for 10 min to prepare the DNA complex. After the incubation, the DNA complex was added dropwise to the cell solution to be transfected and transferred to a shaker at 37 °C containing 8% CO2 for culturing at a rotation speed of 225 rpm. Feed was added 24 h and 48 h after transfection and the culture was continued.

[0104] On the 5th day after transfection, the cell culture supernatant was collected by centrifugation at 4700 rpm for 30 min at 4°C, filtered through a 0.22-μm vacuum filter to remove impurities, and the protein was purified by protein A affinity chromatography. The specific implementation steps are as follows: (1) Column packing: Take a 20-mL empty column, press the lower gasket to the bottom of the column and compact it, rinse the gasket with PBS buffer (pH 7.4), and immediately close the lower outlet after PBS flows out from the lower outlet; suspend the resin, take an appropriate amount of slurry with a pipette and add it to the column (the ratio of the preservation solution to the packing is 1:1), open the lower outlet to drain the protective solution; add an appropriate amount of PBS buffer to rinse the column packing, and close the lower outlet after the column packing drains. (2) Equilibration: Equilibrate with 5 column volumes of equilibration solution (PBS buffer, pH 7.4) to bring the packing into the same buffer system as the target protein. (3) Binding and sample loading: Resuspend the packing with an appropriate amount of sample and transfer it to the sample, bind it on a shaker at 4°C for 3 h; then add the sample and the packing to the equilibrated column, and multiple sample loadings can be performed to increase the binding efficiency and collect the flow-through for subsequent SDS-PAGE to detect the protein binding situation. (4) Equilibration and impurity washing: Wash with 10 column volumes of equilibration / washing solution to remove non-specifically bound impurity proteins, and collect the washing solution. (5) Elution: Finally, elute the target protein with 5 column volumes of elution solution (0.1 M Glycine, pH 3.0), and add 1 / 10 of the neutralization solution (1 M Tris-HCl, pH 9.0) for neutralization, collect in separate tubes and concentrate and change the solution. (6) Quantification: Mix the concentrated protein and perform UV280nm quantification on it, take a small amount of protein for SDS-PAGE detection, and the results are shown in Figure 3 and Figure 4 .

[0105] Example 3: Determination of the binding activity of purified HMPV01 and HMPV02 antibodies The monoclonal antibodies purified in the above case were used to determine the antibody binding ability by direct ELISA. The specific implementation method is as follows: Dilute antigen A-115 to 1 μg / mL, add 100 μl per well, and coat overnight at 4°C. The next day, after patting off the antigen, add 300 μl of 2% BSA solution to each well and block at 37°C for 2 h. After patting off the blocking solution, add the test antibodies HMPV01 and HMPV02 diluted in gradients (starting from 2 μg / mL, 4-fold serial dilution for 12 gradients), 100 μl / well, and incubate at room temperature for 1 h. After patting off the primary antibody, add the secondary antibody labeled with HRP (purchased from Jackson ImmunoResearch Inc.), 100 μl / well, and continue to incubate for 1 h. Then add 100 μl / well of TMB chromogenic solution for color development. After 15 min, add 50 μl / well of stop solution to terminate the color development. Finally, read the absorbance at a wavelength of 450 nm. 2.1 times the reading of the 2% BSA control well is the Cut-off value. If the average reading of the antibody supernatant is greater than this value, it indicates that the antibody can bind to the A-115 protein; if it is less than the Cut-off value, it indicates that the antibody cannot bind to the A-115 protein. The results show that the two antibodies, HMPV01 and HMPV02, can bind to the A-115 protein.

[0106] Example 4: Preparation of horseradish peroxidase-labeled HMPV01 and HMPV02 antibodies and determination of their binding activity

[0107] (1) Antibody labeling

[0108] The two purified antibodies, HMPV01 and HMPV02, were labeled using an HRP labeling kit (purchased from proteintech). The specific implementation steps are as follows: Take out the HRP labeling kit from -20°C and equilibrate at room temperature for 30 min to fully thaw and mix the reaction initiation solution and termination solution for standby; Add 1 μl of the reaction initiation solution to every 10 μl of the antibody solution to be labeled, and pipette several times to mix well to avoid generating bubbles; Open the cap of horseradish peroxidase, directly add the above-initiated antibody solution into the bottle, and pipette several times to mix well to avoid generating bubbles, and place at room temperature for 3 h; Add the reaction termination solution to horseradish peroxidase, at a ratio of 1 μl of reaction termination solution per 10 μl of antibody, mix well, and place at room temperature for 1 h; After the termination is completed, add an equal volume of product protection solution, mix well, and store at -20°C.

[0109] (2) Binding activity assay The binding activities of the two labeled antibodies were detected by direct ELISA as follows: Antigen A-115 was diluted to 1 μg / mL, 100 μL per well, and coated overnight at 4°C. The next day, after discarding the antigen, 300 μL of 2% BSA solution was added to each well and blocked at 37°C for 2 h. After discarding the blocking solution, serially diluted enzyme-labeled antibodies HMPV01-HRP and HMPV02-HRP (starting from 20 μg / mL, 4-fold serial dilution for 12 gradients) were added, 100 μL per well, and incubated at room temperature for 1.5 h. After discarding, 100 μL of TMB chromogenic solution was added to each well for color development. After 15 min, 50 μL of stop solution was added to each well to terminate the color development. Finally, readings were taken at a wavelength of 450 nm. 2.1 times the reading of the 2% BSA control well was defined as the Cut-off value. If the average reading of the antibody supernatant was greater than this value, it indicated that the antibody could specifically bind to the A-115 protein; if it was less than the Cut-off value, it indicated that the antibody could not specifically bind to the A-115 protein. The results showed that the two antibodies, HMPV01 and HMPV02 labeled with HRP, could specifically bind to the A-115 protein.

[0110] Example 5: Paired detection of HMPV01 and HMPV02-HRP and HMPV02 and HMPV01-HRP Taking HMPV01 and HMPV02 as capture antibodies respectively, they were diluted to 2 μg / mL and coated on the ELISA plate, 100 μL per well, and coated overnight at 4°C. The next day, the capture antibodies were discarded, and 2% BSA was added for blocking for 2 h. Subsequently, antigen A-115 was serially diluted from the starting concentration of 1 μg / mL for 12 gradients at 4-fold dilution. After discarding the blocking solution, the diluted antigen was added and incubated at room temperature for 1 h and then discarded. Then, the detection antibody HMPV02-HRP diluted to 2 μg / mL was added corresponding to the capture antibody HMPV01, 100 μL per well. Similarly, the detection antibody HMPV01-HRP diluted to 2 μg / mL was added corresponding to the capture antibody HMPV02, 100 μL per well. After incubating at room temperature for 1 h, they were discarded simultaneously, TMB chromogenic solution was added for color development for 15 min, then the stop solution was added to terminate the reaction and readings were taken at a wavelength of 450 nm using an ELISA reader. The results showed that the two antibodies could be paired. Among them, when HMPV02-HRP was used as the recognition antibody and paired with the coating antibody HMPV01, the sensitivity for quantifying the A-F1115 antigen was the highest; when HMPV01-HRP was used as the recognition antibody and paired with the coating antibody HMPV02, the sensitivity was lower.

[0111] Example 6: Specificity Detection The capture antibody HMPV01 was diluted to 2 μg / mL and then coated on an ELISA plate at 100 μL / well overnight at 4°C. The next day, the capture antibody was discarded, and 2% BSA was added for blocking for 2 h. Subsequently, antigens A-115, SARS CoV-2 RBD, RSV F, MERS S, and Ebola GP proteins were serially diluted 12-fold from an initial concentration of 1 μg / mL. After discarding the blocking solution, the diluted antigens were added and incubated at room temperature for 1 h and then discarded. Then, the detection antibody HMPV02-HRP was diluted to 2 μg / mL and added to the ELISA plate at 100 μL / well. After incubating at room temperature for 1 h, it was discarded simultaneously, and TMB chromogenic solution was added for color development for 15 min. Subsequently, the stop solution was added to terminate the reaction, and the absorbance at 450 nm was read using an ELISA reader. The logarithm of each antigen concentration was used as the abscissa, and the OD450nm value corresponding to the concentration was used as the ordinate to fit the curve. From the experimental results, only the A-115 antigen protein showed an obvious dose-dependent effect, and the results of the other viral proteins were all negative, indicating that the antibody specifically binds to the A-115 antigen and has no cross-reaction with other viral antigens.

[0112] Example 7: Sensitivity Detection The capture antibody HMPV01 was diluted to 2 μg / mL and then coated on an ELISA plate at 100 μL / well overnight at 4°C. The next day, the capture antibody was discarded, and 2% BSA was added for blocking for 2 h. Subsequently, antigen A-115 was serially diluted 12-fold from an initial concentration of 1 μg / mL. After discarding the blocking solution, the diluted antigen was added and incubated at room temperature for 1 h and then discarded. Then, the detection antibody HMPV02-HRP was diluted to 2 μg / mL and added to the ELISA plate at 100 μL / well. After incubating at room temperature for 1 h, it was discarded simultaneously, and TMB chromogenic solution was added for color development. The concentration was used as the abscissa, and the OD450nm value corresponding to each standard concentration was used as the ordinate to plot the standard curve. The standard curve showed that the lowest dilution concentration of A-115 protein was 6.1×10 -5 μg / mL, so the sensitivity of this method was 6.1×10 -5 μg / mL.

[0113] Example 8: Application of the antigen quantitative detection method The established hMPV F protein A-115 antigen quantitative detection method was used to quantify the A-115 antigen contained in the transfection supernatant of the A-115 protein. The cell supernatant was sampled on the 5th day after transfection, and then centrifuged at 4700 rpm for 30 min at 4°C. The cell supernatant was collected and stored at -80°C for later use. By the double sandwich ELISA method, HMPV01 was used as the capture antibody, the diluted standard A-115 was used as the standard curve, and the cell supernatant on the 5th day was diluted by a certain multiple. Finally, HMPV02-HRP was used as the recognition antibody to detect the expression of the antigen in the cell supernatant. The results showed that the concentration of the A-115 protein in the cell expression supernatant on the 5th day after transfection was 0.4 μg / mL.

[0114] Sequence:

[0115] SEQ ID NO:1

[0116] SYELTQPPSVSVSPGQTATITCSGTNLGAKYSCWYQQKPGQSPVLVIFQDNKRASGIPARFSASTSGDTATLTISGTQVMDEADYFCQAWDSRTAVFGGGTTLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS*

[0117] SEQ ID NO:2

[0118] QVQLQESGPGLVKPSQTLSLTCTVSGGSVRGGGDYWSWIRRPPGKGLEWIGHIYNSGTTFYNPSLKSRVTISIDTSKNQFSLKLRSVTAADTAVYYCGRVGSRATGLPDWIDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0119] SEQ ID NO:3

[0120] SYELIQLPSVSVSPGQTARIPCSGDGLPKKYAHWYQQKAGQAPILLMYKDSERPSGIPERFSAFS

[0121] SGTTVTMTISGVQEEDEADYYCQSGDSTDTSVIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQAN

[0122] KATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS*

[0123] SEQ ID NO:4

[0124] QVQLVQSGPALVKSTQTLTLTCTFSGFALTTSGMCVSWVRQPPGKALEWLARIDWEDNTYYS

[0125] TSLKTRVTISKDPSKNQVVLTMTNMDPVDTATYYCARSYITDWKKDWFFDLWGRGTLVTVSSA

[0126] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

[0127] VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT

[0128] LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

[0129] WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI

[0130] AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

[0131] SEQ ID NO:5

[0132] TCTTACGAGCTGACCCAGCCCCCTTCTGTGTCTGTGTCTCCCGGTCAGACCGCCACTATCAC

[0133] CTGTTCTGGTACTAACCTGGGCGCCAAGTACAGTTGCTGGTATCAGCAGAAGCCCGGCCAG

[0134] AGCCCAGTGCTGGTGATTTTCCAGGACAATAAGAGAGCTAGCGGCATCCCCGCCCGGTTTT

[0135] CTGCTTCTACCTCCGGTGACACCGCCACCCTGACTATCTCCGGTACCCAGGTCATGGACGAG

[0136] GCCGATTATTTCTGCCAGGCCTGGGACAGCAGAACCGCTGTGTTCGGAGGCGGTACCACCC

[0137] TGACAGTGCTGGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAG

[0138] GAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCG

[0139] TGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACAC

[0140] CCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCA

[0141] GTGGAAGTCCCACAGGAGCTACAGTTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAA

[0142] GACAGTGGCCCCTACAGAATGTTCA

[0143] SEQ ID NO:6

[0144] CAGGTGCAGCTGCAGGAAAGTGGCCCAGGACTGGTGAAACCCTCCCAGACACTGTCCCTG

[0145] ACCTGTACTGTTTCTGGCGGCTCCGTCAGGGGCGGAGGAGATTACTGGAGCTGGATCAGAA

[0146] GGCCCCCCGGAAAGGGACTGGAGTGGATTGGACACATCTATAACTCCGGCACCACTTTCTA

[0147] TAACCCCTCCCTGAAAAGCCGGGTGACAATTAGTATTGACACCTCAAAGAACCAGTTCTCCC

[0148] TGAAACTGAGATCCGTGACTGCCGCTGACACCGCTGTGTACTATTGCGGCCGGGTGGGAAG

[0149] CAGGGCTACAGGACTGCCTGACTGGATCGACCCCTGGGGACAGGGAACACTGGTGACTGT

[0150] GTCCTCCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCT

[0151] CCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT

[0152] GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT

[0153] CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGAC

[0154] CTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCC

[0155] AAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTT

[0156] CCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCG

[0157] TGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCG

[0158] TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTG

[0159] TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAA

[0160] GGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG

[0161] CCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAG

[0162] GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGA

[0163] GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC

[0164] CTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTC

[0165] TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTC

[0166] TCCCGGTAAA

[0167] SEQ ID NO:7

[0168] TCTTACGAGCTGATCCAGCTGCCCTCCGTCTCCGTGTCTCCTGGACAGACAGCCAGGATTCC

[0169] TTGCTCCGGCGACGGTCTGCCTAAGAAGTACGCCCACTGGTACCAGCAGAAGGCCGGTCAG

[0170] GCACCTATCCTGCTGATGTACAAGGACTCCGAGAGGCCCTCCGGCATTCCAGAAAGATTCTC

[0171] CGCTTTTTCCTCCGGCACCACCGTGACCATGACTATCTCCGGCGTGCAGGAGGAGGACGAG

[0172] GCTGATTACTACTGCCAGTCCGGCGACTCCACCGACACATCTGTCATTTTCGGCGGCGGAAC

[0173] CAAGGTGACCGTGCTGGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCT

[0174] CTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGG

[0175] AGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCA

[0176] CCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCC

[0177] TGAGCAGTGGAAGTCCCACAGGAGCTACAGTTGCCAGGTCACGCATGAAGGGAGCACCGT

[0178] GGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0179] SEQ ID NO:8

[0180] CAGGTGCAGCTGGTGCAGAGTGGACCAGCACTGGTGAAGTCTACCCAGACCCTGACACTG

[0181] ACATGCACATTCTCCGGCTTCGCCCTGACCACATCCGGTATGTGCGTGTCCTGGGTGCGCCA

[0182] GCCTCCTGGAAAAGCTCTGGAGTGGCTGGCCCGAATCGACTGGGAAGACAACACCTACTAC

[0183] AGCACCTCCCTGAAGACCCGGGTGACCATCTCCAAGGACCCCTCTAAGAACCAGGTCGTGC

[0184] TGACTATGACCAACATGGACCCCGTGGACACCGCTACCTACTACTGCGCAAGGTCCTACATC

[0185] ACCGACTGGAAAAAGGACTGGTTCTTCGACCTGTGGGGCAGGGGCACCCTGGTGACCGTG

[0186] TCCTCCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCT

[0187] GGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTG

[0188] TCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTC

[0189] AGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC

[0190] TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCA

[0191] AATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACC

[0192] GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCCGAGGT

[0193] CACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT

[0194] GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCA

[0195] CGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTA

[0196] CAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA

[0197] AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCA

[0198] AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGA

[0199] GTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTC

[0200] CGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG

[0201] AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT

[0202] CTCCCTGTCTCCGGGTAAA

[0203] SEQ ID NO:9

[0204] MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLI

[0205] KTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEV

[0206] TAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRR

[0207] FLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVY

[0208] GSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCE

[0209] TRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSI

[0210] GSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFN

[0211] VALDQVFESIENSQALVDQSNRILSSAEKGNTSGRENLYFQGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGRHHHHHHHHHHSAWSHPQFEK*

[0212] SEQ ID NO:10

[0213] ATGAGTTGGAAGGTGGTGATCATCTTTAGTCTGCTGATCACCCCTCAGCACGGCCTGAAAG

[0214] AATCTTACCTGGAAGAGTCCTGCTCCACAATCACCGAAGGCTATCTGTCCGTTCTGAGAACC

[0215] GGCTGGTACACCAACGTGTTCACTCTGGAGGTGGGCGACGTGGAAAACCTGACATGCGCC

[0216] GATGGCCCAAGCTTGATCAAAACCGAGCTAGACCTGACCAAGTCCGCCCTGCGAGAACTGA

[0217] GAACAGTCTCCGCTGATCAGCTGGCTAGAGAGGAACAGATCGAGAATCCTAGACGGCGGA

[0218] GATTCGTCCTGGGCGCCATCGCACTGGGAGTGGCCACCGCTGCCGCTGTGACCGCCGGCGT

[0219] GGCTATCGCCAAGACCATCAGACTGGAGTCTGAAGTGACCGCCATCAAGAACGCCCTCAAG

[0220] AAGACCAACGAGGCCGTGTCTACCCTGGGCAACGGCGTCAGGGTGCTGGCTACCGCTGTG

[0221] AGAGAGCTGAAGGACTTCGTGTCGAAGAATCTGACCCGGGCCATCAACAAGAACAAGTGC

[0222] GACATCCCCGACCTGAAGATGGCCGTGTCTTTCTCCCAGTTCAACAGACGGTTTCTGAACGT

[0223] GGTGCGGCAGTTCTCCGACAACGCTGGAATCACACCTGCTATCTCCCTTGATCTGATGACCG

[0224] ATGCCGAGCTGGCCAGAGCTGTGTCCAACATGCCTACCTCTGCCGGCCAAATTAAGCTGAT

[0225] GCTGGAGAACAGAGCCATGGTCCGGAGAAAGGGCTTCGGCATTCTGATCGGCGTGTACGG

[0226] CTCCTCCGTGATCTACATGGTGCAGCTGCCAATCTTCGGCGTTATCGACACCCCTTGCTGGAT

[0227] CGTGAAGGCCGCCCCCTCTTGTTCCGAGAAGAAAGGCAACTACGCCTGCCTGCTGCGCGAA

[0228] GATCAAGGATGGTACTGCCAGAACGCCGGCAGCACCGTGTACTACCCTAACGAGAAAGACT

[0229] GCGAGACCAGAGGCGACCACGTGTTCTGCGATACCGCTGCTGGCATCAACGTGGCCGAGC

[0230] AGAGCAAGGAATGCAATATCAACATCTCCACCACCAACTATCCTTGTAAAGTGTCTACCGGC

[0231] AGGCACCCCATCTCCATGGTGGCTCTGAGCCCTCTGGGCGCTCTGGTGGCTTGCTACAAGG

[0232] GCGTGAGCTGTTCTATAGGCTCCAACAGAGTGGGCATCATCAAGCAGCTGAACAAGGGATG

[0233] TAGCTACATCACCAACCAAGACGCTGACACAGTGACCATCGACAACACGGTGTACCAGCTG

[0234] TCTAAGGTGGAAGGAGAGCAGCACGTGATCAAGGGCCGGCCTGTGTCTTCTTCCTTCGACC

[0235] CTGTTAAGTTCCCTGAGGACCAGTTTAATGTCGCCCTGGATCAGGTGTTCGAATCCATCGAG

[0236] AACTCCCAGGCTCTCGTGGACCAGTCTAACCGGATCCTGTCCTCTGCCGAAAAGGGCAATAC

[0237] CTCCGGCCGCGAGAACCTGTACTTTCAGGGCGGTGGCGGCTCTGGCTACATCCCCGAGGCC

[0238] CCTCGGGACGGCCAGGCCTACGTGCGGAAGGACGGCGAGTGGGTGCTGCTGAGCACCTTC

[0239] CTGGGAGGCATCGAGGGCAGACACCATCACCACCACCATCACCATCACCATTCCGCCTGGTC

[0240] CCACCCACAGTTTGAGAAGTGA

[0241] Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen, characterized in that: include: Capture antibody: human metapneumovirus-specific antibody HMPV01, including the HMPV01 antibody light chain amino acid sequence SEQ ID NO: 1, nucleotide sequence SEQ ID NO: 5, HMPV01 antibody heavy chain amino acid sequence SEQ ID NO: 2, nucleotide sequence SEQ ID NO: 6; Identification antibody: an enzyme-labeled antibody HMPV02-HRP specific for human metapneumovirus, including the HMPV02 antibody light chain amino acid sequence SEQ ID NO: 3, the nucleotide sequence SEQ ID NO: 7, the HMPV02 antibody heavy chain amino acid sequence SEQ ID NO: 4, the nucleotide sequence SEQ ID NO:

8.

2. The enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen according to claim 1, characterized in that: The capture antibody and recognition antibody are prepared by a mammalian cell expression system, and the specific steps are as follows: A1. Insert the HMPV01 antibody light chain nucleotide sequence SEQ ID NO: 5 and the HMPV01 antibody heavy chain nucleotide sequence SEQ ID NO: 6 into the eukaryotic expression vector pcDNA3.4, respectively, insert the HMPV02 antibody light chain nucleotide sequence SEQ ID NO: 7 and the HMPV02 antibody heavy chain nucleotide sequence SEQ ID NO: 8 into the eukaryotic expression vector pGS, perform cloning transformation and plasmid extraction, and construct a recombinant expression vector; A2, transfecting the recombinant expression vector into mammalian cells, including Expi293 cells, ExpiCHO cells, and CHO cells, and then using a serum-free transfection reagent for transfection, and culturing the cells in an incubator after transfection; A3. The cell culture supernatant is then collected and initially purified by affinity chromatography, including Protein A and Protein G affinity chromatography columns. The eluate containing the target antibody is then collected and refined and purified by ion exchange chromatography or gel filtration chromatography to remove impurities and impure proteins to obtain capture antibodies and recognition antibodies.

3. The enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen according to claim 1, characterized in that: The human metapneumovirus-specific enzyme-labeled antibody HMPV02-HRP is obtained by labeling the purified HMPV02 antibody using an HRP labeling kit.

4. A quantitative detection method of an enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen, characterized in that: The enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen according to any one of claims 1 to 3 comprises the following steps: S1. Solid-phase antibody coating: dilute the capture antibody with coating buffer, add it to the ELISA plate wells, and incubate overnight to form a solid-phase antibody; S2, blocking: then discard the coating solution, add blocking solution for incubation, and then wash to remove unbound substances; S3, antigen binding: then add gradient diluted standard antigen or test sample in sequence, incubate and wash, and perform specific detection; S4, enzyme-labeled antibody incubation: then add recognition antibody, dilute with PBS solution, incubate, and then wash; S5, color development termination: then add substrate color development solution, color development at room temperature in the dark, and then add stop solution to terminate the reaction; S6. Detection calculation: Then measure the absorbance value at OD450nm, draw a standard curve with the standard antigen concentration as the horizontal axis and the absorbance value as the vertical axis, and calculate the antigen concentration based on the standard curve equation and the absorbance value of the antigen sample to be detected to complete the detection.

5. The quantitative detection method of the enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen according to claim 4, characterized in that: The coating buffer in S1 includes carbonate buffer, and the blocking solution in S2 includes PBS solution containing 2% BSA.

6. The quantitative detection method of the enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen according to claim 4, characterized in that: The washing in S2-S4 is performed by washing with a washing solution, and the washing solution includes a PBS solution containing 0.05% Tween-20.

7. The quantitative detection method of the enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen according to claim 4, characterized in that: The standard antigen in S3 is obtained by recombinant expression and purification through a eukaryotic expression system, including the recombinantly expressed human metapneumovirus preF conformation F glycoprotein A-115, whose amino acid sequence is SEQ ID NO:9 and nucleotide sequence is SEQ ID NO:

10.

8. The quantitative detection method of the enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen according to claim 4, characterized in that: The substrate color developing solution in S5 includes TMB color developing solution, and the stop solution includes H2SO4 solution.

9. The quantitative detection method of the enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen according to claim 4, characterized in that: The specific detection in S3 specifically comprises the following steps: B1. Dilute the capture antibody and coat the ELISA plate overnight. The next day, remove the capture antibody and add blocking solution to block the plate. B2. Then, dilute each antigen in a gradient, including standard antigen, SARS Cov-2 RBD, RSV F, MERS S, and EbolaGP protein, and then discard the blocking solution, add each diluted antigen, incubate at room temperature, and then tap off; B3. Then dilute the detection antibody and add it to the ELISA plate. After incubation at room temperature, shake off the plate and add the color developing solution to develop the color. Then add the stop solution to stop the color development and place the plate in an ELISA reader to read the reading at a wavelength of 450 nm. B4. Then, the logarithm of each antigen concentration is used as the horizontal axis, and the OD450nm value corresponding to the concentration is used as the vertical axis to fit the curve.

10. The quantitative detection method of the enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen according to claim 4, characterized in that: The quantitative detection method of the enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen is applied to the quantitative detection of antigens in samples containing preF conformational F glycoproteins during vaccine production.

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