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

Abstract

The invention relates to the field of biology, and discloses an enzyme linked immunosorbent assay kit for detecting human metapneumovirus antigens and a quantitative detection method.The enzyme linked immunosorbent assay kit for detecting the human metapneumovirus antigens comprises a capture antibody which is a human metapneumovirus specific antibody HMPV01 or an antigen binding fragment thereof and comprises an HMPV01 antibody light chain amino acid sequence SEQ ID NO: 1 and an HMPV01 antibody heavy chain amino acid sequence SEQ ID NO: 2; the recognition antibody is a human metapneumovirus specific enzyme-labeled antibody HMPV02-HRP or an antigen binding fragment of the human metapneumovirus specific enzyme-labeled antibody HMPV02-HRP. A capture antibody HMPV01 and a recognition antibody HMPV02-HRP with specific amino acid sequences are screened, the antibody is efficiently prepared by using a mammalian cell expression system, and an optimized detection method is combined, so that the binding efficiency of the antibody and the antigen is remarkably improved, and the kit can detect the low-concentration human metapneumovirus antigen.

Description

Technical Field

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

[0002] Human metapneumovirus (Hμman metapneμmovirμs, hMPV) belongs to paramyxovirus, subfamily Pneumovirμs, genus Metapneumovirus, and is a single-stranded negative-strand RNA virus. hMPV is divided into two genotypes, A and B, which are further divided into four subtypes, A1, A2, B1 and B2. Its genome is 13kb long and encodes 9 proteins. Among them, F protein is essential for membrane fusion and can mediate cell fusion without the participation of adsorption proteins, that is, the F protein of hMPV can adsorb cell receptors and activate the membrane fusion process mediated by it, with the dual functions of adsorption and fusion. In addition, F protein plays an important role in inducing immune protection, high levels of serum and antibodies. Because the nucleotide sequence of F protein is highly conserved, the homology of strains A and B can reach 95%, and the protective antibodies induced by F protein can resist infection of different subtypes. The high immunogenicity and high conservatism of F protein determine the potential of F protein to become a vaccine target antigen.

[0003] When traditional enzyme-linked immunosorbent assay kits are used to detect human metapneumovirus antigens, the detection sensitivity is low due to the low efficiency of antibody-antigen binding. In the early stages of viral infection, the viral antigen content in the human body is often low, resulting in missed detections and delaying the early diagnosis and treatment of the disease. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an enzyme-linked immunosorbent assay kit and a quantitative detection method for detecting human metapneumovirus antigen, thereby solving the problem that when detecting human metapneumovirus antigen, the traditional enzyme-linked immunosorbent assay kit has low detection sensitivity due to the low binding efficiency between the antibody and the antigen. In the early stage of viral infection, the viral antigen content in the human body is often low, resulting in missed detection and delaying the early diagnosis and treatment of the disease.

[0005] To achieve the above objectives, the present invention is implemented by the following technical scheme: an enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigens, comprising: Capture antibody: human metapneumovirus-specific antibody HMPV01 or an antigen-binding fragment thereof, including the HMPV01 antibody light chain amino acid sequence SEQ ID NO: 1, the nucleotide sequence SEQ ID NO: 5, the HMPV01 antibody heavy chain amino acid sequence SEQ ID NO: 2, the nucleotide sequence SEQ ID NO: 6; Identification antibody: an enzyme-labeled antibody HMPV02-HRP specific for human metapneumovirus or an antigen-binding fragment thereof, 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.

[0006] Preferably, 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, and 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.

[0007] Preferably, the human metapneumovirus-specific enzyme-labeled antibody HMPV02-HRP is obtained by labeling the purified HMPV02 antibody using an HRP labeling kit.

[0008] The quantitative detection method of the enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen 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.

[0009] Preferably, the coating buffer in S1 includes carbonate buffer, and the blocking solution in S2 includes PBS solution containing 2% BSA.

[0010] Preferably, 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.

[0011] Preferably, 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.

[0012] Preferably, the substrate color developing solution in S5 includes TMB color developing solution, and the stop solution includes H2SO4 solution.

[0013] Preferably, 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 Ebola GP 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.

[0014] Preferably, 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 glycoprotein during vaccine production.

[0015] The present invention provides an enzyme-linked immunosorbent assay kit and a quantitative detection method for detecting human metapneumovirus antigens. The present invention has the following beneficial effects: 1. The present invention screens capture antibodies HMPV01 and recognition antibodies HMPV02-HRP with specific amino acid sequences, uses a mammalian cell expression system to efficiently prepare antibodies, and combines them with an optimized detection method to significantly improve the binding efficiency of antibodies and antigens, so that the kit can detect low concentrations of human metapneumovirus antigens, effectively solving the problem of low detection sensitivity of traditional kits, and can detect viral antigens in the human body at the early stage of viral infection, avoiding the occurrence of missed detection, and providing strong support for early diagnosis of the disease.

[0016] 2. The capture antibody and recognition antibody of the present invention are highly specific to the human metapneumovirus preF conformation F glycoprotein A-115. Through the specific detection step, they have no cross-reaction with other viral antigens and can accurately identify the target antigen, greatly reducing the probability of false positive test results and ensuring the accuracy and reliability of the test results.

[0017] 3. The quantitative detection method of the present invention is easy to operate and has good repeatability. It is suitable for the detection of various samples, especially in the vaccine production process. It can accurately detect the antigen quantitatively of samples containing preF conformation F glycoprotein. By drawing a standard curve and calculating the antigen concentration, the antigen content in the vaccine production process can be effectively monitored to ensure the stability and consistency of vaccine quality, which is of great significance to the quality control of vaccine research and development and production.

[0018] 4. The present invention prepares capture antibodies and recognition antibodies by adopting a mammalian cell expression system, constructs a recombinant expression vector, and then obtains high-purity antibodies through a series of operations such as transfection, culture, affinity chromatography, and fine purification, thereby 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

[0019] Figure 1 is the plasmid map of the eukaryotic expression vector pcDNA3.4 of the present invention; Figure 2 This is a schematic diagram of the purification results of the hMPV preF protein A-115 of the present invention; Figure 3 It is a schematic diagram of the purification results of HMPV01 and HMPV02 antibodies of the present invention; Figure 4 It is a schematic diagram of the results of the HMPV01 and HMPV02 antibody binding activity assay of the present invention; Figure 5 It is a schematic diagram of the results of the HMPV01-HRP and HMPV02-HRP binding activity assay of the present invention; Figure 6It is a schematic diagram of the results of paired detection of HMPV0 and HMPV02-HRP and HMPV02 and HMPV01-HRP of the present invention; Figure 7 It is a schematic diagram of the results of the specific detection of the present invention; Figure 8 It is a schematic diagram of the standard curve of the present invention. DETAILED DESCRIPTION

[0020] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Please see attached Figure 1 -Attached Figure 8 The present invention provides an enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigens, comprising: Capture antibody: human metapneumovirus-specific antibody HMPV01 or an antigen-binding fragment thereof, including the HMPV01 antibody light chain amino acid sequence SEQ ID NO: 1, the nucleotide sequence SEQ ID NO: 5, the HMPV01 antibody heavy chain amino acid sequence SEQ ID NO: 2, the nucleotide sequence SEQ ID NO: 6; Identification antibody: an enzyme-labeled antibody HMPV02-HRP specific for human metapneumovirus or an antigen-binding fragment thereof, 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.

[0022] Specifically, the capture antibody HMPV01: a monoclonal antibody was obtained by immunizing rabbits, and its light chain (SEQIDNO: 1) and heavy chain (SEQIDNO: 2) amino acid sequences were obtained after affinity maturation optimization and humanization. X-ray crystal diffraction analysis showed that its CDR region could accurately recognize the conserved epitope (amino acid residues 115-132) of hMPVpreF conformation F glycoprotein A-115, and the dissociation constant was significantly improved compared with the affinity of traditional polyclonal antibodies.

[0023] Identification antibody HMPV02-HRP: Monoclonal antibody was obtained by immunizing rabbits. After affinity maturation optimization and humanization, its light chain (SEQIDNO: 3) and heavy chain (SEQIDNO: 4) amino acid sequences were obtained. After labeling with HRP, more than 95% binding activity was retained, and the enzyme activity recovery rate reached 82%, achieving a signal amplification effect.

[0024] The capture antibody and recognition antibody are prepared by 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, and 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.

[0025] Specifically, the HMPV01 antibody gene was inserted into the pcDNA3.4 vector ( Figure 1 ), using the CMV enhancer 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.

[0026] The light chain and heavy chain of HMPV02 antibody were cloned into the pGS vector at the same time, and the high-expressing cell lines were screened by the glutamine synthetase (GS) pressurization system, and the stable expression level in CHO cells reached 1.8g / L.

[0027] Purification process: Protein A affinity chromatography ( Figure 3 ) to capture antibodies with a purity of 90%; endotoxin was further removed by Q Sepharose ion exchange chromatography (pH 7.0) to <0.1 EΜ / mg, with a final purity of >98% (verified by SDS-PAGE).

[0028] Please refer to the attached Figure 5The human metapneumovirus-specific enzyme-labeled antibody HMPV02-HRP was obtained by labeling the purified HMPV02 antibody using an HRP labeling kit.

[0029] Specifically, the Sμlfo-SMCC crosslinker was used to mediate the coupling of HRP and HMPV02 antibody. The labeling ratio was determined to be 4.2:1 (HRP:antibody) by UV spectrophotometry, and the ELISA confirmed that its detection sensitivity for A-115 antigen was 6.1×10 -5 μg / mL.

[0030] The quantitative detection method of the enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen 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.

[0031] The coating buffer in S1 includes carbonate buffer, and the blocking solution in S2 includes PBS solution containing 2% BSA.

[0032] The washing in S2-S4 is performed by washing with a washing solution, which includes a PBS solution containing 0.05% Tween-20.

[0033] 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.

[0034] In S5, the substrate color developing solution includes TMB color developing solution, and the stop solution includes H2SO4 solution.

[0035] The specific detection in S3 specifically includes 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-2RBD, RSV F, MERS S, and Ebola GP 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.

[0036] The quantitative detection method of the enzyme-linked immunosorbent assay kit for detecting human metapneumovirus antigen is applied to the quantitative detection of antigen in samples containing preF conformation F glycoprotein during vaccine production.

[0037] Specifically, The capture antibody HMPV01 and the recognition antibody HMPV02-HRP with specific amino acid sequences were screened, and they were efficiently expressed using a mammalian cell expression system (Expi293 cells, ExpiCHO cells, CHO cells) combined with a specific eukaryotic expression vector (pcDNA3.4, pGS). High-purity antibodies were obtained through multi-step purification processes such as affinity chromatography (ProteinA, ProteinG affinity chromatography columns) and ion exchange chromatography or gel filtration chromatography, and the purified HMPV02 antibody was labeled with an HRP labeling kit to obtain antibodies with high affinity, high specificity and good labeling effect, which significantly improved the binding efficiency of antibodies and antigens, enhanced the signal intensity during the detection process, and achieved high-sensitivity detection of human metapneumovirus antigens. It can accurately detect low concentrations of viral antigens in the human body in the early stage of viral infection, thereby solving the problem that traditional enzyme-linked immunosorbent assay kits have low detection sensitivity when detecting human metapneumovirus antigens due to low binding efficiency of antibodies and antigens. In the early stage of viral infection, the content of viral antigens in the human body is often low, resulting in missed detection and delaying the early diagnosis and treatment of the disease.

[0038] A human metapneumovirus antigen quantitative detection kit, comprising a capture antibody HMPV01 or an antigen-binding fragment thereof selected from the following sequences: HMPV01 antibody light chain amino acid sequence SEQ ID NO: 1 SYELTQPPSVSVSPGQTATITCSGTNLGAKYSCWYQQKPGQSPVLVIFQDNKRASGIPARFSASTSGDTATLTISGTQVMDEADYFCQAWDSRTAVFGGGTTLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS* HMPV01 antibody heavy chain amino acid sequence SEQ ID NO: 2 QVQLQESGPGLVKPSQTLSLTCTVSGGSVRGGGDYWSWIRRPGKGLEWIGHIYNSGTTFYNPSLKSRVTISIDTSKNQFSLKLRSVTAADTAVYYCGRVGSRATGLPDWIDP WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK* The antibody or antigen-binding fragment comprising HMPV02 is selected from the following sequences: HMPV02 antibody light chain amino acid sequence SEQ ID NO: 3 SYELIQLPSVSVSPGQTARIPCSGDGLPKKYAHWYQQKAGQAPILLMYKDSERPSGIPERFSAFSSGTTVTMTISGVQEEDEADYYCQSGDSTDTSVIFGGGTKVTV LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS* HMPV02 antibody heavy chain amino acid sequence SEQ ID NO: 4 QVQLVQSGPALVKSTQTLTLTCTFSGFALTTSGMCVSWVRQPPGKALEWLARIDWEDNTYYSTSLKTRVTISKDPSKNQVVLTTMTNMDPVDTATYYCARSYITDWKKDWFFDL WGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* A method for quantitatively detecting human metapneumovirus antigens: using human metapneumovirus-specific antibody HMPV01 to coat an ELISA plate to prepare a solid-phase antibody, adding standard antigens or antigen samples to be detected in different concentration gradient dilutions at one time, using human metapneumovirus-specific enzyme-labeled antibody HMPV02-HRP as a recognition antibody, adding a substrate colorimetric solution, adding a 2M H2SO4 solution to terminate color development, measuring the OD450nm value, drawing 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 based on the standard curve equation and the absorbance value of the antigen sample to be detected.

[0039] Example 1: Recombinant expression and purification of hMPV preF protein A-115 protein By genetic engineering technology, the antigen nucleotide sequence SEQ ID NO: 10 was inserted into the eukaryotic expression vector pcDNA3.4 (plasmid map see Figure 1 .), clone and transform the recombinant expression vector and extract the plasmid, which was used to transfect ExpiCHO cells for recombinant protein expression. ExpiCHO cells in the logarithmic growth phase were passaged at high density one day before transfection, and the viable cell density was 3×10 6 After the cells were grown overnight, the cell density was diluted to 6×10 6 / mL. Take 200mL of cell suspension for transfection. Use MetaCell CHO-320 medium to dilute 200μl DNA and 1.4mL PEI40K transfection reagent respectively, and mix gently. Then gently add the mixed transfection reagent to the diluted plasmid and let it stand at room temperature for 10 minutes to prepare the DNA complex. After incubation, add the DNA complex drop by drop to the cell solution to be transfected, and transfer to a shaker at 37℃ with 8% CO2 and culture at 225rpm. Add feed and Enhancer 24h after transfection, and cool to 32℃ for continued culture.

[0040] On the fifth day of transfection, the cell culture supernatant was collected by centrifugation at 4700 rpm for 30 min at 4°C. After impurities were removed with a 0.22 μm filter membrane, the supernatant was concentrated to about 300 mL by TFF and the protein was purified by Strep II affinity chromatography. The specific implementation steps are as follows: (1) Column loading: 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 (μμ pH 7.4), and immediately close the lower outlet after PBS flows out from the lower outlet; suspend the resin, use a pipette to take an appropriate amount of slurry and add it to the column (the ratio of preservation solution to filler is 1:1), open the lower outlet to drain the protective solution; add an appropriate amount of PBS buffer to rinse the column material, and close the lower outlet after the column material flows dry. (2) Equilibration: Use 5 times the column volume of equilibrium solution (PBS buffer, μμ, The column was then equilibrated with 10 columns of equilibration / washing solution to remove non-specifically bound proteins and collect the wash solution. (5) Elution: The target protein was eluted with 5 columns of elution solution (μμD-Biotin PBS buffer, pH 7.4) and the target protein was collected and concentrated in a separate tube for replacement. (6) Quantification: The concentrated protein was mixed and quantified at ΜV280nm. A small amount of protein was taken out for SDS-PAGE detection. The results are shown in the table. Figure 2The remaining aliquots were stored at -80℃.

[0041] Example 2: Recombinant expression and purification of HMPV01 and HMPV02 antibodies By genetic engineering technology, the nucleotide light chain sequence SEQ ID NO: 5 and the heavy chain sequence SEQ ID NO: 6 of the HMPV01 antibody were inserted into the eukaryotic expression vector pcDNA3.4, and the nucleotide light chain sequence SEQ ID NO: 7 and the 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 cloned and transformed, and the plasmids were extracted and used to transfect Expi293 cells for recombinant protein expression. Expi293 cells in the logarithmic growth phase were passaged at high density one day before transfection, and the viable cell density was 2.5×10 6 After the cells were grown overnight, the cell density was diluted to 3×10 6 / mL, take 200mL of cell suspension for transfection. Use Opti-MEM medium to dilute 200μl DNA and 1.4mL PEI40K transfection reagent respectively, and mix gently. Then gently add the mixed transfection reagent to the diluted plasmid and let it stand at room temperature for 10 minutes to prepare the DNA complex. After incubation, add the DNA complex drop by drop to the cell solution to be transfected, and transfer to a shaker at 37℃ with 8% CO2 and culture at 225rpm. Add feed and continue culture 24h and 48h after transfection.

[0042] On the 5th day of transfection, the cell culture supernatant was collected by centrifugation at 4700 rpm for 30 min at 4°C, filtered with a 0.22μm vacuum filter to remove impurities, and then purified by protein A affinity chromatography. The specific implementation steps are as follows: (1) Column loading: Take a 20mL empty column, press the lower gasket to the bottom of the column and compact it, rinse the gasket with PBS buffer (μμpH7.4), and close the lower outlet immediately after PBS flows out from the lower outlet; suspend the resin, use a pipette to take an appropriate amount of slurry and add it to the column (the ratio of preservation solution to filler is 1:1), open the lower outlet to drain the protective solution; add an appropriate amount of PBS buffer to rinse the column material, and close the lower outlet after the column material is dry (2) Balance: Use 5 times the column volume of balance solution (PBS buffer μμ (3) Binding and loading: resuspend the filler with an appropriate amount of sample and transfer it to the sample, and combine it on a shaker at 4°C for 3 hours; then add the sample and filler to the balanced column, and load it multiple times to increase the binding efficiency and collect the flow-through for subsequent SDS-PAGE detection of protein binding (4) Equilibration and washing: wash with 10 times the column volume of equilibration / washing solution to remove non-specifically bound impurities, and collect the washing solution (5) Elution: finally elute the target protein with 5 times the column volume of elution solution (0.1M Glycine, pH3.0), and add 1 / 10 of neutralizing solution (1M Tris-HCl, pH9.0) for neutralization, collect and concentrate the solution in a separate tube (6) Quantification: mix the concentrated protein and quantify it at ΜV280nm, take out a small amount of protein for SDS-PAGE detection, the results are shown in Figure 3 and Figure 4 .

[0043] Example 3: Binding activity assay of purified HMPV01 and HMPV02 antibodies The monoclonal antibodies purified in the above case were used to measure antibody binding capacity by direct ELISA. The specific implementation method is as follows: dilute antigen A-115 to 1μg / mL, 100μl per well, and coat overnight at 4℃. The next day, after the antigen was tapped off, 300μl of 2% BSA solution was added to each well and blocked at 37℃ for 2h. After tapping off the blocking solution, gradient dilutions of the antibodies to be tested HMPV01 and HMPV02 (starting from 2μg / mL, 12 gradients of 4-fold dilution) were added, 100μl / well, and incubated at room temperature for 1h. After tapping off the primary antibody, HRP-labeled secondary antibody (purchased from Jackson ImmμnoResearch Inc.) was added, 100μl / well, and incubated for another 1h. Subsequently, 100μl / well TMB colorimetric solution was added for color development, and 50μl / well stop solution was added after 15min to stop the color development. Finally, the reading was performed at a wavelength of 450nm. The cut-off value is 2.1 times the reading of the 2% BSA control well. If the average reading of the antibody supernatant is greater than this value, it means that the antibody can bind to the A-115 protein. If it is less than the cut-off value, it means 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.

[0044] Example 4: Preparation of horseradish peroxidase-labeled HMPV01 and HMPV02 antibodies and determination of their binding activity (1) Antibody labeling The purified HMPV01 and HMPV02 antibodies were labeled using an HRP labeling kit (purchased from proteintech). The specific implementation steps are as follows: the HRP labeling kit was taken out from -20°C, equilibrated at room temperature for 30 minutes, and the reaction start solution and the stop solution were fully thawed and mixed for use; 1 μl of the reaction start solution was added to every 10 μl of the antibody solution to be labeled, and the mixture was repeatedly blown several times with a pipette to fully mix to avoid bubbles; the cap of the horseradish peroxidase bottle was opened, and the above-mentioned started antibody solution was directly added to the bottle, and the mixture was repeatedly blown several times with a pipette to fully mix to avoid bubbles, and the mixture was placed at room temperature for 3 hours; the reaction stop solution was added to the horseradish peroxidase, with a ratio of 1 μl of the reaction stop solution added to every 10 μl of the antibody, the mixture was fully mixed, and the mixture was placed at room temperature for 1 hour; after the termination was completed, an equal volume of the product protection solution was added, the mixture was fully mixed, and the mixture was stored at -20°C.

[0045] (2) Binding activity assay The binding activity of the two labeled antibodies was detected by direct ELISA. The specific method is as follows: dilute antigen A-115 to 1μg μμg / mL, 100μl per well, and coat overnight at 4℃. The next day, after the antigen was tapped off, 300μl of 2% BSA solution was added to each well and blocked at 37℃ for 2h. After tapping off the blocking solution, add gradient diluted enzyme-labeled antibodies HMPV01-HRP and HMPV02-HRP (starting from 20μg μμg / mL, 12 gradients of 4-fold dilution), 100μl / well, and incubate at room temperature for 1.5h. After tapping off, add 100μl / well TMB colorimetric solution for color development, and add 50μl / well stop solution after 15min to stop color development. Finally, read at a wavelength of 450nm. The Cμt off value is 2.1 times the reading of the 2% BSA control well. If the average reading of the antibody supernatant is greater than this value, it means that the antibody can specifically bind to the A-115 protein. If it is less than the Cμt-off value, it means that the antibody cannot specifically bind to the A-115 protein. The results show that the HRP-labeled HMPV01 and HMPV02 antibodies can specifically bind to the A-115 protein.

[0046] Example 5: Paired detection of HMPV01 and HMPV02-HRP and HMPV02 and HMPV01-HRP HMPV01 and HMPV02 were used as capture antibodies, diluted to 2μμg / mL and coated on the ELISA plate, 100μl / well at 4℃ overnight. The capture antibody was shaken off the next day and 2% BSA was added for blocking for 2h. Then the antigen A-115 was diluted 4-fold from the starting concentration of 1μμg / mL to 12 gradients. The blocking solution was shaken off, the diluted antigen was added, and it was incubated at room temperature for 1h and then tapped off. Then the detection antibody HMPV02-HRP diluted to 2μμg / mL was added to the corresponding capture antibody HMPV01, 100μl / well. Similarly, the detection antibody HMPV01-HRP diluted to 2μμg / mL was added to the corresponding capture antibody HMPV02, 100μl / well. After incubation at room temperature for 1h, shaken off at the same time, TMB colorimetric solution was added for color development for 15min, and then the stop solution was added to stop and placed in the microplate reader to read the reading at a wavelength of 450nm. The results showed that the two antibodies could be paired together. When HMPV02-HRP was used as the recognition antibody and paired with the coating antibody HMPV01, the sensitivity of quantifying 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.

[0047] Example 6: Specificity Detection The capture antibody HMPV01 was diluted to 2μμg / mL and coated on the ELISA plate, 100μl / well at 4℃ overnight. The capture antibody was shaken off the next day and 2% BSA was added for blocking for 2h. Then the antigen A-115, SARS CoV-2 RBD, RSV F, MERS S, and EbolaGP protein were diluted 4-fold in 12 gradients from the starting concentration of 1μμg / mL. The blocking solution was shaken off, the diluted antigen was added, and it was incubated at room temperature for 1h and then tapped off. Then the detection antibody HMPV02-HRP was diluted to 2μμg / mL and 100μl / well was added to the ELISA plate. After incubation at room temperature for 1h, it was shaken off at the same time, TMB colorimetric solution was added for color development for 15min, and then the stop solution was added to stop and placed in the ELISA reader to read the reading at a wavelength of 450nm. The logarithm of each antigen concentration was used as the horizontal axis, and the OD450nm value corresponding to the concentration was used as the vertical axis to fit the curve. Judging from the experimental results, only the A-115 antigen protein has an obvious dose-dependent effect, and the results of other viral proteins are negative, which indicates that the antibody has specific binding to the A-115 antigen and has no cross-reaction to other viral antigens.

[0048] Example 7: Sensitivity Detection The capture antibody HMPV01 was diluted to 2μμg / mL and coated on the ELISA plate, 100μl / well was coated overnight at 4℃. The capture antibody was shaken off the next day and 2% BSA was added for blocking for 2h. Then the antigen A-115 was diluted 4-fold from the starting concentration of 1μμg / mL to 12 gradients. The blocking solution was shaken off, the diluted antigen was added, and it was incubated at room temperature for 1h and then tapped off. Then the detection antibody HMPV02-HRP was diluted to 2μμg / mL and 100μl / well was added to the ELISA plate. After incubation at room temperature for 1h, it was shaken off at the same time, and the concentration of TMB colorimetric solution was added as the horizontal axis, and the OD450nm value corresponding to each standard concentration was used as the vertical axis to draw a standard curve. The standard curve shows that the lowest dilution concentration of A-115 protein is 6.1×10 -5 μgμμg / mL, so the sensitivity of this method is 6.1×10 -5 μg μμg / mL.

[0049] Example 8: Application of the antigen quantitative detection method The hMPV F protein A-115 antigen quantitative detection method established in this case quantified 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 and 4°C for 30 minutes, and the cell supernatant was collected and stored at -80°C for later use. By double sandwich ELISA, 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 a certain multiple, and 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 A-115 protein in the cell expression supernatant on the 5th day after transfection was 0.4μg μμg / mL.

[0050] sequence: SEQ ID NO:1 SYELTQPPSVSVSPGQTATITCSGTNLGAKYSCWYQQKPGQSPVLVIFQDNKRASGIPARFSASTSGDTATLTISGTQVMDEADYFCQAWDSRTAVFGGGTTLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS* SEQ ID NO:2 QVQLQESGPGLVKPSQTLSLTCTVSGGSVRGGGDYWSWIRRPPGKGLEWIGHIYNSGTTFYNPSLKSRVTISIDTSKNQFSLKLRSVTAADTAVYYCGRVGSRATGLPDWIDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK* SEQ ID NO:3 SYELIQLPSVSVSPGQTARIPCSGDGLPKKYAHWYQQKAGQAPILLMYKDSERPSGIPERFSAFSSGTTVTMTISGVQEEDEADYYCQSGDSTDTSVIFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS* SEQ ID NO:4 QVQLVQSGPALVKSTQTLTLTCTFSGFALTTSGMCVSWVRQPPGKALEWLARIDWEDNTYYSTSLKTRVTISKDPSKNQVVLTMTNMDPVDTATYYCARSYITDWKKDWFFDLWGRGTLVTSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK* SEQ ID NO:5 TCTTACGAGCTGACCCAGCCCCCTTCTGTGTCTGTGTCTCCCGGTCAGACCGCCACTATCACCTGTTCTGGTACTAACCTGGGCGCCAAGTACAGTTGCTGGTATCAGCAGAAGCCCGGCCAGAGCCCAGTGCTGGTGATTTTCCAGGACAATAAGAGAGCTAGCGGCATCCCCGCCCGGTTTTCTGCTTCTACCTCCGGTGACACCGCCACCCTGACTATCTCCGGTACCCAGGTCATGGACGAGGCCGATTATTTCTGCCAGGCCTGGGACAGCAGAACCGCTGTGTTCGGAGGCGGTACCACCCTGACAGTGCTGGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGGAGCTACAGTTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA SEQ ID NO:6 SEQ ID NO:7 TCTTACGAGCTGATCCAGCTGCCCTCCGTCTCCGTGTCTCCTGGACAGACAGCCAGGATTCCTTGCTCCGGCGACGGTCTGCCTAAGAAGTACGCCCACTGGTACCAGCAGAAGGCCGGTCAGGCACCTATCCTGCTGATGTACAAGGACTCCGAGAGGCCCTCCGGCATTCCAGAAAGATTCTCCGCTTTTTCCTCCGGCACCACCGTGACCATGACTATCTCCGGCGTGCAGGAGGAGGACGAGGCTGATTACTACTGCCAGTCCGGCGACTCCACCGACACATCTGTCATTTTCGGCGGCGGAACCAAGGTGACCGTGCTGGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGGAGCTACAGTTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA SEQ ID NO:8 SEQ ID NO:9 MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCADGPSLIKTELDLTKSALRELRTVSADQLAREEQIENPRRRRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKKTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPVKFPEDQFNVALDQVFESIENSQALVDQSNRILSSAEKGNTSGRENLYFQGGGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGRHHHHHHHHHHSAWSHPQFEK* SEQ ID NO:10 Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that 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 or an antigen-binding fragment thereof, including the HMPV01 antibody light chain amino acid sequence SEQ ID NO: 1, the nucleotide sequence SEQ ID NO: 5, the HMPV01 antibody heavy chain amino acid sequence SEQ ID NO: 2, the nucleotide sequence SEQ ID NO: 6; Identification antibody: an enzyme-labeled antibody HMPV02-HRP specific for human metapneumovirus or an antigen-binding fragment thereof, 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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