Pref monoclonal antibody combination for metapneumovirus detection and application thereof

Abstract

The application provides a PreF monoclonal antibody combination for metapneumovirus detection and application thereof, the PreF monoclonal antibody combination for metapneumovirus detection comprises a first antibody and a second antibody, the first antibody comprises a first light chain variable region and a first heavy chain variable region, and the second antibody comprises a second light chain variable region and a second heavy chain variable region; wherein the amino acid sequence of the first light chain variable region comprises VL-CDR1-3 shown in SEQ ID NO:1-3; the amino acid sequence of the first heavy chain variable region comprises VH-CDR1-3 shown in SEQ ID NO:4-6; the amino acid sequence of the second light chain variable region comprises VL-CDR1-3 shown in SEQ ID NO:7-9; and the amino acid sequence of the second heavy chain variable region comprises VH-CDR1-3 shown in SEQ ID NO:10-12. The monoclonal antibody combination can improve the sensitivity and accuracy of in vitro detection.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, specifically to a monoclonal antibody combination for detecting metapneumovirus and its application. Background Technology

[0002] Human metapneumovirus (hMPV) is a member of the genus Metapneumovirus in the family Pneumoviridae, first isolated from respiratory infection patients in 2001. The virus particles are spherical or fibrous, approximately 150–600 nm in diameter. Its genome is a single-stranded negative-sense RNA (approximately 13 kb in length), encoding nine proteins, among which the fusion protein (F protein) and attachment protein (G protein) are the main surface antigens. The fusion protein (F protein) is a key molecule for viral invasion of host cells, and its pre-fusion conformation (PreF) is a core target for vaccine development and neutralizing antibody research.

[0003] hMPV is mainly transmitted through respiratory droplets and contact, primarily causing bronchiolitis and pneumonia in infants and young children. The elderly and those with weakened immune systems are more susceptible to developing severe respiratory failure. Its prevalence is global, with late winter and early spring being the peak season. In my country, the detection rate of hMPV in children with respiratory infections is 2%-8%, and the mortality rate in the elderly and immunocompromised individuals exceeds 10%. For the diagnosis of hMPV, because its clinical symptoms are similar to influenza, it is necessary to differentiate it from influenza viruses, parainfluenza viruses, etc., through etiological testing. Currently, there are no specific antiviral drugs or marketed vaccines; clinical treatment relies on "early diagnosis + symptomatic supportive care." Existing detection technologies include virus isolation and culture (culture cycle 7-14 days, time-consuming), nucleic acid detection (high sensitivity, but dependent on specialized equipment and high cost), and existing antigen detection kits (low sensitivity, prone to cross-contamination).

[0004] In summary, developing highly specific monoclonal antibodies is key to solving the clinical diagnosis of hMPV.

[0005] Immunoassay techniques are widely used in pathogen detection due to their high specificity and ease of operation. Existing technologies such as immunochromatography, enzyme-linked immunosorbent assay (ELISA), and chemiluminescent immunoassay can all be used for antigen detection. However, the core of these methods lies in using high-quality antibodies as recognition elements.

[0006] Monoclonal antibodies, due to their targeting of specific antigenic epitopes, high homogeneity, and strong batch stability, have become the foundation of high-performance immunoassays. Specific antibody cell lines are typically obtained through hybridoma technology, and further sequence analysis, expression vector construction, and recombinant expression can be performed to achieve large-scale stable production. However, the lack of a high-affinity, high-specificity PreF monoclonal antibody for human metapneumovirus detection currently limits the development of high-performance diagnostic products based on this target.

[0007] Therefore, a PreF monoclonal antibody with high affinity and high specificity is provided for constructing a high-performance in vitro immunodiagnostic tool to meet the needs of early detection and accurate diagnosis of human metapneumovirus infection. Summary of the Invention

[0008] This application provides a PreF monoclonal antibody combination for the detection of metapneumovirus and its application, aiming to solve the technical problems of low detection sensitivity, poor specificity, easy cross-reactivity and poor batch stability of antibodies in the in vitro diagnosis of human metapneumovirus infection. It is applicable to a variety of immunoassay methods and can be used to improve the early detection rate and diagnostic accuracy of human metapneumovirus infection.

[0009] In a first aspect, this application provides a PreF monoclonal antibody combination for detecting metapneumovirus, comprising a first antibody and a second antibody, wherein the first antibody includes a first light chain variable region and a first heavy chain variable region, and the second antibody includes a second light chain variable region and a second heavy chain variable region; wherein...

[0010] The amino acid sequence of the first light chain variable region includes VL-CDR1 shown in SEQ ID NO:1, VL-CDR2 shown in SEQ ID NO:2 and VL-CDR3 shown in SEQ ID NO:3;

[0011] The amino acid sequence of the first heavy chain variable region includes VH-CDR1 shown in SEQ ID NO:4, VH-CDR2 shown in SEQ ID NO:5, and VH-CDR3 shown in SEQ ID NO:6;

[0012] The amino acid sequence of the variable region of the second light chain includes VL-CDR1 shown in SEQ ID NO:7, VL-CDR2 shown in SEQ ID NO:8, and VL-CDR3 shown in SEQ ID NO:9;

[0013] The amino acid sequence of the variable region of the second heavy chain includes VH-CDR1 shown in SEQ ID NO:10, VH-CDR2 shown in SEQ ID NO:11, and VH-CDR3 shown in SEQ ID NO:12.

[0014] According to this application, the monoclonal antibody combination contains two rabbit-derived antibodies that can recognize PreF, thereby achieving higher binding affinity and stronger specificity, effectively reducing the risk of false negatives and cross-reactions, and improving the sensitivity and accuracy of in vitro detection.

[0015] Specifically, the first and second antibodies each possess independent light and heavy chain variable regions, and their respective CDR1, CDR2, and CDR3 sequences have been validated through animal immunization, screening, and cloning, demonstrating stability and specific recognition capabilities. By clearly defining the six complementarity-determining regions (CDRs) of the two antibodies at the structural level, the precise localization and reproducibility of the antigen recognition sites are ensured, contributing to maintaining the consistency and stability of antibody function during subsequent recombinant expression.

[0016] Furthermore, the first and second antibodies can work synergistically when binding to the PreF antigen, which significantly enhances the antibody combination provided in this application compared to a single antibody in terms of antigen capture, detection signal intensity, and anti-interference ability, thereby effectively improving the sensitivity and accuracy of in vitro detection of human metapneumovirus.

[0017] In some embodiments, the amino acid sequence of the first light chain variable region is shown in SEQ ID NO:13; and the amino acid sequence of the first heavy chain variable region is shown in SEQ ID NO:14.

[0018] In some embodiments, the amino acid sequence of the second light chain variable region is shown in SEQ ID NO:15; and the amino acid sequence of the second heavy chain variable region is shown in SEQ ID NO:16.

[0019] In some of the above embodiments, by defining the full-length variable region sequence, rather than just the complementarity-determining region, the natural combination relationship between the frame region (FR) and the CDR can be fully preserved. The frame region plays an important role in maintaining the spatial conformation of the antibody variable region, stabilizing the correct folding of the CDR, and supporting the formation of the antigen-binding surface. Therefore, further defining the full-length variable region sequence based on the CDR sequence can ensure that the antibody maintains a stable tertiary structure and a high level of specific binding ability in recombinant expression and practical applications. Compared with the scheme that relies solely on the CDR sequence definition, this implementation can reduce structural perturbations or affinity reductions caused by frame region differences, ensure batch-to-batch consistency and reproducibility of antibody function, and thus exhibit higher stability and reliability in practical detection applications.

[0020] In some embodiments, the nucleotide sequence of the first light chain variable region is shown in SEQ ID NO:17; and the nucleotide sequence of the first heavy chain variable region is shown in SEQ ID NO:18.

[0021] In some embodiments, the nucleotide sequence of the second light chain variable region is shown in SEQ ID NO:19; and the nucleotide sequence of the second heavy chain variable region is shown in SEQ ID NO:20.

[0022] In some of the above embodiments, by explicitly defining the nucleotide sequence of the antibody's variable region, it can be ensured that the antibody can be stably expressed in different recombinant expression systems and maintain the correct amino acid sequence. Nucleotide-level definition not only covers the translation products of the amino acid sequence but also ensures the consistency between transcription and translation, thereby avoiding amino acid drift caused by codon differences or potential mutations. Compared to defining only at the amino acid level, nucleotide sequence definition provides a more direct technical basis for subsequent plasmid construction, vector assembly, and expression optimization, improving process controllability and production stability.

[0023] Secondly, this application provides the use of the PreF monoclonal antibody according to any embodiment of the first aspect in the preparation of a tool for detecting human metapneumovirus.

[0024] According to this application, by applying the above-mentioned antibody combination to in vitro diagnostic tools, a highly sensitive and specific human metapneumovirus detection system can be constructed. The antibody combination can simultaneously identify different binding sites of the PreF antigen during the detection process, achieving multiple capture and signal enhancement, thereby significantly improving the sensitivity and accuracy of the detection. Compared with single antibodies, this combination has significant advantages in reducing false negatives, reducing cross-reactivity, and improving the reliability of detection results.

[0025] In some embodiments, the tool for detecting human metapneumovirus is used to detect human metapneumovirus in an in vitro sample, said in vitro sample including at least one of a nasal swab, a throat swab, and a nasopharyngeal swab.

[0026] In some of the above embodiments, specifying the sample type helps to achieve rapid and accurate detection in different clinical scenarios; nasal swabs, throat swabs, and nasopharyngeal swabs expand the scope of application. Compared to detection tools limited to a single type of sample, the tools described in this embodiment have wider applicability in clinical application.

[0027] In some embodiments, the PreF monoclonal antibody is used in an immunoassay method for detecting human metapneumovirus in an in vitro sample.

[0028] In some of the above embodiments, by introducing the antibody combination provided in this application into the immune detection system, the presence of PreF in in vitro samples can be directly detected by utilizing the principle of specific binding between antigen and antibody.

[0029] In some embodiments, the immunoassay method includes at least one of enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, chemiluminescence immunoassay, immunomicrosphere assay, and immunogold assay.

[0030] In some of the above embodiments, different immunoassay methods can utilize the high specificity recognition performance of the antibody combination of this application; enzyme-linked immunosorbent assay (ELISA) is suitable for high-throughput detection in laboratories; immunofluorescence and chemiluminescence methods have higher sensitivity and quantitative detection capabilities; immunomicrosphere methods facilitate multiplexing; and immunogold methods are suitable for rapid and convenient point-of-care testing. Therefore, with the antibody combination of this application, different detection platforms can be flexibly combined to meet the diverse needs of laboratory research and point-of-care testing.

[0031] In some embodiments, the tool for detecting human metapneumovirus includes at least one of reagents, kits, test strips, and antibody chips.

[0032] In some of the above embodiments, the antibody combinations provided in this application can be used to prepare different forms of detection products. The reagents or kits are suitable for standardized laboratory testing, the test strips facilitate rapid screening and point-of-care testing, and the antibody chips can achieve high-throughput, automated parallel analysis. Through the expansion of various tool forms, this embodiment can cover a variety of different application scenarios, greatly improving the flexibility and promotional value of testing.

[0033] Compared with the prior art, the beneficial effects of this application are at least as follows:

[0034] 1. The PreF monoclonal antibody combination provided in this application contains two antibodies obtained through animal immunization and selection. By clearly defining the CDR sequences of their respective light chain and heavy chain variable regions, it achieves efficient recognition of PreF and can obtain higher affinity and specificity in in vitro detection.

[0035] 2. Using two antibodies together in the same detection system can create a synergistic effect during antigen capture. Compared with single antibody detection, this can effectively reduce the risk of false negatives and cross-reactions, thereby improving the sensitivity and accuracy of the test results.

[0036] 3. By further defining the amino acid and nucleotide sequences of the full-length variable region, the structural consistency and functional stability of the antibody in recombinant expression and mass production can be ensured, thereby improving the reproducibility and reliability of the detection tool in clinical and industrial applications. Attached Figure Description

[0037] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0038] Figure 1 This is an SDS-PAGE protein electrophoresis chromatogram of monoclonal antibodies Ab2# and Ab4# in one embodiment of this application. Detailed Implementation

[0039] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0040] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0041] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0042] In this specification, unless otherwise specified, "parts" refers to "parts by weight".

[0043] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0044] The main process route for preparing monoclonal antibodies in this application is as follows: first, antibodies against PreF are obtained through animal immunization; then, monoclonal antibody cell lines are obtained through hybridoma technology; then, monoclonal antibody sequences are obtained through sequencing; and finally, monoclonal antibody plasmids are constructed through genetic engineering technology, and monoclonal antibodies are expressed using a eukaryotic mammalian expression system.

[0045] I. Experimental Materials:

[0046] Pre-fusion conformation of the fusion protein (PreF): self-made. Point mutations and codon optimization were performed based on publicly available data from the GenBank database (GenBank accession number: M14892.1), and gene synthesis and plasmid construction were completed by Nanjing GenScript Biotech Co., Ltd.

[0047] Nickel ion affinity chromatography column, Protein G affinity chromatography column: purchased from GE Healthcare;

[0048] Freund's adjuvant: purchased from SIGMA;

[0049] New Zealand White Rabbit: Purchased from the China National Institutes for Food and Drug Control (Daxing);

[0050] II. Experimental Methods:

[0051] (1) Preparation of PreF:

[0052] A: Protein Expression: The constructed PreF plasmid was added to the culture medium and mixed thoroughly. Then, 4 times the mass of the plasmid transfection reagent PEI was added. After mixing, the mixture was incubated at room temperature in the dark for 10 minutes. Then, while shaking the cell flask, the prepared mixture was added to the 293-F suspension cells (density 2×10⁶). 6 The cells were cultured in a solution of (cells / mL) at 37°C and 5% CO2 for 120 h, and the cell supernatant was collected.

[0053] B: Protein purification: Nickel ion affinity chromatography was used for purification. After sample loading, the column was washed with washing buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) until the UV absorbance returned to baseline. The target protein was then eluted with elution buffer (50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH 8.0), and the elution peak was collected. The collected target protein in the elution peak was dialyzed in 0.02 mol / L PBS (pH 7.4) using a dialysis bag (molecular weight cutoff of 10 kDa). The dialysate was changed 3 times. After dialysis, the protein concentration was measured and stored at -80℃ for later use.

[0054] (2) Animal immunization:

[0055] A: Mix the self-made immunogen with Freund's adjuvant in equal volumes to a suitable volume and emulsify it completely. Immunize the New Zealand white rabbits by subcutaneous injection at multiple points. Each New Zealand white rabbit is injected with 200 μg of immunogen, once every two weeks, for a total of 10 New Zealand white rabbits.

[0056] B: After 10 immunizations, one week after the last immunization, blood was collected from the ear vein of New Zealand white rabbits, and the serum was collected by centrifugation. The antibody titer in the serum was tested by ELISA (using a PreF-coated microplate). The OD values ​​of serum from different New Zealand white rabbits after different dilutions are shown in Table 1.

[0057] Table 1

[0058]

[0059] According to Table 1, rabbits #2, #5, and #8 had low antibody titers and were eliminated; among rabbits #1, #3, #4, #6, #7, #9, and #10, rabbit #6 had the best antibody titer and was selected for monoclonal cell preparation.

[0060] (3) Preparation of monoclonal antibodies:

[0061] A: Screening of hybridoma cells

[0062] a) The spleens of the New Zealand white rabbits obtained through screening were processed, and the isolated spleen cells were fused with myeloma cells. The fused cells were screened and cultured in plates using the limiting dilution method.

[0063] b) Screening and culturing monoclonal cell wells, and detecting the supernatant after cell culture by ELISA. The four monoclonal cell wells with the highest OD values ​​in the cell culture plate were selected as target hybridoma cells (1#, 2#, 3#, 4#) for culture and amplification, which were used for cryopreservation of cell seeds and purification and verification of monoclonal antibodies.

[0064] B: Preparation of monoclonal antibodies

[0065] a) Isolation of antibody variable region genes from hybridoma cells using RT-PCR: After homogenizing the optimal hybridoma cells, add cell lysis buffer for RNA extraction, precipitate the RNA from the aqueous phase with isopropanol, wash the precipitated RNA after centrifugation to remove impurities, resuspend and reverse transcribe to obtain cDNA.

[0066] b) Using existing New Zealand white rabbit-specific primers (synthesized by Genscript Biotech Co., Ltd.), PCR gene amplification was performed. Hybridoma cell cDNA was used as a template to amplify the variable regions of the heavy and light chains of the antibody. A 50 μL system contained 5 μL cDNA, HotStar Taq Plus enzyme, dNTPs, and 0.5 μM specific primers. PCR amplification was performed under the following conditions: pre-denaturation, 94℃, 5 min; amplification, 94℃, 30 s, 55℃, 30 s, 72℃, 50 s, 35 cycles; annealing, 72℃, 7 min. The obtained PCR product was identified by 1% agarose gel electrophoresis, the target fragment was recovered, and the sample was sent for sequencing to obtain the antibody gene sequence.

[0067] c) Constructing expression vectors for monoclonal antibodies:

[0068] Based on the antibody gene sequence obtained from step b, an expression vector for monoclonal antibodies was constructed. Homologous recombination arms were added to both ends of the variable region gene of the antibody heavy chain and the variable region gene of the light chain using homologous recombination primers. The expression plasmid containing the constant regions of the rabbit antibody heavy and light chains IgG1 was linearized using a double enzyme to generate homologous recombination arms. The variable region gene fragment with added homologous recombination arms and the linearized plasmid were ligated together by homologous recombination to form a complete expression vector, pCDNA3.4. The recombination product was transformed into TOP10 E. coli competent cells to amplify the plasmid. Through the above experimental operations, paired pCDNA3.4-antibody heavy chain plasmid and pCDNA3.4-antibody light chain plasmid were obtained.

[0069] d) Expression and purification of monoclonal antibodies:

[0070] The paired monoclonal antibody heavy and light chain expression plasmids obtained in step c were added to the culture medium at a 1:1 ratio. After thorough mixing, PEI transfection reagent (4 times the mass of DNA) was added. After mixing, the mixture was incubated at room temperature in the dark for 10 minutes. Then, the plasmids were added to 293T cells while shaking the cell culture flask. The cells were then incubated in a 5% CO2 incubator at 37°C with shaking for 5 days. The cell culture supernatant was then collected and purified using affinity purification (Protein A) to obtain the monoclonal antibody. The specific steps are as follows:

[0071] (1) Centrifuge the supernatant of the expressed antibody at 10000×g at room temperature for 30 min to remove the precipitate;

[0072] (2) Wash the affinity purification column containing Protein A thoroughly with 10 volumes of binding buffer;

[0073] (3) Pass the expression supernatant through the purification column at a flow rate of 5 mL / min;

[0074] (4) Wash the purification column thoroughly with 20 times the column volume of binding buffer;

[0075] (5) Elute the purification column with 0.1M citrate buffer (pH=3.0-3.5) until the elution peak drops to equilibrium, and adjust the pH to 7.0 with 1M Tris-HCl buffer (pH=9.0).

[0076] (6) The purified monoclonal antibody was concentrated using a concentrated centrifuge column, PBS was used as the antibody preservation buffer, and finally the concentration of the concentrated antibody was determined using an ultra-micro UV spectrophotometer.

[0077] Monoclonal antibodies Ab1#, Ab2#, Ab3#, and Ab4# were thus obtained.

[0078] III. Testing Section

[0079] 1. Screening of monoclonal antibodies:

[0080] a) Coat the ELISA plate with PreF (0.1 μg / mL). Dilute the four antibody strains (Ab1#, Ab2#, Ab3#, Ab4#) to concentrations of 100, 10, 1, and 0 ng / mL, respectively. Add the above dilutions to the ELISA plate and incubate at 37°C for 1 h. After washing the plate, add the ELISA-labeled secondary antibody dilution (5000-fold dilution) and incubate at 37°C for 0.5 h. Add TMB for color development and stop the reaction after 15 min. Measure the absorbance (OD) at wavelengths of 450 / 620 nm. The results are shown in Table 2.

[0081] Table 2

[0082]

[0083] Based on the analysis of Table 2, the recognition of different PreF antibodies by monoclonal antibodies Ab2#, Ab3#, and Ab4# shows that they have better recognition effects on PreF.

[0084] b) Confirmation of antibody pairing combinations

[0085] Nasal swab lysis samples for clinical testing were collected from relevant hospitals, including 30 positive and 30 negative samples. Alternative monoclonal antibodies were used to create sandwich-type combinations (forming coated antibody-antigen-detection antibody complexes). The 60 samples were then tested using the colloidal gold method (coating conditions: 0.1M Tris buffer, coating concentration 1.0 mg / mL, drying temperature 55℃, drying time 16h; labeling conditions: 0.1M Tris buffer, antibody to colloidal gold mass ratio 1:1, labeling for 12h; sample loading volume 100μL, results read 5-10 minutes after loading). Antibody combinations with high positive-negative concordance rates were selected, and the results are shown in Table 3.

[0086] Table 3

[0087]

[0088] As shown in Table 3, the positive and negative concordance rates of samples coated with Ab2# antibody and detected with Ab4# antibody, and coated with Ab3# antibody and detected with Ab4# antibody, are good.

[0089] c) Verification of cross-reactivity of monoclonal antibodies:

[0090] Two pairs of candidate monoclonal antibodies were combined using a sandwich method (forming a coated antibody-antigen-detection antibody complex). Different concentrations of hemoglobin, mucin, and positive samples for respiratory syncytial virus (RSV) and parainfluenza virus (PIV) were detected using a colloidal method to observe the anti-interference ability of different antibodies against these easily cross-reactive antigens. The cross-reactivity is shown in Table 4.

[0091] Table 4

[0092]

[0093] As shown in Table 4, the results indicate that the obtained monoclonal antibody showed the best detection effect against Ab2# antibody-coated + Ab4# antibody, with no cross-reactivity. Therefore, monoclonal antibodies Ab2# and Ab4# were selected for the detection of metapneumovirus.

[0094] 2. Characterization of monoclonal antibodies Ab2# and Ab4#

[0095] a) Monoclonal antibody Ab2#:

[0096] Light chain:

[0097] The nucleotide sequence of the light chain variable region is as SEQ ID Shown in NO.17: GATGTCGTGATGACCCAGACCCCCAGCCCAGTCTCAGCCGCCGTCGGAGATACCGTGACCATCAAGTGTCAGGCTAGCCAGTCTGTGAGCAGCTCCCTCTCCGGGTGGTACCAGCAAAAGCCTGGACAGGCCCCTCGGCTCCTGATCTATGGGACCTC TAAGCTGGCCACCGGGGTGCCTTCTCGCTTTCGGGGCTCCGGCTCCGGAACTCAGTTCACTCTGACAATCTCTGGCATGAAGGCCGAGGACGTGGCCACCTATTACTGCCAGCAGTACGGGAACAGTCTGTGGACCTTCGGCGGCGGCACCAAACTCGAGATCAAG.

[0098] The amino acid sequence of the light chain variable region is shown in SEQ ID NO:13: DVVMTQTPSPVSAAVGDTVTIKCQASQSVSSSLSGWYQQKPGQAPRLLIYGTSKLATGVPSRFRGSGSGTQFTLTISGMKAEDVATYYCQQYGNSLWTFGGGTKLEIK; the amino acid sequence of the complementarity-determining region VL-CDR1 of the light chain variable region is shown in SEQ ID NO:1: QASQSVSSSLS; the amino acid sequence of the complementarity-determining region VL-CDR2 of the light chain variable region is shown in SEQ ID NO:2: GTSKLAT; the amino acid sequence of the complementarity-determining region VL-CDR3 of the light chain variable region is shown in SEQ ID NO:3: QQYGNSLWT.

[0099] Heavy chain:

[0100] The nucleotide sequence of the heavy chain variable region is as SEQ ID Shown in NO.18: CAGCAGCTGAAGGAGAGTGGCGGCGGCCTGGTGAAGCCAGGCGGTTCCCTGAAGCTGTGTTGCAAAGCCAGCGGCGGCTCTATCAACTCCGGTGACTACTACATGTGCTGGGTGCGGCAGGCCCCCGGCAAGGGACTGGAGTGGATTGGCTGTATTTACCACAGCGGAAGCACACACTATGCCT CCTGGGTGAATGGTAGGTTTACCCTGTCCAGAGACAACGCCCAGTCCACAGTGTGCCTGCAGCTGAACAGCCTGACCGCTGCCGACACCGCCGTCTACTTTTGTCAGCAGGATCTGGCCAGCGACCAGGGGCGCAATTACGCTGGGGCTATGGATGTGTGGGGCCAGGGGACCCTGGTGACCGTGTCATCT.

[0101] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.14: QQLKESGGGLVKPGGSLKLCCKASGGSINSGDYYMCWVRQAPGKGLEWIGCIYHSGSTHYASWVNGRFTLSRDNAQSTVCLQLNSLTAADTAVYFCQQDLASDQGRNYAGAMDVWGQGTLVTVSS.

[0102] The amino acid sequence of the complementarity-determining region VH-CDR1 of the heavy chain variable region is shown in SEQ ID NO:4: DYYMC; the amino acid sequence of the complementarity-determining region VH-CDR2 of the heavy chain variable region is shown in SEQ ID NO:5: CIYHSGSTHYASWVNG; and the amino acid sequence of the complementarity-determining region VH-CDR3 of the heavy chain variable region is shown in SEQ ID NO:6: DLASDQGRNYAGAMDV.

[0103] b) Monoclonal antibody Ab4#:

[0104] Light chain:

[0105] The nucleotide sequence of the light chain variable region is as SEQ ID Shown in NO.19: GACCCAATGCTGACCCAGACCGCCAGCCCTGTGAGCGCCGCTGTCGGCAGTACCGTGACCATTAGCTGTCAGGCCTCCCAGTCCGTCAGCAACAACTACTTGGCCTGGTACCAGCAGAAGCCTGGCCAGGCCCCTCGGCTGCTGATCTACGGCGCCAG CAATCTGGCAAGTGGCGTGCCTGATCGATTCTCCGGATCCGGATCCGGAACCCAGTTTACCCTGACCATCTCCGGCGTGCAGTGCGACGACGCAGCTACCTACTACCAGCATTTTGGGGGCAGTCCCATGTACACATTCGGGGGCGGCACCAAGCTGGAGATTAAG.

[0106] The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.15: DPMLTQTASPVSAAVGSTVTISCQASQSVSNNYLAWYQQKPGQAPRLLIYGASNLASGVPDRFSGSGSGTQFTLTISGVQCDDAATYYQHFGGSPMYTFGGGTKLEIK.

[0107] The amino acid sequence of the complementarity-determining region VL-CDR1 of the light chain variable region is shown in SEQ ID NO:7: QASQSVSNNYLA; the amino acid sequence of the complementarity-determining region VL-CDR2 of the light chain variable region is shown in SEQ ID NO:8: GASNLAS; and the amino acid sequence of the complementarity-determining region VL-CDR3 of the light chain variable region is shown in SEQ ID NO:9: HFGGSPMYT.

[0108] Heavy chain:

[0109] The nucleotide sequence of the heavy chain variable region is as SEQ ID NO.20 shows: CAGGAGCAGCTTGAAGAGTCCGGAGGCGGACTGGTGAAGCCTGGGGACACCCTGACACTGACCTGCAAAGCCAGCGGACCCGCCGCCATCAGCGGTGACTACTACATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGGTTTATCCATTCCTCTGGCACCACCTACTACGC CTCATGGGTGAACGGAAGAATTATCATCTCCAGCGATAATACCCAGAACACCGTGTCCCTGCTGATGAACAGCCTGTCCGCCAGAGACAGCGCCGTGTACTTCTGCGCCAGAGATCTGGACTTTGACAATGGGAGAAACTACTACTACGGCCTGGACGTGTGGGGCCAGGGCACCCTGGTGACCGTCTCCAGC.

[0110] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.16: QEQLEESGGGLVKPGDTLTLTCKASGPAAISGDYYMSWVRQAPGKGLEWIGFIHSSGTTYYASWVNGRIIISSDNTQNTVSLLMNSLSARDSAVYFCARDLDFDNGRNYYYGLDVWGQGTLVTVSS.

[0111] The amino acid sequence of the complementarity-determining region VH-CDR1 of the heavy chain variable region is shown in SEQ ID NO:10: SGDYYMS; the amino acid sequence of the complementarity-determining region VH-CDR2 of the heavy chain variable region is shown in SEQ ID NO:11: FHISSGTTYYASWVNG; and the amino acid sequence of the complementarity-determining region VH-CDR3 of the heavy chain variable region is shown in SEQ ID NO:12: DLDFDNGRNYYYGLDV.

[0112] c) Molecular weight determination of monoclonal antibodies Ab2# and Ab4#

[0113] Its SDS-PAGE protein electrophoresis chromatogram is as follows: Figure 1 As shown, according to Figure 1 Lane M is the marker, lane 1 is the monoclonal antibody Ab2#, and lane 2 is the monoclonal antibody Ab4#. Both monoclonal antibodies are reduced to two parts, one with a molecular weight of 50kDa and the other with a molecular weight of 25kDa.

[0114] 3. Development and validation of a human metapneumovirus detection kit based on the obtained anti-PreF monoclonal antibody pairs (monoclonal antibody Ab2# and monoclonal antibody Ab4#).

[0115] Brief description of the reagent kit development process:

[0116] The Ab2# antibody was used as the coating antibody to coat the NC membrane, and the Ab4# antibody, labeled with colloidal gold, was used as the detection antibody. A series of optimization studies were conducted on the coating conditions of the Ab2# antibody (coating buffer, coating concentration, drying temperature and time, etc.), the labeling conditions of the Ab4# antibody (antibody to colloidal gold ratio, labeling buffer, labeling time, etc.), and the sample loading volume. After the kit was finalized, its performance (sensitivity, repeatability, stability, etc.) was also studied.

[0117] The coating conditions were determined as follows: 0.1 M Tris buffer, coating concentration 1.0 mg / mL, drying temperature 55℃, and drying time 16 h.

[0118] Labeling conditions: 0.1M Tris buffer, antibody to colloidal gold mass ratio 1:1, labeling for 12 hours; sample loading volume is 100 μL, and results are read 5 to 10 minutes after loading.

[0119] 1) Sensitivity:

[0120] PreF was diluted to nine concentration levels (100.0, 50.0, 25.0, 10.0, 5.0, 1.0, 0.5, 0.1, and 0.05 ng / mL), and its analytical sensitivity was determined using a human metapneumovirus detection kit (colloidal gold method). The lowest concentration at which the sample tested positive was taken as the analytical sensitivity. The results are shown in Table 5.

[0121] Table 5

[0122]

[0123] According to Table 5, the minimum concentration required for a positive test result is 0.1 ng / mL. Therefore, this invention provides a human metapneumovirus (HMV) detection kit (colloidal gold method) with a detection sensitivity of 0.1 ng / mL for HMV.

[0124] (ii) Repeatability:

[0125] The PreF concentration in sample 1 (PreF concentration of 0.5 ng / mL) and sample 2 (PreF concentration of 10 ng / mL) was determined using a human metapneumovirus detection kit (colloidal gold method). The test results are shown in Table 6.

[0126] Table 6

[0127]

[0128] As can be seen from Table 6, the human metapneumovirus detection kit (colloidal gold method) has good repeatability.

[0129] (iii) Stability:

[0130] The human metapneumovirus detection kit (colloidal gold method) was stored at 2–30°C. The kits were removed after 0, 3, 6, 9, and 12 months post-production, and the PreF concentration in sample 1 (PreF concentration of 0.5 ng / mL) and sample 2 (PreF concentration of 10 ng / mL) was measured. The test results are shown in Table 7.

[0131] Table 7

[0132]

[0133] As can be seen from Table 7, the Human Metapneumovirus Detection Kit (Colloidal Gold Method) has good stability.

[0134] Finally, it should be noted that the above text has described the various embodiments of the present invention in detail. To avoid obscuring the concept of the present invention, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description. The above embodiments are only used to illustrate the technical solutions of this application, and not to limit them;

[0135] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A combination of PreF monoclonal antibodies for metliod of detection of metapneumovirus characterized in that, The first antibody comprises a first light chain variable region and a first heavy chain variable region, and the second antibody comprises a second light chain variable region and a second heavy chain variable region; wherein, the amino acid sequence of the first light chain variable region comprises VL-CDR1 shown in SEQ ID NO:1, VL-CDR2 shown in SEQ ID NO:2, and VL-CDR3 shown in SEQ ID NO:3; the amino acid sequence of the first heavy chain variable region comprises VH-CDR1 shown in SEQ ID NO:4, VH-CDR2 shown in SEQ ID NO:5, and VH-CDR3 shown in SEQ ID NO:6; the amino acid sequence of the second light chain variable region comprises VL-CDR1 shown in SEQ ID NO:7, VL-CDR2 shown in SEQ ID NO:8, and VL-CDR3 shown in SEQ ID NO:9; the amino acid sequence of the second heavy chain variable region comprises VH-CDR1 shown in SEQ ID NO:10, VH-CDR2 shown in SEQ ID NO:11, and VH-CDR3 shown in SEQ ID NO:

12.

2. The PreF monoclonal antibody combination of claim 1, wherein the amino acid sequence of the first light chain variable region is shown in SEQ ID NO:13; and the amino acid sequence of the first heavy chain variable region is shown in SEQ ID NO:

14.

3. The PreF monoclonal antibody combination of claim 1, wherein the amino acid sequence of the second light chain variable region is shown in SEQ ID NO:15; and the amino acid sequence of the second heavy chain variable region is shown in SEQ ID NO:

16.

4. The PreF monoclonal antibody combination of claim 1, wherein the nucleotide sequence of the first light chain variable region is shown in SEQ ID NO:17; and the nucleotide sequence of the first heavy chain variable region is shown in SEQ ID NO:

18.

5. The PreF monoclonal antibody combination of claim 1, wherein the nucleotide sequence of the second light chain variable region is shown in SEQ ID NO:19; and the nucleotide sequence of the second heavy chain variable region is shown in SEQ ID NO:

20.

6. Use of the PreF monoclonal antibody combination according to any one of claims 1-5 in the preparation of a tool for detecting metapneumovirus.

7. Use according to claim 6, characterized in that, The tool for detecting metapneumovirus is used for detecting metapneumovirus in an in vitro sample, wherein the in vitro sample comprises at least one of a nasal swab, a throat swab, and a nasopharyngeal swab.

8. Use according to claim 7, characterized in that, The PreF monoclonal antibody combination is used in an immunoassay method for detecting metapneumovirus in an in vitro sample.

9. Use according to claim 8, characterized in that, The immunoassay method comprises at least one of an enzyme-linked immunoassay, an immunofluorescence assay, a chemiluminescence immunoassay, an immunomicrosphere assay, and an immunocolloidal gold assay.

10. The use according to any one of claims 6 to 9, characterized in that, The tool for detecting metapneumovirus comprises a reagent.

11. Use according to claim 10, characterized in that, The reagent comprises at least one of a kit, a test strip, and an antibody chip.

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