Anti-type 3 human parainfluenza virus monoclonal antibody and its preparation method and application

Abstract

The present invention relates to the field of in vitro detection technology, and more specifically to anti-human parainfluenza virus type 3 monoclonal antibodies, preparation methods, and applications thereof. The monoclonal antibodies provided by the present invention are capable of recognizing human parainfluenza virus F protein and have the characteristics of good specificity and high titer. The double antibody sandwich method, reagents, and kit for detecting human parainfluenza virus type 3 developed based on the anti-human parainfluenza virus type 3 monoclonal antibodies of the present invention are simple to operate, time-saving, and highly efficient, and have broad application prospects in the early diagnosis and clinical diagnosis of respiratory diseases.

Description

Technical Field

[0001] The present invention relates to the field of in vitro detection technology, and in particular to an anti-type 3 human parainfluenza virus monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Human parainfluenza viruses (HPIVs) are common respiratory tract infection pathogens. Studies have shown that 4%-22% of respiratory diseases in children are caused by HPIVs. HPIVs can be divided into four subtypes based on serotype: HPIV1, 2, 3, and 4, which mainly cause lower respiratory tract infections in young children. In adult patients, parainfluenza virus infection can also cause a range of respiratory diseases, including upper respiratory tract infections, acute exacerbations of chronic diseases, and pneumonia. Among these four subtypes, HPIV3 is generally considered to be the most pathogenic.

[0003] Human parainfluenza virus type 3 belongs to the Paramyxoviridae family and is a pleomorphic, enveloped, single-stranded, negative-sense RNA virus with a diameter of approximately 120-180 nm. It contains 15,462 bases. Its genome encodes six mRNAs and expresses eight viral proteins: six structural proteins and two nonstructural proteins. Three viral proteins encapsidate the genomic RNA to form the nucleocapsid: nucleocapsid protein (NP), phosphorylation protein (P), and polymerase protein (L). The viral envelope is composed of lipids and glycoproteins. Two types of membrane glycoproteins exist: the hemagglutinin-neuraminidase protein (HN), which binds to sialic acid receptors on the cell surface, mediating viral attachment and also possesses neuraminidase activity, contributing to viral release. The other, the fusion protein (F protein), promotes cell fusion and hemolytic properties, mediating viral invasion through structural changes. Key biological properties of HPIV3, such as pathogenicity and immunogenicity, are determined by the structure and function of its two envelope glycoproteins: the F and HN proteins. The F protein not only promotes fusion between the viral lipid membrane and the cell membrane but also interacts with the HN protein during viral attachment and invasion of target cells, contributing to viral virulence. Furthermore, antibodies targeting the F protein have a neutralizing effect on the virus, making it a primary target protein for HPIV3 vaccine research. F protein can be detected early in host infection and therefore serves as a target antigen for serological diagnosis of HPIVs, thereby shortening the window for viral detection.

[0004] The current clinical methods for diagnosing HPIVs infection include: 1. Virus isolation combined with hemagglutination inhibition test or hemagglutination test, which is the gold standard for diagnosing respiratory viral infection; 2. Rapid pathogen detection, including the use of immunofluorescence test, enzyme-linked immunosorbent assay and other methods; 3. Detection of specific antibody IgM in a single serum specimen. However, nucleic acid diagnosis requires professional equipment, and the detection time is relatively long. It is greatly affected by the quality of sampling, and there are false positives and false negatives. It is easy to miss the diagnosis by nucleic acid testing alone. Serological diagnosis is the detection of the body's immune response after pathogen infection. It lasts for a long time, the immune response is stable, and the immune response shows a dynamic trend as the disease progresses. Therefore, serological diagnosis is also an important means of diagnosis. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a monoclonal antibody against human parainfluenza virus type 3, and its preparation method and application. The present invention provides a monoclonal antibody for detecting human parainfluenza virus type 3 in a sample, and the application of the antibody in the preparation of a product for parainfluenza virus antigen detection.

[0006] The anti-human parainfluenza virus type 3 monoclonal antibody provided by the present invention is a capture antibody (5D8) and / or a detection antibody (7B12);

[0007] The three CDR regions of the 5D8 light chain have the amino acid sequences of QGIRGN (SEQ ID No: 11), STS, and LQRNAYPYT (SEQ ID No: 12), respectively; the three CDR regions of its heavy chain have the amino acid sequences of GFSLTRYG (SEQ ID No: 13), IWAGGST (SEQ ID No: 14), and ASYYWQRDGMDC (SEQ ID No: 15), respectively;

[0008] The three CDR regions of the 7B12 light chain have the amino acid sequences of KSLLYKDGKTY (SEQ ID No: 16), LMS, and QQLVEYPLT (SEQ ID No: 17), respectively; the three CDR regions of its heavy chain have the amino acid sequences of GFTFSDSY (SEQ ID No: 18), SRNKAYDYST (SEQ ID No: 19), and ARDAGYGNHGGYFDY (SEQ ID No: 20), respectively.

[0009] The anti-type 3 human parainfluenza virus monoclonal antibody provided by the present invention has an amino acid sequence of the light chain variable region as shown in SEQ ID No: 7 or SEQ ID NO: 8; and an amino acid sequence of the heavy chain variable region as shown in SEQ ID No: 9 or SEQ ID NO: 10.

[0010] Specifically, in some embodiments, the anti-type 3 human parainfluenza virus monoclonal antibody 5D8 has an amino acid sequence of the light chain variable region as shown in SEQ ID No: 7; and an amino acid sequence of the heavy chain variable region as shown in SEQ ID No: 9.

[0011] In other embodiments, the anti-human parainfluenza virus type 3 monoclonal antibody 7B12 has an amino acid sequence of the light chain variable region as shown in SEQ ID NO: 8; and an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 10.

[0012] Furthermore, the anti-type 3 human parainfluenza virus monoclonal antibody of the present invention has an IgG subtype and a kappa light chain.

[0013] The present invention provides some biomaterials, which include at least one of the following a) to e):

[0014] a), a nucleic acid encoding the monoclonal antibody of the present invention;

[0015] b) a recombinant vector comprising a vector backbone and a nucleic acid encoding the monoclonal antibody of the present invention;

[0016] c) a host that secretes the monoclonal antibody of the present invention;

[0017] d) chemically labeled or biologically labeled monoclonal antibodies of the present invention;

[0018] e) The monoclonal antibody of the present invention coupled to a carrier.

[0019] Specifically, the nucleic acid encoding the monoclonal antibody of the present invention can be DNA, RNA, cDNA or PNA. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acid can be linear or circular in topology. The nucleic acid can be, for example, a part of a vector (e.g., an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from a natural source, or can be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0020] Specifically, the recombinant vector of the present invention refers to a nucleic acid vector, which is a recombinant DNA molecule that contains a desired coding sequence and suitable nucleic acid sequences or elements necessary for the expression of operably linked coding genes in a specific host organism. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are the most commonly used vector form. However, the present invention is intended to include other forms of expression vectors that function equivalently and are known or will become known in the art, including but not limited to plasmids, phage particles, viral vectors and / or simply potential genomic inserts.

[0021] Specifically, the hosts for secreting monoclonal antibodies described herein include bacteria, fungi, viruses, or animals. Bacteria include Gram-positive and Gram-negative bacteria; Gram-positive bacteria include, but are not limited to, Escherichia coli. Fungi include molds, yeasts, and mushrooms; yeasts include, for example, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Pichia pastoris, and Candida. Viruses include, but are not limited to, adenoviruses, adeno-associated viruses, and lentiviruses. Animals include humans, mice, rabbits, pigs, or zebrafish.

[0022] Specifically, the chemically labeled or biolabeled monoclonal antibodies of the present invention include chemical labels such as isotope labels and fluorescein labels; and biolabels such as enzyme labels and biotin labels, such as fluorescein isothiocyanate, horseradish peroxidase, alkaline phosphatase, β-galactosidase, biotin, and colloidal gold.

[0023] Specifically, the monoclonal antibody coupled to a carrier described in the present invention refers to any carrier capable of carrying the monoclonal antibody, such as magnetic particles, test strips, or ELISA plates.

[0024] The present invention provides use of the monoclonal antibody or the biological material in preparing a type 3 human parainfluenza virus detection reagent or kit.

[0025] The present invention also provides an ELISA kit for detecting type 3 human parainfluenza virus, which comprises at least one of the antibodies of the present invention.

[0026] Furthermore, the ELISA kit uses a double-antibody sandwich method. Specifically, in an embodiment of the present invention, the ELISA kit, using the anti-type 3 human parainfluenza virus monoclonal antibody 5D8 as the capture antibody and the anti-type 3 human parainfluenza virus monoclonal antibody 7B12 as the detection antibody, has extremely high sensitivity and specificity, better detection results, and a negative and positive detection coincidence rate of over 70%, meeting the detection requirements.

[0027] Furthermore, the capture antibody is coupled to magnetic beads, and the detection antibody is modified with horseradish peroxidase.

[0028] In some specific embodiments, the step of coupling the capture antibody to the magnetic beads comprises washing the magnetic microparticle stock solution with PBS buffer, activating the magnetic microparticles with 10% glutaraldehyde, and then adding the monoclonal antibody at 0.3 μg / person to the magnetic beads for coating and blocking;

[0029] The step of detecting antibody-modified horseradish peroxidase comprises activating the horseradish peroxidase with sodium periodate, terminating it with ethylene glycol, dialyzing to remove excess reagents, adding an antibody dialyzed with carbonate buffer for coupling modification, then terminating the reaction with sodium borohydride, dialyzing again, and adding an equal volume of glycerol for storage.

[0030] The present invention also provides a method for detecting type 3 human parainfluenza virus, comprising detecting a sample using the ELISA kit of the present invention.

[0031] The methods provided herein can be used for diagnostic or non-diagnostic purposes. For example, they include methods for detecting a substance in a human or animal body, or in vitro samples derived from a human or animal body, for diagnostic purposes; and methods for detecting a substance in an environmental or simulated sample for scientific research or other non-diagnostic purposes.

[0032] The monoclonal antibody obtained through hybridoma screening in this invention has high titer and can undergo a coupling reaction with a linear epitope of the HPIV3 membrane glycoprotein F protein with strong specificity and high sensitivity. A double-antibody sandwich assay developed using this antibody can specifically detect HPIV3 in serum samples, offering advantages such as simple operation and rapid detection. This assay provides key materials for the subsequent development of commercial kits for HPIV3 antigen detection. DETAILED DESCRIPTION

[0033] The present invention provides anti-type 3 human parainfluenza virus monoclonal antibodies and their preparation methods and applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0034] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0035]

[0036] The amino acid sequence of the F protein is: (SEQ ID No: 2)

[0037] The present invention will be further described below in conjunction with the embodiments:

[0038] Example 1 Construction of a eukaryotic expression vector for parainfluenza virus type 3 F protein and its expression and purification

[0039] 1. Construction of a eukaryotic expression vector for parainfluenza virus type 3 F protein

[0040] 1.1. Construction of eukaryotic expression plasmids

[0041] Primers for eukaryotic expression of the F gene were designed. The upstream primer contained a Bam HI restriction site, and the downstream primer contained a Sph Ⅰ restriction site.

[0042] The primer sequences were: PIV3-FF: TATGG ATCCA TGCCC ACCTC AATCC TG (SEQ ID No: 5);

[0043] PIV3-FR: ACGGC ATGCC TATTA CTTGT TAGTG AGCAC (SEQ ID No: 6).

[0044] The F gene was amplified using Taq enzyme. The PCR reaction protocol was as follows: 94°C for 10 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds, and finally 72°C for 10 minutes. The 1620-bp PCR product was recovered from a gel and digested with BamHI and SphI, ligated into the pFastbac1 vector, and transformed into competent E. coli DH5α cells. After overnight culture, positive cells were screened, and the plasmid was extracted and sequenced to obtain the pFastBac-F plasmid.

[0045] The recombinant plasmid containing the F gene was transformed into DH10Bac competent cells, and the transformation products were screened by blue-white spots on LB solid medium containing Bluo-gal. White colonies were picked and the recombinant Bacmid DNA was extracted. The recombinant plasmid was identified by PCR. The presence of the target gene fragment was a positive recombinant plasmid.

[0046] 1.2 Protein Expression

[0047] Select sf9 cell suspension in logarithmic growth phase and add 1×10 6 100 cells / well were inoculated into 6-well plates and cultured at 27°C for 45 minutes. Recombinant bacmids identified as positive by PCR were transfected into insect cells Sf9 and cultured at 27°C for 72 hours. The supernatant was collected to obtain the P1 virus. The P1 recombinant baculovirus was inoculated at an MOI of 0.1 and cultured at 27°C and 110 rpm for 72 hours. Cell debris was removed by centrifugation at 4°C and 500 × g for 5 minutes, and the supernatant was collected as the P2 recombinant virus.

[0048] The cell culture supernatant, cell lysis supernatant, and cell lysis precipitate were collected for expression identification. Western blot was performed using His monoclonal antibody. A reactive band at around 66 KD was found in the cell lysis supernatant, indicating that F protein was induced and expressed intracellularly. P2 virus was used to infect 100 ml of sf9 cells (2×10 6 / mL), and the cells were collected after 4 days.

[0049] 1.3 Protein Purification

[0050] The collected cells were lysed and centrifuged at 10,000 × g for 10 minutes at 4°C. The precipitate was removed, and the cell lysate supernatant was obtained. The F protein was then purified using nickel affinity chromatography, and the sample was dialyzed into PBS. Protein expression and purification were assessed by SDS-PAGE. The results showed a single protein band at approximately 66 kD in the purified sample, indicating successful induced expression of the F protein and high purity after purification.

[0051] 1.4 Protein antigenicity analysis

[0052] After SDS-PAGE, the expressed F protein was electroporated onto a nitrocellulose (NC) membrane using the wet transfer method. After blocking with blocking buffer at 37°C for 1 hour, a 1:1000 dilution of anti-His tag mouse monoclonal antibody was added and incubated at room temperature for 1 hour. Then, an HRP-conjugated goat anti-mouse secondary antibody (1:4000 dilution) was added and incubated at room temperature for 1 hour. Finally, a luminescent color development solution was added.

[0053] The results showed that the eukaryotically expressed PIV3-F protein could react immune-wise with the tag antibody.

[0054] Example 2 Screening and Preparation of Antibodies Against Parainfluenza Virus F Protein

[0055] 2.1 Animal Immunization

[0056] The purified F protein was subcutaneously injected into 6-8 week old Balb / c mice at a dose of 100 μg / mouse for a total of 3 immunizations, with an interval of 2 weeks between each immunization. Cell fusion was performed 5 days after the three immunizations, and boosted again 3 days before cell fusion at a dose of 100 μg / mouse.

[0057] 2.2 Screening of positive hybridoma cells

[0058] Cell fusion was performed according to conventional methods. Hybridoma cells were screened 7 days after fusion using the following screening method:

[0059] The indirect ELISA method was used to determine the optimal antigen coating concentration and serum dilution using the matrix method to establish an ELISA screening method for monoclonal antibodies. The specific procedures are as follows:

[0060] 1) Use purified F protein as the coating antigen and dilute it to 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, or 0.25 μg / mL in coating solution. Coat ELISA plates at a volume of 50 μL / well. Incubate at 4°C for 12–16 hours, discard the coating solution, and wash the plates twice with PBST.

[0061] 2) Block the ELISA plate with blocking solution containing 1% Casein, 150 μL / well, at 37°C for 2 h, discard the blocking solution, and dry at 37°C.

[0062] 3) Dilute the negative and positive sera at 1:100, 1:200, 1:400, 1:800, and 1:1600, respectively, with 100 μL / well. Incubate at 37°C for 1 h, and wash the plate five times with PBST.

[0063] 4) Add 1:4000 diluted HRP-goat anti-mouse IgG+IgM, 100 μL / well, incubate at 37°C for 1 h, and wash the plate five times with PBST.

[0064] 5) Add 100 μL / well of TMB substrate colorimetric solution and allow color to develop at room temperature for 10 min. Add 50 μL / well of stop solution (2 mol / L H2SO4) to terminate the reaction and read the OD 450 nm using a microplate reader.

[0065] 6) Determine the ELISA results and use the antigen coating concentration and serum dilution with the OD value of the positive well closest to 1, the OD value of the negative well less than 0.1, and the P / N value ≥ 2.1 as the optimal working concentration.

[0066] The cells in the positive culture wells were cloned and purified by limiting dilution until all clones were 100% positive.

[0067] 2.3 Production and purification of monoclonal antibodies

[0068] Balb / c mice were intraperitoneally injected with sterile paraffin oil (0.5 mL / mouse). 7 days later, 10 hybridoma cells of the monoclonal antibody against F protein were intraperitoneally injected. 6 After 7 days, the ascites was collected and purified using the caprylic acid-ammonium sulfate method to obtain monoclonal antibodies, which were then stored at -20°C after concentration determination.

[0069] 2.4 Identification of Monoclonal Antibody Subtypes

[0070] The antibodies of the 12 monoclonal antibodies were all IgG subtypes and the light chains were κ chains, as determined by Beijing Sino-Qiao Antibody Subtyping Kit.

[0071] 2.5. Monoclonal Antibody Potency Detection

[0072] The PIV-3 F recombinant protein obtained in Example 1 was diluted to 1 μg / mL using 0.05 mmol / L CB buffer, pH 9.6. 50 μL was added to each well of a 96-well ELISA plate (Corning) and coated overnight at 4°C. The following day, the plate was washed three times with PBST and blocked with 100 μL / well of 1% Casein at 37°C for 2 hours. Twelve purified monoclonal antibodies (all at a concentration of 5 mg / mL) were serially diluted using 0.05 mmol / L CB buffer, pH 9.6, at ratios of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and so on. The diluted antibodies were added to the ELISA plate coated with the PIV-3 F recombinant protein. 50 μL / well of 0.05 mmol / L CB buffer, pH 9.6, was added to the negative control wells. After incubation at 37°C for 30 minutes, wash the plate five times with PBST, pat dry, add 100 μL / well of a 1:4000 dilution of HRP-goat anti-mouse IgG (SIGMA), and incubate at 37°C for 30 minutes. Wash the plate five times with PBST, pat dry, add 100 μL / well of a standard ELISA substrate, and incubate at room temperature in the dark for 10 minutes. The reaction was terminated by adding 50 μL of 0.1 mol / L sulfuric acid, and the absorbance at 450 nm was measured. The results are shown in Table 1 below.

[0073]

[0074] As shown in Table 1, the average OD value of the negative control was 0.05, and the titers of the 12 monoclonal antibodies all showed high titers.

[0075] 2.6. Site Identification of Monoclonal Antibodies

[0076] To identify the antibody sites, a series of peptides were synthesized based on the epitopes predicted by different software. After coupling, the peptides were coated on enzyme immunoassay plates for reactivity verification. The results showed (Table 2) that all 12 antibodies reacted with peptides coupled to different epitopes of the F protein. The epitopes recognized by 5D8 and 7B12 were:

[0077] Polypeptide 1: 369 TKVINNLVPK FAFINGGVVA 388 (SEQ ID No: 3),

[0078] Polypeptide 2: 502 IIVCILIIII CGILYYLYRV 521 (SEQ ID No: 4),

[0079] This proves that both antibodies are monoclonal antibodies targeting linear epitopes of the F protein. The site identification data are as follows:

[0080]

[0081] Example 3 Application of PIV3-F Antibody in Detecting Parainfluenza Virus Antigens in Human Serum

[0082] 3.1. Detection of F protein by double antibody sandwich method

[0083] (1) ELISA plates were coated with 12 strains of F antibodies at a concentration of 5 μg / mL at 4°C overnight. The plates were washed twice with PBST the next day, patted dry with absorbent paper, and blocked with 1% Casein blocking solution for 2 h.

[0084] (2) Dilute the recombinant F protein to 100 ng / mL, then serially dilute it into 8 concentrations. Add 100 μL of each concentration of antigen to each well of the antibody-coated plate, incubate at 37°C for 1 h, wash the plate 5 times with PBST solution, and pat dry with absorbent paper.

[0085] (3) According to the site classification, add HRP-labeled antibodies (1:1000 dilution) with different sites from the coated antibody, incubate at 37°C for 1 hour, wash 5 times with PBST, add luminescent substrate, and read the luminescence value on a luminometer.

[0086] According to the paired test results, 2F2 and 5D8 as coating antibodies paired with the enzyme-labeled antibody 7B12 have the highest titers and the best correlation. Some paired test data are shown in Table 3:

[0087]

[0088] 3.2 Magnetic Particle Coating

[0089] 30 μL of the mixed magnetic microparticle stock solution was washed five times with 300 μL of PBS buffer. The magnetic particles were then activated with 10% glutaraldehyde for one hour and then washed twice with PBS buffer (pH 7.2). Anti-PIV3-F mouse monoclonal antibodies 2F2 and 5D8 were added to the magnetic beads at a concentration of 0.3 μg / dose and coated at 4°C for two hours. Finally, the beads were blocked with a blocking solution containing BSA for two hours.

[0090] 3.3 Clinical Sample Testing

[0091] Thirty samples that tested positive for PIV3 nucleic acid from a hospital were collected and tested using the double-antibody sandwich method of the present invention. Two paired antibodies were used to detect positive clinical samples from the hospital on a magnetic particle platform. The paired detection of the coated antibody 5D8 and the enzyme-labeled antibody 7B12 had a positive-negative coincidence rate of over 70% with the hospital samples, meeting the testing requirements.

[0092]

[0093] Example 4 Monoclonal Antibody Sequencing

[0094] The following primers were synthesized based on the constant region sequence of the antibody gene:

[0095] LF 5′-GACATTGTGATGACCCAGTCTCCT-3′;

[0096] LR 5′-TGGACACTGTTGGGGCCGCATCGGCCCT-3′

[0097] HF 5′-CAGGTGCAGCTGCAGGAGTCAGGA-3′

[0098] HR 5′-GATAGACAGATGGGGGTGTCGTTTTGGC-3′

[0099] 3×10 6 Total RNA from hybridoma cells 5D8 and 7B12 was reverse transcribed into cDNA. PCR amplification of the heavy chain variable regions of monoclonal antibodies 5D8 and 7B12 was performed using primers HF and HR. PCR amplification of the light chain variable regions of monoclonal antibodies 5D8 and 7B12 was performed using primers LF and LR. Hot-start PCR reactions were performed using the following conditions: 95°C for 5 minutes, followed by 30 cycles of 95°C for 15 seconds, 55°C for 45 seconds, and 72°C for 30 seconds, and 72°C for 7 minutes. PCR products were separated by 1% agarose gel electrophoresis and purified. The fragments were cloned into the PM18-T vector, transformed into Escherichia coli DH5α cells, and screened on LB solid plates. White plaques were then inoculated into LB liquid medium containing ampicillin for amplification. Positive clones were screened, plasmids were extracted using a QIAGEN plasmid extraction kit, and sequencing was performed to determine the heavy and light chain variable region sequences of monoclonal antibodies 5D8 and 7B12.

[0100] The light chain variable region of monoclonal antibody 5D8 has the amino acid sequence shown in SEQ ID NO.7, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.9; the light chain variable region of monoclonal antibody 7B12 has the amino acid sequence shown in SEQ ID NO.8, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.10.

[0101] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Anti-type 3 human parainfluenza virus monoclonal antibody, characterized in that Includes capture and / or detection antibodies: The amino acid sequences of the three CDR regions of the light chain of the capture antibody are QGIRGN, STS and LQRNAYPYT respectively; the amino acid sequences of the three CDR regions of the heavy chain are GFSLTRYG, IWAGGST and ASYYWQRDGMDC respectively; The amino acid sequences of the three CDR regions of the light chain of the detection antibody are KSLLYKDGKTY, LMS and QQLVEYPLT respectively; the amino acid sequences of the three CDR regions of the heavy chain are GFTFSDSY, SRNKAYDYST and ARDAGYGNHGGYFDY respectively.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the capture antibody is shown in SEQ ID No: 7; the amino acid sequence of the heavy chain variable region is shown in SEQ ID No:

9.

3. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the detection antibody is shown in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

10.

4. The monoclonal antibody according to claims 1 to 3, characterized in that Its subtype is IgG, and its light chain is kappa chain.

5. Biomaterial, characterized in that It includes at least one of the following a) to e): a), a nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 4; b) a recombinant vector comprising a vector backbone and a nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 4; c) a host that secretes the monoclonal antibody according to any one of claims 1 to 4; d) A chemically labeled or biologically labeled monoclonal antibody according to any one of claims 1 to 4; e) The monoclonal antibody according to any one of claims 1 to 4 coupled to a carrier.

6. Use of the monoclonal antibody according to any one of claims 1 to 4 or the biomaterial according to claim 5 in the preparation of a reagent or kit for detecting human parainfluenza virus type 3.

7. An ELISA kit for detecting human parainfluenza virus type 3, characterized in that: The method comprises the detection antibody and the capture antibody according to any one of claims 1 to 4.

8. The ELISA kit according to claim 7, wherein The capture antibody is coupled to magnetic beads, and the detection antibody is modified with horseradish peroxidase.

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