Monoclonal antibody against human parainfluenza virus type I and its preparation method and use

Abstract

The present invention relates to the field of in vitro detection technology, specifically to anti-type I human parainfluenza virus monoclonal antibodies, their preparation methods, and applications. The monoclonal antibodies provided by the present invention are capable of recognizing the HN protein of human parainfluenza virus and exhibit excellent specificity and high titer. Double-antibody sandwich methods, reagents, and kits for detecting type I human parainfluenza virus, developed based on the anti-type I human parainfluenza virus monoclonal antibodies of the present invention, are more efficient and time-efficient, and have broad application prospects in the early 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 I human parainfluenza virus monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Parainfluenza virus (PIV) is a pleomorphic, enveloped, single-stranded, negative-sense RNA virus belonging to the paramyxovirus family. It is divided into four subtypes: PIV-1 and PIV-3 belong to the genus Respirovirus, while PIV-2 and PIV-4 belong to the genus Mumpsvirus. The virus has a diameter of 125-250 nm, and its envelope is composed of lipids and glycoproteins. Two membrane glycoproteins are present: the hemagglutinin-neuraminidase protein (HN), which has hemagglutinin and neuraminidase activities; and the fusion protein (F protein), which promotes cell fusion and has hemolytic properties. Parainfluenza virus is a common and often overlooked respiratory pathogen, primarily causing severe respiratory infections in infants, children, and immunosuppressed individuals. In adults, parainfluenza virus infection can also cause a range of respiratory illnesses, including upper respiratory tract infections, acute exacerbations of chronic diseases, and pneumonia. Clinical manifestations vary, but the etiology is similar to that of other common respiratory infections. Furthermore, the lack of serological protection contributes to recurrent parainfluenza virus infection. Since specific therapeutic drugs and effective vaccines are not yet on the market, early diagnosis has become an important measure to prevent infection and obtain timely symptomatic treatment. Early nucleic acid diagnosis and clinical diagnosis are important bases for confirmation.

[0003] Current clinical methods for diagnosing human PIVs infection include: first culturing the tissue, then isolating and identifying the virus in cells, or directly detecting the virus in respiratory secretions. These methods include immunofluorescence testing, PCR, and enzyme-linked immunosorbent assays. However, nucleic acid diagnosis requires specialized equipment, takes a long time to detect, is significantly affected by the quality of the sample, and can result in false positives and false negatives. Nucleic acid testing alone can easily lead to missed diagnoses. Another method is serological diagnosis, which involves detecting specific antibodies (IgM) in a single serum specimen. This method has the advantages of a long duration and a stable immune response.

[0004] At present, most antibody detection kits use the capture ELISA principle to detect specific antibodies. That is, anti-IgM antibodies are used to capture IgM in the sample. If it is pathogen-specific IgM, it will bind to the labeled antigen and be detected. However, since the capture antibody (anti-IgM antibody) will bind to any IgM in the serum, the specificity of this method is only based on the specific recognition and binding of a pair of antigen-antibodies (that is, the antibody in the serum recognizes the viral protein provided by the kit). This results in false positives due to non-specific binding in complex sample components, and the IgM antibody test cannot distinguish between current infection and past infection. Therefore, the current clinical diagnosis of PIVs still requires an antibody with high specificity and high sensitivity. 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 1, as well as a preparation method and application thereof. The present invention provides a monoclonal antibody that specifically recognizes human PIV-1, as well as a preparation method and application of the antibody in detecting parainfluenza virus type 1 antigens in human serum, which can be widely used in clinical diagnosis.

[0006] The present invention provides monoclonal antibodies against human parainfluenza type I virus, including coated antibodies and / or enzyme-labeled antibodies:

[0007] The three CDR regions of the light chain of the coated antibody (5A9) have the amino acid sequences of ESVDSYGNSF (SEQ ID NO: 11), FAS, and HQNNEDPFT (SEQ ID NO: 12), respectively, and the three CDR regions of the heavy chain have the amino acid sequences of GSTFSDYY (SEQ ID NO: 13), ISNGGDST (SEQ ID NO: 14), and ARPSTPTYALDY (SEQ ID NO: 15), respectively;

[0008] The three CDR regions of the enzyme-labeled antibody (7C4) light chain have the amino acid sequences of RSLQHSNGNTY (SEQ ID NO: 16), RMS, and VQHLEYPLT (SEQ ID NO: 17), respectively; the three CDR regions of its heavy chain have the amino acid sequences of GYTFTNYV (SEQ ID NO: 18), INPYNDDT (SEQ ID NO: 19), and ARANWAWSAY (SEQ ID NO: 20), respectively.

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

[0010] Specifically, in some embodiments, the amino acid sequence of the light chain variable region of the anti-human parainfluenza type 1 monoclonal antibody 5A9 is shown in SEQ ID No: 7; and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 9.

[0011] In other embodiments, the amino acid sequence of the light chain variable region of the anti-human parainfluenza type I virus monoclonal antibody 7C4 is shown in SEQ ID NO: 8; and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10.

[0012] Furthermore, the anti-type I 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 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 mammalian cells. 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, lentiviruses, and prions. 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 the use of the monoclonal antibody or the biological material in preparing a type I human parainfluenza virus detection reagent or kit.

[0025] The present invention also provides an ELISA kit for detecting type I human parainfluenza virus, which comprises the coating antibody and enzyme-labeled antibody of the present invention.

[0026] Specifically, in an embodiment of the present invention, the ELISA kit uses the anti-type 1 human parainfluenza virus monoclonal antibody 5A9 as the capture antibody and the anti-type 1 human parainfluenza virus monoclonal antibody 7C4 as the detection antibody, and has extremely high sensitivity and specificity, better detection effect, and the qualified detection rate of negative results is 100%, and the qualified detection rate of positive results is more than 68%, which meets the detection requirements.

[0027] Furthermore, the coated antibody is coupled to magnetic beads, and the enzyme-labeled antibody is modified with horseradish peroxidase;

[0028] Specifically, in some 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 mg / ml EDC, and then adding the monoclonal antibody at 0.3 μg / person to the magnetic beads for coating and blocking;

[0029] The steps of detecting antibody-modified horseradish peroxidase include activating HRP with sodium periodate and then terminating with ethylene glycol, dialyzing to remove excess reagents, reacting with the antibody dialyzed with the same carbonate buffer, then terminating the reaction with sodium borohydride, dialyzing again, and storing with an equal volume of glycerol.

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

[0031] The detection methods described herein include those for diagnostic purposes and those for non-diagnostic purposes. For example, they include detection methods performed on human or animal samples, or on samples derived from human or animal samples in vitro, for diagnostic purposes; and also include detection methods performed on environmental samples or simulated samples for scientific research or other non-diagnostic purposes.

[0032] The present invention uses hybridoma cells produced by immunizing mice with a full-length recombinant protein containing human parainfluenza virus type 1 HN expressed in eukaryotic cells. Subsequently, screening yields monoclonal antibodies against the human PIV-1 HN protein, which exhibit extremely high sensitivity and specificity. A double-antibody sandwich assay developed using the monoclonal antibodies provided by the present invention can be used to detect parainfluenza virus type 1 antigens in human serum with reduced time and increased efficiency, providing key materials for the subsequent development of commercial kits for detecting human PIV-1 antigens and antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 shows the western blot image of HN protein;

[0034] Figure 2 shows the SDS-PAGE image of HN protein;

[0035] Figure 3 Figure 2 shows the antibody site identification data;

[0036] Figure 4 Figure 5 shows some data of HN detection using the double antibody sandwich method. DETAILED DESCRIPTION

[0037] The present invention provides monoclonal antibodies against type I human parainfluenza virus, methods for their preparation, and uses. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention. The methods and uses of the present invention have been described through preferred embodiments, and it is apparent that those skilled in the art can modify or appropriately alter and combine the methods and uses herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0038] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0039] The present invention will be further described below in conjunction with the embodiments:

[0040] Example 1 Construction of a eukaryotic expression vector for parainfluenza virus HN protein type I and its expression and purification 1. Construction of a eukaryotic expression vector for parainfluenza virus HN protein type I

[0041] Primers for eukaryotic expression of the HN gene were designed based on the HN gene sequence (SEQ ID No. 1). The upstream primer contained an EcoRI restriction site, and the downstream primer contained an XbaI restriction site. The primer sequences were: PIV1-HNF: GGAAtCCatggctgaaaaggggaaaacaattagttca (SEQ ID NO: 5); PIV1-HNR: GCTCTAGATTAGTGGTGGTGGTGGTGGTGTCTAGAagatgtgattttaca (SEQ ID NO: 6).

[0042] The HN gene was amplified using EX Taq enzyme. The PCR reaction procedure was: 94°C for 10 minutes, followed by 30 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 90 seconds, and finally 72°C for 10 minutes. The 1728-bp target fragment was recovered from a gel and then digested with EcoRI and XbaI, ligated into the pFastbac1 vector, and transformed into competent E. coli DH5α cells. After overnight culture, single colonies were selected, extracted, and confirmed by PCR and sequencing to obtain the pFastBac-HN plasmid.

[0043] The pFastBac1 plasmid containing the HN gene was used to transform the competent DH10Bac. With the help of the plasmid assistant in the DH10Bac bacteria, the HN gene can be transposed to the Bacmid large plasmid containing the baculovirus genome. Positive clones were obtained through blue-white spot screening and sequencing, namely recombinant Bacmid.

[0044] 2. HN protein expression

[0045] Recombinant bacmids that tested positive for sequencing were transfected into Sf9 insect cells using a Cellfection kit. After 7 days of culture in HyQ liquid medium at 27°C, the supernatant was collected to obtain P0 virus. Expression of the virus in the supernatant and cells was assessed. Western blot analysis using a His monoclonal antibody revealed a reactive band around 60 kD, indicating induced expression of the HN protein (Figure 1). The amino acid sequence of the HN protein is shown in SEQ ID No. 2.

[0046] P0 virus was used to infect 100 mL of sf9 cells (2 × 10 6 / mL), and the supernatant was collected after 4 days to obtain P1 virus.

[0047] 3. HN protein purification

[0048] The cell supernatant was collected and concentrated and filtered using a hollow fiber. The HN protein was then purified using nickel column affinity chromatography and dialyzed into PBS. Finally, protein expression and purification were detected by SDS-PAGE. The SDS PAGE results showed that after cell induction, a clear band appeared at around 60 kD, consistent with the expected molecular weight. The supernatant was concentrated and filtered, and then purified using a nickel column. SDS PAGE revealed a single protein band at the same position in the dissociation buffer ( Figure 1 ). This indicates that HN was successfully induced to express and had a high purity after purification.

[0049] Example 2 Screening and Preparation of Antibodies Against Type 1 Human Parainfluenza Virus HN Protein

[0050] 1. Animal immunization

[0051] BALB / c mice aged 6 to 8 weeks were immunized subcutaneously with HN protein 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 third immunization and the mice were boosted again 3 days before cell fusion. The dose of the booster immunization was 100 μg / mouse.

[0052] 2. Screening of positive hybridoma cells

[0053] Cell fusion was performed according to conventional methods. Hybridoma cells were screened 7 days after fusion using the following screening method:

[0054] The conventional 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 MAbs. The specific procedures are as follows:

[0055] 1) Use purified HN protein as the coating antigen at dilutions of 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, and 16 μg / mL. Coat an ELISA plate at 100 μL / well and incubate at 4°C overnight. The next day, discard the coating solution and wash the plate three times with PBST (5 min each time).

[0056] 2) Add 1% Casein to block the ELISA plate, 200 μL / well, block at 37°C for 2 h, and wash three times with PBST, 5 min each time.

[0057] 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 hour, and wash the plate three times with PBST, 5 minutes each time.

[0058] 4) Add 1:4000 diluted goat anti-mouse HRP-IgG, 100 μL / well, incubate at 37°C for 1 h, and wash the plate three times with PBST, 5 min each time.

[0059] 5) Add TMB substrate colorimetric solution at 100 μL / well and incubate at 37°C for 4 min. Add 2 mol / L H2SO4 to terminate the reaction at 50 μL / well and read the OD at 450 nm.

[0060] 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.

[0061] The cells in the positive culture wells were cloned and purified by limiting dilution until all clones were 100% positive.

[0062] 3. Production and purification of monoclonal antibodies

[0063] Balb / c mice were intraperitoneally injected with sterile paraffin oil (0.5 mL / mouse). 7 days later, 10 hybridoma cell lines with monoclonal antibodies against HN protein were intraperitoneally injected. 6 After 7 days, ascites was collected and purified using the caprylic acid-ammonium sulfate method to obtain monoclonal antibodies, which were stored at -20°C.

[0064] 4. Identification of monoclonal antibody subtypes

[0065] The antibody subtype of the 10 MAbs was determined using the Beijing Sino-Qiao Antibody Subtyping Kit, and the subtypes were all IgG, with a κ light chain.

[0066] 5. Identification of monoclonal antibody sites

[0067] To identify the antibody sites, a series of peptides were synthesized based on the epitopes predicted by various software. After coupling, the peptides were coated on enzyme immunoassay plates for reactivity verification. The results showed that five of the ten antibodies reacted with the two target epitope-coupled peptides from the HN protein. Antibody 5A9 recognized epitope 1, while antibodies 3F4, 6B10, 7A11, and 7C4 recognized epitope 2.

[0068] Polypeptide 1 amino acid sequence: 70TMTVSSVNESAKTIKETITEL 90 (SEQ ID NO: 3),

[0069] Polypeptide 2 amino acid sequence: 211LITQGCADIGKSYQV 225 (SEQ ID NO: 4). Data see Figure 3 .

[0070] Example 3 Application of PIV1-HN Antibody in Detecting Parainfluenza Virus Antigens in Human Serum 1. Antibody Pairing Experiment

[0071] (1) Five anti-human PIV-1 HN antibodies were coated onto ELISA plates at a concentration of 5 μg / mL at 4°C overnight. The plates were washed twice the next day with PBST, patted dry with absorbent paper, and blocked with 1% Casein for 2 h.

[0072] (2) Add 100 ng / mL recombinant HN protein, then dilute to 0.05 ng / mL in eight dilutions, incubate at 37°C for 1 h, wash five times with PBST, and pat dry with absorbent paper.

[0073] (3) Add five HRP-labeled antibodies (1:1000) (divided into two groups according to the site, and the two groups are paired with each other, for a total of four paired groups), incubate at 37°C for 1 h, wash 5 times with PBST, develop with TMB, and read the luminescence value.

[0074] Based on the site identification results, all eight HN antibodies could be paired for HN detection using the double antibody sandwich assay. Among them, 5A9 and 7C4 showed the best pairing, with a detection limit below 0.5 ng / mL.

[0075] Some experimental results are shown as follows Figure 4 5A9 was used as the coating antibody and paired with enzyme-labeled 3F4 and 7C4 to detect the best correlation and titer of recombinant antigens.

[0076] 2. Magnetic particle coating

[0077] 30 μL of the mixed magnetic microparticle stock solution was washed five times with 300 μL of PBS buffer. The magnetic microparticles were then activated with 10 mg / mL EDC for one hour and then washed twice with PBS buffer (pH 7.2). Mouse monoclonal antibody 5A9, directed against human PIV-1 HN protein, was 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 BSA-containing blocking buffer for two hours.

[0078] 3. Positive sample detection rate

[0079] Fifty samples were collected from a hospital for PIV1 nucleic acid testing, and 50 samples were collected for testing using the double-antibody sandwich method of the present invention. Two antibodies were paired on a magnetic microparticle platform to detect gradient recombinant antigens and positive and negative clinical samples from the hospital. The paired detection of the coated antibody 5A9 and the enzyme-labeled antibody 7C4 showed a 100% concordance rate with negative hospital samples and a 68% or higher concordance rate with positive samples, meeting the testing requirements. The results are shown in Table 1.

[0080] Table 1

[0081]

[0082]

[0083]

[0084] Example 4 Monoclonal Antibody Sequencing

[0085] The following primers were synthesized based on the constant region sequence of the antibody gene:

[0086] 4C-LF 5′-GACATTGTGATGACCCAGTCTCCT-3′;

[0087] 4C-LR 5′-TGGACACTGTTGGGGCCGCATCGGGCCT-3′;

[0088] 4C-HF 5′-CAGGTGCAGCTGCAGGAGTCAGGA-3′;

[0089] 4C-HR 5′-GATAGACAGATGGGGGTGTCGTTTTGGC-3′.

[0090] 3×10 6Total RNA from hybridoma cells 5A9 and 7C4 was reverse transcribed into cDNA. PCR was performed using primers 4C-HF and 4C-HR to amplify the heavy chain variable regions of monoclonal antibodies 5A9 and 7C4, while primers 4C-LF and 4C-LR were used to amplify the light chain variable regions of monoclonal antibodies 5A9 and 7C4. Both PCR reactions used a hot start reaction condition: 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 finally 72°C for 7 minutes. PCR products were separated by 1% agarose gel electrophoresis, and the target fragments were recovered 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 inoculated into LB liquid medium containing ampicillin for amplification. Positive clones were screened, and plasmids were extracted using QIAGEN's plasmid extraction kit and sequenced to determine the heavy chain and light chain variable region sequences of monoclonal antibodies 5A9 and 7C4.

[0091] The heavy chain variable region of the monoclonal antibody 5A9 has the amino acid sequence shown in SEQ ID NO.7, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.9; the heavy chain variable region of the murine monoclonal antibody 7C4 has the amino acid sequence shown in SEQ ID NO.8, and the light chain variable region has the amino acid sequence shown in SEQ ID NO.10.

[0092] 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 Ⅰ human parainfluenza virus monoclonal antibody, characterized in that Including coated antibodies and / or enzyme-labeled antibodies: Coating antibody: The amino acid sequences of the three CDR regions of its light chain are ESVDSYGNSF, FAS, and HQNNEDPFT, and the amino acid sequences of the three CDR regions of its heavy chain are GSTFSDYY, ISNGGDST, and ARPSTPTYALDY; The amino acid sequences of the three CDR regions of the light chain of the enzyme-labeled antibody are RSLQHSNGNTY, RMS, and VQHLEYPLT, and the amino acid sequences of the three CDR regions of the heavy chain are GYTFTNYV, INPYNDDT, and ARANWAWSAY.

2. The anti-type I human parainfluenza virus monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the coated 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; The amino acid sequence of the light chain variable region of the enzyme-labeled antibody is shown in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region thereof is shown in SEQ ID NO:

10.

3. The anti-type I human parainfluenza virus monoclonal antibody according to claim 1 or 2, characterized in that Its subtype is IgG, and its light chain is kappa chain.

4. 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 3; b) a recombinant vector comprising a vector backbone and a nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 3; c) a host that secretes the monoclonal antibody according to any one of claims 1 to 3; d) a chemically labeled or biologically labeled monoclonal antibody according to any one of claims 1 to 3; e) The monoclonal antibody according to any one of claims 1 to 3 coupled to a carrier.

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

6. An ELISA kit for detecting type I human parainfluenza virus, characterized in that: The invention comprises the coated antibody and enzyme-labeled antibody according to any one of claims 1 to 3.

7. The ELISA kit according to claim 6, wherein The coated antibody is coupled to the magnetic beads, and the enzyme-labeled antibody is modified with horseradish peroxidase.

Images