CN114149497A

CN114149497A - Porcine parvovirus neutralizing monoclonal antibody, and preparation method and application thereof

Info

Publication number: CN114149497A
Application number: CN202111466368.3A
Authority: CN
Inventors: 刘运超; 张改平; 王聚财; 尚延丽; 杨苏珍; 魏蔷; 陈玉梅; 王方雨; 金前跃
Original assignee: Key Laboratory Of Animal Immunology Henan Academy Of Agricultural Sciences
Current assignee: Key Laboratory Of Animal Immunology Henan Academy Of Agricultural Sciences
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2022-03-08

Abstract

The invention provides a porcine parvovirus neutralizing monoclonal antibody and application thereof. Emulsifying the purified PPV VP2 protein with Freund's adjuvant to prepare PPV VLP vaccine immune BALB/c mouse, after 3 times of immunization, inducing the mouse to generate high-titer antibodies aiming at PPV VP2 protein and HI, the antibody titer is all 2¹⁶. The screening of hybridoma cells by an IPMA method successfully identifies a PPV monoclonal antibody 5F7 with neutralizing activity, the neutralizing titer of the antibody can reach 1: 2048, and the neutralizing monoclonal antibody does not have cross reaction with other swine viruses, which shows that the monoclonal antibody has good activityThe specificity of (A). The kit can be used for the development of an immunodetection reagent, provides biological materials for the development of a PPV immunodetection kit and a specific antibody immunoassay kit, and lays a foundation for the deep research of the structure of PPV and the molecular mechanism of an anti-virus antibody.

Description

Porcine parvovirus neutralizing monoclonal antibody, and preparation method and application thereof

Technical Field

The invention relates to a porcine parvovirus neutralizing monoclonal antibody and application thereof, belonging to the technical field of biology.

Background

Porcine Parvovirus (PPV) is one of the main pathogens causing pig breeding failure, and is first discovered in Germany in 1965, and clinically, the Porcine parvovirus has the main characteristics of stillbirth, mummy, embryonic death, delayed oestrus and the like of pregnant sows, thereby causing considerable economic loss to the pig industry all over the world. In recent years, PPV has received much attention due to mixed infection with other viruses such as porcine circovirus type 2 (PCV2), and is considered to be one of the most common infectious agents that cause reproductive failure in sows.

PPV belongs to parvovirus and parvoviridae, is a single-stranded and non-enveloped icosahedral DNA virus with the diameter of 18-26 nm. The PPV genome has about 5000bp of total length, mainly comprises two Open Reading Frames (ORF), wherein ORF1 encodes non-structural proteins NS1, NS2 and NS3, and is mainly involved in virus DNA replication and expression of regulatory genes; ORF2 encodes the viral structural proteins VP1, VP2 and VP3, and plays an important role in the viral assembly and infection process, and is closely related to attachment, entry, localization and self-assembly of viruses. The ratio of the structural proteins VP1 and VP2 in the capsid of PPV virus is approximately 1: 20. The VP1 protein, which includes the entire VP2 sequence and the N-terminal 150 amino acids, is a minor constituent protein of the PPV capsid. The VP2 protein is the major capsid protein, and self-assembles into virus-like particles (VLPs) in vitro, including the Hemagglutination (HA) active site of PPV and multiple virus-neutralizing antigenic sites, which induce a virus-neutralizing immune response.

The research adopts PPV VP2 protein recombinant expressed by escherichia coli to immunize a BALB/c mouse, prepares a monoclonal antibody with PPV infection neutralizing activity through hybridoma fusion and subclone screening technology, identifies the specificity, sensitivity and neutralizing titer of the monoclonal antibody, researches PPV immunochromatography rapid detection test paper based on the monoclonal antibody, and lays a foundation for the research and development of PPV virus immunodiagnosis reagents.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a porcine parvovirus neutralizing monoclonal antibody and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a neutralizing monoclonal antibody hybridoma cell strain for resisting porcine parvovirus is a hybridoma cell strain 5F7 which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C202180, the preservation address of Wuhan university and the preservation time of 2021, 10 and 19 days.

The monoclonal antibody prepared from the hybridoma cell strain.

The monoclonal antibody is monoclonal antibody 5F 7.

The light chain of the monoclonal antibody 5F7 is Kappa, and the heavy chain is IgG2 a.

The preparation method of the monoclonal antibody comprises the following steps:

(1) animal immunization

Preparing immunogen: mixing 250 mu L of purified PPV VP2 protein with equivalent volume of Freund's complete adjuvant and Freund's incomplete adjuvant, and emulsifying to obtain two PPV VLP immunogens for use;

BALB/C immunization program: dividing 5 BALB/c mice into two groups,

group

1, 3, immunized PPV VLP antigen; group 2, with immune PBS as a negative control; the total immunization is carried out for 3 times, each time is 100 mu L, and subcutaneous multipoint immunization is carried out on the mice every 2 weeks; the primary immunization uses PPV VLP antigen prepared by Freund's complete adjuvant, and the secondary immunization and the tertiary immunization use PPV VLP antigen prepared by Freund's incomplete adjuvant; at 14d after the third immunization, tail vein blood collection is carried out on the mice, and PPV specific antibodies in serum are measured by utilizing a hemagglutination inhibition test;

(2) cell fusion

Selecting mice with the highest antibody titer in the group 1 for boosting immunity, injecting 50 mu L of PPV VP2 protein without adjuvant into abdominal cavity 3d before fusion, removing neck after mouse eyeball bloodletting on the day of fusion, grinding spleen under aseptic condition, and preparing spleen cell suspension; adopting a hybridoma fusion technology to perform fusion of hybridoma cells; at 37 ℃ with 5% CO₂Culturing for 7-10 days in an incubator;

(3) screening and subcloning of hybridoma cells

When obvious cell clusters which can be observed by naked eyes appear at the bottom of the cell plate, detecting cell supernatant by using an immunoperoxidase monolayer cell test; carrying out subcloning on the positive hybridoma cells for 2-3 times by using a limiting dilution method to obtain a monoclonal cell strain;

(4) detecting ELSIA titer and neutralizing antibody titer of the monoclonal antibody, and identifying the monoclonal cell strain subtype to obtain the monoclonal antibody 5F 7.

The monoclonal antibody is applied to preparation of an ELSIA kit for detecting porcine parvovirus.

The monoclonal antibody is applied to preparation of test paper for detecting porcine parvovirus.

The invention has the beneficial effects that:

the invention emulsifies the purified PPV VP2 protein and Freund's adjuvant to prepare PPV VLP vaccine immune BALB/c mouse, and after 3 times of immunization, the invention can induce the mouse to generate high-titer antibodies aiming at PPV VP2 protein and HI, and the antibody titer is all 2¹⁶. By the IPMA methodScreening of hybridoma cells successfully identifies a PPV monoclonal antibody 5F7 with neutralizing activity, the neutralizing titer of the antibody can reach 1: 2048, and the neutralizing monoclonal antibody does not have cross reaction with other swine viruses such as PCV2, PRSV and CSFV, which shows that the monoclonal antibody has good specificity.

The monoclonal antibody with neutralization function and good specificity is successfully screened out, can be used for PPV virus detection test paper, and can be used for rapid, convenient and visual detection.

The monoclonal antibody 5F7 can be used for the development of an immunodetection reagent, provides a biological material for the development of a PPV immunodetection kit and a specific antibody immunoassay kit, and lays a foundation for the deep research on the structure of PPV and the molecular mechanism of an anti-virus antibody.

Drawings

FIG. 1 is a diagram showing the results of identifying a purified PPV VP2 protein;

wherein, A: SDS-PAGE identification; b: HA detection;

FIG. 2 is a graph showing the results of specific reactions between the screened mAbs 5F7 and 11B3 and PPV;

wherein, A: 5F7 monoclonal antibody; b: 11B 3; c: negative control;

FIG. 3 is a diagram showing the result of Western blot identification of a PPV monoclonal antibody;

wherein, A: 5F7 monoclonal antibody; b: 11B 3; c: his monoclonal antibody;

FIG. 4 is a diagram showing the result of subtype identification of the PPV monoclonal antibody.

FIG. 5 is a diagram showing the results of ELISA detection of PPV monoclonal antibody;

wherein, A: VP2 protein as coating antigen; b: PPV virus is a coating antigen;

FIG. 6 is a graph showing the results of measurement of the neutralizing titer of the PPV monoclonal antibody;

FIG. 7 is a diagram showing the results of cross-detection of PPV monoclonal antibody;

FIG. 8 is a schematic diagram of a test strip structure;

wherein, 1 is a support plate, 2 is a nitrocellulose membrane, 3 is a sample pad, 4 is a gold-labeled antibody pad, 5 is absorbent paper, 6 is a detection line T, and 7 is a quality control line C;

FIG. 9 is a diagram showing the test result of the test strip.

Detailed Description

The following examples further illustrate the embodiments of the present invention in detail.

The main experimental materials used in the invention:

1. cell, virus seed and experimental animal

SP2/0, PK15 and Mac145 cells, PPV (porcine circovirus virus), porcine circovirus type 2 (PCV2), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Classical Swine Fever Virus (CSFV), and animal immunology important experimental preservation of agricultural academy of sciences of Henan province. Wherein the SP2/0 cells are used for cell fusion; PK15 cells are used for the proliferation of PPV, CSFV and PCV2 viruses; mac145 cells are used for the propagation of PRRSV virus. Experimental animal BALB/c female mouse is purchased from the center of experimental animals of medical college of Zhengzhou university, and 6-8 weeks old mouse is used for vaccine immunization, and the multiparous mother mouse is used for preparing the ascites of the monoclonal antibody.

2. Primary reagent

A mouse monoclonal antibody subtype identification kit and a horseradish peroxidase (HRP) marked anti-His monoclonal antibody are purchased from Proteintech company; HRP-labeled goat anti-mouse IgG and HRP-labeled goat anti-pig IgG were purchased from Jackson Immuno Research; DMEM medium, FBS fetal bovine serum was purchased from Gibco; HT, HAT medium, fusion agent PEG1500 and Freund's adjuvant are purchased from Sigma company; protein G, Ni column, Superdex 20010/300 GL column was purchased from GE.

EXAMPLE 1 preparation of monoclonal antibody hybridoma cell line of PPV VP2 protein

1. Animal immunization

Antigen sources: the PPV VP2 protein constructed, expressed and purified by this experiment was used as immunogen.

Purification results of PPV VP2 protein: the SDS-PAGE result showed that the purity of VP2 protein after Ni column and Superdex 20010/300 GL purification can reach 80% (FIG. 1A). The purified VP2 protein HAs high HA activity, and the HA titer can reach 2¹⁴(FIG. 1B).

Preparation of immunogen: the purified PPV VP2 protein (250. mu.L)PPV VLPs were prepared by mixing and emulsifying the mixture with an equal amount of Freund's adjuvant (250. mu.L). The HA titer of the vaccine containing the VP2 antigen per milliliter is 2¹³。

BALB/C immunization program: randomly dividing 5 BALB/c mice into two groups,

group

1, 3, immunized PPV VLP antigen; group 2, immune PBS as a negative control. Mice were immunized three times, 100 μ L each, with subcutaneous multiple immunizations every 2 weeks. The primary immunization used the PPV VLP antigen prepared with Freund's complete adjuvant, and the secondary and tertiary immunizations used the PPV VLP antigen prepared with Freund's incomplete adjuvant. At 14d after the triammunization, i.e., 56d after the primary immunization, the mice were bled from the tail vein, and the PPV antibodies in the serum were assayed using enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition assay (HI).

As shown in Table 1, the PPV VLP antigen can stimulate mice to produce high-titer and specific antibodies against PPV VP2 protein and HI, and the antibody titer can reach 2 at most¹⁶It is clearly higher than the negative group, indicating that the PPV VLP vaccine can induce BALB/c mice to generate strong antibody immune response.

TABLE 1 serum titer results for immunized mice

2. Cell fusion

The mice with the highest antibody titer were selected for booster immunization, and 50. mu.L of adjuvant-free PPV VP2 protein (25. mu.g) was intraperitoneally injected 3d before the fusion to perform the fusion of hybridoma cells.

On the day of fusion, the mouse eyeballs were bled, then decapped, and the spleen was ground under aseptic conditions to prepare a spleen cell suspension. The prepared splenocytes and SP2/0 cells with good state are fused by using a fusion agent PEG1500 according to the volume ratio of 5:1 by adopting a classical hybridoma fusion technology, and the fused hybridoma cells are diluted by HAT selective medium and then are paved into a 96-well plate with 300 mu L/well for 20 plates. 37 ℃ and 5% CO₂Culturing for 7-10 days in an incubator.

3. Screening and subcloning of hybridoma cells

Cell supernatants were examined using immunoperoxidase monolayered assay (IPMA) when visually apparent clumps appeared at the bottom of the cell plates. Carrying out subcloning on positive hybridoma cells screened by IPMA for 2-3 times by using a limiting dilution method to obtain a single cell strain.

Positive clones were screened by IPMA method: PK15 cells were cultured at 6X 10⁵The density per well was inoculated into a 96-well plate, and PPV virus was inoculated at a rate of 1% (wt) at 37 ℃ in a volume of 250. mu.L per well and 5% CO₂The incubator is used for 72 h. The plate was washed 3 times with PBS, fixed with 4% (wt) paraformaldehyde for 10min at room temperature, the fixative was discarded, and the plate was blocked with PBST containing 5% (wt) skim milk overnight at 4 ℃. Hybridoma culture supernatant cultured for 7-10 days is used as primary antibody, and the temperature is 37 ℃ for 30 min. PBST was washed 6 times with HRP-labeled goat anti-mouse IgG as a secondary antibody at 37 ℃ for 45 min. PBST washed 6 times, last with ddH₂O washing, adding 100 μ L AEC color developing solution into each well, developing at room temperature for 10min, discarding the color developing solution, adding ddH₂O stop color development, 200. mu.L/well. When observed under an inverted microscope, the positive wells showed specific staining (red color) and the negative wells showed no staining.

The 2 screened monoclonal antibodies 5F7 and 11B3 with neutralizing activity reacted specifically with PPV, and the results are shown in FIG. 2.

4. Preparation of monoclonal antibody ascites

The single cell strain identified as positive is enlarged and cultured, and the cell concentration is adjusted to 0.5 multiplied by 10⁷/mL～1.0×10⁷about/mL, the pregnant BALB/c mother mice were injected intraperitoneally with paraffin pre-treated at a dose of 500. mu.L, and ascites collection was performed after 7 days when the abdomen of the mice had significantly swollen. The collected ascites fluid was purified using a GE Protein G affinity column.

5. Western blot detection

The reactivity of 5F7 and 11B3 antibodies with PPV VP2 expressed by Escherichia coli was examined using hybridoma supernatant as the primary antibody. The purified VP2 protein was subjected to SDS-PAGE and WB analysis. PVDF membrane containing 5% skim milk PBST buffer at 4 degrees C under overnight blocking. PBST membrane washing 6 times, hybridoma cell supernatant as the first antibody, 37 degrees, 30 min. PBST membrane washing 6 times, HRP labeled goat anti-mouse IgG antibody as secondary antibody, 37 degrees, 45 min. PBST membrane washing 6 times, AEC color development. The results are shown in fig. 3, and compared with the His mab positive control, the two neutralizing mabs did not react with the denatured recombinant VP2 protein, indicating that the integrity of the spatial structure of VP2 protein may affect the binding of antibody to protein.

6. Monoclonal antibody subtype identification

The subtype of the monoclonal antibody screened by the invention is identified according to the specification of a mouse monoclonal subtype identification kit of Proteitech company. The results showed that monoclonal antibodies 5F7 and 11B3 belong to two different antibody subtypes, of which the heavy chain of 5F7 is IgG2a, the heavy chain of 11B3 is IgG2B, and the light chains are both Kappa (fig. 4).

The hybridoma cell strain 5F7 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C202180, the preservation address of Wuhan university and the preservation time of 2021 year, 10 months and 19 days.

EXAMPLE 2 ELISA detection of monoclonal antibodies

The reactivity of neutralizing mabs 5F7 and 11B3 with PPV VP2 protein and PPV virus was analyzed by indirect ELISA. The ELISA titer was measured after the plate-coating and blocking with the purified PPV VP2 protein and PPV virus as antigens. Using diluted hybridoma cell supernatant or ascites as primary antibody, using HRP-labeled goat anti-mouse IgG antibody as secondary antibody for detection, adding TMB solution for color development, adding equal volume of 2M H₂SO₄The reaction was terminated. Triplicate for each sample and OD determination₄₅₀ _nmThe absorbance of the antibody was measured.

As shown in FIG. 5, both monoclonal antibodies specifically bind to PPV and the recombinant VP2 protein, and the binding titer of the monoclonal antibody 11B3 ascites to PPV was 1: 10240 (FIG. 5B). When the hybridoma supernatant of the neutralizing monoclonal antibody 5F7 is diluted to 1:6400, the hybridoma supernatant can still be specifically combined with PPV VP2 protein, and the antibody titer of the monoclonal antibody 5F7 ascites can reach 1:4096,000 (Table 2).

TABLE 2 monoclonal antibody cell supernatant and ascites titer

Monoclonal antibody	Hybridoma supernatant	Ascites (ascites)
			5F7	1:6400	1:4096,000
11B3	1:6400	1:4096,000

Example 3 Virus Neutralization (VN) assay for monoclonal antibodies

Mixing hybridoma cell supernatant or purified ascites with 200TCID₅₀Was incubated at 37 ℃ for 1h, and the mixture was added to cells containing PK15 (6X 10)⁵Perwell) 96-well cell plate, 5% CO at 37 ℃₂The incubator continues to culture for 72 h. The plate was washed 3 times with PBS, fixed with 4% (wt) paraformaldehyde for 10min at room temperature, the fixative was discarded, and blocked with 5% (m/v) skim milk in PBST buffer at 4 ℃ overnight. The porcine polyclonal antibody resisting PPV virus is used as a primary antibody, and the temperature is 37 ℃ and the time is 30 min. PBST wash plate 6 times. Using goat anti-pig IgG antibody marked by HRP as a secondary antibody, and performing temperature control at 37 ℃ for 30 min. PBST Wash plates 6 times, last with ddH₂O washing and AEC developing for 10min at room temperature. When the cells are observed under a microscope to be developed and have no specific staining, the antibodies have a neutralization effect, and the highest dilution multiple of the hybridoma clone supernatant or ascites completely neutralized PPV virus is used as the final VN antibody titer.

As shown in FIG. 6, the neutralizing titers of the monoclonal antibodies 5F7 and 11B3 were 1: 2048 and 1: 1024, respectively.

EXAMPLE 4 viral detection of monoclonal antibodies

PPV, PRSV, CSFV and PCV2 viruses are used as antigens to coat ELSIA plates, and the specificity of the monoclonal antibody is identified. Using diluted hybridoma cell supernatant as primary antibody, using HRP-labeled goat anti-mouse IgG antibody as secondary antibody for detection, adding TMB solution for color development, adding equal volume of 2M H₂SO₄The reaction was terminated. Triplicate for each sample and OD determination_450nmThe absorbance value of (c) was measured, and the results were analyzed. The result is shown in fig. 7, the neutralizing monoclonal antibody 5F7 only reacts with PPV virus, but does not react with PCV2, PRSV, CSFV and other viruses, which indicates that the neutralizing single cell strain screened by the invention has good specificity.

Example 5 application of monoclonal antibody 5F7 in PPV Virus test paper

1. Preparation of colloidal gold solution

The colloidal gold is prepared by a trisodium citrate method, which comprises the following steps: putting 99mL of double distilled water into a 200mL triangular flask, heating and continuously stirring on a heating magnetic stirrer, adding 1mL of 1% (wt) chloroauric acid, heating and stirring until the mixture is boiling, then quickly adding 1.6mL of 1% (wt) trisodium citrate, continuously heating and stirring, observing that the color is gradually changed from light yellow to dark red, and then continuously heating and stirring for 5 min. Stopping heating, cooling the triangular flask, recovering to room temperature, adding sterile double distilled water to constant volume of 100mL, determining the wavelength of the maximum absorption peak to be 530nm by ultraviolet scanning, and storing at 4 deg.C for later use.

2. Preparation of colloidal gold-labeled antibody

The monoclonal antibody 5F7 for resisting PPV is used as a gold-labeled antibody, the optimal binding pH value is 7.0, and the ratio of the colloidal gold to the antibody is 30ug of the antibody per ml of the colloidal gold. The labeled colloidal gold is treated by 1% (wt) of Human Serum Albumin (HSA) stabilizer, the labeled solution is uniformly adsorbed on a glass fiber membrane according to the amount of 60uL per square centimeter, and the gold-labeled antibody pad is obtained after vacuum drying for 1 hour at 37 ℃.

3. Preparation of nitrocellulose membranes

The anti-PPV polyclonal antibody is sprayed on a nitrocellulose membrane to be used as a detection line (T line) of the test paper, and staphylococcus aureus protein A (SPA) is used as a quality control line (C line) of the test paper. And (3) airing the quality control line and the detection line in an oven with the humidity of less than 30% and the temperature of 37 ℃ for 10 hours for later use.

4. Sample pads (size 18 × 300mM, glass fiber cotton material, which is soaked in 50mM, pH8.0 Tris-HCl buffer solution for 2h), gold labeled antibody pads (size 7 × 300mM, glass fiber cotton material), nitrocellulose membranes (size 25 × 300mM, nitrocellulose material) and absorbent paper (size 16 × 300mM) are sequentially adhered to a support plate (size 60 × 300mM) in an overlapping manner to obtain test paper boards, and the test paper boards are cut into test strips with different widths according to requirements.

The structure of the test strip is shown in fig. 8.

5. Application method

The invention relates to a colloidal gold test strip for detecting PPV (pentatricopeptide virus), which is assembled in a plastic shell formed by buckling a plastic upper shell and a plastic lower shell when in use, wherein the plastic upper shell is provided with two openings, a sample adding window and a display hole, and the sample adding window corresponds to a sample pad of the colloidal gold test strip for detecting PPV.

The detection result judging method comprises the following steps:

adding a sample to be detected on a sample pad, if the object to be detected contains PPV antigen, combining with the anti-PPV monoclonal antibody marked by colloidal gold on the test strip in the upward process to form a corresponding immune complex, continuously moving the immune complex upward to combine with the anti-PPV polyclonal antibody coated on the detection line on the nitrocellulose membrane, and intercepting at the detection line to form a red indicating line, namely a detection line T line. Whether PPV antigen is contained or not, the colloidal gold labeled monoclonal antibody is combined and intercepted with SPA coated on the membrane at the quality control line on the nitrocellulose membrane in the ascending process, and a red strip is formed at the quality control line. The strip is the quality control line, and if the line does not appear, the colloidal gold test strip is invalid.

As shown in fig. 9, two binding lines appear, and the detection line T and the quality control line C show red color indicating that the sample contains PPV antigen, and the detection result is positive; only the quality control line C is developed, and the T is not developed, which indicates that no PPV exists in the sample, and the result is judged to be negative; if the position of the C strip is not colored, whether the T line is colored or not indicates that the reagent strip is invalid, and the detection result cannot be judged.

While the present invention has been described in detail with reference to the embodiments, those skilled in the art will appreciate that various specific parameters can be adjusted in the above embodiments without departing from the spirit of the present invention, and that various embodiments are formed, which are all the changes of the present invention and will not be described in detail herein.

Claims

1. The neutralizing monoclonal antibody hybridoma cell strain for resisting the porcine parvovirus is characterized in that the hybridoma cell strain is hybridoma cell strain 5F7 which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C202180, the preservation address of Wuhan university and the preservation time of 2021, 10 and 19 days.

2. A monoclonal antibody produced by the hybridoma cell line of claim 1.

3. The monoclonal antibody of claim 2, wherein the monoclonal antibody is monoclonal antibody 5F 7.

4. The monoclonal antibody of claim 3, wherein the monoclonal antibody 5F7 has a light chain of Kappa and a heavy chain of IgG2 a.

5. The method of producing a monoclonal antibody according to claim 2, comprising the steps of:

(1) animal immunization

BALB/C immunization program: dividing 5 BALB/c mice into two groups, group 1, 3, immunized PPV VLP antigen; group 2, with immune PBS as a negative control; the total immunization is carried out for 3 times, each time is 100 mu L, and subcutaneous multipoint immunization is carried out on the mice every 2 weeks; the primary immunization uses PPV VLP antigen prepared by Freund's complete adjuvant, and the secondary immunization and the tertiary immunization use PPV VLP antigen prepared by Freund's incomplete adjuvant; at 14d after the third immunization, tail vein blood collection is carried out on the mice, and PPV specific antibodies in serum are measured by utilizing a hemagglutination inhibition test;

(2) cell fusion

(3) screening and subcloning of hybridoma cells

6. Use of the monoclonal antibody of any one of claims 1-4 in the preparation of an ELSIA kit for detecting porcine parvovirus.

7. Use of the monoclonal antibody of any one of claims 1-4 in the preparation of a test strip for porcine parvovirus detection.