Hybridoma cell secreting monoclonal antibody against n protein of porcine reproductive and respiratory syndrome virus and application thereof

Abstract

The application discloses a hybridoma cell secreting a porcine reproductive and respiratory syndrome virus N protein monoclonal antibody and an application thereof, and the preservation number of the hybridoma cell strain is CGMCC NO. 45603. The application screens a monoclonal antibody 12E1 with a PRRSV positive serum blocking effect, and successfully establishes a PRRSV N protein blocking ELISA antibody detection method. The method has high sensitivity and specificity, and the coincidence rate with an IDEXX PRRSV ELISA antibody detection kit is 94.3%, and the method can be used for PRRSV antibody detection and serological epidemiological investigation.

Description

Technical Field

[0001] This invention belongs to the field of bioimmunotechnology, specifically relating to a hybridoma cell that secretes a monoclonal antibody against the N protein of porcine reproductive and respiratory syndrome virus and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) has become a major pathogen seriously threatening the global swine industry. The virus is prone to mutation, and while vaccination is one of the effective preventative measures against PRRS, it does not induce widespread protection. PRRSV belongs to the genus *Arterivirus* of the family Arteriviridae in the order Nidovirales. It is an enveloped, single-stranded, positive-sense RNA virus. The viral genome is approximately 15 kb in size and contains about 11 open reading frames (ORFs), encoding eight major structural proteins: GP2a, GP3, GP4, and GP5 envelope glycoproteins, the membrane matrix M protein, the non-glycosylated envelope E protein, the ORF5a protein, and the nucleocapsid N protein. The N protein is highly immunogenic and forms the antigenic basis for current PRRS serological diagnostic methods. Several linear epitopes on B cells have been identified, making them ideal targets for developing diagnostic reagents. This study aimed to construct a recombinant expression plasmid for the PRRSV N protein gene using a prokaryotic expression system, prepare and purify the recombinant N protein, develop seven monoclonal antibodies using hybridoma cell fusion technology, and identify three B-cell linear antigenic epitopes located in the 47–57 aa, 57–67 aa, and 67–77 aa regions of the N protein, respectively. Among them, the 57–67 aa region is a newly discovered antigenic epitope, providing new materials for the serological detection of PRRSV and laying the foundation for further investigation of the structure and function of the PRRSV N protein. Summary of the Invention

[0003] The purpose of this invention is to provide a hybridoma cell that secretes a monoclonal antibody against the N protein of porcine reproductive and respiratory syndrome virus and its application.

[0004] The objective of this invention can be achieved through the following technical solutions:

[0005] A hybridoma cell line secreting a monoclonal antibody against porcine reproductive and respiratory syndrome virus N protein, with the accession number CGMCC NO.45603.

[0006] A monoclonal antibody against the N protein of porcine reproductive and respiratory syndrome virus (PRRSV) is disclosed, which is secreted by the aforementioned hybridoma cell line. The N protein antigenic epitope recognized by this monoclonal antibody is located at... 57 LATEDDVRHHF 67 .

[0007] The application of the aforementioned porcine reproductive and respiratory syndrome virus (PRRSV) N protein monoclonal antibody in PRRSV serological detection or in the preparation of PRRSV serological detection kits also falls within the scope of protection of this invention.

[0008] The application of the aforementioned porcine reproductive and respiratory syndrome virus (PRRSV) N protein monoclonal antibody in the detection of PRRSV N protein blocking ELISA antibody or in the preparation of PRRSV N protein blocking ELISA antibody detection kits also falls within the scope of protection of this invention.

[0009] A PRRSV N protein blocking ELISA antibody detection kit, comprising the aforementioned porcine reproductive and respiratory syndrome virus N protein monoclonal antibody. As a preferred embodiment, the kit further comprises: coated antigen PRRSV N recombinant protein, secondary antibody, PBST, antigen coating solution, chromogenic solution, and stop solution.

[0010] Further preferred,

[0011] The secondary antibody is HRP-goat anti-mouse IgG.

[0012] The antigen coating solution (carbonate buffer) formula is as follows: 0.17g anhydrous Na2CO3, 0.28g NaHCO3, pH adjusted to 9.6, and ddH2O brought to a final volume of 100mL.

[0013] PBST formulation: KH2PO4 0.27g, Na2HPO4 1.42g, NaCl 8g, KCl 0.2g, Tween-20 500μL, pH adjusted to 7.4, and deionized water brought to a final volume of 1L.

[0014] The colorimetric solution mentioned is TMB colorimetric solution.

[0015] The termination solution is 2M H2SO4.

[0016] A method for detecting PRRSV N protein blocking ELISA antibodies, using the above-mentioned monoclonal antibody or the above-mentioned kit for detecting PRRSV N protein blocking ELISA antibodies.

[0017] As a preferred technical solution, the method includes the following steps:

[0018] (1) Coating: Dilute the antigen with antigen coating solution and coat the enzyme-labeled strips;

[0019] (2) Washing: Wash with PBST;

[0020] (3) Sealing: The sealing reaction is carried out using a sealing solution;

[0021] (4) Washing: Wash with PBST;

[0022] (5) Treatment of serum to be tested: Dilute serum with PBST and add it, then let it stand to react;

[0023] (6) Washing: Wash with PBST;

[0024] (7) Monoclonal antibody reaction: Dilute the above monoclonal antibody with PBST and add it, then let it stand to react;

[0025] (8) Washing: Wash with PBST;

[0026] (9) Secondary antibody reaction: Dilute HRP-goat anti-mouse-IgG with PBST and add it, then let it stand to react;

[0027] (10) Washing: Wash with PBST;

[0028] (11) Color reaction: Add TMB color reagent to carry out color reaction;

[0029] (12) Termination of reaction: The reaction was terminated with 2M H2SO4 as the termination solution;

[0030] (13) Reading: Set the program of the multi-functional microplate reader, read the absorbance OD450nm value and calculate the blocking rate PI;

[0031] (14) The detection results are judged based on the blocking rate (PI). The judgment criteria are as follows: It is positive. It is negative.

[0032] Those whose results fall between the two are considered suspicious; if repeated testing still yields suspicious results, they are considered positive.

[0033] More preferably, the antigen in step (1) is PRRSV recombinant N protein, diluted to a concentration of 1.0 μg / mL, and the antigen coating condition is 4°C for 12 h.

[0034] The sealing solution mentioned in step (3) is 1% BSA, and the sealing conditions are sealing at 37°C for 1 hour;

[0035] The serum dilution in step (5) is 1:2, and the static reaction conditions are 37°C for 45 min.

[0036] The enzyme-labeled monoclonal antibody 12E1-HRP described in step (7) is diluted 1:800, and 12E1-HRP is incubated at 37°C for 45 min.

[0037] The secondary antibody HRP-goat anti-mouse-IgG mentioned in step (9) is diluted to 1:1000 and incubated at 37°C for 45 minutes.

[0038] The conditions for the colorimetric reaction described in step (11) are 37℃ for 5 min.

[0039] In this study, the ORF7 gene of the PRRSV BB0907 strain was amplified by PCR, and the E. coli expression vector pET-28a-ORF7 was constructed and transformed into E. coli BL21 for induced expression. The recombinant N protein was purified by affinity chromatography. After immunization with BALB / c mice, seven positive hybridoma cell lines (8F1, 5E2, 2B1, 12E1, 4F1, 2H2, and 5A2) were obtained. All seven cells stably secreted monoclonal antibodies after 20 passages. The antibody titers in the cell supernatant via ELISA ranged from 1:800 to 1:25,600, and the titers in mouse ascites fluid ranged from 1:4096,000 to 1:131072,000. The heavy chain type of 8F1, 5E2, 2B1, and 12E1 was IgG1, while the heavy chain type of 4F1, 2H2, and 5A2 was IgG2b. The light chain type of all cells was κ. Three antigenic epitopes recognized by seven monoclonal antibodies were identified through N protein truncation construction and Western blot analysis. 47 KKNPEKPHFPL 57 , 57 LATEDDVRHHF 67 and 67 FTPSERQLCLS 77 Among them, the 12E1, 2H2, and 2B1 monoclonal antibodies recognize antigenic epitopes. 57 LATEDDVRHHF 67 It is a newly discovered antigenic epitope. All seven monoclonal antibodies can be used in PRRSV indirect immunofluorescence assay (IFA) and Western blot assay; 12E1 and 5A2 monoclonal antibodies have good immunohistochemical (IHC) reaction characteristics.

[0040] Using the recombinant N protein as the coating antigen, monoclonal antibody 12E1 with serum blocking effect was screened. After horseradish peroxidase (HRP) labeling and reaction condition optimization, a PRRSV N protein blocking ELISA antibody detection method was successfully established. The cutoff values ​​and judgment criteria are as follows: PI ≥ 60%, considered positive; PI ≤ 48%, considered negative; and PI between the two is considered suspicious. This method shows no cross-reactivity with positive serum antibodies against PRV, CSFV, PCV2, SVA, and PEDV. The coefficient of variation (CV) for intra- and inter-batch repeatability tests is less than 10%, and the concordance rate with the IDEXX PRRSV ELISA detection kit is 94.3%. It can be used for PRRSV antibody detection and serological epidemiological surveys, showing significant application potential.

[0041] Beneficial effects of the present invention

[0042] A monoclonal antibody, 12E1, with blocking effect on PRRSV positive serum was screened, and a PRRSV N protein blocking ELISA antibody detection method was successfully established. This method has high sensitivity and specificity, with a concordance rate of 94.3% with the IDEXX PRRSV ELISA antibody detection kit, and can be used for PRRSV antibody detection and serological epidemiological surveys. Attached Figure Description

[0043] Figure 1 Results of SDS-PAGE (A) and Western blot identification of PRRSV N recombinant protein (B);

[0044] Among them, M. protein molecular weight standard; 1. induction supernatant; 2. flow-through solution; 3-6. washing solution; 7-27. 10-500 mmol / L imidazole gradient elution solution; 28. recombinant N protein; 29. ​​pET-28a empty vector.

[0045] Figure 2 Western blot was used to identify the specificity of the seven monoclonal antibodies in reaction with recombinant N protein (A) and PRRSV (B);

[0046] Among them, M. protein molecular weight standard; 1. recombinant N protein; 2. pET-28a empty vector protein; 3. PRRSV infection of Marc-145 cells; 4. uninfected Marc-145 cells.

[0047] Figure 3 Results of the IFA reaction characteristics of monoclonal antibody with PRRSV-infected cells (200×);

[0048] Among them, A.8F1; B.5E2; C.2B1; D.12E1; E.4F1; F.2H2; G.5A2; H.PRRSV N monoclonal antibody; I.SP2 / 0 supernatant; J.uninfected Marc-145 cells.

[0049] Figure 4 Western blot was used to identify the PRRSVN protein antigenic epitopes recognized by the seven monoclonal antibodies.

[0050] Among them, A. Construction strategy of PRRSVN protein truncated variant and results of monoclonal antibody reaction; B. Western blot identification of N protein truncated variant expression; C. Reaction characteristics of 7 monoclonal antibodies with F1 and F2 truncated variants; D. Reaction characteristics of 2 monoclonal antibodies with F5 and F6 truncated variants; E. Reaction characteristics of 2 monoclonal antibodies with F3 and F4 truncated variants.

[0051] Figure 5 Results of RT-PCR for lung tissue;

[0052] Wherein, M: Marker DL 2000; 1: 1 lung of a diseased pig; 2: 2 lungs of a diseased pig; 3: Positive control; 4: 1 lung of a healthy pig; 5: 2 lungs of a healthy pig; 6: Negative control.

[0053] Figure 6 IHC results of PRRSV N protein monoclonal antibody in lung tissue.

[0054] Figure 7 The results show the comparison of the blocking rates of monoclonal antibodies.

[0055] Figure 8 SDS-PAGE analysis of purified monoclonal antibody 12E1 (A) and UV quality control of 12E1-HRP monoclonal antibody (B);

[0056] Wherein, M: protein molecular weight standard; 1: ascites fluid of monoclonal antibody 12E1 before purification; 2: ascites fluid of monoclonal antibody 12E1 after purification.

[0057] Figure 9 The ELISA antibody titer for 12E1-HRP.

[0058] Figure 10 Normal distribution analysis of PRRSV-negative serum blocking rate.

[0059] Figure 11 For sensitivity testing analysis.

[0060] Figure 12 For specificity testing analysis. Detailed Implementation

[0061] The present invention will now be described in detail with reference to specific embodiments. Based on the following description and these embodiments, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the invention.

[0062] Example 1: Preparation of monoclonal antibodies against porcine reproductive and respiratory syndrome virus nucleocapsid protein and identification of its antigenic epitopes

[0063] 1. Materials and Methods

[0064] 1.1 Main Materials

[0065] The PRRSVBB0907 strain was isolated, identified, and preserved in our laboratory. pET-28a(+) plasmid, *E. coli* DH5α and BL21(DE3), and mouse myeloma cells SP2 / 0 were preserved in our laboratory. SPF-grade female BALB / c mice (6–8 weeks old) were purchased from the Experimental Animal Center of Yangzhou University; 2×Phanta Max Master Mix and 180kDa Prestained Protein Marker were purchased from Novizan Biotechnology Co., Ltd.; HRP-goat anti-mouse-IgG (H+L) was purchased from Beyotime Biotechnology Co., Ltd.; RPMI-1640 medium and fetal bovine serum were purchased from Gibco; FITC-goat anti-mouse-IgG and mouse monoclonal antibody subtype identification ELISA kits were purchased from Proteintech Biotechnology Co., Ltd.; Tanon... TM High-sig ECL Western blot substrate kit was purchased from Tianneng Technology Co., Ltd.; isopropyl thiogalactoside (IPTG), HAT and HT were purchased from Sigma.

[0066] 1.2 Construction of Recombinant Plasmids

[0067] Primers were designed based on the ORF7(N) gene sequence of PRRSV BB0907 strain (GenBank: ADY86738.1), NF: 5'-CCC GGATCC ATGCCAAATAATAACG-3', contains a BamHI restriction site (underlined); NR: 5'-CGC AAGCTT The TCATGCTGAGGGTGAC-3' fragment, containing a Hind III restriction site (underlined), was used to amplify the ORF7(N) gene, with a size of 372 bp. The target fragment and the pET-28a(+) vector were simultaneously digested with BamHI and HindIII and then ligated. The ligation product was transformed into DH5α, and after confirmation by bacterial PCR and double enzyme digestion, it was sent to General Biosystems for sequencing.

[0068] To identify the antigenic epitopes of the N protein monoclonal antibody, a series of truncated primers were designed based on the PRRSV ORF7(N) gene sequence. The primer sequences are shown in Table 1. The truncated expression vector was pET-32a(+).

[0069] Table 1. Primer sequences for N protein truncated variant PCR

[0070]

[0071] Note: The underscore “” indicates the base sequence of the enzyme cleavage site.

[0072] 1.3 Induction and purification of recombinant N protein

[0073] The recombinant plasmid pET-28a-N was transformed into fresh BL21 competent cells and cultured in LB liquid medium containing kanamycin. When the bacterial culture OD... 600nm When the pH was 0.4–0.6, IPTG was added to a final concentration of 1 mmol / L, and expression was induced for 6 h at 37 °C and 160 r / min. The induced bacterial cells were then sonicated, and the precipitate and supernatant were collected by centrifugation. The expression pattern of the recombinant N protein was identified by SDS-PAGE. Protein purification was performed using a pre-packed Ni-filled HisTrap FF column: a low-pressure chromatography system was used according to the manufacturer's instructions, and the purified N protein was finally obtained by continuous gradient elution.

[0074] 1.4 Preparation of recombinant N protein monoclonal antibody

[0075] 1.4.1 Mouse Immunization

[0076] The purified recombinant N protein was emulsified with ISA206 adjuvant and subcutaneously injected into 6-8 week old female BALB / c mice using a standard method, with an immunization dose of 100 μg / mouse. Two to three days after the third immunization, the serum antibody titer of the mice was measured using an indirect ELISA method. Cell fusion was performed when the titer was higher than 1:12,800. Three days before fusion, a pulse immunization was administered via intraperitoneal injection of 100 μg of purified recombinant N protein.

[0077] 1.4.2 Cell Fusion and Establishment of Positive Hybridoma Cells

[0078] Three days after the initial immunization, mouse spleen lymphocytes and SP2 / 0 cells were fused at a ratio of 5:1 to 10:1 using PEG4000. The fused cells were cultured in HAT selective medium. After 5–7 days, single clusters of hybridoma cells were selected and, when they reached approximately 1 / 5 of the well bottom area, the cell supernatant was detected using an indirect ELISA method. Positive hybridoma cells required at least two subcloning processes until all subcloned cell clusters were positive, at which point the cell line could be established. The hybridoma cell line that stably secreted anti-N protein antibodies was then cultured on a large scale.

[0079] 1.4.3 Indirect ELISA Antibody Detection Method

[0080] Recombinant N protein was used as a known antigen, diluted to a concentration of 2 μg / mL with carbonate coating buffer, and used to coat ELISA strips. Hybridoma cell supernatant was used as the primary antibody, with SP2 / 0 cell culture supernatant as a negative control and pulse-immunized mouse serum as a positive control; HRP-goat anti-mouse IgG was used as the secondary antibody. TMB chromogenic solution was used for color development in the dark, and the reaction was terminated with 2 mol / L H2SO4. The OD of the test wells was measured using an ELISA reader. 450nm Calculate the OD values ​​of the holes to be inspected. 450nm / Negative sample OD 450nm The P / N value is used to determine a positive result when P / N > 2.1.

[0081] 1.4.4 Ascites preparation

[0082] Eight-week-old female BALB / c mice were selected. They were injected intraperitoneally with 500 μL of sterile liquid paraffin beforehand, and 7–10 days later, they were injected intraperitoneally with 2 × 10⁻⁶ g of paraffin. 6 Approximately 100 hybridoma cells were injected. Ascites fluid was collected from mice approximately 7–14 days after cell injection, when the abdomen was significantly distended. The collected ascites fluid was centrifuged at 5,000 rpm for 5 minutes, and the supernatant was stored at -20°C.

[0083] 1.4.5 Determination of ascites titer

[0084] The recombinant N protein was diluted to 2 μg / mL using carbonate coating buffer to coat ELISA strips. The ascites fluid containing the seven monoclonal antibodies prepared was serially diluted 2-fold starting from 1:200 and used as primary antibodies. SP2 / 0 cell culture supernatant was set up as a negative control. The ascites fluid titer was detected by indirect ELISA antibody detection method. The highest dilution corresponding to a positive detection result was the mouse ascites fluid titer.

[0085] 1.5 Stability test of antibody secretion by hybridoma cell lines

[0086] Positive hybridoma cell lines were expanded and cultured, and cell supernatants were collected at passages of 5, 10, 15, and 20. The antibody titers of the cell supernatants at different passages were detected using an indirect ELISA antibody detection method.

[0087] 1.6 Determination of monoclonal antibody subtype

[0088] Monoclonal antibody subtype identification was performed in ascites fluid according to the instructions of the commercially available mouse monoclonal antibody subtype identification kit. 1.7 Indirect Immunofluorescence Assay (IFA)

[0089] PRRSV was seeded into a monolayer of Marc-145 cells in 96-well plates, with an uninoculated group also included. The cells were incubated at 37°C in a 5% CO2 incubator. Obvious cytopathic effects were observed after 48 hours. The culture supernatant was discarded, and the cells were fixed with 4% (g / 100ml) paraformaldehyde for 20–30 min, followed by clearing with 0.1% (v / v) Triton X-100 for 25–30 min. The hybridoma cell supernatant was used as the test antibody, the previously stored N monoclonal antibody was used as a positive control, the SP2 / 0 cell culture supernatant was used as a negative control, and FITC-goat anti-mouse IgG was used as the secondary antibody. The results were observed under a fluorescence inverted microscope.

[0090] 1.8 Western blot

[0091] The recombinant N protein expressed in prokaryotes was used as an antigen for SDS-PAGE detection, with pET-28a empty vector protein set as a negative control. The gel protein bands were transferred onto a nitrocellulose membrane (NC membrane). PRRSV-positive serum and His monoclonal antibody were used as primary antibodies, and HRP-goat anti-mouse IgG as a secondary antibody. Chemiluminescence was used for imaging, and the protein bands were observed to identify the antigenicity of the recombinant N protein.

[0092] Recombinant N protein and PRRSV-infected Marc-145 cell samples were used as antigens, with irrelevant proteins as negative controls. The culture supernatant of seven positive hybridoma cell lines was used as the primary antibody, and HRP-goat anti-mouse-IgG was used as the secondary antibody. The specificity of the monoclonal antibody response was identified by Western blot.

[0093] 1.9 Overlay ELISA

[0094] The antigenic epitopes of seven monoclonal antibodies were analyzed using a superimposed ELISA antibody detection method. Recombinant N protein was diluted to a concentration of 2 μg / mL with carbonate coating buffer and used to coat ELISA strips. Supernatants from identical or different hybridoma cells were paired and used as primary antibodies, with equal amounts of both monoclonal antibodies added (total 100 μL). HRP-goat anti-mouse-IgG was used as the secondary antibody. The reaction was performed using TMB chromogenic buffer in the dark, and terminated with 2 mol / L H2SO4. The OD of each well was measured using an ELISA reader. 450nm Value. The superposition factor (AI) is calculated using the following formula: AI = [A 1+2 [ / (A1+A2)]×100%, where A1 and A2 are the OD values ​​of the cell supernatants of the two monoclonal antibodies reacting individually. 450nm Value, A 1+2 OD of the superposition reaction of cell supernatants of two monoclonal antibodies 450nm An AI greater than 50% indicates that the two tested monoclonal antibodies have different antigen-binding sites, while an AI less than 50% indicates that the two tested monoclonal antibodies have the same antigen-binding sites.

[0095] 1.10 Identification of antigenic epitopes

[0096] The target gene fragments were amplified using the primer sequences for the truncated N protein fragments in Table 1 and inserted into the prokaryotic expression vector pET-32a(+) to construct six recombinant plasmids containing truncated N protein fragments. These plasmids were then transformed into *E. coli* BL21 and expressed by IPTG induction. The culture supernatants of seven positive hybridoma cell lines were used as primary antibodies, and Western blot analysis was performed to identify the antigenic epitopes recognized by the monoclonal antibodies.

[0097] 2. Results

[0098] 2.1 Expression, purification and identification of recombinant N protein

[0099] The PRRSV ORF7(N) gene fragment was amplified by PCR and inserted into the pET-28a(+) expression vector. The successfully constructed recombinant plasmid pET-28a-N was transformed into E. coli BL21, and expression was induced by IPTG. SDS-PAGE results showed that the target protein was approximately 15 kDa, consistent with the expected location, and the recombinant N protein was soluble. The supernatant recombinant N protein was purified according to the AKTA Pure instrument chromatography system operating manual. SDS-PAGE results showed a single band (…). Figure 1 A). Western blot analysis of His monoclonal antibody and PRRSV-positive serum demonstrated that the recombinant protein had good antigen specificity. Figure 1 B).

[0100] 2.2 Establishment of hybridoma cell lines that stably secrete monoclonal antibodies

[0101] BALB / c mice were immunized three times, and the antibody titers detected by indirect ELISA were all greater than 1:12800. Mouse spleens were collected and fused with SP2 / 0 cells. After screening and subcloning by ELISA, a total of seven monoclonal antibody hybridoma cell lines were obtained, named as follows: 8F1, 5E2, 2B1, 12E1, 4F1, 2H2, and 5A2.

[0102] 2.3 Identification of Monoclonal Antibody Cell Stability

[0103] Seven hybridoma cell lines were passaged to 20 generations, and the antibody levels detected by indirect ELISA were basically consistent, demonstrating good stability. The titers of the prepared mouse ascites fluid ranged from 1:4096,000 to 1:131,072,000 (Table 2).

[0104] Table 2. Results of indirect ELISA antibody titers in hybridoma cell supernatant and ascites.

[0105]

[0106] 2.4 Monoclonal antibody subtype identification

[0107] The results are shown in Table 3. The heavy chain type of monoclonal antibodies 8F1, 5E2, 2B1, and 12E1 is IgG1 subclass, and the heavy chain type of monoclonal antibodies 4F1, 2H2, and 5A2 is IgG2b subclass. The light chain type of all of them is Kappa type.

[0108] Table 3. Monoclonal antibody subtype identification (OD) 450nm )

[0109]

[0110] 2.5 Western blot identification of monoclonal antibody specificity

[0111] Western blot analysis was performed using recombinant N protein and PRRSV-infected cell samples as antigens. The results showed that all seven monoclonal antibodies specifically reacted with recombinant N protein and N protein in PRRSV-infected cells. Figure 2 ).

[0112] 2.6 Identification of the specificity of monoclonal antibody IFA reaction

[0113] IFA results show ( Figure 3 All seven monoclonal antibodies prepared were able to specifically react with PRRSV-infected Marc-145 cells, producing green fluorescence.

[0114] 2.7 Monoclonal antibody identification of N protein epitopes

[0115] Monoclonal antibody OD was detected using stacked ELISA. 450nm AI was calculated, and the results (Table 4) showed that there were both AI < 50% and AI ≥ 50% among the 7 monoclonal antibodies, suggesting that among the 7 monoclonal antibodies, there may be monoclonal antibodies that recognize the same antigenic epitopes and those that recognize different antigenic epitopes.

[0116] Table 4. Analysis of Monoclonal Antibody Stacking Coefficient

[0117]

[0118] To further identify and analyze the antigenic epitopes recognized by the monoclonal antibody, six truncated variants of the PRRSV N protein were constructed. Western blot results of the His monoclonal antibody showed that all truncated variants expressed the target protein. Figure 4 A, B).

[0119] Seven monoclonal antibodies against the N protein were identified by two rounds of Western blot analysis. The results showed that antibodies 5A2 and 4F1 reacted only with F1 and not with F2; antibodies 8F1 and 5E2 reacted only with F2 and not with F1; antibodies 12E1, 2H2, and 2B1 reacted with both F1 and F2, confirming that the antigenic epitope was located in the 57–67 aa region. Figure 4 C). The monoclonal antibodies 8F1, 5E2, 4F1, and 5A2 underwent a second round of evaluation. Figure 4 Results D and 4E showed that the 4F1 and 5A2 antigenic epitopes were located in the 47–57aa region; the 8F1 and 5E2 antigenic epitopes were located in the 67–77aa region.

[0120] Example 2. Monoclonal antibody against porcine reproductive and respiratory syndrome virus nucleocapsid protein for IHC

[0121] Of the seven PRRSV N protein-specific monoclonal antibodies, monoclonal antibodies 12E1 and 5A2 were found to be usable in the PRRSV IHC assay. The antigenic epitope recognized by monoclonal antibody 12E1 is described below. 57 LATEDDVRHHF 67 This newly discovered N protein antigenic epitope provides a new target for understanding the structure and related functions of the N protein.

[0122] 1. Materials and Methods

[0123] 1.1 Materials

[0124] 1.1.1 Cells, Viruses and Laboratory Animals

[0125] The Marc-145 cell line was preserved in our laboratory. The PRRSV BB0907 strain was isolated, identified, and preserved in our laboratory. SPF-grade female BALB / c mice (6–8 weeks old) and female ICR mice were obtained from the Experimental Animal Center of Yangzhou University.

[0126] Main reagents: 2×Phanta Max Master Mix, 2×Taq Master Max, HiScript III Reverse Transcriptase, and 180kDa Preserved Protein Marker (Nanjing Novizan Biotechnology Co., Ltd.); Fast Digest BamHI, Fast Digest Hind III, and T4 DNA Ligase (Thermo Fisher Scientific); HRP-SPA and hematoxylin staining solution (Beyotime Biotechnology Co., Ltd.); soluble TMB substrate powder (Sigma-Aldrich, USA); commercially available PRRSV antibody detection kit, commercially available CSFV antibody detection kit, and commercially available PRV gB / gE antibody detection kit (IDEXX, USA); Total RNA extraction kit (OMEGA, USA); DAB chromogenic reagent kit (Zhongshan Jinqiao Biotechnology Co., Ltd.). All other reagents were domestically produced analytical grade.

[0127] 1.2 Methods

[0128] 1.2.1 Preparation of PRRSV-infected piglet lung tissue sections

[0129] 1.2.1.1 Piglet challenge test and sample collection

[0130] Select two healthy PRRSV-negative piglets aged 30-35 days and inject them intramuscularly with PRRSV BB0907 strain (10). 6.0 TCID 50 / ml), with 2 healthy piglets serving as a blank control. Pigs were isolated and observed until obvious clinical symptoms of PRRS appeared. Blood samples were collected from affected pigs for pathological necropsy, and the visual lesions of the lung tissue were observed.

[0131] After the blood was incubated at 37°C for 30 minutes, serum was separated and then transferred to a 4°C refrigerator and left to stand overnight. The blood was then centrifuged at 5,000 rpm for 4 minutes at 4°C. The supernatant was collected for PRRSV, PRRSV gE / gB, and CSFV serum antibody detection to determine the infection status of the pigs.

[0132] Two lung tissue samples were collected. One sample (approximately 2g) was placed in a 5mL centrifuge tube, minced, and diluted with 1×PBS solution to 4mL. One 4mm diameter magnetic bead and two 2mm diameter magnetic beads were then added. The samples were homogenized in a tissue homogenizer at 60Hz for 2 minutes, followed by three freeze-thaw cycles at -40℃ for PRRSV nucleic acid detection. The other tissue sample was cut into several pieces and fixed in 4% paraformaldehyde for 48 hours. Normal lung tissue was used as a negative control. After fixation, paraffin sections of the lung tissue were prepared using standard methods.

[0133] 1.2.1.2 PRRSV detection in lung tissue

[0134] The lung tissue treated in 1.2.1.1 was centrifuged at 6,000 rpm for 3 min, and 200 μL of the homogenate supernatant was collected. Total RNA was extracted from the tissue according to the OMEGATotal RNA Extraction Kit instructions. The specific steps are as follows:

[0135] (1) Add 500 μL of TRK lysis buffer to the supernatant (add 20 μL of β-mercaptoethanol to each 1 mL of TRK lysis buffer in advance), centrifuge at 13,000 g for 5 min at room temperature, and transfer the supernatant to the Homogenization Spin column;

[0136] (2) Centrifuge at 13,000g for 5 min at room temperature, and transfer the collected liquid to a new 2mL centrifuge tube;

[0137] (3) Add 250 μL of anhydrous ethanol to the above pyrolysis liquid and vortex mix for 20 s;

[0138] (4) Transfer the above liquid to a Hibind RNA Mini column, centrifuge at 10,000g for 1 min at room temperature, and discard the lower layer effluent;

[0139] (5) Transfer the upper Hibind RNA Mini column to a new 2mL centrifuge tube, add 500μL RNAWash Buffer I (previously mixed with anhydrous ethanol), centrifuge at 10,000g for 30s at room temperature, and discard the lower layer effluent;

[0140] (6) Add 500 μL RNA Wash Buffer II (with anhydrous ethanol added beforehand), centrifuge at 10,000 g for 30 s at room temperature, and discard the lower layer effluent;

[0141] (7) Repeat (6), centrifuge at 13,000g for 2 min in an empty tube at room temperature;

[0142] (8) Transfer the upper Hibind RNA Mini column to a new 1.5 mL centrifuge tube and let it stand at room temperature for 5-10 min to allow the ethanol to evaporate as much as possible;

[0143] (9) Preheat RNase-free ddH2O in a 70℃ water bath, then add 50μL to the center of the Hibind RNA Mini column and let it stand at room temperature for 1-2 min;

[0144] (10) Centrifuge at 13,000g for 1 min at room temperature. The liquid collected in the lower layer is the tissue total RNA sample. Store in a -40℃ refrigerator for later use.

[0145] RT-PCR detection. The presence of PRRSV ORF7(N) gene nucleic acid in lung tissue samples was detected according to the HiScript III Reverse Transcriptase instructions. The specific procedure is as follows:

[0146] (1) Template RNA denaturation: Add 3 μg of total RNA from lung tissue to an RNase-free centrifuge tube, and bring the RNase-free ddH2O to a final volume of 10 μL. Heat the above system in a 65°C water bath for 5 min, then quickly cool it on ice and let it stand for 2 min.

[0147] (2) Preparation of first-strand cDNA synthesis reaction solution: Take 10 μL of the mixture in (1), 4 μL of 5×HiScript III Buffer, 1 μL of dNTP Mix, 1 μL of HiScript III Reverse Transcriptase, 1 μL of RNase inhibitor, and Oligo(dT). 20 VN 1μL, RNase-free ddH2O 2μL, mix well by pipetting;

[0148] (3) First-strand cDNA synthesis reaction: Place the above system in a PCR instrument, set the program to 37℃ for 45 min, 85℃ for 5 s, and the product can be immediately subjected to PCR reaction.

[0149] (4) Design PCR amplification primers: Based on the PRRSV BB0907 ORF7(N) gene sequence, a pair of specific primers were designed. The upstream primer F is 5'-ATGGCCAGCCAGTCAATCA-3', and the downstream primer R is 5'-TCGCCCTAATTGAATAGGTG-3'. The amplified gene fragment size is 372bp.

[0150] (5) PCR amplification of the target gene fragment: Add 12.5 μL of 2×Taq Master Mix, 1 μL each of primers F and R, 1 μL of template from (3) to a centrifuge tube, and ddH2O to a final volume of 25 μL. Gently pipette the prepared PCR reaction mixture and place it in a PCR instrument for amplification. The reaction program is as follows: 95℃ for 3 min; 95℃ for 15 s, 54℃ for 15 s, 72℃ for 30 s, 35 cycles; 72℃ for 5 min.

[0151] (6) Result identification: The PCR products in (5) were separated by electrophoresis in a 1% nucleic acid gel, and negative and positive controls were set up at the same time.

[0152] 1.2.1.3 Preparation of lung tissue sections

[0153] The specific steps are as follows:

[0154] (1) Tissue trimming: Trim the fixed lung tissue with scissors. The trimmed tissue block should have a neat cut, be about the thickness of a one-yuan coin, and have a regular shape. Note that the tissue block should not be squeezed with tweezers to avoid damaging the morphological integrity of the lung tissue;

[0155] (2) Rinsing: Place the prepared tissue block in a plastic embedding box and put it in a large beaker filled with tap water. Cover it with a gauze and rinse it under running tap water for 24-48 hours to remove formaldehyde from the lung tissue.

[0156] (3) Gradient dehydration: The lung tissue blocks treated above were sequentially placed in 70%, 80%, 90%, 95%, and 100% (I and II) alcohol for dehydration. The specific procedure was as follows: soaking in 70% alcohol overnight → soaking in 80% alcohol for 2 hours → soaking in 90% alcohol for 2 hours → soaking in 95% alcohol for 1-2 hours → soaking in 100% I for 45 minutes → soaking in 100% II for 45 minutes (100% alcohol was strictly carried out according to the time and should not be soaked for too long).

[0157] Note that normal lung tissue and diseased lung tissue should be handled separately. At the beginning of the dehydration process, the wax melting box should be opened and the temperature set to 65°C. An appropriate amount of wax block should be placed in the wax melting box and melted completely.

[0158] (4) Clearing: After dehydration, gently place the lung tissue on a paper towel to remove excess alcohol, and then immerse it in xylene for clearing. The clearing time varies depending on the tissue block (generally 30-60 seconds; the clearing time should not be too long to prevent the tissue block from drying and cracking). During clearing, a colored oily substance can be seen spreading around the tissue; this is a mixture of alcohol and xylene.

[0159] (5) Paraffin infiltration: Place the transparent tissue block in a plastic embedding box and then in pre-melted liquid paraffin. Note that the paraffin must completely immerse the lung tissue. The infiltration time is 4-6 hours, and the temperature is always kept at 65℃.

[0160] (6) Embedding: After the paraffin is impregnated, the lung tissue is removed from the plastic embedding box and placed horizontally in the iron embedding frame. The pre-melted liquid paraffin is quickly poured in and the paraffin is trimmed after solidification at room temperature (30 min). The normal lung tissue and the diseased lung tissue are marked.

[0161] (7) Sectioning: Set the microtome to 3mm section thickness, use the old blade to roughly cut the paraffin block, and replace the blade with a new one when the tissue is exposed to begin preparing serial sections;

[0162] (8) Prepare the water bath and oven: Add deionized water to the water bath and adjust the temperature to 45℃. Set the oven temperature to 60℃.

[0163] (9) Spreading and mounting: Gently take the continuous lung tissue slices onto a glass slide, spread the tissue slices horizontally in a 45°C water bath, and let stand for 1 minute to fully spread the slices.

[0164] (10) Baking: Select relatively intact lung tissue slices, and use positively charged adsorption slides to pick them up. Place them in a 60℃ oven and let them stand for 30-60 minutes. Then transfer them to a 37℃ incubator overnight and dry them.

[0165] (11) Observe the sections, select relatively intact paraffin tissue sections, and place them in a 4°C refrigerator for later use.

[0166] 1.2.2 Specificity identification of monoclonal antibodies

[0167] 1.2.2.1 Immunohistochemical (IHC) identification

[0168] Monoclonal antibodies 5E2, 12E1, and 5A2, prepared in this chapter and exhibiting good IFA reactivity and different antigenic epitopes, were selected for IHC detection. Normal lung tissue and diseased lung tissue should be processed separately. The specific experimental steps are as follows:

[0169] (1) Prepare paraffin sections of lung tissue according to the steps in 1.2.1.3 of this chapter;

[0170] (2) Dewaxing and hydration: Place all sections on a vertical sectioning rack and immerse them completely in xylene I for 10 min → xylene II for 10 min → alcohol / xylene for 10 min → 100% alcohol for 10 min → 95% alcohol for 10 min → 90% alcohol for 10 min → 80% alcohol for 10 min → 70% alcohol for 10 min → distilled water for 5 min, repeating the immersion process 3 times;

[0171] (3) Antigen retrieval: Pour 0.01M sodium citrate buffer into a large beaker, place a vertical slide rack containing lung tissue slices in it, and heat it in a microwave oven until the buffer boils. Remove the large beaker and let it cool naturally to room temperature. Repeat the above operation 3 times.

[0172] (4) Washing: Immerse lung tissue sections in 1×PBS three times, 5 min each time;

[0173] (5) Blocking endogenous peroxidase activity: Place the washed lung tissue slices on a wet slide box and wait for them to dry naturally. Then add 3% H2O2-methanol solution and place them in a 37°C incubator in the dark for 1 hour.

[0174] (6) Washing: Same as (4);

[0175] (7) Blocking: Place lung tissue slices on a humidified box, add PBS solution containing 50% normal fetal bovine serum, and incubate at 37°C for 2 hours;

[0176] (8) Incubation of monoclonal antibodies: The blocking solution on the slides is directly removed without washing. The ascites fluid of monoclonal antibodies 5E2, 12E1 and 5A2 is diluted 1:50 in 1×PBS solution and dropped onto normal lung tissue and diseased lung tissue slides respectively. At the same time, 1×PBS is set as a negative antibody control. The slides are incubated at 4°C for 12 h and then incubated at 37°C for 45 min.

[0177] (9) Washing: Same as (4);

[0178] (10) Secondary antibody incubation: Use absorbent paper to remove the water around the tissue section, dilute HRP-goat anti-mouse-IgG in 1×PBS solution at 1:100, add it to the lung tissue section and incubate at 37℃ for 1.5-2h;

[0179] (11) Washing: Same as (4), wash again in distilled water for 5 minutes;

[0180] (12) DAB substrate development: Use absorbent paper to remove the water around the tissue section, add 50 μL of the prepared DAB development solution (prepare fresh according to the instructions), develop the color at room temperature in the dark, pay attention to the color change of the tissue, and stop the development in time when the tissue turns yellow after about 1 minute to avoid the dark background caused by over-staining.

[0181] (13) Termination of color development: Soak lung tissue slices in distilled water 3 times, 2 minutes each time;

[0182] (14) Hematoxylin counterstaining: Immerse in hematoxylin for 30s, then differentiate with hydrochloric acid alcohol for 2-5s;

[0183] (15) Rinsing: Rinse lung tissue sections under running tap water for 15 minutes;

[0184] (16) Dehydration and clearing: 70% alcohol, 80% alcohol, 85% alcohol, 90% alcohol, 95% alcohol, 100% alcohol, xylene I, xylene II each for 5 min;

[0185] (17) Mounting: Before the xylene has evaporated, drop an appropriate amount of neutral resin onto the lung tissue and cover it with a clean coverslip. Place it in a 60°C oven for 30 minutes to complete the mounting.

[0186] (18) Microscopic examination and result interpretation: Observe the lung tissue under an optical microscope to see if brown staining is present. If the cells are brown, it is judged as PRRSV antigen positive, otherwise it is negative.

[0187] 2 Results and Analysis

[0188] 2.1 Preparation of lung tissue sections

[0189] Serum samples were collected from piglets challenged with PRRSV and tested for PRRSV, PRRSV gE / gB, and CSFV serological parameters. The results showed that PRRSV antibody was positive, while CSFV and PRRSV antibodies were negative (see Table 5). Lung tissue RT-PCR testing was positive for PRRSV in all samples. Figure 5 As shown, it can therefore be used as a lung tissue sample for subsequent IHC.

[0190] Table 5 Results of serum antibody detection in experimental pigs

[0191]

[0192]

[0193] 2.2 PRRSV IHC Identification in Lung Tissue

[0194] PRRSV N protein monoclonal antibodies 5E2, 12E1, and 5A2 were used as primary antibodies, and IHC was performed according to the experimental procedure in 1.2.2.1. The results are as follows. Figure 6 As shown, monoclonal antibody 5E2 did not stain brown in the lung tissue of diseased pigs, while the other two monoclonal antibodies, 12E1 and 5A2, both showed obvious brown staining, indicating a PRRSV-specific positive reaction.

[0195] In this study, we selected three PRRSV N protein monoclonal antibodies with good IFA reaction and different antigenic epitopes as the core reagents for IHC antigen detection. The results showed that monoclonal antibodies 12E1 and 5A2 can be used for IHC detection of PRRSV pathogen, providing important materials for studying antigen localization and dynamic distribution in infected pigs.

[0196] Example 3. Monoclonal antibody used in PRRSV blocking ELISA antibody detection method

[0197] This study used recombinant PRRSV N protein as an antigen to screen a monoclonal antibody 12E1 that has PRRSV serum blocking effect. Finally, a PRRSV N protein blocking ELISA antibody detection method was successfully established. This method has high sensitivity and specificity, and the concordance rate with commercial kits produced by IDEXX is as high as 94.3%, providing an effective method for the clinical sample diagnosis and epidemiological investigation of PRRS.

[0198] 1. Materials and Methods

[0199] 1.1 Proteins, Antibodies, and Serum

[0200] Protein: The PRRSV purified recombinant N protein was prepared and preserved in our laboratory.

[0201] Antibody: Monoclonal antibody 12E1 was prepared, identified and preserved in our laboratory.

[0202] Serum: PRRSV negative and positive sera were collected, tested, and preserved by our laboratory. Standard positive sera for porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2), porcine Seneca virus type A (SVA), and porcine epidemic diarrhea virus (PEDV) were all preserved by our laboratory. Clinical swine serum was obtained from pig farms in Jiangsu, Shandong, Anhui, Henan, and Zhejiang provinces.

[0203] 1.2 Main Reagents

[0204] Main reagents: HRP-goat anti-mouse-IgG and TMB chromogenic solution, Shanghai Beyotime Biotechnology Co., Ltd.; BSA and gelatin, Sigma Reagents; skim milk, Nestlé (China) Co., Ltd.; commercial PRRSV antibody detection kit, IDEXX, USA; detachable 96-well ELISA strips, Wuxi Nice Biotechnology Co., Ltd. All other reagents were domestically produced analytical grade.

[0205] Antigen coating solution (carbonate buffer) formulation: anhydrous Na₂CO₃ 0.17g, NaHCO₃ 0.28g, pH adjusted to 9.6, ddH₂O to bring volume to 100mL. PBST formulation: KH₂PO₄ 0.27g, Na₂HPO₄ 1.42g, NaCl 8g, KCl 0.2g, Tween-20 500μL, pH adjusted to 7.4, deionized water to bring volume to 1L.

[0206] 1.3 Experimental Animals

[0207] SPF-grade female 8-week-old BALB / c mice, Experimental Animal Center of Yangzhou University.

[0208] 1.4 Methods

[0209] 1.4.1 Experimental Procedure for Blocking ELI SA

[0210] (15) Coating: Dilute recombinant N protein to 0.5 μg / mL with antigen coating solution and coat the enzyme-labeled strips, 100 μL / well, and let stand at 4℃ for 12 h;

[0211] (16) Washing: Discard the supernatant, wash 3 times with PBST, 200 μL / well / wash, and pat dry;

[0212] (17) Sealing: Prepare 5% skim milk, 200 μL / well, and let stand in an incubator at 37°C for 2 hours;

[0213] (18) Washing: Same as (2);

[0214] (19) Serum preparation: Dilute serum with PBST (1:1), 100 μL / well, and incubate at 37°C for 1 h;

[0215] (20) Washing: Same as (2);

[0216] (21) Monoclonal antibody activity: Dilute the monoclonal antibody with PBST (1:1,000), 100 μL / well, and incubate at 37°C for 1 h;

[0217] (22) Washing: Same as (2);

[0218] (23) Secondary antibody activity: HRP-goat anti-mouse-IgG was diluted with PBST (1:1,000), 100 μL / well, and incubated at 37℃ for 45 min;

[0219] (24) Washing: Same as (2);

[0220] (25) Colorimetric reaction: Add TMB colorimetric solution at 100 μL / well and incubate at 37°C in the dark for 10-15 min.

[0221] (26) Termination of reaction: 2M H2SO4 was used as the termination solution, 50μL / well;

[0222] (27) Reading: Set the program of the multi-functional microplate reader and read the absorbance value at OD450nm;

[0223] (28) Calculate the blocking rate: Calculate the blocking rate according to the following formula (3-1).

[0224]

[0225] 1.4.2 Screening of Antibodies for Blocking ELISA Detection

[0226] Following the operating steps in Example 1.4.1, the serum blocking effect of the seven PRRSV N protein monoclonal antibodies prepared in Example 2 was simultaneously tested, and the monoclonal antibody with the highest blocking rate was selected as the detection antibody for establishing the blocking ELISA method.

[0227] 1.4.3 Ascites purification and HRP labeling

[0228] 10 mL of monoclonal antibody 12E1 ascites fluid was prepared and sent to Jiangsu Aibosen Biotechnology Co., Ltd. for protein G affinity purification and HRP labeling. The purified ascites fluid samples were subjected to SDS-PAGE electrophoresis to determine the purification effect, and the labeled ascites fluid samples were analyzed for labeling using UV technology. The antibody titer of 12E1-HRP was detected by direct ELISA, as follows:

[0229] (1) Dilute the antigen coating solution to purify the recombinant N protein to 1.0 μg / mL to coat the enzyme-labeled strips, 100 μL / well, and incubate at 37℃ for 2 h;

[0230] (2) Discard the coating solution, wash 3 times with PBST, 200 μL / well / wash, and spin dry;

[0231] (3) Prepare a PBST solution containing 5% skim milk, 200 μL / well, and incubate at 37°C for 2 h;

[0232] (4) Discard the blocking solution, wash 3 times with PBST, 200 μL / well / wash, and spin dry;

[0233] (5) 12E1-HRP was serially diluted 2-fold with PBST (1:200-1:12,800), 100 μL / well, and reacted in an incubator at 37℃ for 1 h;

[0234] (6) Add TMB colorimetric solution, 100 μL / well, and react in a 37℃ incubator in the dark for 10-15 min;

[0235] (7) Add 2M H2SO4 stop solution, 50μL / well, to terminate the colorimetric reaction;

[0236] (8) Read the OD450nm value and calculate the S / N value. S is the OD450nm value of 12E1-HRP at different dilution ratios, and N is the OD450nm value of the negative control. When S / N>2.1, it is positive.

[0237] 1.4.4 Optimization of the best reaction conditions for blocking ELISA

[0238] 1.4.4.1 Optimization of antigen coating solution concentration and serum dilution

[0239] Refer to the operating method in 1.4.1 of this embodiment. Dilute the recombinant N protein to 1.0, 0.5, 0.25, and 0.1 μg / mL with PBST, respectively. At the same time, serially dilute PRRSV positive serum from 1:1 to 1:2 to 1:16. Set up PBST as a negative control to determine the optimal antigen coating concentration and serum dilution.

[0240] 1.4.4.2 Optimization of Antigen Coating Conditions

[0241] Refer to the operating method in section 1.4.1 of this embodiment. Based on the above optimization, three commonly used antigen coating conditions were screened: 37℃ for 2 hours, 4℃ for 12 hours, and 37℃ for 2 hours followed by 4℃ for another 12 hours. The condition corresponding to the maximum blocking rate is the optimal antigen coating condition.

[0242] 1.4.4.3 Optimization of Sealing Fluid Selection

[0243] Refer to the operating method in 1.4.1 of this embodiment. Based on the above optimization conditions, six commonly used blocking solutions were screened: PBST solutions containing 2% (g / 100ml) skim milk, 5% (g / 100ml) skim milk, 10% (g / 100ml) skim milk, 1% (g / 100ml) BSA, 2% (g / 100ml) BSA, and 2% (g / 100ml) gelatin. The condition corresponding to the maximum blocking rate is the optimal blocking solution.

[0244] 1.4.4.4 Optimization of Closure Time

[0245] Referring to the operation method in 1.4.1 of this embodiment, based on the above-mentioned optimized conditions, the sealing time was screened, specifically 1 hour, 2 hours, and 3 hours at 37°C. The condition corresponding to the maximum blocking rate is the optimal sealing time.

[0246] 1.4.4.5 Optimization of Serum Reaction Time

[0247] Refer to the operating method in 1.4.1 of this embodiment. Based on the above optimization conditions, the treatment time of the serum to be tested was screened, namely 30 min, 45 min, and 60 min at 37℃. The condition corresponding to the maximum blocking rate is the optimal blocking time.

[0248] 1.4.4.6 Optimization of Working Concentration of Enzyme-Labeled Monoclonal Antibody

[0249] Refer to the operating method in 1.4.1 of this embodiment. Based on the initially screened working concentration of 12E1-HRP and the above-mentioned optimized conditions, dilute it with PBST to 1:200, 1:400, 1:800, 1:1,000, 1:1,600, and 1:2,000, respectively. The condition corresponding to the maximum blocking rate is the optimal working concentration of the enzyme-labeled monoclonal antibody.

[0250] 1.4.4.7 Optimization of enzyme-labeled monoclonal antibody reaction time

[0251] Refer to the operating method in 1.4.1 of this embodiment. Based on the above optimization conditions, the reaction time of the enzyme-labeled monoclonal antibody was screened, and the reaction times were 37℃ for 30 min, 45 min, 60 min, 75 min, and 90 min, respectively. The condition corresponding to the maximum blocking rate is the optimal enzyme-labeled monoclonal antibody reaction time.

[0252] 1.4.4.8 Optimization of substrate TMB development time

[0253] Refer to the operation method in 1.4.1 of this embodiment. Based on the above optimization, the development times for four common substrates (TMB) were screened, namely 5 min, 10 min, 15 min, and 20 min at 37℃ in the dark. The condition corresponding to the maximum blocking rate is the optimal development time.

[0254] 1.4.5 Determination of the cutoff value for blocking ELISA

[0255] The blocking rate of 63 PRRSV-negative swine serum samples (detected using a PRRSV antibody detection kit manufactured by IDEXX) stored in our laboratory was determined according to the established PRRSV N protein blocking ELISA antibody detection method, and the average blocking rate (PI) was calculated. The standard deviation (SD) is used to determine the critical value of this method. The judgment criteria are defined as follows: It is positive. If it is negative, and it falls between the two, it is suspicious.

[0256] 1.4.6 Sensitivity Test

[0257] Five PRRSV positive sera and three PRRSV negative sera were randomly selected and serially diluted 2-fold (1:2 to 1:256) using PBST. The blocking rate PI was calculated according to the PRRSV N protein blocking ELISA antibody detection method established in this study, and the maximum serum dilution factor corresponding to a positive detection result was statistically analyzed.

[0258] 1.4.7 Specificity test

[0259] The PRRSV N protein blocking ELISA antibody detection method established in this study was used to detect positive sera for common pathogens in pig farms, including PRRSV, PRV, CSFV, PCV2, SVA, and PEDV, and to verify the specificity of the method.

[0260] 1.4.8 Repeatability Test

[0261] The PRRSV N protein blocking ELISA antibody detection method was established in this study.

[0262] (1) Intra-batch repeatability test: The same batch of recombinant N protein expressed and purified was used to coat the enzyme-labeled strips. Six positive sera and four negative sera were tested. Three replicate wells were set for each test, and three parallel replicates were performed. The coefficient of variation (CV) was calculated.

[0263] (2) Inter-batch repeatability test: Three batches of recombinant N protein purified at different times were coated with three different enzyme-labeled strips, and the 10 serum samples in (1) were tested to calculate the coefficient of variation (CV).

[0264] 1.4.9 Compliance Rate Analysis

[0265] The PRRSV N protein blocking ELISA antibody detection method established in this study was used to detect 282 clinical pig serum samples, and the results were compared with those of the IDEXX commercial PRRSV ELISA antibody detection kit. The concordance rate was calculated according to the following formula (3-2):

[0266]

[0267] 2 Results

[0268] 2.1 Screening of monoclonal antibodies for use in blocking ELISA methods

[0269] Following the operating steps in 1.4.1 of this embodiment, the seven PRRSV N protein monoclonal antibodies prepared in Example 2 were subjected to blocking ELISA detection, as follows: Figure 7 As shown, the blocking rate of 12E1 monoclonal antibody against PRRSV-positive serum was significantly higher than that of the other 6 monoclonal antibodies.

[0270] 2.2 Purification and HRP labeling of monoclonal antibody 12E1 ascites fluid

[0271] After purification of the monoclonal antibody 12E1 ascites fluid, SDS-PAGE was performed for identification, and the results are as follows: Figure 8 As shown in Figure A, the purified monoclonal antibody exhibits high purity and distinct bands at the 50kDa heavy chain and 25kDa light chain positions, indicating that the purified monoclonal antibody still contains antibody active sites and can therefore be used for subsequent studies. The HRP-labeled 12E1 monoclonal antibody was subjected to UV quality control, and the results are shown in Figure A. Figure 8 As shown in B, the presence of two peaks before and after labeling indicates that the 12E1 monoclonal antibody was successfully labeled.

[0272] The antibody titer of 12E1-HRP was detected by direct ELISA, and the results are as follows: Figure 9 As shown, the antibody titer of 12E1-HRP is greater than 1:6,400, and the OD450nm value is approximately equal to 1.0 when the antibody dilution is 1:1,600. Therefore, when establishing the blocking ELISA method, the initial working concentration of 12E1-HRP is selected as 1:1,600.

[0273] 2.3 Screening for optimal reaction conditions for blocking ELISA

[0274] 2.3.1 Screening of antigen coating solution concentration and serum dilution

[0275] The results are shown in Table 6. The highest blocking rate was achieved when the antigen coating concentration was 1.0 μg / mL and the serum dilution was 1:2.

[0276] Table 6 Screening based on antigen coating concentration and serum dilution

[0277]

[0278] Note: "PI" stands for blocking rate, the same applies below.

[0279] 2.3.2 Screening of antigen coating conditions

[0280] Recombinant N protein purified at a concentration of 1.0 μg / mL was used as the antigen to coat the enzyme-labeled strips. The blocking ELISA detection was performed at a serum dilution of 1:2. The results are shown in Table 7. The serum blocking rate was the highest when the antigen coating conditions were 4℃ for 12 h.

[0281] Table 7 Screening for optimal antigen coating conditions

[0282]

[0283]

[0284] 2.3.3 Screening of blocking solutions

[0285] Based on the above optimization conditions, the optimal blocking solution was screened, and the results are shown in Table 8. The serum blocking rate was highest when the blocking solution was 1% BSA.

[0286] Table 8 Screening of Optimal Blocking Fluids

[0287]

[0288] 2.3.4 Screening of Closure Time

[0289] Based on the above optimization conditions, the blocking time was screened, and the results are shown in Table 9. The serum blocking rate was highest when the blocking time was 1 hour at 37℃.

[0290] Table 9 Screening of Closure Time

[0291]

[0292] 2.3.5 Screening of serum reaction time

[0293] Based on the above optimized conditions, the reaction time of the serum to be tested was selected. The results are shown in Table 10. The serum blocking rate was the highest when the serum to be tested was reacted at 37℃ for 45 min.

[0294] Table 10 Screening of serum treatment time

[0295]

[0296] 2.3.6 Screening of working concentrations of enzyme-labeled monoclonal antibodies

[0297] Based on the above optimization conditions, the optimal working concentration of the enzyme-labeled monoclonal antibody was screened. The results are shown in Table 11. The serum blocking rate was highest when the dilution factor of 12E1-HRP was 1:800.

[0298] Table 11 Screening of enzyme-labeled monoclonal antibody dilutions

[0299]

[0300] 2.3.7 Screening of enzyme-labeled monoclonal antibody reaction time

[0301] Based on the above optimized conditions, the reaction time of the enzyme-labeled monoclonal antibody was screened. The results are shown in Table 12. The serum blocking rate was highest when 12E1-HRP was incubated at 37℃ for 45 min.

[0302] Table 12 Screening of enzyme-labeled monoclonal antibody reaction time

[0303]

[0304]

[0305] 2.3.8 Screening of substrate TMB development time

[0306] Based on the above optimized conditions, the substrate color development time was selected. The results are shown in Table 13. The serum blocking rate was highest when the color development reaction was carried out at 37℃ for 5 min.

[0307] Table 13 Screening of Substrate Development Time

[0308]

[0309] The results obtained from all the screenings are summarized in Table 14, which is the PRRSV N protein blocking ELISA detection method established in this study.

[0310] Table 14 Results of Optimization of Reaction Conditions for PRRSV N Protein Blockade ELISA Detection Method

[0311]

[0312] 2.4 Determination of the cutoff value for blocking ELISA method

[0313] The PRRSV N protein blocking ELISA detection method established in this study was used to detect 63 PRRSV negative serum samples, with an average blocking rate of [missing data]. The PI was 24%, and the standard deviation (SD) was 12%. Based on the cutoff value calculation formula, the criteria for this blocking ELISA were determined as follows: X + 2SD = 48%, X + 3SD = 60%. A PI ≥ 60% was considered PRRSV antibody positive, and a PI ≤ 48% was considered PRRSV antibody negative. A PI between these two values ​​was considered suspicious; if a repeat test remained suspicious, it was considered positive (e.g., ...). Figure 10 ).

[0314] 2.5 Sensitivity Test

[0315] Sensitivity test results as follows Figure 11 As shown, the antibody dilution titers of the five positive sera were 1:64, 1:32, 1:32, 1:8, and 1:2, respectively, indicating that the blocking ELISA method has good sensitivity and can effectively detect PRRSV serum antibodies.

[0316] 2.6 Specificity test

[0317] Blocking ELISA antibody detection was performed on positive sera from five common swine pathogens found in pig farms. The results are as follows: Figure 12As shown, except for PRRSV positive serum antibody reaction, PRV, CSFV, PCV2, SVA and PEDV positive serum antibody reactions were all negative, indicating that the PRRSV N protein blocking ELISA antibody detection method established in this study has good specificity and no cross-reaction with antibodies against other swine pathogens.

[0318] 2.7 Repeatability Test

[0319] Six PRRSV-positive sera and four PRRSV-negative sera were selected for detection using the blocking ELISA method. The results are shown in Table 15. The coefficients of variation for intra-assay and inter-assay repeatability tests were 2%–8% and 2%–6%, respectively, both less than 10%, indicating that the PRRSV N protein blocking ELISA antibody detection method established in this study has good repeatability.

[0320] Table 15 Results of Intra-batch and Inter-batch Repeatability Tests

[0321]

[0322] 2.8 Compliance Rate

[0323] The PRRSV N protein blocking ELISA antibody detection method established in this study was used to simultaneously detect 282 clinical swine serum samples with the commercially available PRRSV ELISA antibody detection kit manufactured by IDEXX (USA). The results are shown in Table 16. The commercial kit showed 247 positive samples and 35 negative samples, while the blocking ELISA method showed 241 positive samples and 41 negative samples. Statistical analysis showed that the relative sensitivity of the blocking ELISA antibody detection method established in this study was 97.9%, the relative specificity was 73.2%, and the overall concordance rate was 94.3%.

[0324] Table 16 Compliance Rate Test Results

[0325]

[0326] In this study, seven monoclonal antibodies were screened using a blocking ELISA method. The results showed that the blocking effect of monoclonal antibody 12E1 on PRRSV-positive serum was significantly stronger than that of the other six antibodies. Therefore, this monoclonal antibody was used to establish a blocking ELISA method. In the previous study, the N protein antigenic epitope recognized by this monoclonal antibody was identified as located at... 57 LATEDDVRHHF 67The antigenicity of this epitope may be stronger than that of other antigenic epitopes, which is a key factor affecting the sensitivity of blocking ELISA antibody detection methods. The hybridoma cell line secreting monoclonal antibody 12E1 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on April 27, 2023, with accession number CGMCC NO.45603, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0327] This study established a PRRSV N protein blocking ELISA antibody detection method through a series of condition screenings. It is important to note that during reaction condition screening, it is crucial to strictly maintain a single variable and set relevant replicate groups to avoid inaccuracies in experimental results due to operational or environmental factors. The established blocking ELISA was tested on 63 PRRSV-negative sera. The criteria for this method were: PI ≥ 60% for positive; PI ≤ 48% for negative; and PI between these two values ​​for suspicious results. This method exhibits high sensitivity and can effectively detect PRRSV serum antibodies. It shows no cross-reactivity with positive serum antibodies against common swine pathogens such as PRV, CSFV, PCV2, SVA, and PEDV, demonstrating high specificity. Six PRRSV-positive sera and four PRRSV-negative sera were tested using the blocking ELISA method, and the results showed that the ELISA method established in this study has good reproducibility. The blocking ELISA method, when used in conjunction with the IEDXX commercial PRRSV ELISA kit to detect 282 clinical porcine serum samples, showed a high relative sensitivity of 97.9%, a relatively low relative specificity of 73.2%, and a high overall concordance rate of 94.3%. The relatively low specificity may be due to the HRP labeling of monoclonal antibody 12E1 affecting its immunomodulatory activity and thus interfering with the specificity of the blocking ELISA antibody detection method. Monoclonal antibody affinity is also a major factor affecting specificity.

[0328] The PRRSV N protein blocking ELISA antibody detection method established in this study has high sensitivity, specificity, and good reproducibility, and has strong clinical application and development value. It can be applied to the clinical serological detection of PRRSV, which provides an important method for monitoring the prevalence of PRRSV in my country.

[0329] Invention points: Recombinant PRRSV N protein was expressed and prepared using a prokaryotic expression system. This protein was expressed in a soluble form, and the purified protein still exhibited good immunogenicity. Seven monoclonal antibodies against PRRSV N protein were developed. Among them, the heavy chain type of antibodies 8F1, 5E2, 2B1, and 12E1 was IgG1 subclass, and the heavy chain type of antibodies 4F1, 2H2, and 5A2 was IgG2b subclass. The light chain type was Kappa. The antibodies from the seven positive hybridoma cell lines showed good secretory stability, with antibody titers in the cell culture supernatant ranging from 1:800 to 1:25,600. All seven monoclonal antibodies exhibited PRRSV Western blot and IFA reactivity characteristics. Monoclonal antibodies 12E1 and 5A2 can be used in the PRRSV VHC assay.

[0330] Western blot identified three linear B-cell antigenic epitopes recognized by the seven monoclonal antibodies mentioned above, namely: 47 KKNPEKPHFPL 57 , 57 LATEDDVRHHF 67 and 67 FTPSERQLCLS 77 .in, 57 LATEDDVRHHF 67 This is a newly discovered antigenic epitope.

[0331] A monoclonal antibody, 12E1, with blocking effect on PRRSV positive serum was screened, and a PRRSV N protein blocking ELISA antibody detection method was successfully established. This method has high sensitivity and specificity, with a concordance rate of 94.3% with the IDEXX PRRSV ELISA antibody detection kit, and can be used for PRRSV antibody detection and serological epidemiological surveys.

Claims

1. A hybridoma cell line that secretes a monoclonal antibody against the N protein of porcine reproductive and respiratory syndrome virus (PRRSV). The preservation number of this hybridoma cell line is CGMCC NO.45603.

2. A monoclonal antibody against the N protein of porcine reproductive and respiratory syndrome virus, characterized in that, The monoclonal antibody against the porcine reproductive and respiratory syndrome virus N protein was obtained by secretion from the hybridoma cell line described in claim 1.

3. The use of the porcine reproductive and respiratory syndrome virus N protein monoclonal antibody as described in claim 2 in the preparation of a PRRSV serological detection kit.

4. The use of the porcine reproductive and respiratory syndrome virus (PRRSV) N protein monoclonal antibody as described in claim 2 in the preparation of a PRRSV N protein blocking ELISA antibody detection kit.

5. A PRRSV N protein blocking ELISA antibody detection kit, characterized in that, The kit contains the porcine reproductive and respiratory syndrome virus N protein monoclonal antibody as described in claim 2.

6. The PRRSV N protein blocking ELISA antibody detection kit according to claim 5, characterized in that, The kit also includes: coated antigen PRRSV N recombinant protein, secondary antibody, PBST, antigen coating solution, chromogenic solution, and stop solution.

7. A non-diagnostic ELISA antibody detection method for PRRSV N protein blocking, characterized in that, The PRRSV N protein blocking ELISA antibody detection was performed using the monoclonal antibody as described in claim 2, or using the kit as described in claim 5 or 6.

8. The method according to claim 7, characterized in that, The method includes the following steps: (1) Coating: Dilute the antigen with antigen coating solution and coat the enzyme-labeled strips; (2) Washing: Wash with PBST; (3) Sealing: The sealing reaction is carried out using a sealing solution; (4) Washing: Wash with PBST; (5) Treatment of the serum to be tested: Dilute the serum with PBST and add it, then let it stand to react; (6) Washing: Wash with PBST; (7) Monoclonal antibody reaction: Dilute the monoclonal antibody according to claim 2 with PBST and add it, then let it stand to react; (8) Washing: Wash with PBST; (9) Secondary antibody reaction: Dilute HRP-goat anti-mouse-IgG with PBST and add it, then let it stand to react; (10) Washing: Wash with PBST; (11) Color reaction: Add TMB color reagent to carry out color reaction; (12) Termination of reaction: The reaction was terminated with 2M H2SO4 as the termination solution; (13) Reading: Set the program of the multi-functional microplate reader, read the absorbance OD450nm value and calculate the blocking rate PI; PI=[(OF) 450nm阴性血清 -OF 450nm被检血清 ) / OF 450nm阴性血清 ]×100% (14) The detection result is judged based on the blocking rate (PI). The judgment criterion is: PI ≥ +3SD is positive, PI≤ +2SD is negative, and anything in between is suspicious. If a repeat test is still suspicious, then it is considered positive.

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