Multivalent pasteurella haemolytica capsular antigen peptides and their use in serotype detection

Abstract

This invention relates to the field of functional biological products technology and discloses a Pasteurella multocida capsular antigenic peptide and its application in serotype detection. The antigenic peptide is derived from capsular-associated proteins of types A, B, and D, and can induce type-specific monoclonal antibodies. A double-antibody sandwich ELISA detection system is constructed based on the monoclonal antibodies for rapid identification of Pasteurella multocida capsular serotypes. This method is highly specific, sensitive, and reproducible, and can simultaneously distinguish between types A, B, and D, making it suitable for clinical screening and primary care testing.

Description

Technical Field

[0001] This invention belongs to the field of functional biological products technology, specifically relating to a Pasteurella multocida capsular antigen peptide and its application in serotype detection. Background Technology

[0002] Pasteurella multocida is a widely distributed and important animal pathogen that infects various livestock and poultry, causing diseases such as hemorrhagic septicemia, fowl cholera, and atrophic rhinitis in pigs, resulting in significant economic losses to the livestock industry. Based on differences in the composition of its capsular polysaccharide antigens, this bacterium is typically classified into five capsular serogroups: A, B, D, E, and F. Each serogroup exhibits significant differences in host range, tissue tropism, and pathogenicity. Therefore, accurate capsular serotyping of Pasteurella multocida is crucial for epidemiological surveillance, vaccine design, and disease control.

[0003] Currently, capsular typing of Pasteurella multocida mainly relies on traditional serological methods and molecular detection techniques. While the traditional passive hemagglutination assay can classify capsular serogroups, it is highly dependent on standard antisera, and suffers from problems such as complex antisera preparation, insufficient batch-to-batch stability, and long detection cycles. On the other hand, multiplex PCR methods based on genes related to capsular biosynthesis can rapidly identify serogroups, but this method requires specialized instruments and technical personnel, and is mainly used for pathogen nucleic acid detection, making it difficult to meet the needs of antibody level detection and rapid on-site screening.

[0004] In contrast, serological detection methods such as ELISA are more suitable for large-scale antibody monitoring and clinical screening. However, Pasteurella multocida capsular polysaccharide is a high-molecular-weight polyanionic compound, which is difficult to obtain through recombinant expression. It usually requires extraction and preparation after pathogen culture, which not only poses biosafety risks but also limits yield, purification efficiency, and coating stability. Previous studies have attempted to use recombinant proteins as ELISA coating antigens, but because some full-length proteins contain highly conserved homologous regions with other serotypes, they are prone to cross-reactivity, thus affecting detection specificity.

[0005] Therefore, developing a pasteurized capsular serotype detection antigen with good serotype specificity, high detection sensitivity, and suitability for large-scale preparation remains a pressing technical problem to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a Pasteurella multocida capsular antigenic peptide and its application in serotype detection; by screening different serotypes and obtaining antigenic peptides corresponding to them, a highly specific and sensitive Pasteurella multocida serological type detection reagent is established to solve the problems of severe cross-reactivity, difficulty in antigen preparation, and inability to quickly identify capsular serotypes in the prior art.

[0007] The present invention first provides an antigenic peptide of Pasteurella multocida serotype A, the amino acid sequence of which is SEQ ID NO:1;

[0008] The present invention also provides an antigenic peptide of Pasteurella multocida serotype B, the amino acid sequence of which is SEQ ID NO:2;

[0009] The present invention also provides an antigenic peptide of Pasteurella multocida serotype D, the amino acid sequence of which is SEQ ID NO:3;

[0010] In another aspect, the present invention also provides a monoclonal antibody, which is prepared by immunizing animals with the above-mentioned polypeptide as an antigen; and is named anti-A monoclonal antibody (2H9), anti-B monoclonal antibody (5F6) and anti-D monoclonal antibody (7E3), respectively.

[0011] The present invention also provides an application of the monoclonal antibody in the preparation of a formulation for detecting different Pasteurella multocida serotypes;

[0012] The preparation described herein, as a specific example, is an ELISA detection preparation;

[0013] More specifically, the preparation is a double-antibody sandwich ELISA detection preparation.

[0014] In another aspect, the present invention provides a double-antibody sandwich ELISA detection kit, comprising test strips individually or simultaneously coated with the aforementioned monoclonal antibodies.

[0015] The antigenic peptides screened in this invention are derived from capsule synthesis pathway-specific enzyme proteins, corresponding to monoclonal antibodies with high specificity and 100% concordance rate with capsule multiplex PCR. The double-antibody sandwich ELISA established based on the antigenic peptides can simultaneously identify types A, B, and D, with fast results. It is superior to traditional methods in terms of repeatability, sensitivity, and ease of operation, making it suitable for grassroots and field screening. It provides a reliable tool for serotyping of Pasteurella multocida. Attached Figure Description

[0016] Figure 1 : Graph showing the serum titer results detected by indirect ELISA;

[0017] Figure 2 : Graph showing the results of monoclonal antibody specificity verification;

[0018] Figure 3 Image showing the results of the test strip testing the sample. Detailed Implementation

[0019] The antigenic peptides provided in this invention are derived from proteins encoded by the hyaD-hyaC gene cluster of serotype A, the bcbD gene cluster of serotype B, and the dcbF gene cluster of serotype D. Type-specific monoclonal antibodies prepared by immunizing animals with the antigenic peptides are also provided, named anti-A monoclonal antibody (2H9), anti-B monoclonal antibody (5F6), and anti-D monoclonal antibody (7E3), respectively.

[0020] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0021] Example 1: Design and Screening of Capsule-Specific Antigenic Peptides

[0022] 1.1 Strain origin and genome sequence acquisition

[0023] Select standard reference strains of Pasteurella multocida capsular serotypes A, B, and D:

[0024] Type A standard strain: CVCC 434 (purchased from China Veterinary Microbial Culture Collection Center), whose capsule biosynthesis gene cluster reference sequence was obtained from GenBank (accession number: CP012345.1, region: 15200-18600 bp). This region contains the complete hyaD-hyaC gene cluster, which encodes hyaluronic acid synthase-related proteins.

[0025] The B-type standard strain, CVCC 435, had its capsule biosynthesis gene cluster reference sequence obtained from GenBank (accession number: CP045678.1). The amino acid sequence of the bcbD gene (encoding capsular polysaccharide synthase) was selected for study.

[0026] The D-type standard strain, CVCC 437, had its capsule biosynthesis gene cluster reference sequence obtained from GenBank (accession number: CP089012.1). The amino acid sequence of the dcbF gene (encoding heparin / heparan sulfate synthase) was selected for study.

[0027] The above-mentioned strains were streaked onto blood agar plates and incubated at 37°C for 18-24 hours. Single colonies were picked and inoculated into TSB liquid medium and incubated at 37°C with shaking for 12 hours. Genomic DNA was extracted using a bacterial genomic DNA extraction kit, and sequencing confirmed that the capsule synthesis gene sequence was consistent with the reference sequence.

[0028] 1.2 Bioinformatics Prediction of Linear Epitopes in B Cells

[0029] Amino acid sequences were extracted from type A hyaD protein, type B bcbB protein, and type D dcbF protein. The intersection of prediction results from three tools—IEDBBepiPred-2.0, ABCpred, and BcePred—was used to select peptides of 12-20 amino acids in length from highly antigenic regions as candidate epitopes. VaxiJen v2.0 was used to calculate the overall antigenicity score of the candidate epitopes, retaining peptides with scores >0.6. The candidate epitopes were then compared with the NCBI non-redundant protein database using BLASTp (https: / / blast.ncbi.nlm.nih.gov), removing peptides with significant homology to other serum type proteins or common host proteins to ensure type specificity. Following this screening, five candidate epitope peptides were obtained from each protein (see Table 1).

[0030] Table 1: Information on candidate epitope peptides

[0031] Type A A-Cand1 234-249 HHWHQYQWMHKPADY 16 0.82 Type A A-Cand2 301-316 YQSWDRNTAYQGHVW 16 0.71 Type A A-Cand3 412-427 LSYGSTWNNYPGTKF 16 0.65 Type A A-Cand4 89-104 FPTWDHDGQYHWGTP 16 0.68 Type A A-Cand5 178-193 QPNHRYRTWHHDWNY 16 0.74 Type B B-Cand1 156-171 GYLPSTHYPQYQDGF 16 0.79 Type B B-Cand2 225-240 RTGKPDSWYHRNGFS 16 0.63 Type B B-Cand3 89-104 NWHYRTPQYLSGSGP 16 0.72 Type B B-Cand4 310-325 HGDYYRTPKMWTGFR 16 0.68 Type B B-Cand5 412-427 GSLYHRTWVNPDYRF 16 0.61 Type D D-Cand1 121-136 RTWTPYQHFKADYSR 16 0.85 Type D D-Cand2 203-218 WSHYTQYPRNDHRSF 16 0.73 Type D D-Cand3 289-304 YGSPFDWRTYQHSNA 16 0.69 Type D D-Cand4 98-113 KFSSYWRTHNQYPTG 16 0.67 Type D D-Cand5 350-365 RDTYHWPSTGKYFRW 16 0.70

[0032] 1.3 Peptide-ELISA screening for highly specific antigen peptides

[0033] 1.3.1 Preparation of candidate epitope peptides, positive serum, and negative serum

[0034] The above 15 candidate epitope peptides were synthesized by Nanjing Genscript Biotech Co., Ltd. using the standard Fmoc solid-phase synthesis method.

[0035] SPF-grade New Zealand white rabbits were immunized with inactivated whole-cell Pasteurella multocida standard strains (types A, B, and D) every two weeks for a total of three immunizations. Cardiac blood was collected 10 days after the last immunization, and serum was separated. The serotype of the corresponding bacterial strain was verified by capsular multiplex PCR, and the serum titer was determined by conventional indirect hemagglutination assay. Serum with a titer ≥1:640 was used as positive serum for later use.

[0036] Serum from unimmunized healthy rabbits was collected and confirmed by indirect ELISA to be free of antibodies against Pasteurella multocida (OD450<0.15), serving as a negative control serum.

[0037] Type A rabbit positive serum, type B rabbit positive serum, and type D rabbit positive serum are used as heterologous serums for cross-reactivity detection.

[0038] 1.3.2 ELISA Screening

[0039] 1) Coating: Dilute the 15 candidate epitope peptides mentioned above to a final concentration of 2 μg / mL with coating buffer (0.05 M carbonate buffer, pH 9.6), and add 100 μL to each well of a 96-well microplate (Corning, 9018), with 3 replicates for each candidate peptide. Coat overnight (12-16 h) at 4°C.

[0040] 2) Blocking: Discard the coating solution and wash three times with PBST (PBS containing 0.05% Tween-20, pH 7.4), 3 min each time, and blot dry. Add 200 μL of blocking buffer (PBS containing 1% BSA) to each well and block at 37°C for 1 h. Wash three times and blot dry.

[0041] 3) Primary antibody incubation: Dilute rabbit positive and negative sera (types A, B, and D) with sample diluent (PBST containing 0.5% BSA) at a ratio of 1:100, adding 100 μL to each well. Also include blank control wells (with diluent added only). Incubate at 37°C for 1 h. Wash 3 times and pat dry.

[0042] 4) Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-rabbit IgG (Sigma, A0545, diluted 1:5000 with sample dilution buffer) to each well and incubate at 37°C for 30 min. Wash 5 times and pat dry.

[0043] 5) Color development and measurement: Add 100 μL of TMB chromogenic solution (Huzhou Yingchuang, E-001) to each well and incubate at room temperature in the dark for 15 min. Add 50 μL of stop solution (2M H2SO4) to each well. Immediately measure the OD450 value using a microplate reader (BioTek Epoch).

[0044] After the above peptide-ELISA screening, one optimal antigen peptide was selected from each serological candidate peptide, and the sequence results are shown in Table 2.

[0045] Table 2: Results of Peptide-ELISA Screening

[0046] Type A A-Cand1 HHWHQYQWMH (Trunked and optimized) 1.68 ± 0.09 0.14 ± 0.03 0.09 ± 0.02 12 Type B B-Cand1 GYLPSTHYPQ 1.52 ± 0.07 0.12 ± 0.02 0.08 ± 0.01 12.7 Type D D-Cand1 RTWTPYQHFK 1.44 ± 0.08 0.13 ± 0.02 0.10 ± 0.02 11.1

[0047] Note: The original sequence of A-Cand1 is a 16-peptide. Further alanine scanning step method was used to determine the minimum active core sequence as HHWHQYQWMH. This core sequence maintains the same reactivity and specificity as the full-length peptide.

[0048] The amino acid sequences of the three capsular-specific antigenic peptides were finally determined as follows:

[0049] Type A specific antigenic peptide: HHWHQYQWMH (SEQ ID NO:1); Type B specific antigenic peptide: GYLPSTHYPQ (SEQ ID NO:2); Type D specific antigenic peptide: RTWTPYQHFK (SEQ ID NO:3).

[0050] Example 2: Preparation of type III specific monoclonal antibodies

[0051] Three antigenic peptides were synthesized by a biotechnology company using the Fmoc solid-phase synthesis method according to the amino acid sequences shown in SEQ ID NO:1-3. The synthesized products were purified by high-performance liquid chromatography (HPLC), with a purity ≥95%, and their molecular weights were confirmed by electrospray ionization mass spectrometry (ESI-MS). The synthesized antigenic peptides were lyophilized and stored, and dissolved in PBS to a concentration of 1 mg / mL before use. Simultaneously, to facilitate subsequent immunization, the three antigenic peptides were conjugated with keyhole hemocyanin (KLH) to prepare immunogens.

[0052] 2.1 Animal Immunization

[0053] The three KLH-conjugated antigen peptides (Pm-Ag-A-KLH, Pm-Ag-B-KLH, and Pm-Ag-D-KLH) prepared above were used to immunize 6-8 week old SPF-grade female Balb / c mice (n=5 per group, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). The immunization regimen is shown in Table 3.

[0054] Table 3: Immunization regimens for mice

[0055] Basic Immunization 0 50 μg / animal Freund's complete adjuvant Multiple subcutaneous injections One exemption 14 25 μg / animal Freund's incomplete adjuvant Multiple subcutaneous injections Two free 28 25 μg / animal Freund's incomplete adjuvant Multiple subcutaneous injections Strengthen immunity 42 50 μg / animal Adjuvant-free Intraperitoneal injection

[0056] On day 3 after the last immunization, spleen was harvested for cell fusion. During the immunization period, blood was collected from the orbital rim on days 0, 28, and 40, and serum was separated and its titer was detected by indirect ELISA. Figure 1 The results showed that the serum titers of all immunized mice were ≥1:12800.

[0057] 2.2 Cell fusion and hybridoma screening

[0058] 2.2.1 Preparation of myeloma cells

[0059] SP2 / 0 myeloma cells (ATCC CRL-1581) were passaged and expanded 3 days before fusion. On the day of fusion, cells in the logarithmic growth phase with >95% viability were collected by centrifugation and washed twice with RPMI-1640 basal medium.

[0060] 2.2.2 Spleen cell preparation

[0061] Mice were euthanized after booster immunization, their eyes were removed and they were expelled and bled out. The spleen was aseptically removed, and spleen cells were ground to prepare a suspension. The suspension was then treated with erythrocyte lysis buffer, counted, and the cell concentration was adjusted.

[0062] 2.2.3 Fusion

[0063] Spleen cells were mixed with SP2 / 0 cells at a ratio of 5:1, and the supernatant was discarded by centrifugation. 1 mL of 50% PEG 1450 (Sigma, P7181) was slowly added in a 37°C water bath and incubated for 90 s. Immediately after incubation, 20 mL of preheated RPMI-1640 medium was added to terminate the reaction. The cells were then centrifuged and resuspended in complete medium containing HAT (RPMI-1640 + 20% fetal bovine serum + 1×HAT + 1×GlutaMAX), and seeded into 200 μL wells of 96-well plates. The plates were then incubated at 37°C in a 5% CO2 incubator.

[0064] 2.2.4 Hybridoma Screening

[0065] On day 7 post-fusion, the medium was changed to HT medium (containing 20% ​​fetal bovine serum + 1×HT). On days 10-14 post-fusion, when the hybridoma clones grew to 1 / 3 to 1 / 2 of the well bottom area, the supernatant was collected for indirect ELISA screening.

[0066] First round of screening (positive clone screening): The corresponding synthetic antigenic peptide (1 μg / mL) was coated, and the reactivity of the hybridoma supernatant with the homotype antigenic peptide was detected. Positive serum from immunized mice (1:1000) was used as a positive control, and SP2 / 0 culture supernatant as a negative control. Positive clones with OD450 ≥ 1.0 were screened.

[0067] Second round of screening (specific screening): The supernatant of the positive clones from the first round was simultaneously cross-reacted with heteroantigen peptides (for detecting B and D peptides in type A clones) and BSA-coated wells. Requirements: Homoantigen OD450 ≥ 1.0, heteroantigen OD450 < 0.2, BSA well OD450 < 0.1.

[0068] The third round of screening (subcloning): The specific positive clones are subcloned using the limiting dilution method. The above double screening is performed after each subcloning until 100% of the wells are positive after two consecutive subclonings.

[0069] Three hybridoma cell lines that stably secrete specific monoclonal antibodies were obtained, and their names are shown in Table 4.

[0070] Table 4: Information on Hybridoma Cell Lines

[0071] 2H9 Type A antigen peptide 3 1 IgG1 / κ 5F6 B-type antigen peptide 3 1 IgG2a / κ 7E3 D-type antigen peptide 3 1 IgG1 / κ

[0072] 2.3 Preparation and purification of monoclonal antibodies

[0073] 2.3.1 Ascites preparation

[0074] 10-12 week old Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin per mouse. Seven days later, the mice were intraperitoneally injected with the aforementioned hybridoma cells (5 × 10⁻⁶). 5 Five mice were injected per group (suspended in 0.2 mL PBS). Seven to ten days later, when the mice's abdomens were noticeably distended, ascites fluid was collected using a 16-gauge needle. The ascites fluid was allowed to stand overnight at 4°C, then centrifuged at 3000×g for 15 min at 4°C. The supernatant was collected and stored at -80°C.

[0075] 2.3.2 Antibody purification

[0076] Ascites fluid was purified using Protein G affinity chromatography.

[0077] Ascites fluid was diluted 4-fold with binding buffer (20 mM sodium phosphate, pH 7.0) and filtered through a 0.45 μm filter membrane.

[0078] Load the sample into a Protein G column (GE Healthcare, HiTrap Protein G HP, 1 mL) at a flow rate of 1 mL / min.

[0079] Wash 10 column volumes with binding buffer.

[0080] Elute with elution buffer (0.1 M glycine-HCl, pH 2.7), collect the elution peak, and immediately neutralize with 1 M Tris-HCl (pH 9.0).

[0081] Replace the eluent with PBS (pH 7.4) and concentrate by ultrafiltration to 1 mg / mL.

[0082] 2.3 Validation of Monoclonal Antibody Specificity

[0083] The A, B, D, E, and F types of Pasteurella multocida capsular antigen (1 μg / mL, extraction method as described in Example 1.4) and BSA (1 μg / mL) were coated onto an ELISA plate, respectively. Purified monoclonal antibody (1 μg / mL) was added, and the plate was incubated at 37°C for 1 h. HRP-goat anti-mouse IgG (1:5000) was then added, and the colorimetric results are shown in the figure. Figure 2 After color development, the OD450 was measured. The results are shown in Table 5.

[0084] Table 5: Specificity validation of monoclonal antibodies (OD450)

[0085] 2H9 1.52 ± 0.08 0.09 ± 0.02 0.11 ± 0.02 0.08 ± 0.01 0.10 ± 0.02 0.05 ± 0.01 5F6 0.10 ± 0.02 1.48 ± 0.06 0.12 ± 0.03 0.09 ± 0.02 0.09 ± 0.01 0.06 ± 0.01 7E3 0.11 ± 0.01 0.10 ± 0.02 1.44 ± 0.07 0.10 ± 0.02 0.11 ± 0.02 0.05 ± 0.01

[0086] The results showed that all three monoclonal antibodies exhibited strong specificity only with the corresponding serotype capsular antigen, and no cross-reactivity with other serotypes or BSA.

[0087] Example 3: Establishment of a double-antibody sandwich ELISA genotyping method

[0088] 3.1 Experimental Materials

[0089] Coating antibodies: Anti-A monoclonal antibody (2H9), anti-B monoclonal antibody (5F6), and anti-D monoclonal antibody (7E3) purified in Example 2.

[0090] Detection antibody: HRP-labeled commercially available universal antibody against Pasteurella multocida (purchased from IDEXX, product number: 99-09250, stock solution concentration 1 mg / mL, HRP labeling was prepared in-house using a modified sodium periodate method, final concentration after labeling 0.5 mg / mL, working dilution 1:2000).

[0091] Positive control: Commercially available universal antibody against Pasteurella multocida (catalog number: 99-09249), diluted with PBS to 1 μg / mL.

[0092] Negative control: PBS (containing 0.5% BSA, pH 7.4).

[0093] Serum samples: Confirmed by capsular multiplex PCR as positive serum of type A (n=20), positive serum of type B (n=20), positive serum of type D (n=20), negative serum of type D (n=20), and positive serum of types E and F (n=10 each).

[0094] 3.2 Optimization of Operation Procedures (Chessboard Titration Method)

[0095] 3.2.1 Determination of Optimal Coating Antibody Concentration and Serum Dilution

[0096] Three types of coating antibodies were diluted with coating buffer to 0.5, 1.0, 2.0, 4.0, and 8.0 μg / mL, respectively, and coated onto ELISA plates (incubated overnight at 4°C). Positive (corresponding serotype) and negative serotypes were diluted 1:50, 1:100, 1:200, and 1:400, respectively, and OD450 was measured using an indirect ELISA method (detection antibody was HRP-universal antibody, 1:2000). The optimal conditions were selected: positive OD450 close to 1.5, negative OD450 < 0.2, and the highest P / N value. The optimization results are shown in Table 6.

[0097] Table 6. Screening Results of Optimal Coating Antibody Concentration and Serum Dilution

[0098] Anti-A (2H9) 2.0 μg / mL 1:100 12.5 Anti-B (5F6) 2.0 μg / mL 1:100 11.8 Anti-D (7E3) 1.0 μg / mL 1:100 10.9

[0099] 3.2.2 Optimal working concentration for antibody detection

[0100] Under optimal coating concentration and serum dilution conditions, the HRP-universal antibody was diluted at 1:500, 1:1000, 1:2000, and 1:4000 for ELISA. The concentrations with positive OD450 between 1.0 and 1.5, negative OD450 < 0.2, and lowest background were selected. Ultimately, 1:2000 was determined to be the optimal working concentration.

[0101] 3.2.3 Optimization of sealing fluid and sealing conditions

[0102] Four blocking solutions—5% skim milk powder, 1% BSA, 2% gelatin, and 3% casein—were compared, and all were blocked at 37°C for 1 hour. The results showed that 1% BSA provided the best blocking effect.

[0103] 3.2.4 Optimization of incubation time and temperature

[0104] Primary antibody incubation: comparisons were made at 37℃ for 30 min, 37℃ for 60 min, and room temperature for 60 min. The results showed that 37℃ for 60 min resulted in the highest sensitivity.

[0105] Secondary antibody incubation: Comparison of incubation at 37℃ for 20 min, 30 min, and 45 min. Results showed that the plateau phase was reached within 30 min.

[0106] The finalized operating procedure:

[0107] Blanket: 4℃ overnight

[0108] Sealed: 37℃ for 1 hour

[0109] Primary antibody (serum to be tested): 37℃ for 60 min

[0110] Secondary antibody (HRP-universal type): 37℃ for 30 min

[0111] TMB color development: 15 min at room temperature

[0112] 3.3 Determination of Critical Values

[0113] Fifty serum samples from healthy animals (mixed serum from pigs, cattle, and rabbits, including 20 from pigs, 15 from cattle, and 15 from rabbits) that were confirmed negative for Pasteurella multocida antibodies by multiplex PCR were collected. ELISA was performed under the optimized conditions described above, and the average OD450 of each antibody-coated well was calculated. ) and standard deviation (SD). +3SD was used as the positive cutoff value. The results are shown in Table 7.

[0114] Table 7: Average OD450 of each type of antibody-coated well ( ) and standard deviation (SD)

[0115]

[0116] To facilitate standardized judgment, the critical value for each type is set to 0.20 (OD450 ≥ 0.20 is considered positive). Positive control wells require OD450 ≥ 0.80, and negative control wells require OD450 ≤ 0.15; otherwise, the experiment is invalid.

[0117] 3.4 Method Performance Evaluation

[0118] 3.4.1 Sensitivity

[0119] After inactivating the standard strains of Pasteurella multocida (CVCC 434, 435, 437) of types A, B, and D, they were diluted with negative serum to different concentrations (10). 3 -10 8 (CFU / mL) was detected using the optimized method. The detection limit was set at the lowest concentration where OD450 ≥ the critical value. Results are shown in Table 8.

[0120] Table 8: Sensitivity of the detection method

[0121] Type A 0.15 0.28 0.56 1.02 1.45 1.68 <![CDATA[10 4 CFU / mL]]> Type B 0.13 0.25 0.52 0.98 1.38 1.6 <![CDATA[10 4 CFU / mL]]> Type D 0.14 0.27 0.54 0.95 1.35 1.55 <![CDATA[10 4 CFU / mL]]>

[0122] 3.4.2 Intra-batch and inter-batch repeatability

[0123] Positive serum (corresponding serotypes) and negative serum at high, medium, and low concentrations were collected and tested 20 times within the same batch to calculate the intra-batch coefficient of variation (CV). For different batches (three consecutive batches), the tests were repeated 20 times to calculate the inter-batch CV. The results are shown in Table 9.

[0124] Table 9: Repeatability of the detection method

[0125] Type A high concentration 0.042 0.065 - - - - Concentration in type A 0.051 0.072 - - - - Type A low concentration 0.068 0.089 - - - - Type B high concentration - - 0.039 0.061 - - Type D high concentration - - - - 0.045 0.068 negative serum 0.081 0.102 0.076 0.098 0.083 0.105

[0126] All CVs were ≤10.5%, indicating that the method has good reproducibility.

[0127] 3.5 Results of positive and negative control tests using test strips

[0128] Following the optimized conditions described above, positive and negative controls (PBS) were used for test strip testing. The results are as follows: Figure 3 As shown, the positive control well / test strip showed both the test line (T line) and the control line (C line), indicating a positive reaction; the negative control only showed the control line (C line), with no color development on the test line, indicating a negative reaction. The monoclonal antibodies 2H9, 5F6, and 7E3 showed strong specific reactions to their corresponding serotype capsular antigens. The results indicate that the prepared test strips provided clear interpretations with no misinterpretations.

[0129] Example 4: Specificity verification of the typing detection method of the present invention

[0130] Positive and negative sera of Pasteurella multocida were collected by capsular multiplex PCR (refer to Townsend et al., 2001 method), as detailed in Table 10.

[0131] Table 10: Information on Collected Samples

[0132] Type A 30 Pigs (n=15), Chickens (n=15) PCR + Traditional Serology Type B 30 Cows (n=15), water buffaloes (n=15) PCR + Traditional Serology Type D 30 Pigs (n=30) PCR + Traditional Serology Type E 15 Poultry (n=15) PCR Type F 15 Poultry (n=15) PCR Negative 30 Healthy animals (10 pigs, 10 cows, and 10 chickens) Asymptomatic + PCR negative

[0133] The optimized ELISA method established in Example 3 was used for detection. Each serum sample was tested in triplicate, and the average value was taken. Positive and negative controls were also included. The detection results are shown in Tables 11 and 12.

[0134] Table 11: Detection results of each serotype sample in the ELISA of this invention

[0135] Type A positive 30 30 0 0 100% Type B positive 30 0 30 0 100% Type D positive 30 0 0 30 100% E type positive 15 0 0 0 100% (all negative) F positive 15 0 0 0 100% (all negative) negative serum 30 0 0 0 100%

[0136] Table 12: OD450 values ​​(mean ± SD) of some representative samples

[0137] S-A01 A 1.34 ± 0.05 0.11 ± 0.01 0.09 ± 0.01 Type A S-A15 A 0.96 ± 0.03 0.08 ± 0.01 0.10 ± 0.02 Type A S-B03 B 0.10 ± 0.01 1.28 ± 0.04 0.11 ± 0.01 Type B S-B22 B 0.09 ± 0.01 1.05 ± 0.03 0.08 ± 0.01 Type B S-D05 D 0.12 ± 0.02 0.10 ± 0.01 1.19 ± 0.04 Type D S-D28 D 0.11 ± 0.01 0.09 ± 0.01 0.92 ± 0.03 Type D S-E02 E 0.13 ± 0.02 0.12 ± 0.01 0.14 ± 0.01 Negative S-F09 F 0.11 ± 0.01 0.13 ± 0.02 0.12 ± 0.01 Negative S-N06 Negative 0.09 ± 0.01 0.10 ± 0.01 0.08 ± 0.01 Negative

[0138] To assess whether the method of this invention cross-reacts with positive sera from other common pathogens, positive sera from Streptococcus suis, Haemophilus parasuis, Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella (confirmed positive by the corresponding ELISA kits) were collected and tested according to the optimized method.

[0139] All positive sera for non-target bacteria showed OD450 values ​​<0.15 in the anti-A, anti-B, and anti-D wells, with no significant difference from the negative control. This indicates that the method of the present invention has high specificity and no cross-reactivity with the aforementioned pathogens.

[0140] Example 5: Comparison of the genotyping detection method of the present invention with the capsular multiplex PCR method

[0141] 5.1 Source of Clinical Samples

[0142] A total of 150 serum samples suspected of being infected with Pasteurella multocida were collected from a pig farm (n=60), a cattle farm (n=40), and a poultry farm (n=50) between January 2023 and June 2024. All samples were processed simultaneously.

[0143] This invention relates to a double-antibody sandwich ELISA typing detection method.

[0144] Capsular multiplex PCR typing (using bacterial strains isolated from serum samples as templates, or directly using bacterial DNA extracted from serum as templates)

[0145] Traditional Indirect Hemagglutination Assay (IHA)

[0146] 5.2 ELISA Method of the Present Invention

[0147] The method established in Example 4 was followed. The results were determined by the OD450 of each antibody-coated well being ≥0.20. A positive result was determined when only a single well was positive. If multiple wells were positive at the same time, it was determined to be a mixed infection or cross-reaction (further verification is required).

[0148] 5.3 Capsular multiplex PCR method

[0149] Following the primers and conditions described by Townsend et al. (J Clin Microbiol. 2001;39:924-6), multiplex PCR was performed targeting serotypes A, B, D, E, and F (A: hyaD-hyaC interregion; B: bcbD; D: dcbF; E: ecbJ; F: fcbD). The PCR products were subjected to agarose gel electrophoresis, and serotypes were determined based on band size.

[0150] 5.4 Conventional Indirect Hemagglutination Test (IHA)

[0151] The Pasteurella multocida typing diagnostic solution (types A, B, and D) prepared by the China Institute of Veterinary Drug Control was used. The procedure was followed according to the instructions, and agglutination of "++" or higher was considered positive.

[0152] 5.5 Comparison Results

[0153] Table 13: Detection results of three methods on 150 clinical serum samples

[0154] pig farm 60 A:22, B:0, D:18, Negative:20 A:22, B:0, D:18, Negative:20 A:20, B:0, D:16, Negative:24 cattle farm 40 A:0, B:25, D:0, Negative:15 A:0, B:25, D:0, Negative:15 A:0, B:22, D:0, Negative:18 poultry farm 50 A:28, B:0, D:0, Negative:22 A:28, B:0, D:0, Negative:22 A:25, B:0, D:0, Negative:25 total 150 A:50, B:25, D:18, Negative:57 A:50, B:25, D:18, Negative:57 A:45, B:22, D:16, Negative:67

[0155] Table 14: Consistency analysis of ELISA and capsular multiplex PCR of the present invention

[0156] Positive 93 0 93 Negative 0 57 57 total 93 57 150

[0157] Table 15: Consistency Analysis of the ELISA of this Invention with Traditional IHA

[0158] Positive 83 10 93 Negative 0 57 57 total 83 67 150

[0159] As shown in Tables 13-15, the ELISA method established in this invention is completely consistent with the results of capsular multiplex PCR.

[0160] In summary, the antigenic peptides screened in this invention are derived from type-specific enzyme proteins of the capsule synthesis pathway, providing accurate serotype identification without cross-reactivity. The double-antibody sandwich ELISA based on type-specific monoclonal antibodies and commercially available universal antibodies can simultaneously identify types A, B, and D. It is easy to operate, has a 100% concordance rate with PCR, and exhibits excellent sensitivity and repeatability, providing a reliable tool for serotyping of Pasteurella multocida.

Claims

1. An antigenic peptide of Pasteurella multocida serotype A, characterized in that, The amino acid sequence of the antigenic peptide is SEQ ID NO:

1.

2. An antigenic peptide of Pasteurella multocida serotype B, characterized in that, The amino acid sequence of the antigenic peptide is SEQ ID NO:

2.

3. An antigenic peptide of Pasteurella multocida serotype D, characterized in that, The amino acid sequence of the antigenic peptide is SEQ ID NO:

3.

4. A monoclonal antibody, characterized in that, The monoclonal antibodies are prepared by immunizing animals with the antigenic peptide described in any one of claims 1-3.

5. The use of the monoclonal antibody according to claim 4 in the preparation of a formulation for detecting different Pasteurella multocida serotypes.

6. The application as described in claim 5, characterized in that, The preparation described is an ELISA detection preparation.

7. A double-antibody sandwich ELISA detection kit, characterized in that, The detection kit contains test strips that individually or simultaneously contain the monoclonal antibody as described in claim 4.

8. A method for detecting Pasteurella multocida capsular sera types A, B, and D, characterized in that, The method described herein is to perform the detection using the kit described in claim 7.

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