Lawsonia intracellularis monoclonal antibody, hybridoma cell line and application thereof

Abstract

The invention discloses a Lawsonia intracellularis monoclonal antibody, a hybridoma cell line and application thereof, and relates to the technical field of biological detection. The lawsonia intracellularis monoclonal antibody comprises a light chain CDR1 of which the amino acid sequence is shown as SEQ ID NO.3, a light chain CDR2 of which the amino acid sequence is shown as SEQ ID NO.4, a light chain CDR3 of which the amino acid sequence is shown as SEQ ID NO.5, a heavy chain CDR1 of which the amino acid sequence is shown as SEQ ID NO.6, a heavy chain CDR2 of which the amino acid sequence is shown as SEQ ID NO.7 and a heavy chain CDR3 of which the amino acid sequence is shown as SEQ ID NO.8. The lawsonia intracellularis monoclonal antibody can be specifically combined with lawsonia intracellularis, so that the lawsonia intracellularis monoclonal antibody can be applied to preparation of a detection product of a lawsonia intracellularis antigen or antibody.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to an intracellular Lawsonia monoclonal antibody, a hybridoma cell line, and their applications. Background Technology

[0002] Lawsonia intracellularis ( Lawsonia intracellularis Lawsonia intracellularis (LI) is a strictly intracellular, Gram-negative bacterium belonging to the genus Lawsonia in the family Desulfovibrioceae. It has a wide host range, exhibiting high pathogenicity, particularly in livestock such as pigs, horses, and rabbits, as well as some wild animals. Pigs are the most common natural host, and porcine proliferative enteropathy (PPE) caused by infection is a serious gastrointestinal infectious disease in the pig industry. This disease primarily affects the ileum and colonic mucosa of pigs, leading to abnormal proliferation, necrosis, and hemorrhage of intestinal epithelial cells. Clinical manifestations include stunted growth, emaciation, diarrhea, and bloody stools in finishing pigs, and in severe cases, acute death. Even if infected pigs survive, they may experience decreased growth performance and reduced feed conversion rates, prolonging their time to market and increasing farming costs. In recent years, with the widespread adoption of large-scale, intensive pig farming, the transmission routes of Lawsonia intracellularis have further expanded. It spreads rapidly via the fecal-oral route, easily forming persistent infections in farmed populations, and often co-infects with other intestinal pathogens, exacerbating the complexity of the disease and posing a severe challenge to disease control.

[0003] Currently, detection methods for Lawsonia intracellularis mainly fall into three categories: etiological detection, serological detection, and molecular biological detection. Etiological detection, such as cell isolation and culture, and pathological histological observation, while accurate, are complex, time-consuming, and require stringent experimental conditions, making them difficult to implement in grassroots farms. Molecular biological detection, such as PCR and nested PCR, offers advantages in specificity and sensitivity, but requires specialized equipment and technicians, is costly, and cannot distinguish between live and dead bacteria, thus failing to accurately reflect the infection status and immune level of the organism. Serological detection, due to its simplicity, speed, and low cost, is suitable for large-scale sample screening and has become a commonly used clinical detection method, with enzyme-linked immunosorbent assay (ELISA) being the most widely used.

[0004] Monoclonal antibodies possess advantages such as high specificity, high purity, and large-scale production capability. They can accurately identify specific epitopes of antigens and effectively avoid cross-reactivity, making them crucial for improving the accuracy of serological detection methods. Currently, some research has been conducted both domestically and internationally on the preparation of monoclonal antibodies against Lawsonia intracellularis. However, most monoclonal antibodies suffer from insufficient affinity, poor specificity, or limited applicability, making it difficult to meet the needs of clinical testing. Therefore, developing a monoclonal antibody with high specificity and affinity, and establishing a sensitive and accurate detection method based on it, is of great significance for the early diagnosis, epidemic monitoring, and prevention and control of Lawsonia intracellularis infection. Summary of the Invention

[0005] The purpose of this invention is to provide an intracellular Lawsonia monoclonal antibody, a hybridoma cell line, and their applications, to address the problems existing in the prior art. This intracellular Lawsonia monoclonal antibody can specifically bind to intracellular Lawsonia, and thus can be used to prepare detection products for Lawsonia intracellular antigens or antibodies.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides an intracellular Lawsonia monoclonal antibody comprising a light chain CDR1 with an amino acid sequence as shown in SEQ ID NO.3, a light chain CDR2 with an amino acid sequence as shown in SEQ ID NO.4, a light chain CDR3 with an amino acid sequence as shown in SEQ ID NO.5, a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO.6, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO.7, and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO.8.

[0007] Furthermore, the amino acid sequence of the light chain variable region of the intracellular Lawsonia monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2.

[0008] The present invention also provides a hybridoma cell line that secretes monoclonal antibodies against Lawsonia intracellularis. The hybridoma cell line was deposited on December 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46760.

[0009] The present invention also provides a gene encoding the above-mentioned intracellular Lawsonia monoclonal antibody.

[0010] Furthermore, the coding gene includes a light chain variable region coding gene and a heavy chain variable region coding gene; The nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO.9; The nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO.10.

[0011] The present invention also provides a biomaterial, wherein the biomaterial is the substance described in (1) or (2): (1) A recombinant expression vector containing the above-mentioned coding genes; (2) Recombinant non-plant host cells containing the recombinant expression vector.

[0012] The present invention also provides the application of the above-mentioned hybridoma cell lines, encoding genes or biological materials in the preparation of intracellular Lawsonia monoclonal antibodies.

[0013] The present invention also provides the application of the above-mentioned intracellular Lawsonia monoclonal antibody in the preparation of an ELISA detection kit for detecting intracellular Lawsonia antigen or antibody.

[0014] The present invention also provides an ELISA detection kit for detecting Lawsonia intracellularis antigen, comprising the above-mentioned Lawsonia intracellularis monoclonal antibody.

[0015] The present invention also provides an ELISA detection kit for detecting Lawsonia intracellularis antibodies, comprising the above-mentioned Lawsonia intracellularis monoclonal antibodies.

[0016] The present invention discloses the following technical effects: This invention uses whole-cell protein of *L. intracellularis* as an immunogen to immunize BALB / c mice to prepare a monoclonal antibody, obtaining a monoclonal antibody 3A3 targeting *L. intracellularis*. IFA specificity identification showed that monoclonal antibody 3A3 specifically reacts with *L. intracellularis*, with its subclass being IgG2a / κ and a relative affinity constant of 2.5 mol / L. Using porcine polyclonal antibody as a capture antibody and purified monoclonal antibody 3A3 labeled with HRP as a detection antibody, the application effect of 3A3 as a detection antibody in the detection method of *L. intracellularis* antigen was preliminarily explored. The results showed that monoclonal antibody 3A3 can be used for the specific detection of *L. intracellularis* antigen. To verify the application value of monoclonal antibody 3A3 in establishing an antibody detection method for *L. intracellularis*, this invention uses whole-cell protein of *L. intracellularis* as an antigen and HRP-labeled 3A3 as a detection antibody to establish an ELISA method, detecting serially diluted negative and positive sera of *L. intracellularis*, and calculating the inhibition rate of the reaction. The results showed that the ELISA method established using monoclonal antibody 3A3 could specifically distinguish between Lawsonia intracellularis negative and positive sera. Therefore, the monoclonal antibody 3A3 prepared in this invention can be used to prepare detection products for Lawsonia intracellularis antigens or antibodies. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is an indirect immunofluorescence assay of the reaction between monoclonal antibodies and intracellular Lawsonia strains. Figure 2 The graph shows the percentage inhibition rate of strongly positive, weakly positive, and negative serum for ELISA detection of Lawsonia intracellularis. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Biological Preservation: The hybridoma cell line PE-3A3, which contains the intracellular Lawsonia monoclonal antibody 3A3 obtained by screening in this invention, was deposited on December 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46760.

[0025] Example 1 1. Materials and Methods 1.1 Experimental Materials 1.1.1 Bacteria, cells, strains, and laboratory animals Intracellular Lawsonia lactiflora, IPEC cells, and SP2 / 0 myeloma cells were provided by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences; clean-grade female BALB / c mice were purchased from the Experimental Animal Center of the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences.

[0026] 1.1.2 Main Reagents Fusion agent PEG / DMSO (Mw, 1450), HAT salt (50×), HT salt (50×), HRP or FITC-labeled goat anti-mouse IgG, Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma-Aldrich; monoclonal antibody subclass identification kit was purchased from Southern Biotech; L-glutamine and glycine were purchased from Amresco; dimethyl sulfoxide (DMSO), o-phenylenediamine (OPD), and PEG6000 were purchased from Solarbio; high-glucose DMEM dry powder was purchased from GIBCO; imported premium fetal bovine serum (PAA) was purchased from Nalgene, Spain; and 96-well cell culture plates were purchased from JET Biochemical, Canada.

[0027] 1.2 Experimental Methods 1.2.1 Mouse Immunization and Preparation of Monoclonal Antibodies (1) Mouse immunization Purified Lawsonia intracellulare whole-cell protein antigen (100 µg / 200 μL) was emulsified with an equal volume of Freund's complete adjuvant and subcutaneously injected into the back of 6-week-old female BALB / c mice. Two weeks later, a second immunization was performed using Freund's incomplete adjuvant. Two weeks later, a third immunization was performed using an equal volume of antigen without adjuvant. To stimulate a rapid and strong immune response in the mice, a booster immunization was performed 3 days before fusion using a combination of tail vein and spleen injection, employing a double dose of antigen.

[0028] (2) Cell fusion a) Preparation of feeder cells: Feeder cells were prepared the day before cell fusion. Ophthalmic scissors, forceps, petri dishes, and other experimental equipment were sterilized by dry heat before use. HAT complete culture medium was tested for sterility. Two BALB / c mice were enucleated to expel blood, and negative serum was separated using standard methods. Mice were euthanized by cervical dislocation and immersed in 75% alcohol for 10 minutes before being transferred to a clean bench. The mice were fixed to a rack with their abdomens facing upwards. The skin in the midline of the abdomen was lifted with forceps, and a small horizontal incision was made with ophthalmic scissors, being careful not to puncture the peritoneum. The skin was then torn open from top to bottom with scissors and forceps to fully expose the peritoneum. The peritoneum was gently lifted with forceps, and 10 mL of HAT complete culture medium (drawn from a 10 mL syringe) was injected into the peritoneal cavity, being careful not to puncture organs or intestines. The syringe was not removed. The fluid was aspirated back and forth in the peritoneal cavity 5 times. Then, the abdomen was massaged on both sides with forceps for about 30 seconds, and aspiration was repeated 3 times. The fluid in the peritoneal cavity was then aspirated back with a syringe. Care should be taken to avoid the mesentery and adipose tissue to prevent clogging of the needle. Peritoneal cells taken from two mice were added to 55 mL of HAT culture medium, the cells were dispersed and mixed, and then dispensed into six 96-well culture plates at 100 µL / well. The plates were then incubated at 37°C in a 5% CO2 incubator.

[0029] b) Preparation of myeloma cells: 36-48 h before fusion, expand the myeloma cell culture to induce logarithmic growth. On the day of fusion, use 15 mL of DMEM basal culture medium to aspirate the cells from the flask wall and collect them in a 50 mL centrifuge tube. Centrifuge at 1000 rpm for 10 min. Resuspend the cell pellet in 20 mL of DMEM basal culture medium and mix well. Take a small amount of the myeloma cell suspension, stain with trypan blue, and count the cells for later use.

[0030] c) Preparation of immune spleen cells: Blood was collected from the eyes of mice before fusion to prepare positive serum. Mice were euthanized by cervical dislocation and immersed in 75% alcohol for 10 min, then placed in a clean bench. The peritoneal cavity was aseptically opened, connective tissue was separated, and the spleen was removed. The spleen was placed in a petri dish containing a sterile nylon mesh and 15 mL of DMEM basal culture medium. The spleen was ground using the core of a sterile glass syringe to ensure all spleen cells passed through the mesh into the petri dish. The spleen cell solution was transferred to a 50 mL centrifuge tube, and DMEM basal culture medium was added to a final volume of 30 mL. The mixture was mixed, centrifuged at 1000 rpm for 8 min, and the supernatant was discarded. The cell pellet was resuspended in 10 mL of DMEM basal culture medium and mixed well. The cell suspension was then stained with trypan blue for counting and prepared for later use.

[0031] d) Fusion of spleen cells and myeloma cells: Preheat 65 mL of HAT culture medium, 15 mL of basal DMEM culture medium, and 1 mL of 50% PEG in a 37°C water bath. Prepare a 200 mL beaker containing 37°C water. Add the corresponding cell suspension volume (5 parts spleen cells to 1 part myeloma cells) to a 50 mL glass centrifuge tube, and add DMEM basal culture medium to a final volume of 30 mL. Mix well. Centrifuge at 1000 rpm for 10 min, discard the supernatant, and drain as much as possible. Gently tap the bottom of the centrifuge tube with your palm to loosen the cell pellet into a uniform paste. Place the centrifuge tube in a 200 mL beaker containing 37°C water. While rotating the centrifuge tube evenly with one hand, use a 1 mL Pasteur pipette to add 1 mL of 37°C 50% PEG solution over 1 min, and let it stand for 2 min. Then, slowly add DMEM basal culture medium, gradually increasing the speed, to stop the reaction. Incubate at 37°C for 10 min. Centrifuge at 1000 rpm for 10 min, discard the supernatant, add 65 mL of HAT medium, gently disperse the cells, and seed 0.1 mL per well into six 96-well culture plates containing feeder cells. Incubate at 37°C with 5% CO2. Replace half the medium after 5 days, and completely after 8 days. When the colonies have grown to 1 / 4-1 / 3 of the bottom area of ​​each well, collect the supernatant for analysis and replace with HT medium.

[0032] 1.2.2 Antibody Detection via Indirect ELISA The purified Lawsonia intracellularis whole-cell protein antigen was used to coat a 96-well plate. 100 μL of hybridoma cell culture supernatant was added, and the plate was incubated at 37°C for 1 h. After washing, HRP-labeled goat anti-mouse secondary antibody diluted 1:5000 was added. After washing, the substrate TMB was added and the plate was developed in the dark for 10 min. The absorbance was measured at a wavelength of 450 nm.

[0033] 1.2.3 Indirect Immunofluorescence IPEC cells were cultured into a monolayer in 96-well microplates, inoculated with Lawsonia intracellularis, and fixed with cold anhydrous ethanol after 48 h. 50 μL of hybridoma cell culture supernatant was added, and the cells were incubated at 37 °C for 40 min. After washing three times with PBS, FITC-labeled goat anti-mouse IgG antibody diluted 1:200 was added, and the cells were incubated at 37 °C for 40 min. After washing, the fluorescence intensity was observed under an inverted fluorescence microscope.

[0034] 1.2.4 Purification of Monoclonal Antibodies The ascites fluid was purified using the octanoic acid-saturated ammonium sulfate method. The procedure is briefly described below: (1) Take 3 mL of pretreated ascites fluid and add 6 mL of acetate buffer (0.06 mol, pH 4.8); add 99 μL of caprylic acid to the ascites fluid and stir at room temperature for 30 minutes, then let stand at 4℃ for more than 2 hours; take it out and centrifuge at 11000 rpm for 30 minutes, and discard the precipitate; adjust the pH of the supernatant to 7.4 with 2 mol / L NaOH; (2) Add saturated ammonium sulfate to the supernatant at 4℃ to 50% saturation, stir in an ice bath for 30 minutes, and let stand at 4℃ for more than 2 hours; centrifuge at 11000 rpm for 30 minutes and discard the supernatant; dissolve the precipitate in 5 mL PBS buffer (0.1M, pH7.4) to obtain the precipitate suspension; (3) Add an appropriate amount of saturated ammonium sulfate solution to the precipitate suspension while stirring, so that the concentration of ammonium sulfate solution is 30%-40%. Stir in an ice bath for 30 minutes, and let stand at 4℃ for more than 2 hours. Centrifuge at 11000 rpm for 30 minutes, take the precipitate, and dissolve the precipitate in 2 mL of PBS buffer to obtain the precipitate suspension. (4) The precipitate suspension was placed in a dialysis bag and dialyzed with PBS at 4°C, with the solution changed every 2 hours. Dialysis was performed for 2 days. After dialysis, the purified ascites fluid in the dialysis bag was collected, the protein concentration was determined by UV spectrophotometer, and the purity was detected by SDS-PAGE.

[0035] The monoclonal antibody was further purified by DEAE-Sephadex A-50 (GE) column chromatography. The purified monoclonal antibody was recovered, the protein concentration was determined by UV spectrophotometer, and the purity was detected by SDS-PAGE.

[0036] 1.2.5 Sequence identification of monoclonal antibodies The purified monoclonal antibody was sequenced.

[0037] 2. Experimental Results 2.1 Screening and identification of monoclonal antibodies against Lawsonia intracellularis Hybridoma cells derived from the fusion of spleen cells and SP2 / 0 cells from immunized mice were used to verify the presence of *L. intracellularis*-specific antibodies in their culture supernatant using indirect ELISA and IFA methods. The positive criterion was determined by the OD value of the hybridoma cell culture supernatant against *L. intracellularis* antigen. 450 Light absorbance values ​​and OD values ​​of intracellular Lawsonia antigen in normal BALB / c mouse serum and SP2 / 0 cell culture supernatant 450 The ratio of the values ​​was greater than 2.1, and the hybridoma cell supernatant did not produce a fluorescent reaction with normal IPEC cells, thus indicating a positive result. Antibody-positive hybridoma cells were subcloned using a three-stage limiting dilution method to obtain a hybridoma cell line that stably secretes antibodies, named PE-3A3, PE-3B7, and PE-4D5, respectively. The corresponding monoclonal antibodies were named 3A3, 3B7, and 4D5.

[0038] Immunoglobulin subclass identification showed that monoclonal antibody 3A3 has a heavy chain type of IgG2a and a light chain type of κ. Monoclonal antibody 3B7 has a heavy chain type of IgG2a and a light chain type of κ. Monoclonal antibody 4D5 has a heavy chain type of IgG2b and a light chain type of κ.

[0039] The culture supernatant of monoclonal antibody 3A3 was used to perform indirect immunofluorescence detection on IPEC cells infected with Lawsonia intracellularis. Figure 1 The results showed that 3A3 reacted positively with Lawsonia intracellularis, indicating that 3A3 is a Lawsonia intracellularis-specific monoclonal antibody. Monoclonal antibodies 3B7 and 4D5, however, did not react positively with Lawsonia intracellularis.

[0040] 2.2 Purification and labeling of monoclonal antibody ascites fluid Given the specific reactivity of monoclonal antibody 3A3, this invention further investigates its diagnostic value in the detection of intracellular Lawsonia solani antigen and antibody. The ascites fluid containing monoclonal antibody 3A3 prepared according to this invention was purified using an octanoic acid-saturated ammonium sulfate method, and the concentration of 3A3 was determined to be 6.47 mg / mL using a UV spectrophotometer. Further purification was performed by DEAE-Sephadex a-50 ion exchange chromatography, and HRP was labeled using a modified periodate method.

[0041] 2.3 Sequence identification results of monoclonal antibody 3A3 The amino acid sequence of the variable region of the light chain is: ELVMTQSPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLICLVSKLDSGVPDRFTGSGSGTDFTLKISRVQAEDLGIYYCVQGTHFPYTFGGGTKLEL (SEQ ID NO.1).

[0042] The amino acid sequence of the variable region of the heavy chain is: VHCEVQLQQSGPELVKPGASVKMSCKASGYIFTDHAMSWVRQSHGKRLEWIGDIIPYNDDTTFNQKFRAKATLTVDKSSSTTYMQLNSLTSEDSAVYYCTLEGSYDYDGAFTYWGQGTMVTI (SEQ ID NO.2).

[0043] Light chain CDR1: KSSQSLLYSNGKTYLN (SEQ ID NO.3); Light chain CDR2: LVSKLDS (SEQ ID NO.4); Light chain CDR3: VQGTHFPYT (SEQ ID NO.5); Heavy chain CDR1: DHAMS (SEQ ID NO.6); Heavy chain CDR2: DIIPYNDDTTFNQKFRA (SEQ ID NO.7); Heavy chain CDR3: EGSYDYDGAFTY (SEQ ID NO.8).

[0044] Nucleotide sequence of the light chain variable region encoding gene: GAGCTCGTGATGACCCAGTCTCCACTCACTTTGTCGGTAACCATTGGACAACCAGCCTCTATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTAATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTGTCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGCAGGCTGAGGATTTGGGAATTTATTACTGCGTGCAAGGTACACATTTTCCATACACGTTCGGAGGGGGGACCAAGCTGGAGCTG (SEQ IDNO.9).

[0045] Nucleotide sequence of the heavy chain variable region encoding gene: GTCCACTGCGAGGTTCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACATATTCACTGACCACGCCATGAGCTGGGTGAGGCAGAGCCATGGAAAGCGCCTCGAATGGATTGGAGATATTATTCCCTACAACGATGATACTACCTTCAATCAGAAGTTCAGGGCCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAACCTACATGCAGCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTACATTAGAGGGGTCCTATGATTACGACGGGGCCTTTACTTACTGGGGCCAAGGAACTATGGTCACAATC (SEQ ID NO.10).

[0046] Example 2 1. Detection method for intracellular Lawsonia lactiflora antigen – Establishment of antigen capture ELISA method 1) Coat the captured porcine anti-Lawsonia intracellularis polyclonal antibody with carbonate buffer (pH 9.6) and incubate overnight at 4°C. Wash the plate three times with PBS containing 0.05% Tween-20, 3 min each time. 2) Block the ELISA plate with 5% skim milk PBST, incubate at 37°C for 1 h, and wash the plate as above; 3) Add the antigen to be tested and the positive and negative control antigens, incubate at 37°C for 1 hour, and wash as above; 4) Add HRP-labeled detection monoclonal antibody 3A3, incubate at 37°C for 1 h, and wash as above; 5) Add the substrate and incubate at room temperature in the dark for 10 minutes; 6) Stop the reaction by adding 2 M H2SO4 (50 μL / well), and measure the absorbance A value (OD) at 450 nm using a microplate reader. 450 nm).

[0047] 2. Determination of the concentration of polyclonal antibodies used for coating and detection The concentrations of the coating polyclonal antibody and the detection monoclonal antibody were titrated using square titration, and the results are shown in Table 1. When the porcine anti-Lawsonia intracellularis polyclonal antibody was diluted 1:800 (3.1 µg / mL) and the HRP-labeled detection monoclonal antibody 3A3 was diluted 1:2000 (0.93 µg / mL), the OD... 450nm The value is close to 1.0, and the P / N ratio is the largest.

[0048] Table 1. Optimal coating concentration of polyclonal antibodies and optimal concentration of monoclonal antibodies for detection, obtained from square matrix titration. 3. Sensitivity test Lawsonia intracellularis antigen (2.3 mg / mL) was serially diluted and detected by ELISA. The results are shown in Table 2. The lowest detection limit for Lawsonia intracellularis antigen was 7.6 μg / mL, and the lowest detection limit for Lawsonia intracellularis cytotoxicity was 5.9 × 10⁻⁶. 3 TCID 50 .

[0049] Table 2. Results of Sensitivity Test Example 3 To verify the application value of monoclonal antibody 3A3 in the detection of Lawsonia intracellularis antibodies, a competitive ELISA method for detecting Lawsonia intracellularis antibodies was initially established using monoclonal antibody 3A3. Serially diluted negative sera, strongly positive sera, and weakly positive sera were detected, and the percentage inhibition rate was calculated.

[0050] The competing ELISA detection methods are as follows: 1) Dilute the purified Lawsonia intracellularis whole cell protein antigen with carbonate buffer (pH 9.6) at the concentration used for coating onto an ELISA plate, 50 μL / well, and incubate overnight at 4°C. The next day, remove the ELISA plate, discard the liquid, wash 5 times with pH 7.4 PBST, and shake dry. 2) Negative serum and positive serum were serially diluted from 1:8 to 1:512, 50 μL / well, and added to the ELISA plate; 3) Immediately add 50 μL of HRP-3A3 diluted with diluent to each well of the ELISA plate, shake thoroughly to mix, and incubate at 37°C for 1 h. At this point, the reaction volume in the ELISA plate is 100 μL. 4) Wash the plate 5 times as above, add 50 μL of TMB substrate solution per well, and incubate at room temperature in the dark for 15 min. After 15 min, add 50 μL of 2M H2SO4 stop solution per well to stop the reaction, and read the OD value on a microplate reader. 450nm value.

[0051] Result Interpretation: The inhibition percentage was calculated by testing known negative and positive sera: PI = 100% - OD of the tested serum. 450nm / Blank control OD 450nm .

[0052] Test results as follows Figure 2 As shown, the ELISA method was used to detect strongly positive, weakly positive, and negative serum of Lawsonia intracellularis, and the percentage inhibition curves were good, which can specifically distinguish between positive and negative serum of Lawsonia intracellularis.

[0053] The above experimental results confirm that monoclonal antibody 3A3 has application value in establishing methods for detecting intracellular Lawsonia antigen and its antibodies.

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody to L. intracellularis, characterized in that, the light chain CDR1 comprising the amino acid sequence as shown in SEQ ID NO. 3, the light chain CDR2 comprising the amino acid sequence as shown in SEQ ID NO. 4, the light chain CDR3 comprising the amino acid sequence as shown in SEQ ID NO. 5, the heavy chain CDR1 comprising the amino acid sequence as shown in SEQ ID NO. 6, the heavy chain CDR2 comprising the amino acid sequence as shown in SEQ ID NO. 7, and the heavy chain CDR3 comprising the amino acid sequence as shown in SEQ ID NO.

8.

2. The monoclonal antibody of L. intracellularis according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the L. intracellularis monoclonal antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

2.

3. A hybridoma cell line secreting monoclonal antibodies to L. intracellularis, characterized in that, The hybridoma cell line is deposited in China General Microbiological Culture Collection Center on December 3, 2025, at the address of No. 1, Beichen West Road, Hua-yang District, Beijing, with the preservation number of CGMCC No. 46760.

4. A coding gene of the L. intracellularis monoclonal antibody according to claim 1.

5. The genetic code according to claim 4, wherein, The coding gene comprises a light chain variable region coding gene and a heavy chain variable region coding gene; The nucleotide sequence of the light chain variable region coding gene is shown in SEQ ID NO. 9; The nucleotide sequence of the heavy chain variable region coding gene is shown in SEQ ID NO.

10.

6. A biomaterial, characterized by, The biological material is the substance according to (1) or (2): (1) a recombinant expression vector comprising the coding gene according to claim 4 or 5; (2) a recombinant non-plant host cell comprising the recombinant expression vector.

7. Use of the hybridoma cell line according to claim 3, the coding gene according to claim 4 or 5, or the biological material according to claim 6 in the preparation of a L. intracellularis monoclonal antibody.

8. Use of the L. intracellularis monoclonal antibody according to claim 1 or 2 in the preparation of an ELISA detection kit for detecting L. intracellularis antigens or antibodies.

9. An ELISA test kit for detecting L. intracellularis antigen, characterized in that, The L. intracellularis monoclonal antibody according to claim 1 or 2.

10. An ELISA test kit for detecting antibodies to Lawsonia intracellularis, characterized in that The L. intracellularis monoclonal antibody according to claim 1 or 2.

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