Preparation of a hybridoma cell and use thereof

Abstract

The application belongs to the field of animal bacteriology and molecular biology technology, and relates to preparation of a hybridoma cell and application thereof. A monoclonal antibody is prepared through intracellular Lawsonia intracellularis outer membrane protein OmpA expressed by prokaryotes, and IFA, WB and blocking ELISA and other immunological detection methods for detecting or diagnosing Lawsonia intracellularis are established through the expressed intracellular Lawsonia intracellularis outer membrane protein OmpA and the prepared monoclonal antibody. The blocking ELISA for detecting serum antibodies of Lawsonia intracellularis can detect serum samples of suspected Lawsonia intracellularis infection from different species, and does not exist cross reaction with positive serum of common pig pathogens, has high sensitivity and specificity, is good in repeatability, and is suitable for high-throughput detection of serum samples.

Description

Technical Field

[0001] This invention belongs to the fields of animal bacteriology and molecular biology, and relates to the preparation and application of a hybridoma cell. Specifically, it relates to a monoclonal antibody specific to the OmpA outer membrane protein of Lawsonia lactiflora, its preparation method, and its application. Background Technology

[0002] Lawsonia intracellularis (LI) primarily causes porcine proliferative enteropathy (PPE), characterized by proliferative hemorrhagic enteropathy (PHE) and porcine intestinal adenomatosis (PIA). LI mainly infects weaned piglets and finishing pigs, presenting with acute, chronic, and subclinical clinical symptoms, with the chronic form being the most common. In severe cases, it can lead to diarrhea and even death. Porcine proliferative enteropathy is a common disease in intensive pig farms, widely prevalent in all major pig-producing countries worldwide. Besides occasionally causing acute mortality in medium and large pigs, its main harm to the pig industry is the widespread chronic infection of LI, which severely impacts feed conversion ratios and causes significant economic losses to the global pig farming industry.

[0003] To date, six *L. li* genome sequences are deposited in the National Center for Biotechnology Information (NCBI) database, including the complete genome sequences of two porcine pathogenic isolates. The complete genome of the *L. li* strains comprises a 1.4 Mb chromosome and three plasmid genomes of 27, 39, and 194 kb, containing 1324 ORFs. Molecular studies of *L. li* isolates from various animal species, including horses and hamsters, showed 98% homology with the 16S-RNA of porcine isolates. Furthermore, phenotypic characterization of outer membrane proteins and immunoblotting of different *L. li* isolates showed only minor differences between the isolates, and antigenic relevance was unaffected. This demonstrates that there is little or no genetic variation among *L. li* isolates.

[0004] The outer membrane protein OmpA of *L. intracellularis* is composed of 340 amino acids. It is a hydrophilic protein with good immunogenicity and conservation. Although the molecular mechanisms underlying OmpA in *L. intracellularis* have not been characterized, in many pathogenic bacteria, due to its high extracellular expression, OmpA-like proteins participate in various important pathogenic effects, including bacterial adhesion, invasion, intracellular survival, and evasion of host defenses or stimulation by pro-inflammatory cytokines. These pathogenic effects are most commonly associated with diseases of the central nervous system, respiratory system, and genitourinary system. Therefore, OmpA protein has also become a potential candidate antigen for subunit vaccines.

[0005] Currently, the main methods used domestically and internationally for diagnosing Lawsonia intracellularis infection are serological testing and various PCR methods for detecting fecal samples. PCR is the most commonly used pathogen detection method in laboratories and clinics. However, PCR is only suitable for detecting fecal samples from infected animals during the spore-shedding period. The genome extraction process from feces is relatively cumbersome and not suitable for testing large numbers of samples. Serological diagnosis mainly relies on enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA). These two methods not only have high sensitivity and specificity, but also allow specific antibodies against Lawsonia intracellularis to persist in vivo for a longer period. The disadvantage is that antibodies cannot be detected for at least two weeks after infection. In recent years, the economic losses caused by PPE in pig farms in my country have become increasingly severe. Therefore, there is an urgent need for differential diagnostic methods for Lawsonia intracellularis to conduct epidemiological surveys, evaluate vaccine immunization efficacy, and assess the effectiveness of prevention and control measures in Chinese pig populations.

[0006] Since there are currently no commercially available ELISA test kits for serological diagnostics in China, there is a need to provide a mature ELISA detection method to fill this gap in the field. This invention prepares a specific monoclonal antibody against the intracellular Lawsonia outer membrane protein OmpA using cell fusion hybridoma technology and establishes immunological detection methods such as WB, IFA, and blocking ELISA. Summary of the Invention

[0007] This invention addresses the technical problem at hand and overcomes the shortcomings of existing technologies by providing a method for preparing and applying a hybridoma cell. Specifically, it relates to a monoclonal antibody specific to the intracellular Lawsonia outer membrane protein OmpA, its preparation method, and its application.

[0008] Preparation and application of a monoclonal antibody specific to the outer membrane protein OmpA of Lawsonia intracellularis: The monoclonal antibody specific to the outer membrane protein OmpA of Lawsonia intracellularis was prepared using cell fusion hybridoma technology, and the prepared monoclonal antibody specific to the outer membrane protein OmpA of Lawsonia intracellularis was successfully applied to various immunological assays. Based on the prepared recombinant antigen and its monoclonal antibody, a blocking ELISA detection method for detecting the outer membrane protein OmpA of Lawsonia intracellularis was established, which can be used for the development of commercial kits.

[0009] One of the technical solutions of this invention is to provide a preparation of a monoclonal antibody against the outer membrane protein OmpA of Lawsonia intracellularis. The monoclonal antibody against OmpA is produced by hybridoma cell line 5F2, which is named hybridoma cell line 5F2. Hybridoma cell line 5F2 was deposited with Wuhan University, Wuhan, China on September 15, 2023; the deposit number is CCTCC NO: C2023263.

[0010] The monoclonal antibody against OmpA, an outer membrane protein of Lawsonia intracellularis, of the present invention can be used for the immunological detection of OmpA, an outer membrane protein of Lawsonia intracellularis.

[0011] Furthermore, the monoclonal antibody against the intracellular Lawsonia outer membrane protein OmpA can be used in ELISA, WB, or IFA experiments to detect the intracellular Lawsonia outer membrane protein OmpA.

[0012] The second technical solution of the present invention is to provide an antibody preparation comprising the above-mentioned monoclonal antibody against the outer membrane protein OmpA of Lawsonia intracellularis, or the antibody preparation comprising a fragment of the monoclonal antibody.

[0013] The antibody preparation described further is used for the effective diagnosis or detection of Lawsonia intracellularis.

[0014] The third technical solution of the present invention is to provide the application of the monoclonal antibody against the outer membrane protein OmpA of Lawsonia intracellularis in blocking the detection of Lawsonia intracellularis by ELISA.

[0015] This invention also provides the application of the above-mentioned monoclonal antibody and antibody preparation of Lawsonia intracellularis outer membrane protein OmpA in the preparation of diagnostic or detection methods for Lawsonia intracellularis.

[0016] This invention prepares two monoclonal antibodies using the prokaryotically expressed Lawsonia intracellularis outer membrane protein OmpA. These two monoclonal antibodies can be used for various immunological assays, including ELISA, WB, and IFA, demonstrating wide applicability. Simultaneously, based on the prepared recombinant antigen and one of the monoclonal antibodies, a blocking ELISA method for detecting Lawsonia intracellularis was explored and optimized, achieving a higher concordance rate compared to existing commercial kits.

[0017] The monoclonal antibody specific to the outer membrane protein OmpA of Lawsonia intracellularis provided by this invention is a particularly useful tool for Lawsonia intracellularis research and has been successfully applied to various immunological assays, including enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), and immunofluorescence assay (IFA). Simultaneously, this invention establishes a blocking ELISA method for detecting Lawsonia intracellularis, providing a new reference for the detection of Lawsonia intracellularis.

[0018] Explanation of Preservation Certificate and Visibility Information

[0019] The strain deposited is named Hybridoma cell line 5F2, with accession number CCTCCNO: C2023263, deposited on September 15, 2023, by the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. Attached Figure Description

[0020] Figure 1 This is a diagram illustrating the construction of the pET30a-ompA plasmid of this invention.

[0021] Figure 2 This is a diagram illustrating the identification of the target fragment of ompA amplified by PCR in this invention.

[0022] Figure 3 This is a double enzyme digestion identification diagram of the pET30a-ompA plasmid of the present invention.

[0023] Figure 4 This is a schematic diagram showing the SDS-PAGE Coomassie Brilliant Blue staining results of the recombinant purified LIOmpA protein in this invention.

[0024] Figure 5 This is a schematic diagram of the Western blotting identification results of the recombinant purified LIOmpA protein in this invention.

[0025] Figure 6 This is a schematic diagram of the Western blotting identification results of the LIOmpA monoclonal antibody 5F2 clone in this invention.

[0026] Figure 7 This is a schematic diagram of the Western blotting identification results of the LIOmpA monoclonal antibody 12F6 clone in this invention.

[0027] Figure 8 This is a schematic diagram of the IFA detection results of the 5F2 monoclonal antibody in this invention.

[0028] Figure 9 This is a schematic diagram of the IFA detection results of the 12F6 monoclonal antibody in this invention.

[0029] Figure 10 This is a schematic diagram of the detection results of the 5F2 and 12F6 monoclonal antibody subclasses in this invention.

[0030] Figure 11 This is a schematic diagram of the SDS-PAGE Coomassie Brilliant Blue staining results of the purified 5F2 ascites monoclonal antibody in this invention.

[0031] Figure 12 This is a schematic diagram of the ROC curve analysis of the blocking ELISA detection results in this invention. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0033] Explanation of the sequence list:

[0034] (1) SEQ ID NO:1 is the nucleotide sequence of ompA in the pET30a-ompA recombinant plasmid in Example 1 of this invention. The specific sequence is as follows.

[0035] Atgaaaattatccattctgcaatttttgctgttacattattaacagcatggtcaacagtttgctttgctgcagaagttac

[0036] agctagttgtactaaacgtgttgaaagctataattatcttgtggattattcaggctctatgatgatgaaacatgttgctg

[0037] ttagagagcctaaaatagaattagcaaaagaagctatattaaaaattaatgcggcaatgcctaaaatgtcatatcaaggt

[0038] ggattatatacttttgcaccttattctgtaattattccccaaggttcttggaattcatgtgttgccgaatgtgcggttaa

[0039] tacaattaagtctgatttagaaatttttggtcgtcttactcctatggggagacggcataaaaatgcatgaaacagtcatta

[0040] atcaaatgccccctcaggcagccgttattcttctcactgatggtcataataatttagggatgaatcctgttgaggaagta

[0041] aaatctatatatcaaacaaatcctaatgtttgttttcatgtagtttcatttgcagatgatgctgaaggcaaagcaataat

[0042] tgatcaaattgttgcacttaatagtggaagtgttcttgttgatggtttacagcttctacaaaatcctgctgtttgccaag

[0043] aatttgttaatagtgttttttgtcaagagcaaattcttgttacagaagaagttgttgtacttcgtggtgtaaactttgct

[0044] tttgattcttttgcattagatgatactgctaaagctattttagaagaaacagttcgtcttatcagagcaaatccagattt

[0045] taatgttcgtttgcttgggtggacagatagtactggtcctgatgcatataacttgcgtttatcacaagaacgtgctgatg

[0046] cagttaaaaacttcttggttaaaatgggtataccttcaaatcgtttatttgctaaaggaatgggtaaatcctatcagtat

[0047] aataatgctacaaaagaaggacgatatatgaatcgtcgtacagaacttgtcttttttgattag

[0048] (2) SEQ ID NO:2 is the specific sequence of the amino acid sequence list of ompA in the pET30a-ompA recombinant plasmid in Example 1 of the present invention, which is described as follows.

[0049] MKIIHSAIFAVTLLTAWSTVCFAAEVTASCTKRVESYNYLVDYSGSMMMKHVAVREPKIELAKEAILKINAAMPKMSYQG

[0050] GLYTFAPYSVIIPQGSWNSCVAECAVNTIKSDLEIFGRLTPMGDGIKMHETVINQMPPQAAVILLTDGHNNLGMNPVEEV

[0051] KSIYQTNPNVCFHVVSFADDAEGKAIIDQIVALNSGSVLVDGLQLLQNPAVCQEFVNSVFCQEQILVTEEVVVLRGVNFA

[0052] FDSFALDDTAKAILEETVRLIRANPDFNVRLLGWTDSTGPDAYNLLRLSQERADAVKNFLVKMGIPSNRLFAKGMGKSYQY

[0053] NNATKEGRYMNRRTELVFFD

[0054] Example 1: LiOmpA gene cloning and expression, protein purification, and preparation of monoclonal antibodies

[0055] 1.1 Screening and preparation of strains, plasmids, cells, and experimental animals

[0056] The pET30a-ompA plasmid was preserved in our laboratory (prepared using standard experimental methods) and used to express the OmpA recombinant protein; SP2 / 0 myeloma cells were revived and preserved in our laboratory; BALB / c mice used for cell fusion were purchased from Henan Skebers Biotechnology Co., Ltd.

[0057] The encoding nucleotide sequence of the ompA gene is shown in SEQ ID NO:1 of the sequence listing.

[0058] 1.2 Main experimental reagents and consumables

[0059] SDS-PAGE kit purchased from Yageo; RPMI Medium 1640 basic (1x) purchased from Gibco; Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), PEG 50, HAT Media Supplement, HT Media Supplement, and DMSO purchased from Sigma-Aldrich; FBS serum purchased from Yeasen; TMB two-component chromogenic solution and stop solution purchased from Solarbio; HRP-labeled goat anti-mouse IgG antibody, BCA protein quantification kit, and BeyoGold... TMHis-tag Purification Resin (reduction-resistant chelating type) was purchased from Beyotime Biotechnology Co., Ltd.; Tween-20 was purchased from Nanjing Yifeixue Biotechnology Co., Ltd.; LB solid powder was purchased from Oxoid; IPTG, ampicillin, and NAD coenzyme powder were purchased from Biosharp; monoclonal antibody subtype identification kit was purchased from Proteintech; cell culture plates were purchased from Biofil; other chemical reagents were commercially available and commonly used. All reagents were prepared fresh for use by the laboratories of the College of Animal Science and Technology and the College of Veterinary Medicine, where the applicant is located, and were of analytical grade. 1.3 Construction of LI-ompA prokaryotic expression plasmid

[0060] 1.3.1 Cloning of the LI ompA gene

[0061] Based on the bioinformatics prediction results of LI ompA, a signal-free peptide and transmembrane region ompA (uompA) were designed, and a recombinant plasmid map was constructed using snapGene 3.2.1 (see [link to article]). Figure 1 Upstream and downstream primers were designed: uompA-F and uompA-R (the primers contain BamH-Ⅰ and Not-Ⅰ restriction sites, see Table 1 for details). The primers were synthesized by Wuhan Qingke Biotechnology Co., Ltd. See Tables 1 and 2:

[0062] Table 1. Primer information for amplifying the ompA gene.

[0063]

[0064] The target gene was synthesized using porcine LI attenuated live vaccine DNA as a template. The reaction system is shown in Table 2.

[0065] Table 2. PCR reaction system for amplifying the ompA gene

[0066]

[0067] Following the above reaction system, the target fragment was amplified using a reaction program of 98℃ pre-denaturation for 3 min, 98℃ denaturation for 15 s, 50℃ annealing for 15 s, 72℃ extension for 15 s, repeated 30 times, and a final extension at 72℃ for 3 min. The amplified fragment size was 1023 bp (see [link]). Figure 2 ).

[0068] 1.3.2 Construction of recombinant plasmid pET30a-ompA

[0069] After a positive result was obtained from 1% agarose gel electrophoresis, the ompA fragment was excised and recovered. The reaction sample was mixed with 5× Loading Buffer, and after identification by 1% agarose gel electrophoresis, the target band was excised, and the target gene was recovered and purified according to the DNA purification kit instructions. Using pET30a as a vector, pET30a and the above PCR amplification product were digested with BamH-I and Not-I enzymes and ligated to construct the recombinant plasmid pET30a-ompA. Double enzyme digestion identification results showed that the recombinant plasmid was successfully constructed (see...). Figure 3 ).

[0070] 1.4 Expression and purification of recombinant OmpA protein from Lawsonia intracellularis

[0071] First, *E. coli* containing the pET30a-OmpA recombinant plasmid, stored in an ultra-low temperature freezer, were revived. 100 μL of this bacterial culture was evenly spread onto a Kana-resistant plate and incubated overnight at 37°C. Single colonies were picked and inoculated into 700 μL of LB broth containing Kana resistance, and shaken at 37°C for approximately 8-12 hours. 100 μL of the above bacterial culture was then inoculated into 10 ml of LB broth containing Kana resistance and shaken at 37°C for approximately 8-12 hours to obtain the revived recombinant expression bacteria. The revived bacteria were then inoculated into 1 L of LB broth containing Kana resistance and shaken at 37°C until OD (dose elapsed). 450nm The value was adjusted to 0.6-0.8. OmpA recombinant protein was expressed by induction with 0.8 mmol / L IPTG at 25°C for 16 h. The purified recombinant antigen was obtained by His-tag affinity chromatography; SDS-PAGE (see [link to SDS-PAGE]) was then performed. Figure 4 ) and Western-Blot (see Figure 5 Verify the correctness and immunogenicity of the recombinant protein. After verifying the correctness of the recombinant protein, dialyze and concentrate it, then determine the protein concentration using the BCA method, aliquot it into 1.5 ml Eppendorf tubes, and store it at -80°C for later use.

[0072] 1.5 Mouse Immunization

[0073] The immunogenic antigen was the prepared LIOmpA protein. The immunization procedure was as follows: 200 μg of recombinant protein was emulsified with Freund's complete adjuvant at a 1:1 ratio and administered intraperitoneally to Balb / C mice. On days 14 and 28, the same dose of recombinant protein was emulsified with Freund's incomplete adjuvant at a 1:1 ratio for the second and third immunizations. Seven days after the third immunization, blood was collected to detect serum antibody titers. Once the mouse serum antibody titer reached 1:12800, the mice with the highest antibody titer were selected for a shock immunization. Cell fusion was performed 3 days after the shock immunization.

[0074] 1.6 Antibody Detection Using Indirect ELISA Method

[0075] 1.6.1 Preparation of serum samples for testing

[0076] Five mice had their blood collected from their tail tips. The blood was incubated at 37°C for 30 minutes, then transferred to a 4°C freezer and left to stand overnight. The blood was then centrifuged at 4000 rpm for 5 minutes, and the supernatant was collected as the serum for testing. Any remaining serum after testing could be stored at -80°C.

[0077] 1.6.2 Preparation of antigen-coated plates and determination of serum titer

[0078] The LIOmpA protein concentration was adjusted to 1 μg / mL using 0.05 mol / L carbonate buffer (pH 9.6). 100 μL of antigen coating solution was added to each ELISA well using a multi-channel pipette, and the plate was incubated overnight at 4°C. The liquid was discarded, and the plate was washed five times with 300 μL of PBST per well, then blotted dry. Subsequently, 100 μL of 2% BSA was added to each well for blocking, and the plate was incubated at 37°C for 2 hours. The blocking solution was then discarded, and the plate was washed with PBST again as described above, followed by blotting dry. For titer assay, 200 μL of 1:100 diluted immunopositive mouse serum and unimmunized mouse serum were added to the first well of each row. For the remaining wells, 100 μL of PBST was added first, and then 100 μL of the mixed solution was added to the second well, and so on, serially diluted until the sixth well. The remaining 100 μL of diluted solution was discarded, and the plate was incubated. After 1 hour, discard the liquid and wash the plate. Add HRP-labeled goat anti-mouse IgG antibody, diluted 1:5000 with PBST, and add to the wells. Incubate for 1 hour, then wash and pat dry. Add 100 μL TMB to the wells for color development. Incubate in the dark for 10 minutes. Adjust the microplate reader to OD500. 450nm Prepare for reading. After color development, add 50 μL of 2M sulfuric acid stop solution and take the reading. When the P / N value is ≥2.1 and the OD value is ≥2.1, the reading is taken. 450nm A value >0.4 indicates a positive result. The highest dilution at which a serum antibody is considered positive is the serum antibody titer.

[0079] 1.7 Cell Fusion

[0080] 1.7.1 Preparation of materials, instruments and equipment, and reagents

[0081] Materials: 96-well cell culture plate, sterile 50mL centrifuge tubes, sterile 15mL centrifuge tubes, sterile filter paper, sterile grinder, sterile rodenticide, sterile 10mL pipettes; 40μm cell sieve, cell counting plate, dissection plate and fixation needle, alcohol swabs.

[0082] Instruments and equipment: water bath, centrifuge, balance, 37℃ constant temperature chamber.

[0083] Reagents: 75% ethanol, 100mL RPMI 1640 medium (containing 1% antibiotics), 100mL HAT medium (1% antibiotics, 2% HAT, 20% newborn fetal bovine serum, RPMI 1640 added to 100mL).

[0084] 1.7.2 Preparing feeder cells

[0085] One day before fusion, mature and healthy Balb / C mice were euthanized by cervical dislocation, immersed in 75% ethanol for 5 minutes, and then placed in a glass dish sterilized in a laminar flow hood. After sterilizing the dissection plate with 75% ethanol, fix it on the plate and, under aseptic conditions, cut along the midline of the abdomen, bluntly separating the skin and peritoneum to fully expose the abdomen. Using a sterile syringe, inject an appropriate amount of HAT into the mouse's peritoneal cavity (avoiding penetration into internal organs). Gently massage the mouse's abdomen and aspirate the culture medium used to rinse the abdomen. Repeat this step twice. Aseptically open the peritoneal cavity and remove the spleen. Place it in a grinder containing 5 mL of serum-free RPMI 1640 medium and slowly grind it 6-8 times using the provided grinding rod. Add 5 mL of serum-free RPMI 1640 medium and let it stand for 3 minutes. Transfer 5 mL of the upper cell suspension to a 50 mL centrifuge tube. Repeat the above operation 2-3 times to fully release the spleen cells. Filter the lower cell suspension through a 40 μm cell sieve and transfer the filtrate to a 50 mL centrifuge tube. Finally, add serum-free RPMI 1640 to make up to 30 mL, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, and use 10 mL of... Resuspend the precipitate in HAT, then add HAT to make up to 60 mL, mix thoroughly, and add to a 96-well cell culture plate at 100 μL / well. Incubate overnight at 37°C with 5% CO2. Observe the bottom of the cell culture plate the next day. If it is clear and transparent without turbidity, cell fusion can be performed. If contamination occurs, prepare feeder cells again according to the above steps.

[0086] 1.7.3 Collection of myeloma cells (SP2 / 0)

[0087] Two weeks prior to fusion, SP2 / 0 cells were revived, and the medium was changed daily according to cell growth. Subculture was performed approximately every two days to ensure a cell mass exceeding 2 × 10⁶ cells before fusion. 7 Excess cells in good condition can be cryopreserved. When preparing for fusion, select SP2 / 0 cells with good morphology and logarithmic growth, wash them with serum-free RPMI 1640 and blow them off, transfer them to a pre-autoclaved 50mL centrifuge tube, and finally add serum-free RPMI 1640 to make up to 30mL. Incubate at 1000r / 10min, discard the supernatant, resuspend the pellet with 10mL of serum-free RPMI 1640, and place in a beaker at 37℃ for water bath.

[0088] 1.7.4 Collection of immune spleen cells

[0089] On the third day after booster immunization, blood was collected from the eyeballs of immunized mice and serum was separated as an antibody positive control. The serum was then aliquoted and stored in a -80°C freezer. Mice euthanized by cervical dislocation were immersed in 75% alcohol for 5 minutes, then placed abdomen-up. The limbs were pinned to a sterile rodenticide board. The abdominal cavity was aseptically opened, and the spleen was removed. The spleen was placed in a grinder containing 5 mL of serum-free RPMI 1640 medium and slowly ground 6-8 times using the provided grinding rod. Another 5 mL of serum-free RPMI 1640 medium was added, and the mixture was allowed to stand for 3 minutes. 5 mL of the supernatant cell suspension was transferred to a 50 mL centrifuge tube. This process was repeated 2-3 times to fully release the spleen cells. The supernatant was then filtered through a 40 μm cell sieve, and the filtrate was transferred to a 50 mL centrifuge tube. Finally, serum-free RPMI 1640 was added to bring the volume to 30 mL. The mixture was centrifuged at 1000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in 10 mL of serum-free RPMI 1640. The mixture was then placed in a beaker at 37°C for water bath preparation.

[0090] 1.7.5 Cell Fusion Methods

[0091] (1) Preheat HAT and RPMI 1640 culture media for 30 minutes in advance; separately aspirate the prepared spleen cell and SP2 / 0 cell suspensions and add them to 50 mL centrifuge tubes, then gently mix. Add serum-free RPMI 1640 to bring the suspension volume to 30 mL, centrifuge at 1000 r / min for 10 minutes, and discard as much supernatant as possible. Gently tap the bottom of the tube with your fingers and gently tap the bottom against the edge of the laminar flow hood to loosen the cell pellet. Place the centrifuge tubes in a beaker at 37°C for later use.

[0092] (2) Preheat a 1.5 mL centrifuge tube containing 1 mL of PEG50 in a 37°C water bath. Use a Pasteur pipette to add 1 mL of PEG50 to the centrifuge tube while gently stirring. Add the PEG50 dropwise over an average of 1 minute. After adding the PEG50, gently stir for 30 seconds to ensure thorough mixing with the cells, then let it stand for 30 seconds. Slowly add 10 mL of preheated serum-free RPMI 1640 over 5 minutes. Add one drop every 2 seconds for the first 30 seconds of the first minute, and one drop every 1 second for the next 30 seconds. Add 2 mL at the second minute and 3 mL at the third minute, continuing until all 10 mL of RPMI 1640 culture medium has been added. Incubate at 37°C for 10 minutes, then centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Add an appropriate amount of HAT medium, pipette and resuspend the confluent cells, gently shake to mix the precipitated cells evenly, and then plate the cells. The next day, observe the board for any signs of contamination. If contamination occurs, seal the hole and the surrounding 8 holes with 1M NaOH.

[0093] (3) After culturing the cell plates in a 37°C, 5% CO2, saturated humidity incubator for 5 days, observe the cell growth under a microscope. Record the growth of cell clusters in each well in a timely manner. On days 6-8 of fusion, use HAT medium for half-medium replacement. On day 9, start using HT medium. Observe the growth rate and status of cell clusters in the wells daily. When the cell clusters occupy about 1 / 10 of the bottom of the well, positive hybridoma cell clones can be screened for the first time.

[0094] 1.7.6 Screening and subcloning of antibody-secreting hybridoma cell clusters

[0095] Hybridoma cell clones secreting antibodies were screened using an indirect ELISA method. 96-well plates were coated with purified LIOmpA protein. Positive serum separated from ocular blood was diluted 1:100 as a positive control; the negative control was SP2 / 0 supernatant. The ELISA reader was set to OD... 450nm After reading the values, a P / N ratio ≥ 2.1 indicates a positive result. Subcloning of the positive hybridoma is performed at least three times using limiting dilution, and the supernatant is examined. If there are many positive wells, wells with higher P / N ratios and better cell growth are selected for subsequent subcloning operations.

[0096] The subcloning process in this invention employs the limiting dilution method. The preparation method for feeding cells the previous day is described in section 1.6.2. The optimal time for subcloning is when the clonal cluster in the well is approximately 1 / 4 to 1 / 2 of the total well size. Generally, three subcloning operations are required to obtain a stable antibody-secreting hybridoma cell line. After each subcloning, when the cell cluster reaches 1 / 4 of its original size, the supernatant is aspirated for testing, following the method described in section 1.6.6 of this embodiment for screening and subcloning antibody-secreting hybridoma cell clusters. Positive wells with a single cell cluster are selected for each subcloning operation. Subcloning is considered complete when all subcloned cell clusters are positive. Two monoclonal antibodies were prepared and named 5F2 and 12F6.

[0097] Example 2: Identification of LIOmpA protein monoclonal antibody

[0098] 1.1 Western blot specificity analysis of monoclonal antibodies

[0099] Take 80 μL of OmpA protein solution, add 20 μL of 5×SDS PAGE Loading Buffer, mix well, and boil at 100℃ for 10 min. Perform 12.5% ​​SDS PAGE gel electrophoresis at a loading rate of 20 μg / well. After transferring to a PVDF membrane, place the PVDF membrane in 5% skim milk (prepared with PBST) and incubate overnight at 4℃. Dilute the hybridoma cell supernatant 10-fold with PBST and incubate with the PVDF membrane at room temperature for 2 h. Add a 1:5000 diluted secondary antibody (HRP-goat anti-mouse IgG antibody) and incubate at 37℃ with a shaker at 60 rpm for 1 h. Discard the secondary antibody, wash as above, and mix equal volumes of 500 μL each of solution A and solution B from the ECL chemiluminescent substrate development solution (prepare fresh for use). Drop an appropriate amount of the above liquid onto the membrane and expose it in a gel imaging system for analysis to identify the reactivity of the two monoclonal antibodies to LIOmpA (see...). Figure 6 , Figure 7 ).

[0100] 1.2 IFA-specific analysis of monoclonal antibodies

[0101] McCoy cells in good growth condition were divided into groups of 1×10 6 Cells were seeded per well into 6-well cell culture plates, 1 mL / well, and cultured at 37°C in a 5% CO2 incubator for 12 h. The cell culture medium was discarded, and 1 mL of a 1:10 diluted live porcine ileitis vaccine was seeded into 3 wells, with the remaining 3 wells serving as negative controls. The plates were then incubated in a tri-gas incubator for 24 h. The cell supernatant was discarded, and the cells were washed three times with PBS for 5 min each time. Then, 1 mL of PBS was added to each well. Fix with 4% paraformaldehyde at room temperature for 10 min, wash three times with PBS, 5 min each time; add 500 μL of pre-chilled methanol to each well and incubate at room temperature for 20 min, wash three times with PBS, 5 min each time; add 1:200 diluted 12F6 and 5F2 monoclonal antibodies (500 μL / well) to the corresponding positive and negative control wells, incubate at 37°C for 1 h, wash as above; add 1:5000 diluted FITC-labeled goat anti-mouse fluorescent secondary antibody (500 μL / well) in the dark, incubate at 37°C for 45 min, wash as above; add 100 μL of DAPI staining solution to each well, react at room temperature in the dark for 15 min, wash as above; discard DAPI, wash as above, add PBS to cover the bottom of the wells for preservation; place the cell culture plate in the dark and observe under an inverted fluorescence microscope to identify the reactivity of the two monoclonal antibodies to LI IFA (see Figure 8 , Figure 9 ).

[0102] 1.3 Monoclonal antibody stability analysis

[0103] Stability analysis was performed on monoclonal antibodies 5F2 and 12F6 by expanding culture and passage. The antibody titers in the supernatant were measured by indirect ELISA (refer to the 1.5 indirect ELISA method of Example 1) when the 1st, 10th, 15th and 20th generation monoclonal antibodies were selected. The results showed that the 5F2 and 12F6 monoclonal antibodies had good stability in the first 20 generations (see Table 3).

[0104] Table 3. Potency of supernatant from culture of different passages of 5F2 and 12F6 monoclonal antibodies

[0105]

[0106] 1.4 Monoclonal Antibody Subtype Identification Analysis

[0107] Follow the instructions for the Proteintech KMIA-2 kit, specifically as follows:

[0108] (1) Take 10 μL of 12F6 and 5F2 cell supernatant and 990 μL of 1×PBST in a 1.5 mL centrifuge tube, mix well and add 50 μL to each well of the plate.

[0109] (2) Add goat anti-mouse 1×IgG+IgA+IgM-HRP to the detection plate at a rate of 50 μL / well. Gently tap the plate rack to mix well, and react at room temperature in the dark for 1 h. Then wash with 1×PBST and blot dry.

[0110] (3) Add 100 μL of freshly prepared AB solution to it and develop color in the dark for 15 min.

[0111] (4) The reaction was terminated with 100 μL of the stop solution.

[0112] (5) OD reading from the microplate reader 450nm Numerical determination.

[0113] Subclass identification revealed that the 5F2 positive hybridoma cell line subclass was IgG2b / κ, and the 12F6 positive hybridoma cell line subclass was IgG1 / κ. The identification results are shown below. Figure 10 .

[0114] 1.5 Analysis of monoclonal antibody antigen binding sites using additive assays

[0115] The saturation working concentration of the two monoclonal antibodies was determined using a serial dilution method. The determination method was based on the change in OD value of the microplate reader as the antibody concentration increased. 450nm When the value remains essentially unchanged, the monoclonal antibody dilution at this point is the saturated working concentration; then, an ELISA plate coated with LIOmpA protein is used, with 50 μL of each of two different McAbs as the primary antibody. The steps are the same as the indirect ELISA method in 2.1 of Example 1. Finally, the color development is terminated by reading the OD value using a microplate reader. 450nmAI = [2A1+2 / (A1+A2)-1]×100% can be used to calculate the superposition rate: AI represents the superposition rate, and A1+2 is the OD of the enzyme-labeled wells containing the two monoclonal antibodies. 450nm The numerical values, A1 and A2, represent the OD values ​​of the wells labeled with only one monoclonal antibody. 450nm The AI ​​value is used to determine whether two monoclonal antibodies target the same antigenic epitope. This is achieved by calculating an AI value greater than 50%.

[0116] After detection using the superimposed ELISA method, the AI ​​value of the two monoclonal antibodies was calculated. If it was less than 50%, the result showed that the two monoclonal antibodies may target the same antigenic epitope (see Table 4).

[0117] Table 4. Antigen epitope targeting of the two monoclonal antibodies

[0118]

[0119] 1.6 Preparation and enzyme labeling of ascites monoclonal antibodies

[0120] 1.6.1 Preparation of ascites

[0121] Ascites fluid was prepared by immunizing mice with monoclonal antibody 5F2. Newly purchased mice needed to be fed in the animal facility for about a week to avoid stress-induced mortality. One week before intraperitoneal injection of cells, each mouse needed to be injected intraperitoneally with 500 μL of Freund's incomplete adjuvant to induce immune stimulation. At this time, the monoclonal antibody cells could be passaged so that they were in the logarithmic growth phase at the time of immunization. Before injection, the cells were washed twice with RPMI 1640, and after being blown off with a bent pipette, 0.5 mL of cell suspension was injected into each mouse, with a cell suspension density of 5 × 10⁻⁶. 6 Five 5-6 week old female BALB / c mice were injected with a dose of [number] per mL. The mice were observed daily after injection. After approximately 1-2 weeks, their fur became disheveled, their abdomens became distended, and a fluctuating sensation could be felt upon palpation. Ascites fluid was collected by puncturing the peritoneal cavity with a 12-gauge needle, 2-3 times per mouse. The collected ascites fluid was centrifuged at 1000 rpm for 10 minutes, and the supernatant was collected. A portion was aliquoted for ELISA titer detection, and the remainder was stored at -80°C.

[0122] 1.6.2 Labeling of Monoclonal Antibody 5F2: Weigh 5 mg of horseradish peroxidase (HRP) and dissolve it in 1 ml of double-distilled water. Add 500 μl of freshly prepared NaIO4 and react at 2–8 °C for 30 min. The solution turns grass green. Then add 0.5 ml of ethylene glycol, mix well, and react at room temperature in the dark for 30 min. Then add 5 mg of purified 5F2 monoclonal antibody and dialyze in carbonate buffer (pH 9.5) at 2–8 °C for 15 h. The next day, add 0.2 ml of freshly prepared NaBH4 to the solution, mix well, and react at 2–8 °C for 2 h. Then add an equal volume of saturated ammonium sulfate solution, let stand at 2–8 °C for 30 min, centrifuge at 7000 r / min for 10 min, discard the supernatant, resuspend in PB, and dialyze in PB at 2–8 °C for 1.5 h. Collect the contents of the dialysis bag, adjust the enzyme-labeled reagent concentration to 4 mg / ml, add an equal volume of glycerol as the enzyme-labeled reagent stock solution, aliquot 1 ml / tube, and store at -70℃ or below for later use. Using a purified protein-coated detection plate at a concentration of 1 μg / mL, the titer of the purified antibody was determined by indirect ELISA to be >1:512000; protein electrophoresis confirmed that the purified antibody bands were clear and the purity was >85%, indicating high antibody purity. SDS-PAGE results are shown below. Figure 11 As shown.

[0123] Two hybridoma cell lines were obtained in this embodiment, which the applicant named 5F2 and 12F6, respectively. The titers of the primary supernatant after in vitro expansion culture were 1:25600 and 1:51200, respectively.

[0124] The monoclonal antibody 5F2 was preliminarily identified as having a blocking effect. Western blot analysis showed that the LIOmpA protein specifically reacted with the antibody supernatant but not with SP2 / 0, indicating that the hybridoma cells prepared by this invention have good specificity. When the 5F2 and 12F6 monoclonal antibodies were passaged continuously for 20 generations in vitro, the antibody titer remained unchanged, demonstrating relatively stable antibody secretion. Monoclonal antibody subclass identification results showed that antibody 5F2 belongs to the IgG2b / κ chain; 12F6 belongs to the IgG1 / κ chain.

[0125] Ascites fluid was prepared by immunizing mice with monoclonal antibody 5F2, and after purification and labeling, it was made into enzyme-labeled monoclonal antibody, which provided the necessary conditions for establishing an intracellular Lawsonia monoclonal antibody blocking ELISA detection method.

[0126] Example 3: Establishment of an ELISA method for detecting intracellular Lawsonia monoclonal antibody blocking

[0127] 1.1 Laboratory animals and reagents

[0128] Two healthy 3-month-old Landrace pigs; live ileitis vaccine Ileitis was purchased from Boehringer Ingelheim; enzyme-labeled monoclonal antibody 5F2; ELISA test plate was purchased from Biofil Biotechnology; TMB chromogenic solution was purchased from Solarbio; His-tag Purification Resin (reduction-resistant chelating type) and BCA detection kit were purchased from Beyotime Biotechnology; other chemical reagents were domestically produced analytical grade.

[0129] Commercially available indirect ELISA kits were used to screen for clinical Lawsonia intracellularis standard negative serum; prepared porcine polyclonal antibodies were used as standard positive controls; and laboratory-preserved reference positive serum was also used.

[0130] 1.2 Preparation of antigen protein and standard positive serum

[0131] 1.2.1 Preparation of antigen proteins

[0132] After reviving laboratory-preserved glycerol bacteria, LIOmpA protein was induced to express. The induction effect was assessed using SDS-PAGE, followed by purification. The purity was then assessed again using SDS-PAGE. The purified protein antigen was mixed with glycerol and a protease inhibitor, aliquoted, and cryopreserved in an ultra-low temperature freezer for coating detection plates and preparing standard positive sera. For specific procedures, please refer to section 1.3, Expression and Purification of Recombinant Protein, in Example 1.

[0133] 1.2.2 Preparation of Standard Positive Serum

[0134] Blood was collected from the anterior vena cava before immunization, and negative control serum was obtained. Porcine ileitis live vaccine was administered, diluted according to the instructions, with 2 mL administered orally to each pig. A total of three immunizations were given, with each immunization spaced three weeks apart. Seven days after the third immunization, blood was collected from the anterior vena cava, and the titer was measured using an indirect ELISA method. If the titer was high, the serum obtained from the anterior vena cava served as the standard positive control.

[0135] 1.3 Preparation of enzyme-labeled monoclonal antibodies

[0136] Ascites was prepared by immunizing mice with monoclonal antibody 5F2 cells, and then purified and HRP-labeled, as described in 1.6 Preparation and enzyme-labeled purification of monoclonal antibody ascites in Example 1.

[0137] 1.4 Blocking ELISA Procedure

[0138] Purified LIOmpA protein was coated onto 96-well microplates with 0.05 mol / L carbonate buffer (pH 9.6), 100 μL per well, and incubated overnight at 4°C. The liquid was discarded, and the plates were washed 5 times with 300 μL PBST per well, then blotted dry. The plates were then blocked with PBST containing 5% BSA at 37°C for 2 h. The liquid was discarded again, and the plates were washed 5 times with 300 μL PBST per well, then blotted dry. 100 μL of the serum to be tested was added to each well, with standard positive and negative porcine serum used as controls, and incubated for 1.5 h. The enzyme-labeled monoclonal antibody was diluted 1:20000, 100 μL per well, and incubated for 1 h. The liquid was discarded, and the plates were washed 5 times with 300 μL PBST per well, then blotted dry. Single-component TMB chromogenic solution was added, and the reaction was allowed to proceed for 10 min. Finally, 2M sulfuric acid was added to terminate the reaction, and the OD values ​​were read using a microplate reader. 450nm The PI value is calculated after numerical analysis. The formula for calculating the PI value is: PI = (Negative serum OD) / (OD0.05) 450nm Value - OD of the tested serum 450nm Value) / Negative serum OD 450nm Value × 100%.

[0139] 1.5 Selection of optimal reaction conditions for blocking ELISA

[0140] 1.5.1 Screening of the optimal concentration of coating antigen and dilution ratio of enzyme-labeled monoclonal antibody

[0141] Selection was performed using a matrix titration method. Vertically, purified proteins were coated onto ELISA plates at concentrations of 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, and 0.03125 μg / mL per column using carbonate coating buffer at pH 9.6. LI antibody-positive and negative sera were then separated and coated into plates at 2... 0 2 1 2 2 2 3 Perform four serial dilutions, adding the strips to four wells per column of coated plate, and incubate at 37°C for 1.5 hours. Subsequent steps are performed according to the blocking ELISA procedure. Finally, place the strips into an OD reader. 450nm After reading the values, calculate the PI value. The highest PI value represents the subsequent coating antigen concentration and serum dilution (see Table 5).

[0142] Table 5. N / P values ​​of ELISA detection in the optimization experiment of antigen coating concentration and serum dilution.

[0143]

[0144] Note: Bold values ​​correspond to optimal conditions.

[0145] 1.5.2 Optimization of antigen coating conditions

[0146] Based on the optimal antigen coating concentration selected from the above experimental results, subsequent experimental conditions were further optimized. Four conditions were set for the experiments: 37℃ for 2 hours, 37℃ for 3 hours, and 37℃ for 4 hours. Subsequent steps were performed according to the blocking ELISA procedure in Example 3, Section 1.4. Finally, the OD values ​​were read using a microplate reader. 450nm Numerical analysis involves calculating the N / P ratio and selecting the condition with the larger N / P value as the optimal condition (see Table 6).

[0147] Table 6. N / P values ​​for ELISA detection in antigen coating condition experiment.

[0148]

[0149] Note: Bold values ​​correspond to optimal conditions.

[0150] 1.5.3 Optimization of enzyme-labeled monoclonal antibody dilution

[0151] Following the blocking ELISA procedure in Example 3, section 1.4, proceed to primary antibody incubation, plate washing, and blotting. Dilute the enzyme-labeled monoclonal antibody at four gradients: 1:2000, 1:5000, 1:10000, and 1:20000, and continue with the remaining steps. Calculating the N / P ratio, the 1:20000 dilution yielded an N / P value of 9.60, higher than the corresponding N / P values ​​for the other dilutions. Therefore, 1:20000 was selected as the optimal dilution (see Table 7).

[0152] Table 7. N / P values ​​of ELISA detection in the experiment of optimizing enzyme-labeled monoclonal antibody dilution.

[0153]

[0154] Note: Bold values ​​correspond to optimal conditions.

[0155] 1.5.4 Selection of Sealing Fluid

[0156] According to the conditions determined in section 1.5.1 of this embodiment for screening the concentration of the coating antigen and the optimal dilution ratio of serum, the ELISA plate was coated and diluted with serum, then incubated at 37°C for 2 hours. After washing with PBST, 5% skim milk, 1% BSA, 2% BSA, and 5% BSA were added for blocking for 1 hour, respectively. Each blocking solution was used in triplicate. Subsequent steps were performed according to the blocking ELISA procedure in section 1.4 of Example 3. OD values ​​were read. 450nm The optimal sealing solution is the one that produces the maximum N / P value (see Table 8).

[0157] Table 8. N / P values ​​detected by ELISA in the blocking solution screening experiment.

[0158]

[0159] Note: Bold values ​​correspond to optimal conditions.

[0160] 1.5.5 Selection of Closure Conditions

[0161] Based on the blocking liquid coating of the detection plate determined by the above screening, three blocking conditions were adopted to determine the optimal blocking conditions: 37℃ for 1 hour, 37℃ for 2 hours, and 37℃ for 3 hours. Subsequent steps were performed according to the blocking ELISA procedure in 1.4 of Example 3, and OD was read. 450nm The maximum value of N / P is calculated, and the corresponding closure condition is the optimal closure condition (see Table 9).

[0162] Table 9. ELISA detection N / P values ​​in the closed-condition optimization experiment.

[0163]

[0164] Note: Bold values ​​correspond to optimal conditions.

[0165] 1.5.6 Screening for the optimal incubation time of the serum to be tested

[0166] After coating and blocking the ELISA plate with the determined antigen concentration, negative and positive sera were added to the coated and blocked plate at the optimal dilution. The plates were incubated at three different conditions: 37°C for 30 min, 37°C for 1 h, and 37°C for 1.5 h. Subsequent steps were performed according to the blocking ELISA procedure in Example 3, Section 1.4, and OD values ​​were read. 450nm The maximum value of N / P is calculated, and the corresponding closure condition is the optimal closure condition (see Table 10).

[0167] Table 10. ELISA N / P values ​​in the serum incubation condition optimization experiment.

[0168]

[0169] Note: Bold values ​​correspond to optimal conditions.

[0170] 1.5.7 Screening for optimal enzyme-labeled monoclonal antibody reaction conditions

[0171] After coating and blocking the ELISA plate with the determined antigen concentration, add 100 μL of negative and positive serum to each well of the coated and blocked plate, incubate for 1.5 h, then add 1:5000 enzyme-labeled monoclonal antibody, and incubate at 37℃ for 30 min, 37℃ for 1 h, and 37℃ for 1.5 h respectively. Subsequent steps are performed according to the blocking ELISA procedure in 1.4 of Example 3. OD values ​​are then read. 450nmThe maximum N / P value is calculated, and the corresponding enzyme-labeled monoclonal antibody incubation time is the optimal enzyme-labeled monoclonal antibody incubation time (see Table 11).

[0172] Table 11. ELISA detection N / P values ​​in the optimal enzyme-labeled monoclonal antibody reaction conditions optimization experiment.

[0173]

[0174] Note: Bold values ​​correspond to optimal conditions.

[0175] 1.5.8 Screening for the optimal reaction time of TMB colorimetric solution

[0176] To determine the color development time, the coated and sealed ELISA plate was washed and dried according to the blocking ELISA procedure in Example 3, Section 1.4, as determined above. TMB single-component colorimetric solution was added, and the plate was incubated at room temperature in the dark for 5 min, 10 min, 15 min, and 20 min, respectively, before adding the stop solution. OD values ​​were read within 10 min. 450nm The optimal color development time is determined by the color development time corresponding to the maximum N / P value (see Table 12).

[0177] Table 12. ELISA detection N / P values ​​in the optimal color development time optimization experiment.

[0178]

[0179]

[0180] Note: Bold values ​​correspond to optimal conditions.

[0181] 1.6 Determination of the cutoff value for blocking ELISA and the criteria for result interpretation

[0182] 150 clinical serum samples were tested using a commercially available indirect ELISA kit and the established ELISA antibody detection method. The results were plotted as ROC curves, with an area under the ROC curve (AUC) of 0.917, indicating good method accuracy. The Youden index was calculated using the curve coordinates. The cut-off point was set at 29.5%, corresponding to a maximum Youden index of 0.757. With a cut-off rate of 29.5%, the sensitivity was 0.857 and the specificity was 0.90, both showing good sensitivity and specificity. For ease of calculation, the cutoff criteria were determined as follows: a pI value ≥ 30% was considered positive, and a pI value < 30% was considered negative (see [link to relevant documentation]). Figure 12 (Tables 13 and 14).

[0183] Table 13 ROC Curve Analysis

[0184]

[0185] Table 14 Analysis of ROC Curve Coordinates

[0186]

[0187]

[0188]

[0189] 1.7 Repeatability Test

[0190] 1.7.1 Intra-batch repeatability test

[0191] The proteins used to coat the ELISA strips were purified from the same batch. After optimal coating and blocking conditions, eight serum samples (negative, weakly positive, and strongly positive) were selected from clinical samples. Each serum sample was tested in triplicate, and the blocking rate was calculated. The coefficient of variation (CV) was calculated based on the PI value, which indicates whether the intra-assay reproducibility was good. The CV of the blocking rate for the three replicates of different serums within the same batch was less than 10%, indicating good intra-assay CV (see Table 15).

[0192] Table 15 Intra-batch Repeatability Tests

[0193]

[0194] 1.7.2 Inter-batch repeatability test

[0195] The antigens used to coat the ELISA strips were proteins purified from three batches at different times. After optimal coating and blocking conditions, eight serum samples (negative, weakly positive, and strongly positive) were clinically screened. Each sample was subjected to three replicates, and the blocking rate was calculated. The coefficient of variation (CV) was calculated based on the PI value, indicating good batch-to-batch reproducibility. The CVs of the blocking rates from different serum samples across three replicates were all below 15%, indicating good batch-to-batch CV (see Table 16).

[0196] Table 16 Inter-batch Repeatability Tests

[0197]

[0198]

[0199] 1.8 Sensitivity test for blocking ELISA

[0200] Based on optimal experimental conditions, the intracellular Lawsonia solani positive serum was tested from 2... 0 Serial dilution to 2 4 The established blocking ELISA method was used to detect the OD450nm value of corresponding serum dilutions and calculate the blocking rate. The results showed that at a serum dilution of 2... 3The blocking rate was 48.13%, which was considered a positive result. Therefore, this method can detect up to 8-fold dilutions (see Table 17).

[0201] Table 17 Sensitivity tests for blocking ELISA

[0202]

[0203] 1.9 Specific assays for blocking ELISA

[0204] Positive sera for classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), porcine circovirus type I (PCV-I), Pasteurella multocida (PM), Actinobacillus pleuropneumoniae (APx), Escherichia coli (E. coli), and Streptococcus suis type II (SS-2) were collected from the laboratory. The OD450nm value of the corresponding serum dilutions was detected using the established blocking ELISA method, and the blocking rate was calculated. Except for serum positive for Lawsonia intracellularis antibody, the blocking rate of all other pathogen antibody-positive sera was <30% (see Table 18).

[0205] Table 18 Specific assays for blocking ELISA

[0206]

[0207]

[0208] 1.10 Concordance rate between blocking ELISA and commercial ELISA detection

[0209] 150 clinical serum samples were simultaneously tested using a commercially available indirect ELISA kit and the blocking ELISA method established in this invention.

[0210] Table 19. Sample concordance rates between blocking ELISA and commercial ELISA detection.

[0211]

[0212] The determination of the results of the blocking ELISA method established in this invention is carried out in accordance with the determination of the blocking ELISA threshold and the result standard judgment in Example 1.6, and is compared with the detection results of commercial indirect ELISA to calculate the concordance rate (the results are shown in Table 19).

Claims

1. A hybridoma cell line 5F2 that secretes a monoclonal antibody against the intracellular Lawsonia outer membrane protein OmpA, characterized in that, The hybridoma cell line 5F2 is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei Province, with accession number CCTCCNO: C2023263.

Images