Anti-tcdatoxin hybridoma cell lines, monoclonal antibodies and uses

Abstract

The application relates to an anti-TcdA toxin hybridoma cell strain, a monoclonal antibody and application. Two anti-TcdA toxin hybridoma cell strains are constructed and screened by taking anti-TcdA toxin as an antigen, and are named as 2EC5 and 2JC2. Antibodies 2EC5 and 2JC2 expressed by the two anti-TcdA toxin hybridoma cell strains can specifically recognize the anti-TcdA toxin, the antibody subtype of the antibody 2EC5 is IgG 2b, the antibody subtype of the antibody 2JC2 is IgG 2a, and the titers are all above 1:2560000. The antibodies 2EC5 and / or 2JC2 can be made into a kit for detecting the anti-TcdA toxin, and can be used for detecting the anti-TcdA toxin and have high specificity and sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular relates to an anti-TcdA toxin hybridoma cell line, a monoclonal antibody and their applications. Background Technology

[0002] Clostridium difficile ( Clostridioides difficile *Clostridium difficile* is an obligate anaerobic, Gram-positive spore-forming bacillus. It was first isolated from neonatal feces by Hall and O'Toole in 1935. In 1978, Bartlett et al. reported that cytotoxins secreted by *Clostridium difficile* were a major cause of antibiotic-associated diarrhea and pseudomembranous colitis. In the past 20 years, due to the widespread use of antibiotics and the increasing proportion of the elderly population, *Clostridium difficile* infection (…) has become increasingly common. Clostridioides difficile infection The incidence, recurrence rate, and severity of Clostridium difficile infection (CDI) are on the rise. In particular, in recent years, with the emergence of highly virulent strains of Clostridium difficile, the incidence of CDI has increased significantly, seriously threatening public health security.

[0003] Clostridium difficile infection (CDI) has become a significant cause of hospital-acquired infections. Early and accurate diagnosis of CDI based on relevant symptoms and test results not only aids in clinical diagnosis and disease prevention but also guides the rational use of antibiotics and reduces unnecessary medical expenses. In 2021, the "Diagnosis Standard for Clostridium difficile Infection (T / CPMA 008-2020)" formulated by the Chinese Preventive Medicine Association proposed a rapid diagnostic method for clinical samples, requiring the detection of glutamate dehydrogenase (GDH) and toxins A / B in the samples. Obtaining a highly sensitive and specific reagent for detecting toxin A is essential for the diagnosis of CDI. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an anti-TcdA toxin hybridoma cell line, a monoclonal antibody, and their applications.

[0005] The technical solution adopted in this invention is as follows: A hybridoma cell line resistant to TcdA toxin, named 2EC5, with accession number CGMCC No. 46721, classified as a hybridoma cell, deposited at the China General Microbiological Culture Collection Center, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of November 18, 2025, and tested as viable; or named 2JC2, with accession number CGMCC No. 46726, classified as a hybridoma cell, deposited at the China General Microbiological Culture Collection Center, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of November 18, 2025, and tested as viable.

[0006] Anti-TcdA toxin antibody, antibody 2EC5 includes a light chain variable region and a heavy chain variable region. The light chain variable region includes CDRL1 as shown in SEQ ID NO:1, CDRL2 as shown in SEQ ID NO:2, and CDRL3 as shown in SEQ ID NO:3. The heavy chain variable region includes CDRH1 as shown in SEQ ID NO:4, CDRH2 as shown in SEQ ID NO:5, and CDRH3 as shown in SEQ ID NO:6.

[0007] or,

[0008] Antibody 2JC2 includes a light chain variable region and a heavy chain variable region. The light chain variable region includes CDRL1 as shown in SEQ ID NO:11, CDRL2 as shown in SEQ ID NO:12, and CDRL3 as shown in SEQ ID NO:13. The heavy chain variable region includes CDRH1 as shown in SEQ ID NO:14, CDRH2 as shown in SEQ ID NO:15, and CDRH3 as shown in SEQ ID NO:16.

[0009] Preferably, the amino acid sequence of the light chain variable region of antibody 2EC5 is SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is SEQ ID NO:9;

[0010] Alternatively, the amino acid sequence of the light chain variable region of antibody 2JC2 is SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region is SEQ ID NO:19.

[0011] Preferably, monoclonal antibody 2EC5 is produced by the anti-TcdA toxin hybridoma cell line with accession number CGMCC No. 46721; or, monoclonal antibody 2JC2 is produced by the anti-TcdA toxin hybridoma cell line with accession number CGMCC No. 46726.

[0012] Nucleic acid molecules, which are polynucleotides encoding antibodies against TcdA toxin.

[0013] Preferably, the nucleotide sequence encoding the variable region of the light chain of antibody 2EC5 is shown in SEQ ID NO:8, and the nucleotide sequence encoding the variable region of the heavy chain of antibody 2EC5 is shown in SEQ ID NO:10;

[0014] Alternatively, the nucleotide sequence encoding the variable region of the light chain of antibody 2JC2 is shown in SEQ ID NO:18, and the nucleotide sequence encoding the variable region of the heavy chain of antibody 2JC2 is shown in SEQ ID NO:20.

[0015] Application of anti-TcdA toxin antibody in the preparation of reagents for detecting anti-TcdA toxin.

[0016] A kit for detecting anti-TcdA toxin, comprising anti-TcdA toxin antibody.

[0017] Preferably, antibody 2EC5 and / or antibody 2JC2 are fabricated into a colloidal gold immunoassay kit, a chemiluminescent immunoassay kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit, or a fluorescent immunoassay kit, or into a microfluidic chip.

[0018] Preferably, when preparing a colloidal gold immunoassay kit, antibody 2EC5 is used as the coating antibody and antibody 2JC2 is used as the gold-labeled antibody; or, antibody 2JC2 is used as the coating antibody and antibody 2EC5 is used as the gold-labeled antibody.

[0019] The advantages and positive effects of this invention are as follows: This invention provides a hybridoma cell line capable of producing antibodies against TcdA toxin. The produced antibodies 2EC5 and 2JC2 can specifically recognize anti-TcdA toxin. The antibody subtype of antibody 2EC5 is IgG2b, and the antibody subtype of antibody 2JC2 is IgG 2a, with titers exceeding 1:2,560,000. A kit made from antibodies 2EC5 and 2JC2 can be used for the detection of anti-TcdA toxin, exhibiting high specificity and sensitivity. Attached Figure Description

[0020] Figure 1 Tcd A antigen was identified by SDS-PAGE electrophoresis; TA is the purified protein, and M is a marker of the protein's relative molecular mass.

[0021] Figure 2 Electrophoresis image of antibody purification; where 1 is 2EC5 monoclonal antibody, 2 is 2JC2 monoclonal antibody, and M is a marker of protein relative molecular mass;

[0022] Figure 3 Results of Tcd A toxin antigen detection using a colloidal gold method Tcd A toxin detection card;

[0023] Figure 4 The colloidal gold method for detecting Tcd A toxins: Detection results of samples containing Tcd A toxin;

[0024] Figure 5 Sensitivity analysis of colloidal gold method for Tcd A toxin detection card;

[0025] Biological material: 2EC5, classified as hybridoma cells, deposited on November 18, 2025, deposited 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. 46721;

[0026] Biological material: 2JC2, classified as hybridoma cell, deposited on November 18, 2025, deposited 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. 46726. Detailed Implementation

[0027] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0028] This invention relates to an anti-TcdA toxin hybridoma cell line, a monoclonal antibody, and their applications. Using anti-TcdA toxin as an antigen, two anti-TcdA toxin hybridoma cell lines were constructed and screened, named 2EC5 and 2JC2, respectively. Antibodies 2EC5 and 2JC2 expressed by these two anti-TcdA toxin hybridoma cell lines can specifically recognize anti-TcdA toxin. Antibodies 2EC5 and / or 2JC2 can be used to formulate a kit for detecting anti-TcdA toxin, which can be used to assist in determining whether Clostridium difficile infection is present.

[0029] The antibody 2EC5 produced by the anti-TcdA toxin hybridoma cell line 2EC5 includes a light chain variable region and a heavy chain variable region. The light chain variable region includes CDRL1 as shown in SEQ ID NO:1, CDRL2 as shown in SEQ ID NO:2, and CDRL3 as shown in SEQ ID NO:3. The heavy chain variable region includes CDRH1 as shown in SEQ ID NO:4, CDRH2 as shown in SEQ ID NO:5, and CDRH3 as shown in SEQ ID NO:6.

[0030] SEQ ID NO:1 RASQDIGSSLN

[0031] SEQ ID NO:2 ATSSLDS

[0032] SEQ ID NO:3 LQYASSPPT

[0033] SEQ ID NO:4 GFIFSDYWMN

[0034] SEQ ID NO:5 EIRLKSNNYATHYAESVKG

[0035] SEQ ID NO:6 VYYGSSYSAMDY

[0036] The amino acid sequence of the light chain variable region of antibody 2EC5 is SEQ ID NO:7, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region of antibody 2EC5 is SEQ ID NO:9, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:10.

[0037] The amino acid sequence of the light chain variable region is SEQ ID NO:7

[0038] DIVLTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPPTFGGGTKLEIKRADA

[0039] Light chain variable region base sequence SEQ ID NO:8

[0040] GACATTGTGCTCACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGACATTGGTAGTAGCTTAAACTGGCTTCAGCAGGAACCAGATGGAACTATTAAACGCCTGATCTACGCCACATCCAGTTTAGATT CTGGTGTCCCCAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGCCTTGAGTCTGAAGATTTTGTAGACTATTACTGTCTACAATATGCTAGTTCTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCT

[0041] Heavy chain variable region amino acid sequence SEQ ID NO:9

[0042] GGGLVRPGGSMKLSCVVSGFIFSDYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLEMNNLRAEDTGIYYCTRVYYGSSYSAMDYWGQGTSVTVSAAK

[0043] Heavy chain variable region base sequence SEQ ID NO:10

[0044] GGAGGAGGCTTGGTGCGACCTGGAGGATCCATGAAACTCTCCTGTGTTGTCTCTGGATTCATTTTTAGTGACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACACATTATGCGGAGTCTGTG AAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGGAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGGGTTTTACTACGGTAGTAGCTACTCTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCTGCAGCCAAA

[0045] The antibody 2JC2 produced by the anti-TcdA toxin hybridoma cell line 2JC2 includes a light chain variable region and a heavy chain variable region. The light chain variable region includes CDRL1 as shown in SEQ ID NO:11, CDRL2 as shown in SEQ ID NO:12, and CDRL3 as shown in SEQ ID NO:13. The heavy chain variable region includes CDRH1 as shown in SEQ ID NO:14, CDRH2 as shown in SEQ ID NO:15, and CDRH3 as shown in SEQ ID NO:16.

[0046] SEQ ID NO:11 RASQSVDYNGISYVH

[0047] SEQ ID NO:12 ATSNLES

[0048] SEQ ID NO:13 QQSVEDPFT

[0049] SEQ ID NO:14 GLNIKDFYMH

[0050] SEQ ID NO:15 WIDPDNGHTRYDPKFRD

[0051] SEQ ID NO:16 SPRMDY

[0052] The amino acid sequence of the light chain variable region of antibody 2JC2 is SEQ ID NO:17, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:18; the amino acid sequence of the heavy chain variable region of antibody 2JC2 is SEQ ID NO:19, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:20.

[0053] The amino acid sequence of the light chain variable region is SEQ ID NO:17

[0054] DIVLTQSPASLAVSVGQRATIFCRASQSVDYNGISYVHWFQQKPGQPPKLLIYATSNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSVEDPFTFGSATKLEIKRADA

[0055] Light chain variable region base sequence SEQ ID NO:18

[0056] GACATTTGTGCTCACCCAGTCTCCAGCTTCTTTGGCTGTGTCTGTAGGACAGAGAGCCACTATCTTCTGCAGAGCCAGCCAGAGTGTCGATTATAATGGAATTAGTTATGTGCACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTACATCCAACC TCGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAAGATGCTGCAACCTATTACTGTCAGCAAAGTGTTGAGGATCCATTCACGTTCGGCTCGGCGACAAAGTTGGAAATAAAACGGGCTGATGCT

[0057] Heavy chain variable region amino acid sequence SEQ ID NO:19

[0058] GAELVRPGALVKLSCKASGLNIKDFYMHWVKQRPEQGLEWIGWIDPDNGHTRYDPKFRDKASITADTSSNTAYLQLSSLTSEDTAVYFCTRSPRMDYWGQGTSVTVSSAKTTA

[0059] Heavy chain variable region base sequence SEQ ID NO:20

[0060] GGGGCTGAGCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCCTCAACATTAAAGACTTCTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGACAATGGTCATACTAGATATGACCCGAAGT TCCGGGACAAGGCCAGTATAACAGCAGACACGTCCTCCAACACAGCCTACCTGCAACTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTTCTGTACTCGATCCCCGAGGATGGACTACTGGGGTCAAGGAACCTCAGTCACTGTCTCTTCAGCCAAAACAACAGCC

[0061] Detection of antibodies 2EC5 and 2JC2 revealed that antibody 2EC5 is IgG 2b and antibody 2JC2 is IgG 2a, with titers exceeding 1:2,560,000. Sequencing of the genes of antibodies 2EC5 and 2JC2 showed that the heavy and light chain variable region sequences of both antibodies conformed to typical antibody variable region sequence characteristics. Systematic evaluation of the anti-TcdA toxin monoclonal antibodies, including assessment of antibody subtype and titer, kit sensitivity, specificity, and stability, demonstrated superior performance in all aspects, making them suitable as immunodiagnostic reagents for preparing in vitro diagnostic kits. Antibodies 2EC5 and 2JC2 can be fabricated into colloidal gold immunoassay kits, chemiluminescent kits, radioimmunoassay kits, enzyme-linked immunosorbent assay (ELISA) kits, or fluorescent immunoassay kits, or into microfluidic chips; the resulting kits can detect TcdA toxin antigen. In some embodiments of the present invention, antibodies 2EC5 and 2JC2 can be prepared into a double-antibody sandwich immunogold test strip. Antibody 2EC5 can be used as the gold-labeled antibody, and antibody 2JC2 as the coating antibody, or antibody 2JC2 can be used as the gold-labeled antibody, and antibody 2EC5 as the coating antibody. Verification has shown that the colloidal gold method for preparing TcdA toxin detection cards exhibits good specificity and sensitivity.

[0062] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0063] Example 1: Construction of a hybridoma cell line resistant to TcdA toxin

[0064] 1.1 Preparation of TcdA toxin antigen

[0065] The Tcd A toxin protein gene sequence was obtained from NCBI, and the gene fragment was chemically synthesized and ligated into the pet-28a plasmid to construct a recombinant plasmid. This plasmid was then transformed into *E. coli* BL21(DE3) competent cells and cultured overnight at 37°C. Single colonies were picked and cultured in LB medium until OD200 reached. 600 The concentration of the sample was 0.6-0.8. IPTG was added to a final concentration of 1 mM, and the mixture was induced overnight at 18°C. After induction, the bacterial cells were collected by centrifugation, sonicated, and the supernatant was collected by centrifugation for nickel column purification. First, the nickel column was equilibrated with equilibration buffer. Then, the supernatant sample was added to the nickel column. After complete sample entry, the target protein, Tcd A toxin, was collected by elution with washing buffer containing 20, 50, 100, and 200 mmol / L imidazole, respectively. The prepared protein was detected by SDS-PAGE electrophoresis. The results are shown below. Figure 1 As shown, the obtained protein has good purity, a size of 54 kDa, and a concentration of 1.6 mg / mL as determined by the BCA protein quantification kit. The prepared Tcd A toxin protein was aliquoted and stored at -80°C.

[0066] 1.2 Mouse Immunization

[0067] Antibody preparation was performed by immunizing approximately 6-week-old female Balb / c mice with purified Tcd A toxin antigen. The Tcd A toxin antigen content in the immunoglobulin preparation was 0.1 mg / mL. Mice were divided into two groups of 5 mice each, according to the immunization dose. The immunization dose for the first group was 25 μg / mouse, and the immunization dose for the second group was 50 μg / mouse.

[0068] For the initial immunization, the corresponding dose of Tcd A toxin antigen was diluted with distilled water to 300 μL, and an equal volume of Freund's complete adjuvant (300 μL) was added. After emulsification, the mice were immunized subcutaneously at multiple sites. Two weeks later, the same dose of Tcd A toxin antigen was used for a second immunization, administered via intraperitoneal injection. Two weeks later, a second immunization was administered, also via intraperitoneal injection. Seven days later, blood was collected from the mouse tails, and the serum titer was measured by ELISA to evaluate its suitability for cell fusion assays.

[0069] 1.3 Cell Fusion

[0070] Three days prior to fusion, mice were boosted with the same dose as the previous immunization, but without adjuvant, via intraperitoneal injection. One day before fusion, feeder cells were prepared. One 6-8 week old Balb / c mouse was euthanized by exsanguination of the eyeball followed by cervical dislocation. The mouse was then sterilized in 75% alcohol for 5 minutes, fixed on a plate, and its abdominal skin was aseptically cut open in a laminar flow hood. 10 mL of HAT selection medium was injected into the mouse's peritoneum using a sterile syringe. The abdomen was gently rubbed with an alcohol swab, and the medium was aspirated. This medium was added to 40 mL of HAT medium and plated into four 96-well cell culture plates (100 μL / well). The plates were incubated at 37°C in a 5% CO2 incubator. One week before fusion, myeloma cells (Sp2 / 0 cells) were resuscitated and cultured in PRMI-1640 medium containing 10% fetal bovine serum. The cells were passaged at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were collected into centrifuge tubes, counted, and diluted to 10-1. 7 Units / mL are available for use.

[0071] Balb / c mice that had received a booster 3 days prior to death underwent ocular avulsion to extract positive serum. The mice were then euthanized by cervical dislocation and disinfected with 75% alcohol for 5 minutes. The spleen was aseptically removed in a laminar flow hood and rinsed several times in a sterile petri dish to remove connective tissue. The spleen was placed on a microporous copper grid, and fresh RPMI-1640 culture medium was added. The culture medium was first injected into the spleen from one end using a syringe, and spleen cells were blown off. This process was repeated several times. The remaining spleen was then gently ground with the syringe stopper until no obvious red tissue clumps remained. The spleen cell suspension in the petri dish was gently pipetted and transferred to a 50 mL centrifuge tube. The tubes were centrifuged at 1000 rpm for 5 minutes, and the spleen cells were collected, counted, and used for later use.

[0072] Immunized mouse spleen cells and Sp2 / 0 cells were mixed at a ratio of 10:1 and added to a 50 mL centrifuge tube. The mixture was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The two cell lines were thoroughly mixed, and the centrifuge tube was placed in a container of 37°C hot water. 1 mL of preheated DMSO / PEG was added dropwise over 1 min, starting slowly and gradually increasing the speed while gently rotating the centrifuge tube. The reaction was stopped by adding antibiotic-free and blood-free RPMI-1640 culture medium, adding 1 mL in the first minute, 2 mL in the second minute, 3 mL in the third minute, and 4 mL in the fourth minute. The mixture was incubated at 37°C for 5 min, followed by centrifugation at 800 rpm for 5 min. The supernatant was discarded, and the pellet was resuspended in HAT and mixed into 40 mL of HAT selective culture medium containing 20% ​​fetal bovine serum preheated at 37°C. This mixture was then seeded into 96-well cell culture plates containing feeder cells at a density of 100 μL / well. The culture plates were incubated at 37°C in a 5% CO2 incubator. After 7 days, the cell plate will be partially replaced with fresh HAT medium, and after 10 days, the medium will be completely replaced with HT medium.

[0073] Subcloning of positive cells in 96-well plates was performed using the limiting dilution method: Feeder cells were prepared as described above. Hybridoma cells to be cloned were counted, and the cells were diluted to 5-8 cells / mL with HT medium. 100 μL / well was added to each pre-coated feeder cell incubator, with one 96-well plate per hybridoma cell line. The plates were incubated at 37°C in a 5% CO2 cell culture incubator. After approximately 5 days, the number of clones in each well was counted and labeled. On day 7, the medium was replaced. Cells were tested when they covered 1 / 3 to 1 / 2 of the bottom of each well. After 2-3 cycles of cloning, when all wells in the 96-well plate showed positive results, the plates were expanded, established, and cryopreserved.

[0074] Two hybridoma cell lines, named 2EC5 and 2JC2, were constructed and screened using the above method. The identified hybridoma cells were then expanded and cryopreserved. Vigorously growing, healthy hybridoma cells were gently removed from the cell culture flask using antibiotic-free and blood-free DMEM, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Cryopreservation buffer (containing 40% RPMI-1640 medium, 50% fetal bovine serum, and 10% DMSO) was added, and the cells were dispersed and aliquoted into cryovials. The cryovials were placed in a cryopreservation box at -70°C. After one day, the cryovials were transferred to liquid nitrogen and the data were recorded.

[0075] Example 2: Preparation and analysis of monoclonal antibodies against TcdA toxin

[0076] 2.1 Ascites preparation

[0077] The two hybridoma cell lines obtained in Example 1 were cultured to the logarithmic growth phase. Female Balb / c mice aged 10-12 weeks were intraperitoneally injected with sterile liquid paraffin (0.5 mL / mouse). Seven days later, hybridoma cells cultured to the logarithmic growth phase were injected intraperitoneally at a dose of 5 × 10⁻⁶. 6 Cells / mouse. Observe daily for approximately 7-10 days. Once a noticeable bulge appears in the mouse's abdomen, disinfect the lower abdominal skin with a 75% alcohol swab, insert a 16-gauge needle into the peritoneal cavity, and collect the ascites fluid. Collect the ascites fluid again after it regenerates and accumulates. Centrifuge the collected ascites fluid at 3000 rpm for 10 minutes, collect the clear intermediate fraction, filter it through filter paper, aliquot, and store at -70℃.

[0078] 2.2 Antibody purification

[0079] Ascites fluid was purified using a Protein-G column. 2 mL of ascites fluid was centrifuged at 10000 g, and the clear fraction was collected. 2 mL of (1:1) wash buffer was added and mixed well. The Protein-G column was flushed with 20% ethanol and then equilibrated with 8 mL of wash buffer. The sample was passed through the Protein-G column at a flow rate of 8 s / drop, repeated three times. The sample was then washed with 15 mL of wash buffer at a flow rate of 8 s / drop. After washing, 10 mL of elution buffer was used for elution. The pH was adjusted to 7.4 with 1 M Tris (pH=9). The solution was concentrated using a concentrate syringe and dialyzed overnight in a 50 kDa dialysis bag with PBS at 4°C.

[0080] 2.3 Antibody subtype identification

[0081] Following the SIGMA kit instructions, monoclonal antibody subclass identification was performed using a capture ELISA method. The monoclonal antibody subclass identification reagent was diluted 1:1000 and added to each well (100 μL / well), incubated at 37°C for 1 h; washed three times with PBST and blotted dry; the antibody was diluted 1:1000 and added to each well (100 μL / well), incubated at 37°C for 1 h; washed three times with PBST and blotted dry; HRP-labeled goat anti-mouse IgG secondary antibody was diluted 1:6000 and added to each well (100 μL / well), incubated at room temperature for 30 min; color development took 10–20 min. The OD value was calculated as follows: 450 The readings significantly higher than those of other wells indicated the subclass of the monoclonal antibody. The identification results are shown in Table 1: antibody 2EC5 was of the IgG2b subtype, and antibody 2JC2 was of the IgG2a subtype.

[0082] Table 1

[0083]

[0084] 2.4 Antibody titer determination

[0085] The purified antibodies 2EC5 and 2JC2 were determined using an indirect ELISA method. TcdA toxin was diluted to 0.2 μg / mL, 100 μL / well, with an uncoated control included. Coating was performed overnight at 4°C. The ELISA plates were then centrifuged, washed three times with PBST, and 200 μL / well of 5% skim milk powder was added. Blocking was performed at 37°C for 2 h. The plates were then centrifuged again, washed three times with PBST, and antibody (1 mg / mL) was serially diluted from 1:1000, for a total of 12 gradients. An uncoated control was also included, 100 μL / well, and incubated at 37°C for 1 h. The plates were centrifuged again, washed three times with PBST, and 100 μL / well of goat anti-mouse secondary antibody diluted 1:6000 in PBS was added. Incubation was performed at 37°C for 45 min. The plates were centrifuged again, washed five times with PBST, and 100 μL / well of TMB was added. Incubation was performed at 37°C for 10 min, and the plate was then read. The antibodies at different dilution gradients were tested, and the results are shown in Table 2. After purification, the antibody was diluted to 1 mg / ml, and the titer reached more than 1:2560000.

[0086] Table 2

[0087]

[0088] 2.5 Identification of antibody purity and molecular weight

[0089] The molecular weight and purity of antibodies 2EC5 and 2JC2 were determined using SDS-PAGE. Figure 2 As shown, the purified monoclonal antibody was identified by SDS-PAGE, and the bands were clear and free of impurities, with clear bands at 50 kDa and 25 kDa.

[0090] Example 3: Genetic verification of anti-TcdA toxin monoclonal antibody

[0091] The Ig variable region gene was cloned using RT-PCR to identify monoclonal antibodies against TcdA toxin 2EC5 and 2JC2. Total RNA was extracted from the 2EC5 and 2JC2 hybridoma cell lines using the Trizol method (kit purchased from Invitrogen), and the total RNA was reverse-transcribed into cDNA libraries using M-MLV reverse transcriptase (purchased from Invitrogen). RT-PCR primer information is as follows.

[0092] Upstream primer for the heavy chain backbone region:

[0093] P1:5'SAGGTGGMAGCTKCASSARTCWGG3' (SEQ ID NO:21)

[0094] Downstream primers for the heavy chain variable region:

[0095] P2: 5'TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3' (SEQ ID NO: 22)

[0096] upstream primer of light chain leader peptide:

[0097] P3: 5'GACATTGTGCTCACCCAGTCTCCA3' (SEQ ID NO: 23)

[0098] Downstream primers for the light chain variable region:

[0099] P4: 5'GGATACAGTTGGTGCAGCATCAGCCCGTTT3' (SEQ ID NO:24)

[0100] PCR reaction mixture (50 μL): cDNA: 2 μL; upstream primer (10 μM): 2 μL; downstream primer (10 μM): 2 μL; dNTP mixture: 2 μL; pfu DNA polymerase (5 U / μL): 1 μL; 10×pfu Buffer II: 5 μL; ddH2O: bring to 50 μL. Reaction conditions: 95℃ pre-denaturation for 5 min; repeat the following cycle 35 times: 95℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min; finally, 72℃ extension for 10 min.

[0101] The VL and VH fragments of the antibody were separated and recovered by agarose gel electrophoresis. The recovered VL and VH fragments were then ligated into the pMD19-T (sKPCle) vector (Takara), respectively. The ligation system consisted of 70 ng each of VL and VH PCR products, 1 μL of pMD19-T (sKPCle) vector, 5 μL of Solution I ligation reaction solution, and ddH2O to a final volume of 10 μL. The mixture was incubated overnight at 4°C.

[0102] The ligation product was transformed into E. coli DH5α competent cells and cultured overnight at 37°C. Single colonies were picked, and after shaking at 37°C for 2 hours, bacterial culture was performed for identification by PCR. cDNA containing the corresponding antibody was used as a positive control. The reaction mixture (25 μL) consisted of: bacterial culture: 1 μL; upstream primer (10 μM): 1 μL; downstream primer (10 μM): 1 μL; dNTP Mixture (2.5 μM each): 2 μL; Taq DNA polymerase (5 U / μL): 0.5 μL; 10×Taq Buffer (MgCl2). 2+ (plus): 2.5 μL; add water to 25 μL. Reaction conditions are the same as before.

[0103] PCR-positive clones were selected and expanded for culture. Plasmids from the positive clones were extracted using a plasmid extraction kit (Takara) and sent for sequencing. At least five clone samples were sent for sequencing of each chain of each antibody until at least two samples yielded identical sequencing results. The heavy and light chain variable region sequences of antibodies 2EC5 and 2JC2 were successfully cloned and matched the characteristics of typical antibody variable region sequences.

[0104] Example 4: Double-antibody sandwich immunochromatographic colloidal gold Tcd A toxin detection card

[0105] Antibodies 2EC5 and 2JC2 were used to prepare TcdA toxin detection cards via the colloidal gold method, and double-antibody sandwich immunogold test strips were prepared. Antibody 2JC2 was used as the gold-labeled antibody, and antibody 2EC5 was used as the coating antibody. 0.1M K₂CO₃ solution was added to the colloidal gold solution while stirring. After adjusting the pH, anti-TcdA toxin monoclonal antibody 2JC2 was added, followed by stirring and the addition of 10% bovine serum albumin solution and 2% PEG. 20000 After stirring, the supernatant was collected by low-speed centrifugation, followed by high-speed centrifugation and collection of the precipitate. The precipitate was then brought to a final volume with colloidal gold resuspension to form a gold-labeled antibody. The gold-labeled antibody was sprayed onto a glass fiber membrane and dried to prepare a gold-labeled pad. 1% sodium thimerosal solution was added to anti-Tcd A toxin monoclonal antibody 2EC5 and mixed to form a detection line coating solution. PBS and 1% sodium thimerosal solution were added to goat anti-mouse IgG and mixed to form a control line coating solution. The control line and detection line coating solutions were streaked onto a nitrocellulose membrane and dried to obtain the coated membrane. The coated membrane was attached to a substrate, and the gold-labeled pad and absorbent paper were placed over the coated membrane. After lamination, the membrane was cut to obtain a colloidal gold-based Tcd A toxin detection card.

[0106] The blank sample (sample processing solution without sample matrix) and the Tcd A toxin antigen sample prepared in Example 1 were tested separately, and the results are as follows: Figure 3 As shown, the left side shows the test results of a blank sample, and the right side shows the test results of three Tcd A toxin antigen samples, proving that the prepared colloidal gold method Tcd A toxin detection card can produce a positive reaction to Tcd A toxin.

[0107] Negative samples (serum samples without Tcd A toxin) and positive samples (serum samples containing Tcd A toxin) were then prepared separately and detected using a colloidal gold Tcd A toxin detection card. The results are as follows: Figure 4 As shown, the three results on the left are negative sample test results, and the three results on the right are positive sample test results. This demonstrates that the colloidal gold method Tcd A toxin detection card can accurately identify samples containing Tcd A toxin. The sensitivity of the prepared colloidal gold method Tcd A toxin detection card was tested, and the results are as follows... Figure 5As shown, the serum samples tested from left to right include Tcd A toxin at concentrations of 2 ng / ml, 1 ng / ml, and 0.5 ng / ml, respectively. This indicates that the limit of detection for this colloidal gold Tcd A toxin detection card is 1 ng / ml.

[0108] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made in accordance with the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A hybridoma cell line resistant to TcdA toxin, characterized in that: It is named 2EC5 with accession number CGMCC No.46721; or named 2JC2 with accession number CGMCC No.46726.

2. Anti-TcdA toxin antibody, characterized in that: Antibody 2EC5 includes a light chain variable region and a heavy chain variable region. The light chain variable region includes CDRL1 as shown in SEQ ID NO:1, CDRL2 as shown in SEQ ID NO:2, and CDRL3 as shown in SEQ ID NO:

3. The heavy chain variable region includes CDRH1 as shown in SEQ ID NO:4, CDRH2 as shown in SEQ ID NO:5, and CDRH3 as shown in SEQ ID NO:

6. or, Antibody 2JC2 includes a light chain variable region and a heavy chain variable region. The light chain variable region includes CDRL1 as shown in SEQ ID NO:11, CDRL2 as shown in SEQ ID NO:12, and CDRL3 as shown in SEQ ID NO:

13. The heavy chain variable region includes CDRH1 as shown in SEQ ID NO:14, CDRH2 as shown in SEQ ID NO:15, and CDRH3 as shown in SEQ ID NO:

16.

3. The anti-TcdA toxin antibody according to claim 2, characterized in that: The amino acid sequence of the light chain variable region of antibody 2EC5 is SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is SEQ ID NO:9; Alternatively, the amino acid sequence of the light chain variable region of antibody 2JC2 is SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region is SEQ ID NO:

19.

4. The anti-TcdA toxin antibody according to claim 2 or 3, characterized in that: Monoclonal antibody 2EC5 was produced from the anti-TcdA toxin hybridoma cell line with accession number CGMCC No. 46721; or monoclonal antibody 2JC2 was produced from the anti-TcdA toxin hybridoma cell line with accession number CGMCC No. 46726.

5. A nucleic acid molecule, characterized by: It is a polynucleotide encoding the anti-TcdA toxin antibody as described in claim 2 or 3.

6. The nucleic acid molecule according to claim 5, characterized in that: The nucleotide sequence encoding the variable region of the light chain of antibody 2EC5 is shown in SEQ ID NO:8, and the nucleotide sequence encoding the variable region of the heavy chain of antibody 2EC5 is shown in SEQ ID NO:

10. Alternatively, the nucleotide sequence encoding the variable region of the light chain of antibody 2JC2 is shown in SEQ ID NO:18, and the nucleotide sequence encoding the variable region of the heavy chain of antibody 2JC2 is shown in SEQ ID NO:

20.

7. The use of the anti-TcdA toxin antibody according to any one of claims 2-4 in the preparation of a reagent for detecting anti-TcdA toxin.

8. A kit for detecting anti-TcdA toxin, characterized in that: Includes the anti-TcdA toxin antibody as described in any one of claims 2-4.

9. The kit for detecting anti-TcdA toxin according to claim 8, characterized in that: Antibodies 2EC5 and / or 2JC2 can be fabricated into colloidal gold immunoassay kits, chemiluminescent kits, radioimmunoassay kits, enzyme-linked immunosorbent assay kits, or fluorescent immunoassay kits, or fabricated into microfluidic chips.

10. The kit for detecting anti-TcdA toxin according to claim 8, characterized in that: When preparing a colloidal gold immunoassay kit, antibody 2EC5 is used as the coating antibody and antibody 2JC2 is used as the gold-labeled antibody; or, antibody 2JC2 is used as the coating antibody and antibody 2EC5 is used as the gold-labeled antibody.

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