Anti-TcdA toxin hybridoma cell strain, monoclonal antibody and application

Abstract

The invention relates to an anti-TcdA toxin hybridoma cell strain, a monoclonal antibody and application. According to the invention, anti-TcdA toxin is used as an antigen, and two anti-TcdA toxin hybridoma cell strains are constructed and screened, and are respectively named as 2EC5 and 2JC2. The antibody 2EC5 and the antibody 2JC2 obtained through expression of 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 of the antibody 2EC5 and the antibody 2JC2 both reach 1: 2560000 or above; the antibody 2EC5 and / or the antibody 2JC2 can be prepared into a kit for detecting the anti-TcdA toxin, and the kit can be used for detecting the anti-TcdA toxin and has relatively 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] The anti-TcdA toxin antibody, antibody 2EC5 comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises 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 comprises 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] Alternatively,

[0008] The antibody 2JC2 comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises 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 comprises 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 antibody 2EC5 light chain variable region amino acid sequence is SEQ ID NO: 7, and the heavy chain variable region amino acid sequence is SEQ ID NO: 9;

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

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

[0012] A nucleic acid molecule is a polynucleotide encoding an anti-TcdA toxin antibody.

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

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

[0015] The anti-TcdA toxin antibody is used for preparing a detection reagent for detecting an anti-TcdA toxin.

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

[0017] Preferably, the antibody 2EC5 and / or the antibody 2JC2 are made into a colloidal gold immunoreagent kit, a chemiluminescence reagent kit, a radioimmunoassay kit, an enzyme-linked immunoreagent kit or a fluorescent immunoreagent kit, or into a microfluidic chip.

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

[0019] The present application has the advantages and positive effects that the present application provides a hybridoma cell strain capable of producing anti-TcdA toxin antibody, the produced antibody 2EC5 and the antibody 2JC2 can specifically recognize anti-TcdA toxin, the antibody subtype of the antibody 2EC5 is IgG2b, the antibody subtype of the antibody 2JC2 is IgG2a, and the titers are all above 1:2560000; the antibody 2EC5 and the antibody 2JC2 are made into a kit, which can be used for detecting anti-TcdA toxin, and have high specificity and sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 SDS-PAGE electrophoresis identification of Tcd A antigen; TA is the protein after purification, and M is a protein relative molecular mass marker;

[0021] Figure 2 Antibody purification electrophoresis map; wherein, 1 is 2EC5 monoclonal antibody, 2 is 2JC2 monoclonal antibody, and M is a protein relative molecular mass marker;

[0022] Figure 3 Detection results of Tcd A toxin detection card by colloidal gold method on Tcd A toxin antigen;

[0023] Figure 4 Detection results of Tcd A toxin detection card by colloidal gold method on samples containing Tcd A toxin;

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

[0025] Biological material: 2EC5, classified as hybridoma cell, preservation date is 2025.11.18, preservation unit is China General Microbiological Culture Collection Center (CGMCC), address is No. 1, Beichen West Road, Hua-yuan Community, Chaoyang District, Beijing, and preservation number is CGMCC No. 46721;

[0026] Biological material: 2JC2, classified as hybridoma cell, preservation date is 2025.11.18, preservation unit is China General Microbiological Culture Collection Center (CGMCC), address: No. 1, Beichen West Road, Hua-yuan District, Beijing, preservation number is CGMCC No. 46726. DETAILED DESCRIPTION

[0027] The embodiments of the present application will be described below in conjunction with the accompanying drawings.

[0028] The present application relates to an anti-TcdA toxin hybridoma cell strain, a monoclonal antibody and an application. Two anti-TcdA toxin hybridoma cell strains are constructed and screened by using anti-TcdA toxin as an antigen, and are named as 2EC5 and 2JC2 respectively. The antibodies 2EC5 and 2JC2 expressed by the two anti-TcdA toxin hybridoma cell strains can specifically recognize anti-TcdA toxin, and the antibodies 2EC5 and / or 2JC2 can be made into a kit for detecting anti-TcdA toxin, which is used for assisting in judging whether there is Clostridium difficile infection.

[0029] The antibody 2EC5 produced by the anti-TcdA toxin hybridoma cell strain 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; and 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 as 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 as SEQ ID NO: 10;

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

[0038] DIVLTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPPTFGGGTKLEIKRADA

[0039] The nucleotide sequence of the light chain variable region is SEQ ID NO: 8

[0040] GACATTGTGCTCACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAGTCAGTCTCACTTGTCGGGCAAGTCAGGACATTGGTAGTAGCTTAAACTGGCTTCAGCAGGAACCAGATGGAACTATTAAACGCCTGATCTACGCCACATCCAGTTTAGATTCTGGTGTCCCCAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGCCTTGAGTCTGAAGATTTTGTAGACTATTACTGTCTACAATATGCTAGTTCTCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCT

[0041] The amino acid sequence of the heavy chain variable region is SEQ ID NO: 9

[0042] GGGLVRPGGSMKLSCVVSGFIFSDYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLEMNNLRAEDTGIYYCTRVYYGSSYSAMDYWGQGTSVTVSAAK

[0043] The nucleotide sequence of the heavy chain variable region is SEQ ID NO: 10

[0044] GGAGGAGGCTTGGTGCGACCTGGAGGATCCATGAAACTCTCCTGTGTTGTCTCTGGATTCATTTTTAGTGACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGGAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGGGTTTACTACGGTAGTAGCTACTCTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCTGCAGCCAAA

[0045] The antibody 2JC2 produced by the anti-TcdA toxin hybridoma cell line 2JC2 includes a light chain variable region including a CDRL1 as set forth in SEQ ID NO: 11, a CDRL2 as set forth in SEQ ID NO: 12, and a CDRL3 as set forth in SEQ ID NO: 13, and a heavy chain variable region including a CDRH1 as set forth in SEQ ID NO: 14, a CDRH2 as set forth in SEQ ID NO: 15, and a CDRH3 as set forth 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 as 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 as SEQ ID NO: 20;

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

[0054] DIVLTQSPASLAVSVGQRATIFCRASQSVDYNGISYVHWFQQKPGQPPKLLIYATSNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSVEDPFTFGSATKLEIKRADA

[0055] The nucleotide sequence of the light chain variable region is SEQ ID NO: 18

[0056] GACATTGTGCTCACCCAGTCTCCAGCTTCTTTGGCTGTGTCTGTAGGACAGAGAGCCACTATCTTCTGCAGAGCCAGCCAGAGTGTCGATTATAATGGAATTAGTTATGTGCACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTACATCCAACCTCGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAAGATGCTGCAACCTATTACTGTCAGCAAAGTGTTGAGGATCCATTCACGTTCGGCTCGGCGACAAAGTTGGAAATAAAACGGGCTGATGCT

[0057] The amino acid sequence of the heavy chain variable region is SEQ ID NO: 19

[0058] GAELVRPGALVKLSCKASGLNIKDFYMHWVKQRPEQGLEWIGWIDPDNGHTRYDPKFRDKASITADTSSNTAYLQLSSLTSEDTAVYFCTRSPRMDYWGQGTSVTVSSAKTTA

[0059] The nucleotide sequence of the heavy chain variable region is SEQ ID NO: 20

[0060] GGGGCTGAGCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCCTCAACATTAAAGACTTCTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGACAATGGTCATACTAGATATGACCCGAAGTTCCGGGACAAGGCCAGTATAACAGCAGACACGTCCTCCAACACAGCCTACCTGCAACTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTTCTGTACTCGATCCCCGAGGATGGACTACTGGGGTCAAGGAACCTCAGTCACTGTCTCTTCAGCCAAAACAACAGCC

[0061] Through detection of antibody 2EC5 and antibody 2JC2, the antibody subtype of antibody 2EC5 is IgG 2b, and the antibody subtype of antibody 2JC2 is IgG 2a, and the titers are all above 1:2560000. Sequencing of the genes of antibody 2EC5 and antibody 2JC2, the sequences of the heavy chain and light chain variable regions of antibody 2EC5 and antibody 2JC2 are consistent with the typical antibody variable region sequence characteristics. Through systematic evaluation of the anti-TcdA toxin monoclonal antibody, including evaluation of the antibody subtype and titer, kit sensitivity, specificity and stability, the anti-TcdA toxin monoclonal antibody has better performance in all aspects, and is thus suitable for being used as an immunodiagnostic reagent for preparing an in-vitro diagnostic kit. Antibody 2EC5 and antibody 2JC2 can be made into a colloidal gold immunoreagent kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit or a fluorescent immunoreagent kit, or into a microfluidic chip; the prepared kit can detect TcdA toxin antigen. In some embodiments of the present application, antibody 2EC5 and antibody 2JC2 can be prepared into a double-antibody sandwich method immunocolloidal gold test strip, antibody 2EC5 can be used as a gold-labeled antibody, antibody 2JC2 can be used as a coated antibody, or antibody 2JC2 can be used as a gold-labeled antibody, and antibody 2EC5 can be used as a coated antibody. It has been verified that the TcdA toxin detection card prepared by the colloidal gold method has better specificity and sensitivity.

[0062] The present application will be described in detail below with reference to the accompanying drawings, wherein the experimental methods of the operation steps not specifically described are performed according to the corresponding product instructions. The instruments, reagents and consumables used in the examples can be purchased from commercial companies if not specifically stated.

[0063] Example 1: Construction of anti-TcdA toxin hybridoma cell strain

[0064] 1.1 Preparation of Tcd A toxin antigen

[0065] The Tcd A toxin protein gene sequence was obtained from NCBI, and the gene fragment was chemically synthesized and then linked to a pet-28a plasmid to construct a recombinant plasmid, which was transformed into E. coli BL21 (DE3) competent cells and cultured at 37°C overnight. A single colony was picked and cultured in LB medium to an OD 600 of 0.6-0.8, and 1 mM IPTG was added, and the cells were induced at 18°C overnight. The induced cells were collected by centrifugation, broken by ultrasonication, and the supernatant was collected and purified by nickel column. First, the nickel column was equilibrated with equilibration buffer, then the supernatant sample was added to the nickel column, and after the sample was completely introduced, the target protein Tcd A toxin protein was eluted and collected using washing solution containing 20, 50, 100, and 200 mmol / L imidazole. The prepared protein was detected by SDS-PAGE electrophoresis, and the results are shown in Figure 1 . The obtained protein had good purity and a size of 54 KD, and the concentration was 1.6 mg / mL as determined by BCA protein quantification kit. The prepared Tcd A toxin protein was aliquoted and stored at -80°C.

[0066] 1.2 Mouse immunization

[0067] Purified Tcd A toxin antigen was used to immunize 6-week-old female Balb / c mice for antibody preparation. The Tcd A toxin antigen content in the immunization protein preparation was 0.1 mg / mL, and the mice were divided into two groups according to the immunization dose, with 5 mice in each group. According to the antigen content, the first group was immunized at a dose of 25 μg per mouse, and the second group was immunized at a dose of 50 μg per mouse.

[0068] At the first immunization, the corresponding dose of Tcd A toxin antigen was diluted to 300 μL with distilled water, and an equal amount of Freund's complete adjuvant 300 μL was added, and the mixture was emulsified uniformly and injected subcutaneously into the mice at multiple points. Two weeks later, the same dose of Tcd A toxin antigen was used for the second immunization, which was injected intraperitoneally into the mice. Two weeks later, the mice were immunized again, which was also injected intraperitoneally. Seven days later, the mouse tail blood was collected, and the serum titer of the mice was determined by ELISA to evaluate whether it could be used for cell fusion test.

[0069] 1.3 Cell fusion

[0070] The mice were boosted three days before fusion, and the inoculation amount was the same as the previous immunization, without adjuvant, intraperitoneal injection. One day before fusion, the feeder layer cells were prepared, and one 6-8 week old Balb / c mouse was taken, and the eyeball was bled after cervical dislocation, and the mouse was placed in 75% alcohol for 5 minutes for disinfection, and fixed on a plate, and the abdominal skin was cut aseptically in a clean bench. 10 mL of HAT selection medium was sucked into a sterile syringe and injected into the mouse's abdominal cavity, and the abdomen was gently rubbed with alcohol cotton ball, and the medium was withdrawn. Add to 40 mL of HAT medium, and plate into 4 96-well cell culture plates, 100 μL / well, and place in a 37°C, 5% CO2 cell incubator. The myeloma cells (Sp2 / 0 cells) were resuscitated one week before fusion, cultured with PRMI-1640 medium containing 10% fetal bovine serum, and placed in a 37°C, 5% CO2 incubator for subculture. The cells in the logarithmic growth phase were collected into a centrifuge tube, and the cells were counted, and the cells were diluted to 10 7

[0071] The Balb / c mouse boosted for 3 days was taken, and the positive serum was prepared by eyeball bleeding, and the mouse was killed by cervical dislocation, and the spleen was aseptically taken out in a clean bench, and washed several times in a sterile dish, and the connective tissue was peeled off. The spleen was placed on a microporous copper mesh, and fresh RPMI-1640 culture medium was added. First, the medium was sucked into a syringe and injected from one end of the spleen, and the spleen cells were blown off, and repeated several times. Then the remaining spleen was gently ground with the inner plug of the syringe until there was no obvious red tissue mass. After the spleen cell suspension in the dish was gently blown, it was transferred to a 50 mL centrifuge tube, centrifuged at 1000 r / min for 5 min, and the spleen cells were collected and used after counting.

[0072] The immune mouse spleen cells were mixed with Sp2 / 0 cells at a cell number of 10:1, added to a 50 mL centrifuge tube, centrifuged at 1000 r / min for 5 min, and the supernatant was discarded; the two kinds of cells were mixed thoroughly, and the centrifuge tube was placed in a container containing 37°C hot water, and 1 mL of preheated DMSO / PEG was added dropwise into the centrifuge tube within 1 min, first slowly and then quickly, and the centrifuge tube was rotated gently. Add 1 mL of RPMI-1640 culture medium without antibody and blood to terminate the reaction, add 2 mL in the second minute, add 3 mL in the third minute, and add 4 mL in the fourth minute. 37°C water bath for 5 min, then centrifuge at 800 r / min for 5 min, discard the supernatant, suspend the precipitate with HAT, mix well into 40 mL of HAT selection medium containing 37°C preheated 20% calf serum, and plate into 96-well cell plates with feeder cells, 100 μL / well, and place the culture plates in a 37°C, 5% CO2 incubator for culture. After 7 days, the cell plates were half-replaced with fresh HAT medium, and after 10 days, the cell plates were fully replaced with HT medium.

[0073] ​The positive cells in the 96-well plate are subcloned by limiting dilution method: the feeder layer cells are prepared according to the above method, the hybridoma cells to be cloned are counted, and the cells are diluted to 5-8 cells / mL with HT medium, and then added to the 96-well cell plate with feeder cells at 100 μL / well. Each hybridoma cell clones one 96-well cell plate, and is cultured in a 37°C, 5% CO2 cell incubator. After about 5 days, the number of clones in the cell wells is counted and labeled, and the medium is changed on the 7th day. When the cells cover 1 / 3-1 / 2 of the entire well bottom, detection is performed. After 2-3 times of cloning, when all the cell wells in the 96-well plate are positive, the culture is expanded, the strain is determined, and the cells are frozen.

[0074] Two hybridoma cell strains are constructed and screened by the above method, and are named 2EC5 and 2JC2, respectively. The determined hybridoma cells are expanded and frozen. The hybridoma cells growing vigorously and in good condition are blown off the cell bottle gently, centrifuged at 1000 r / min for 5 min, and the supernatant is discarded. The freezing solution (containing 40% RPMI-1640 culture solution, 50% fetal bovine serum, and 10% DMSO) is added, the cells are blown apart, and then are divided into cell freezing tubes. The freezing tubes are placed in a freezing box and put into a -70°C refrigerator, and after one day, the freezing tubes are transferred into liquid nitrogen and recorded.

[0075] Example 2: Preparation and analysis of anti-TcdA toxin monoclonal antibody

[0076] 2.1 Preparation of ascites

[0077] The two hybridoma cell strains screened in Example 1 are cultured to the logarithmic phase. 10-12-week-old female Balb / c mice are injected intraperitoneally with sterile liquid paraffin, 0.5 mL per mouse, and after 7 days, the hybridoma cells cultured to the logarithmic phase are injected intraperitoneally at a dose of 5×10 6 Each day, the mice are observed, and after about 7-10 days, when the mice have obvious abdominal bulges, the lower abdominal skin is disinfected with a 75% alcohol cotton ball, a 16-gauge needle is inserted into the abdominal cavity, and the ascites is collected. When the ascites accumulates again, it is collected again. The collected ascites is centrifuged at 3000 r / min for 10 min, the middle clear part is taken, filtered with filter paper, and then is divided and stored at -70°C.

[0078] 2.2 Antibody purification

[0079] Ascites purification with Protein-G column. Take 2 mL ascites, centrifuge at 10000 g, take the clear part, add 2 mL (1:1) washing buffer, mix well, equilibrate the Protein-G column with 8 mL washing buffer after the column is run dry with 20% ethanol, then put the sample on the Protein-G column, flow rate is 8 S / drop sediment, repeat 3 times, then wash the column with 15 mL washing buffer, flow rate is 8 S / drop sediment, after washing, elute with 10 mL elution buffer, after elution, adjust the pH to 7.4 with 1 M Tris PH=9, concentrate with a concentrator, dialyze in a 50 kd dialysis bag with PBS at 4°C overnight.

[0080] 2.3 Antibody subtype identification

[0081] According to the SIGMA kit instructions, the antibody subclass identification reagent was diluted 1:1000, then added to the enzyme-labeled hole, 100 μL / hole, 37°C incubation for 1 h; PBST wash three times, pat dry; the antibody was diluted 1:1000, then added, 100 μL / hole, 37°C incubation for 1 h; PBST wash three times, pat dry; HRP enzyme-labeled goat anti-mouse IgG secondary antibody was diluted 1:6000, then added, 100 μL / hole, room temperature incubation for 30 min; color development for 10-20 min. The OD 450 The OD value of the well with the antibody subclass reagent was significantly higher than that of the other wells, which was the antibody subclass type. The identification results are shown in Table 1, the antibody subtype of antibody 2EC5 is IgG2b, and the antibody subtype of antibody 2JC2 is IgG 2a.

[0082] Table 1

[0083]

[0084] 2.4 Antibody titer determination

[0085] The antibody 2EC5 and the antibody 2JC2 after purification were subjected to antibody titer determination by indirect ELISA. The Tcd A toxin was diluted to 0.2 μg / mL, 100 μL / well, and a non-coating control was set up, and was coated at 4°C overnight. The ELISA plate was spun dry, and was washed with PBST for 3 times; 5% skim milk powder was added, 200 μL / well, and was blocked at 37°C for 2 h; the ELISA plate was spun dry, and was washed with PBST for 3 times, and the antibody was added in a dilution gradient starting from 1:1000 (the concentration was 1 mg / mL), a total of 12 gradients, and a non-coating control was set up, 100 μL / well, at 37°C for 1 h. Spinning dry, washing with PBST for 3 times, adding a goat anti-mouse secondary antibody diluted by 1:6000 in PBS, 100 μL / well, at 37°C for 45 min. Spinning dry, washing with PBST for 5 times, adding 100 μL TMB / well, developing at 37°C for 10 min, stopping, and reading. The antibody in different dilution gradients was subjected to detection, 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 Antibody Purity and Molecular Weight Identification

[0089] The antibody 2EC5 and the antibody 2JC2 were subjected to antibody molecular weight and purity identification by SDS-PAGE. As shown in Table 2, the purified monoclonal antibody was identified by SDS-PAGE, and the bands were clear without impurity bands, and there were clear bands at 50 KDa and 25 KDa. Figure 2

[0090] Example 3: Gene Verification of Anti-Tcd A Toxin Monoclonal Antibody

[0091] The anti-Tcd A toxin monoclonal antibodies 2EC5 and 2JC2 were identified by cloning Ig variable region genes by RT-PCR. The total RNA of the 2EC5 and 2JC2 hybridoma cell strains was extracted by the Trizol method (the kit was purchased from Invitrogen), and the total RNA was reversely transcribed into a cDNA library by using an M-MLV reverse transcriptase (purchased from Invitrogen). The RT-PCR primer information is as follows.

[0092] Heavy chain framework region upstream primer:

[0093] P1: 5'SAGGTGMAGCTKCASSARTCWGG 3' (SEQ ID NO: 21)

[0094] Heavy chain variable region downstream primer:

[0095] ​P2: 5' TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA 3' (SEQ ID NO: 22)

[0096] Light chain leader peptide upstream primer:

[0097] P3: 5' GACATTGTGCTCACCCAGTCTCCA 3' (SEQ ID NO: 23)

[0098] Light chain variable region downstream primer:

[0099] P4: 5' GGATACAGTTGGTGCAGCATCAGCCCGTTT 3' (SEQ ID NO: 24)

[0100] Prepare a PCR reaction system (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 x pfu Buffer II: 5 μL; ddH2O: make up to 50 μL. Reaction conditions: 95 °C pre-denaturation for 5 min; repeat the following cycle 35 times: 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min; finally, 72 °C extension for 10 min.

[0101] Separate and recover the VL, VH fragments of the antibody by agarose gel electrophoresis. Recovered VL, VH fragments are respectively connected with pMD19-T (sKPCle) vector (Takara Company) with the following connection system: 70 ng of VL PCR product / VH PCR product, 1 μL of pMD19-T (sKPCle) vector, 5 μL of Solution I connection reaction solution; ddH2O makes up to 10 μL, 4 °C connection overnight.

[0102] Transform the connection product into E. coli DH5α competent bacteria, after overnight culture at 37 °C, pick single colonies, shake at 37 °C for 2 hours, and then perform bacterial liquid PCR identification with the corresponding antibody cDNA as a positive control. Prepare a reaction system (25 μL): bacterial liquid: 1 μl, upstream primer (10 μM): 1 μL; downstream primer (10 μM): 1 μl; dNTP Mixture (2.5Mm each): 2 μL; Taq DNA polymerase (5 U / μL): 0.5 μL; 10 x Taq Buffer (Mg 2+ plus): 2.5 μL; make up to 25 μL. Reaction conditions are the same as before.

[0103] The positive clones were expanded and the plasmid was extracted by using a plasmid extraction kit (Takara) and sent for sequencing. At least 5 clone samples of each chain of each antibody were sent for sequencing, and the sequencing was continued until at least 2 samples had the same sequencing result. The variable region sequences of the heavy chain and light chain of the antibodies 2EC5 and 2JC2 were successfully cloned, and the sequences were consistent with the typical antibody variable region sequence characteristics after comparison.

[0104] Example 4: Double antibody sandwich method immunocolloidal gold Tcd A toxin detection card

[0105] The antibodies 2EC5 and 2JC2 were used to prepare a Tcd A toxin detection card by the colloidal gold method, and a double antibody sandwich method immunocolloidal gold test strip was prepared. The antibody 2JC2 was used as the gold-labeled antibody, and the antibody 2EC5 was used as the coating antibody. The colloidal gold solution was stirred, and 0.1M K2CO3 solution was added to adjust the pH value. Then the anti-Tcd A toxin monoclonal antibody 2JC2 was added, followed by the addition of 10% bovine serum albumin solution and 2% PEG 20000 . After stirring, the supernatant was obtained by low-speed centrifugation, and the precipitate was obtained by high-speed centrifugation. The colloidal gold resuspension solution was used to make the gold-labeled antibody. The gold-labeled antibody was sprayed on the glass cellulose membrane, and the gold-labeled pad was prepared after drying. The anti-Tcd A toxin monoclonal antibody 2EC5 was mixed with 1% sodium thiomersalate solution to form the detection line coating solution. The PBS and 1% sodium thiomersalate solution were added to the goat anti-mouse IgG to form the quality control line coating solution. The quality control line coating solution and the detection line coating solution were drawn on the nitrocellulose membrane, and the coated membrane was obtained after drying. The coated membrane was attached to the bottom plate, and the gold-labeled pad and the absorbent paper were attached to the coated membrane. After lamination, the colloidal gold method Tcd A toxin detection card was obtained.

[0106] The blank sample (sample processing solution without sample matrix) and the Tcd A toxin antigen sample prepared in Example 1 were detected, and the results are shown in Figure 3 . The left side is the detection result of the blank sample, and the right side is the detection result of the three Tcd A toxin antigen samples. It is proved that the prepared colloidal gold method Tcd A toxin detection card can produce a positive reaction to Tcd A toxin.

[0107] The negative sample (serum sample without Tcd A toxin) and the positive sample (serum sample containing Tcd A toxin) of Tcd A toxin were prepared, and the colloidal gold method Tcd A toxin detection card was used for detection, and the results are shown in Figure 4 . The left 3 are the detection results of the negative samples, and the right 3 are the detection results of the positive samples. It can be seen that the colloidal gold method Tcd A toxin detection card can accurately identify the detection sample containing Tcd A toxin. The sensitivity of the prepared colloidal gold method Tcd A toxin detection card was detected, and the results are shown in Figure 5As shown, from left to right, the serum samples of Tcd A toxin in the detection samples sequentially include 2 ng / ml, 1 ng / ml and 0.5 ng / ml, and it can be seen that the minimum detection limit of the colloidal gold method Tcd A toxin detection card is 1 ng / ml.

[0108] The above detailed description of the embodiments of the present application, but the content is only the preferred embodiments of the present application, and cannot be considered as used to limit the scope of the present application. Any equivalent changes and improvements made in the scope of the present application shall still belong to the scope of the present application.

Claims

1. Anti-TcdA toxin hybridoma cell line, characterized by the fact that: It is named 2EC5 with accession number CGMCC No.46721; or named 2JC2 with accession number CGMCC No.46726.

2. Anti-TcdA toxin antibodies 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 of claim 2, wherein: 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 of 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 in that: It is a polynucleotide encoding the anti-TcdA toxin antibody as described in claim 2 or 3.

6. The nucleic acid molecule of claim 5, wherein: 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 by: 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 by: 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 by: 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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