Hybridoma cell strain secreting CFP10 antibody, antibody of hybridoma cell strain and application of hybridoma cell strain in magnetic particle chemiluminescence detection

Abstract

The invention discloses a hybridoma cell strain secreting a CFP10 antibody, the antibody and application of the hybridoma cell strain in magnetic particle chemiluminescence detection, the hybridoma cell strain is 2E7 and is preserved in the China Center for Type Culture Collection on May 14, 2025, and the preservation number is CCTCC NO: C2025162. The monoclonal antibody 2E7 provided by the invention has the advantages of high titer, good specificity and strong affinity with natural antigens and recombinant antigens, and can specifically recognize recombinant antigens rHis-CFP10 and rGST-CFP10 and a CFP10 antigen in a natural antigen H37Ra strain. The magnetic particle chemiluminescence detection method established by the invention is simple to operate, low in cost and wider in applicability.

Description

Technical Field

[0001] This invention relates to a hybridoma cell line that secretes CFP10 antibody, its antibody, and its application in magnetic microparticle chemiluminescence detection, belonging to the field of biotechnology. Background Technology

[0002] Tuberculosis (TB) is a chronic zoonotic infectious disease caused by the Mycobacterium tuberculosis complex (MTC), posing serious health problems to humans and livestock. The MTC includes Mycobacterium tuberculosis (Mtb), Mycobacterium africanum, and Mycobacterium bovis, among others, with Mtb and Mycobacterium bovis being the primary sources of harm. In addition, some nontuberculous mycobacteria (NTM), such as Mycobacterium vaccae, Mycobacterium phlei, and Mycobacterium smegmatis, not only pose health threats but also interfere with TB detection, increasing the difficulty of TB eradication.

[0003] Human tuberculosis (TB) is a global public health problem caused by Mycobacterium bovis and Mycobacterium vesiculum, and is most severe in developing countries. In 2023, the estimated number of deaths worldwide was 1.25 million, and the number of cases was 10.8 million, making it the world's leading cause of death from a single infectious source. In addition, Mycobacterium bovis can cause bovine tuberculosis. Inadequately sterilized milk and livestock workers frequently exposed to bovine tuberculosis can be infected through respiratory droplets formed by the coughs of infected cattle, leading to disease when their immunity is relatively weakened. Bovine tuberculosis is listed as a reportable infectious disease by the World Organisation for Animal Health (OIE) and is classified as a Class II animal disease in my country. It has a global distribution; for example, approximately 2.5 million livestock in Brazil are affected, impacting about 15% of the cattle industry. It is also a common and frequently occurring disease in my country, especially in dairy cows, with an incidence rate as high as 9% in some dairy farms. The disease can cause a 10%-20% reduction in milk production, weight loss, decreased reproductive capacity, and reduced meat quality. Currently, the number of tuberculosis patients is enormous and has not been effectively contained. The co-infection of tuberculosis and HIV, as well as the continuous emergence of drug-resistant tuberculosis strains, pose serious challenges to its prevention and control. Therefore, tuberculosis testing has become particularly important.

[0004] Currently, laboratory methods for detecting tuberculosis mainly include bacteriological methods, molecular biological methods, and immunological methods. Bacteriological methods are simple and rapid, but they cannot effectively distinguish between Mtb and NTM, and have poor specificity and low accuracy. Molecular biological methods are important for distinguishing between Mtb and NTM, laying the foundation for further tuberculosis research. Immunological methods include the tuberculin skin test (TST), enzyme-linked immunosorbent assay (ELISA), and interferon-γ release assay (IGRA). TST is a standardized Mtb detection method with high sensitivity and is widely used in biopsies, but it lacks specificity and cannot accurately identify allergic reactions caused by other mycobacteria (such as BCG), and is prone to false positives. ELISA is highly sensitive, specific, simple, and rapid, but its detection range is relatively narrow. IGRA can reduce false positives / false negatives, and since this method is performed in vitro, it does not interfere with the bovine immune status, but it cannot distinguish between latent infection and active tuberculosis.

[0005] Magnetic microparticle chemiluminescence technology is a next-generation diagnostic technology platform that combines immunomagnetic bead systems, chemiluminescence systems, and immunoreaction systems. It possesses both the high sensitivity of radioimmunoassay and the ease and speed of enzyme-linked immunosorbent assay (ELISA), making it easily automated. It avoids the use of harmful reagents, has a long reagent shelf life, and is applied in biological and medical research and clinical diagnostic work, making it one of the most promising and advanced non-radioactive immunoassay methods. The use of magnetic microparticles allows the reaction to proceed under near-homogeneous conditions, resulting in rapid and reproducible reactions and enabling fully automated detection. Acridinium ester labeling exhibits low interference, low background signal, high luminescence efficiency, and requires no enzyme catalysis, making it less susceptible to environmental interference. It holds promise as a replacement for radioimmunoassay products, meeting the needs of clinical diagnosis.

[0006] Culture Filtrate Protein 10 (CFP-10) is a secreted protein encoded by Rv3875 in the RD1 region of the Mtb genome, with a molecular weight of approximately 10-11 kDa. The CFP10 antigen possesses immunoprotective and memory functions, is closely related to Mtb virulence, and can induce a specific Th1 immune response. It has significant diagnostic value during both the active and latent infection phases of Mtb. The CFP10 antigen is present only in pathogenic Mtb and not in non-pathogenic Mtb such as BCG, exhibiting good specificity.

[0007] In conclusion, since traditional detection methods still have shortcomings in detecting Mycobacterium tuberculosis infection, there is an urgent need for a rapid, fully automated, and accurate method for detecting tuberculosis. Summary of the Invention

[0008] Purpose of the invention: The purpose of this invention is to provide a hybridoma cell line that secretes a monoclonal antibody against Mycobacterium tuberculosis CFP10, its antibody, and its application.

[0009] Technical solution: The present invention describes a hybridoma cell line that secretes monoclonal antibodies against Mycobacterium tuberculosis CFP10. The hybridoma cell line is 2E7 and was deposited at the China Center for Type Culture Collection on May 14, 2025, with accession number CCTCC NO:C2025162.

[0010] The monoclonal antibody against Mycobacterium tuberculosis CFP10 described in this invention is secreted by the above-mentioned hybridoma cell line.

[0011] The present invention discloses an anti-Mycobacterium tuberculosis CFP10 monoclonal antibody 2E7, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The light chain variable region comprises amino acid sequences as shown in SEQ ID NO.1, CDR-L2 as shown in SEQ ID NO.2, and CDR-L3 as shown in SEQ ID NO.3. The heavy chain variable region comprises amino acid sequences as shown in SEQ ID NO.5, CDR-H1 as shown in SEQ ID NO.6, CDR-H2 as shown in SEQ ID NO.7, and CDR-H3 as shown in SEQ ID NO.7.

[0012] Furthermore, the full-length amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.4, and the full-length amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8.

[0013] The application of the anti-tuberculosis CFP10 monoclonal antibody and anti-tuberculosis CFP10 monoclonal antibody 2E7 described in this invention in the preparation of a kit for detecting Mycobacterium tuberculosis.

[0014] Furthermore, the kit is a magnetic microparticle chemiluminescence detection kit.

[0015] The present invention discloses a magnetic microparticle chemiluminescence assay kit for detecting pathogenic Mycobacterium tuberculosis, the kit comprising magnetic microparticle-labeled Mycobacterium tuberculosis-specific protein antigen, the aforementioned monoclonal antibody 2E7, and sample diluent.

[0016] Furthermore, the Mycobacterium tuberculosis-specific protein antigen is the rHis-CFP10-ESAT6 (rHis-CE) fusion protein.

[0017] Furthermore, the pathogenic tuberculosis mycobacteria include Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium bovis (M. bovis).

[0018] Furthermore, the sample diluent contains any one or more combinations of bovine serum albumin, bromonitol, surfactant brij35, chitosan, or hydrolyzed soybean protein.

[0019] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: The monoclonal antibody 2E7 secreted by the hybridoma cell line with accession number CCTCC NO: C2025162 has the advantages of high titer, good specificity, and strong affinity for natural antigens and recombinant antigens. It can specifically recognize recombinant antigens rHis-CFP10 and rGST-CFP10, as well as the natural antigen CFP10 antigen in strain H37Ra. The magnetic particle chemiluminescence detection method established in this invention is simple to operate, low in cost, and has wider applicability. Attached Figure Description

[0020] Figure 1 : Schematic diagram of the magnetic particle chemiluminescence method in this invention.

[0021] Figure 2 : The reactivity of 2E7 monoclonal antibodies with natural antigens.

[0022] Figure 3 Western blotting analysis of 2E7 monoclonal antibody.

[0023] Figure 4 Analysis of the detection limit of magnetic particle chemiluminescence method. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0025] Example 1: Obtaining the 2E7 hybridoma cell line containing anti-CFP10 monoclonal antibody

[0026] Obtaining hybridoma cell line with accession number CCTCC NO:C2025162.

[0027] 1. Animal immunization

[0028] Using rHis-CFP10 (Shen Junsong. Prokaryotic expression of Mycobacterium tuberculosis Ihp gene and development of monoclonal antibodies against its products. Master's thesis, Yangzhou University, 2008) as an immunogen, 6-8 week old BALB / c mice (purchased from the Comparative Medicine Center of Yangzhou University, weighing 18-22g) were immunized with an equal amount of Freund's complete adjuvant (FCA) emulsion, 100μg / mouse, for the first immunization. Two weeks later, 100μg of rHis-CFP10 and an equal amount of Freund's incomplete adjuvant (FIA) emulsion protein were injected subcutaneously for the second immunization. Two weeks later, 100μg of incomplete Freund's adjuvant emulsion protein was injected subcutaneously again for the third immunization. One week later, the serum antibody titer of the mice was measured, and mice with a titer of 1:10000 or higher were selected for tail vein injection of 100μg of purified protein without adjuvant to complete the booster immunization.

[0029] 2. Cell fusion

[0030] Three days after tail vein booster immunization, blood was collected from the eyes of immunized mice, and the serum was separated and frozen at -20℃ as a positive control for screening. Immunized mice were euthanized by cervical dislocation, disinfected by immersion in 75% alcohol for 10 min, and spleen cells were aseptically harvested and fused with myeloma cells SP2 / 0 in logarithmic growth phase under PEG (MW4000) conditions. Peritoneal macrophages of ICR mice were used as feeder cells. The fused cells and feeder cells were suspended in HAT medium, dispensed into 96-well plates, and cultured in a 37℃, 6% CO2 incubator. Fresh HAT medium was added after 5 days, and HT medium was used after 10 days. The medium was changed and tested regularly.

[0031] 3. Establishment of the Indirect ELISA Method

[0032] Hybridoma cell lines specifically secreting CFP10 antibodies were screened using indirect ELISA to obtain positive clones. A checkerboard assay was used to determine the optimal coating concentration for the detection antigen rGST-CFP10 protein. The antigen was serially diluted with coating buffer (pH 9.6 carbonate buffer) at 10×, 20×, 40×, 80×, and 160×, with 50 μL per well, and incubated overnight at 4°C. The plates were washed three times with PBST for 3-5 min each time and then blotted dry. The plates were then blocked with PBS containing 10% fetal bovine serum (200 μL / well) at 37°C for 2 h, followed by four washes with PBST for 3-5 min each time and then blotted dry. Immune mouse serum was longitudinally diluted with PBS at 100×, 200×, 400×, 800×, 1000×, and 2000×. As a positive control, 50 μL / well was used. SP2 / 0 cell serum, diluted at the same multiple as the negative control, was used. The mixture was incubated at 37°C for 2 hours, followed by 5 washes with PBST, 3-5 min each time. Then, 100 μL / well of HRP-goat anti-mouse IgG (1:1000 PBST diluted to the working concentration) was added, and the mixture was incubated at 37°C for 1 hour, followed by 7 washes with PBST, 3-5 min each time. For TMB color development, 100 μL / well was added, and after 5 min at 37°C, 50 μL / well of 2 mmol / L concentrated H2SO4 was added to terminate the reaction. OD was measured using an ELISA reader. 450 The value is used to determine the optimal coating concentration for detecting the antigen.

[0033] 4. Screening for positive clones

[0034] The established indirect ELISA method was used to detect the antibody secretion of hybridoma cells. Hybridoma cell culture supernatant was added to pre-coated ELISA plates (50 μL / well), with SP2 / 0 cell supernatant as a negative control and immunized mouse polyclonal antibody serum as a positive control. The plates were incubated at 37°C for 2 h. After washing three times with PBST, 50 μL / well of working concentration HRP-goat anti-mouse IgG antibody was added, and the plates were incubated at 37°C for 1 h. TMB was allowed to develop for 10-15 min, followed by the addition of 50 μL / well of 2 mmol / L concentrated H2SO4 to terminate the reaction. OD was measured using an ELISA reader. 450 The value of the measured hole OD. 450 Reading. OD of the measured orifice. 450 A reading more than twice that of the negative control was considered positive. One positive clone selected was named positive cell clone 2E7.

[0035] 5. Cloning of positive hybridoma cells

[0036] The selected positive cell clone 2E7 was subcloned 3-4 times using the limiting dilution method.

[0037] Example 2: Large-scale preparation and purification of anti-CFP-10 monoclonal antibody 2E7

[0038] Produced by hybridoma cell line or its passaged cell line with accession number CCTCC NO:C2025162.

[0039] 1. Large-scale preparation of monoclonal antibody 2E7

[0040] Ascites fluid containing monoclonal antibodies was prepared using an in vivo induction method in female BALB / c mice over 10 weeks of age. 0.4 mL of liquid paraffin was injected intraperitoneally per mouse. 7-10 days later, hybridoma cells cultured to the logarithmic growth phase and diluted with PBS were inoculated intraperitoneally at a concentration of 5 × 10⁻⁶ cells / mL. 5 1 cell / animal; after 7 days, ascites fluid was collected, centrifuged to remove the precipitate, the supernatant was collected, the antibody titer was determined by indirect ELISA, aliquoted, and stored at -70℃.

[0041] 2. Purification of monoclonal antibody 2E7

[0042] Ascites fluid purification was performed using an rProteinA Sepharose 4B affinity chromatography column. First, 2-3 mL of ascites fluid was placed in a dialysis bag treated with NaHCO3 and EDTA and dialyzed overnight at 4°C in PBS. The purification column was equilibrated with 10 column volumes of sterile PBS at a flow rate of approximately 1 mL / min. The dialyzed 2E7 ascites fluid was added to the column at a flow rate of approximately 1 mL / min, repeated 2-3 times. For the final addition, the column was gently shaken at 4°C for 30 min to allow for more effective antibody binding. After antibody binding, the column was washed with 20 column volumes of sterile PBS at a flow rate of approximately 1 mL / min. After washing, 100 mmol / L glycine (pH 2.7) elution buffer was added, and the eluent was collected in a neutralization buffer containing 1 mol / L Tris HCl (pH 9.0). 50 μL of neutralization buffer was added to every 1 mL of collected elution buffer. Finally, SDS-PAGE was performed to analyze the purification effect.

[0043] Example 3: Characterization of anti-CFP-10 monoclonal antibody 2E7

[0044] 1. Monoclonal antibody 2E7 subclass determination

[0045] Follow the instructions for the monoclonal antibody subclass identification kit. Add 100 μL / well of 1:5000 diluted monoclonal antibody ascites fluid to each well of the ELISA plate, incubate at 37°C for 1 h, and wash three times with PBST for 3-5 min each time. Add 100 μL / well of 1:1000 diluted goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA subclass serum to each well, incubate at 37°C for 0.5 h, adding two wells for each subclass of each monoclonal antibody, and wash three times with PBST for 3-5 min each time. Add 100 μL / well of 1:5000 diluted goat anti-mouse ELISA-labeled secondary antibody, incubate at 37°C for 15 min, and wash three times with PBST. Add 100 μL / well of o-phenylenediamine (OPD) chromogenic reagent, incubate at 37°C in the dark for 5-10 min, and terminate the reaction with 100 μL / well of 2M H2SO4. Measure the OD. 450 Value, in OD 450 The serum subclass added to patients with significantly higher values ​​than those in other wells was a monoclonal antibody subclass.

[0046] The results showed that the subclass of monoclonal antibody 2E7 was IgG2b.

[0047] Identification results showed that the amino acid sequence of the complementarity-determining region 1 (CDR1) of the light chain variable region of monoclonal antibody 2E7 is as shown in SEQ ID NO.1, specifically:

[0048] QSLVNSNGNTY.

[0049] The amino acid sequence of the complementarity-determining region 2 (CDR2) of the light chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.2, specifically as follows:

[0050] KVS.

[0051] The amino acid sequence of complementarity-determining region 3 (CDR3) of the light chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.3, specifically as follows:

[0052] SQSSHVPLT.

[0053] The amino acid sequence of the light chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.4, specifically as follows:

[0054] DIVITQSPLSLPVSLGDQASISCRSS QSLVNSNGNTY LHWYLQKPGQPPKLLIY KV S NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSSHVPLT FGAGTKLELK. That is, the light chain variable region of monoclonal antibody 2E7 contains 112 amino acids.

[0055] The amino acid sequence of the complementarity-determining region 1 (CDR1) of the heavy chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.5, specifically as follows:

[0056] GFSLTNYG.

[0057] The amino acid sequence of the complementarity-determining region 2 (CDR2) of the heavy chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.6, specifically:

[0058] IWSDGRT.

[0059] The amino acid sequence of the complementarity-determining region 3 (CDR3) of the heavy chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.7, specifically:

[0060] ARQSTATMDY

[0061] The amino acid sequence of the heavy chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.8, specifically:

[0062] QVQLKQSGPGLVAPSQSLSITCTIS GFSLTNYG VHWVRQPPGKGLEWLVV IWSD GRT TYNSALKSRLTISKVNSKSQVFLKMNSLQTDDTAMYYC ARQSTATMDY WGQG TTVTVSS. That is, the heavy chain of monoclonal antibody 2E7 contains 116 amino acids.

[0063] Correspondingly, the nucleotide sequence of the complementarity-determining region 1 (CDR1) of the light chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO. 9, specifically as follows:

[0064] CAGAGCCTTGTAAACAGTAATGGAAACACCTAT.

[0065] The nucleotide sequence of the complementarity-determining region 2 (CDR2) of the light chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.10, specifically as follows:

[0066] AAAGTTTCC.

[0067] The nucleotide sequence of complementarity-determining region 3 (CDR3) of the light chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.11, specifically as follows:

[0068] TCTCAAAGTTCACATGTTCCTCTCACG.

[0069] The nucleotide sequence of the light chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.12, specifically as follows:

[0070] GATATTGTGATCACCCAGTCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGT CAGAGCCTTGTAAACAGTAATGGAAACACCTAT TTACACTGGTACCTGCAGAAGCCAGGCCAGCCTCCAAAGCTCCTGATCTAC AAAGTTTCC AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGC TCTCAAAGTTCACATGT TCCTCTCACG TTCGGTGCTGGGACCAAGCTGGAGCTGAAA. That is, the light chain of monoclonal antibody 2E7 contains 336 nucleotides.

[0071] The nucleotide sequence of the complementarity-determining region 1 (CDR1) of the heavy chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.13, specifically as follows:

[0072] GGATTCTCATTAACCAACTATGGT.

[0073] The nucleotide sequence of the complementarity-determining region 2 (CDR2) of the heavy chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.14, specifically as follows:

[0074] ATATGGAGTGATGGAAGGACA.

[0075] The nucleotide sequence of the complementarity-determining region 3 (CDR3) of the heavy chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.15, specifically as follows:

[0076] GCCAGACAATCTACGGCTACGATGGACTAC.

[0077] The nucleotide sequence of the heavy chain variable region of monoclonal antibody 2E7 is shown in SEQ ID NO.16, specifically as follows:

[0078] CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCATCTCA GGATTCTCATTAACCAACTATGGTGTACACTGGGTTCCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGTAGTG ATATGGAGTGATGGAAGGACA ACCTATAATTCAGCTCTCAAATCCAGACTGACCATCAGCAAGGTCAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTCCAAACTGATGACACAGCCATGTACTACTGT GCCAGACAATC TACGGCTACGATGGACTAC TGGGGTCAAGGAACCACGGTCACCGTCTCGAGC. That is, the heavy chain of monoclonal antibody 2E7 contains 348 nucleotides.

[0079] 2. Monoclonal antibody titer determination in cell supernatant and ascites fluid

[0080] The rHis-CFP10 antigen was diluted to 2 μg / mL using coating buffer, and 50 μL / well was used to coat the plate. The plate was incubated overnight at 4°C. After washing three times with PBST, 200 μL of blocking buffer was added to each well, and the plate was incubated overnight at 4°C. The cell supernatant and ascites fluid of the monoclonal antibody were serially diluted to 100 μL / well. SP2 / 0 cell supernatant and ascites fluid were also serially diluted as negative controls, and the plates were incubated at 37°C for 2 h. After washing three times with PBST, 100 μL / well of the working concentration of enzyme-labeled secondary antibody was added, and the plate was incubated at 37°C for 1 h. After washing with PBST, TMB was displayed, and OD was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 450 The value of P / N value ≥ 2.1 was used as the criterion for determining the titer of monoclonal antibody in cell supernatant and ascites.

[0081] The results showed that the monoclonal antibody titer in the 2E7 cell supernatant was 1,024,000, and the ascites titer was 1 × 10⁻⁶. 10 .

[0082] 3. Monoclonal antibody specificity identification

[0083] 3.1 Indirect ELISA Experiment

[0084] Multiple recombinant proteins, including rGST-ESAT6, rHis-ESAT6, rGST-CFP10, rHis-CFP10, and tag proteins His and GST, were used as detection precursors to coat ELISA plates, indirectly detecting the reactivity between the recombinant and tag proteins and the monoclonal antibody 2E7. The recombinant proteins were diluted to the optimal coating concentration, 50 μL / well, and coated onto the plate overnight at 4°C. After washing three times with PBST, 200 μL of blocking buffer was added to each well, and the plate was blocked at 37°C for 2 h. After washing four times with PBST, the monoclonal antibody ascites fluid or cell supernatant was serially diluted to 50 μL / well. SP2 / 0 cell supernatant was also serially diluted as a negative control, and PBS was added as a blank control. The plates were incubated at 37°C for 2 h. After washing four times with PBST, 100 μL of the working concentration of the enzyme-labeled secondary antibody was added to each well, and the plate was incubated at 37°C for 1 h. After washing with PBST, TMB was displayed, and OD was measured using an ELISA reader. 450 The value of .

[0085] The results are shown in Table 1. The 2E7 antibody only reacted with rGST-CFP10 and rHis-CFP10, but not with other recombinant proteins rGST-ESAT6 and rHis-ESAT6, including the tag proteins His and GST.

[0086] Table 1.2 Results of E7 Monoclonal Antibody Specificity Identification

[0087]

[0088] +: Positive; -: Negative

[0089] Natural strains, including H37Ra, BCG, and nontuberculous mycobacteria (M. smegmatis, M. abscessus, M. fortuitum, M. marinum, M. kansasii), were used as assay precursors and serially diluted 2-fold to coat ELISA plates to indirectly detect the reactivity between natural strains and 2E7 monoclonal antibodies.

[0090] The results are as follows Figure 2 As shown, the monoclonal antibody 2E7, which is specific to the CFP10 protein of Mycobacterium tuberculosis, can specifically identify the antigen in H37Ra without reacting with BCG strains and non-tuberculous mycobacteria, indicating that the antibody has good specificity in the detection of natural antigens.

[0091] 3.2 Western Blotting Experiment

[0092] The procedure was performed according to the method described in *Molecular Cloning: A Laboratory Manual*. First, SDS-PAGE electrophoresis was performed, with a stacking gel of 5% and a separating gel of 12%. pET-30a(+) / BL21(DE3), pGEX-6P-1 / BL21, H37Ra whole bacterial lysis buffer, BCG whole bacterial lysis buffer, rHis-CFP10, rGST-CFP10, and rHis-CFP10-ESAT6 (rHis-CE) were added at a 1:1 ratio to 2×SDS sample processing solution (4% SDS, 20% glycerol, 5% 2-mercaptoethanol, and 0.002% bromophenol blue in 0.1M Tris, pH 6.8). The mixture was incubated in a 100°C water bath for 5 min and electrophoresed in a vertical electrophoresis system until the bromophenol blue just emerged from the gel surface. After electrophoresis, excess gel was removed, and the gel was transferred in the following order from bottom to top: two filter papers - NC membrane - gel - two filter papers. Both the filter papers and the NC membrane were soaked in transfer buffer at room temperature for 10 min and then tightly adhered without any air bubbles. The gel was transferred in a Bio-Rad semi-dry transfer cell system (NC membrane on the positive electrode side, gel on the negative electrode side); after transfer, it was blocked with 5% skim milk and gently shaken overnight at room temperature; after thorough washing, 1:2000 diluted ascites monoclonal antibody was added and incubated for 2 hours; washed 3 times with PBST for 3-5 minutes each time, and 1:5000 diluted HRP-labeled goat anti-mouse IgG antibody was added and incubated for 1 hour; washed, developed with DAB chromogenic solution, and the reaction was terminated with distilled water.

[0093] The results are as follows Figure 3 As shown, the monoclonal antibody 2E7 specifically binds to rHis-CFP10, rGST-CFP10, and rHis-CE, with specific bands appearing at 17kDa, 38kDa, and 35kDa, respectively. It also recognizes the CFP10 antigen in the H37Ra strain, with a size of approximately 10kDa. However, it does not cross-react with empty vector bacteria that do not express CFP10 or with the BCG strain, indicating that the 2E7 monoclonal antibody is a monoclonal antibody specifically targeting CFP10.

[0094] Example 4: Establishment of Magnetic Particle Chemiluminescence Method

[0095] 1. Steps of Magnetic Particle Chemiluminescence Method

[0096] Step 1: Preparation of magnetically labeled Mycobacterium tuberculosis-specific protein antigen (magnetic beads-CE):

[0097] 1) Use a vortex mixer to thoroughly mix and suspend the carboxyl magnetic microspheres, and take 0.5 mg into a centrifuge tube; place the centrifuge tube on a magnetic rack for 1-2 min, and remove the supernatant;

[0098] 2) Add 1.0 mL of coupling buffer, vortex to disperse the magnetic microspheres, remove the supernatant, and repeat twice;

[0099] 3) Add 10 μL of EDC solution and 10 μL of Sulfo-NHS solution (10 mg / mL, freshly prepared with coupling buffer), and add coupling buffer to a total volume of 1.0 mL. Vortex disperse the magnetic microspheres. Keep the magnetic microspheres suspended and activate the reaction at room temperature for 30 min. Remove the supernatant and wash twice with coupling buffer to remove the activator from the mixture.

[0100] 4) Add antigen rHis-CE, add coupling buffer to a total volume of 0.5 mL, vortex disperse the magnetic microspheres; keep the magnetic microspheres in suspension, react at room temperature for 4 h, and then remove the supernatant.

[0101] 5) Add 0.5 mL of blocking solution and vortex to disperse the magnetic microspheres; keep the magnetic microspheres in suspension and allow them to stand at room temperature or 37°C for 2 hours, then remove the supernatant; wash twice with 0.5 mL of the solution to remove the blocking solution from the mixture.

[0102] 6) Add 0.5 mL of preservation solution, vortex to mix, and obtain a magnetic bead working solution with a concentration of 1 mg / mL. Store at 2-8℃.

[0103] The amount of carboxyl magnetic beads was 0.5 mg, and the amount of antigen labeling was 60 μg / mL.

[0104] Step 2: Preparation of acridinium ester-labeled Mycobacterium tuberculosis CFP10 antibody 2E7 (AE-2E7):

[0105] 1) Add 5 μL of 10 mM biotinylated solution to 0.5 mg 2E7 solution and incubate the mixture at room temperature for 30 minutes.

[0106] 2) Remove unreacted and hydrolyzed reagents using a desalting column or dialysis (PBS ultrafiltration).

[0107] 3) Add the preservation solution to 500 μL and store at 2-8℃ away from light.

[0108] The amount of acrylamide labeled was 5 μL.

[0109] Step 3: Following the reaction principle of the competitive method, add 50 μL of the sample to be tested, 50 μL of magnetic bead solution, and 50 μL of tracer solution to the reaction tube. After reacting at 37°C for 15 min, wash the tube 5 times with washing solution, add 100 μL of pre-excitation solution and 100 μL of excitation solution, measure the relative luminescence count (RLU), and calculate the sample inhibition rate. The measurement process is automatically completed on the fully automated magnetic particle chemiluminescence immunoassay analyzer.

[0110] 2. Determination of the dilution concentration of magnetic beads-CE and AE-2E7

[0111] Magnetic beads-CE (1:100, 1:200, 1:300, 1:400, 1:500) and AE-2E7 (1:500, 1:1000, 1:2000, 1:400, 1:8000) were diluted to different concentrations using sample diluents (containing bovine serum albumin 10 mg / mL, bromonidol 0.2 mg / mL, surfactant brij35 1 mg / mL, chitosan 0.5 mg / mL, and soy hydrolyzed protein 5 mg / mL). Simultaneously, negative PBS samples and positive rHis-CE samples were tested, and chemiluminescence values ​​were read. The optimal dilution combination was determined when the N / P ratio reached its maximum.

[0112] The results showed that when the magnetic bead-CE dilution concentration was 1:100 and the AE-2E7 dilution concentration was 1:1000, the chemiluminescence value N / P of the sample reached its maximum. Therefore, 1:100 and 1:1000 are the optimal dilution concentration combinations.

[0113] 3. Determination of the threshold for magnetic particle chemiluminescence method

[0114] A total of 110 nasal swab samples from healthy cattle collected from a cattle farm in Henan Province were tested to determine the cutoff point. Results were calculated based on the luminescence values ​​of the blank control (B) and the test samples (S) using the following formula: Inhibition percentage = [(BS) / B] × 100%. The mean (X) and standard deviation (SD) of the inhibition percentages were calculated, with X + 3SD representing the cutoff value (C). n If the inhibition rate is ≥ C n The bovine tuberculosis antigen reaction was positive; if the inhibition rate... <C n The bovine tuberculosis antigen reaction was negative.

[0115] The results showed that the average inhibition rate (X) of the luminescence values ​​detected in 110 healthy bovine nasal swab samples was 7.23%, the standard deviation (SD) was 7.48%, and the C... n The value was 22.19%. Therefore, the criteria for determining the result were: when the sample inhibition rate was ≥22.19%, the bovine tuberculosis antigen reaction was positive; when the sample inhibition rate was <22.19%, the bovine tuberculosis antigen reaction was negative.

[0116] 4. Sensitivity test of magnetic particle chemiluminescence method

[0117] The established method was used to detect rHis-CE fusion protein. The initial concentration was 40 μg / mL, which was serially diluted 2-fold to 0.001 μg / mL. The luminescence value was read using a fully automated magnetic microparticle chemiluminescence analyzer. The inhibition percentage was calculated as [(BS) / B]×100%, and a curve was plotted to obtain the formula. The cut-off value was substituted into the formula to calculate the limit of detection, where B is the blank control and S is the test sample.

[0118] The results are as followsFigure 4 As shown, y = 103.27615 / [1 + (x / 0.01248)] -0.83029 -7.45323, R 2 =0.99552. Substituting the Cut-off value into the equation, we get LOD = 4.4 ng / mL, which means that the sensitivity of the established magnetic microparticle chemiluminescence method is 4.4 ng / mL.

[0119] Example 5: Detection of bovine nasal swab samples using magnetic microparticle chemiluminescence method

[0120] The magnetic particle chemiluminescence method established in this experiment was used in conjunction with the Mycobacterium tuberculosis fluorescent PCR kit produced by Zhengzhou Hexu Biotechnology Co., Ltd. in Henan Province to detect 34 positive samples and 102 negative samples. As shown in Table 2, the number of positive samples detected by both the magnetic particle chemiluminescence method and the fluorescent PCR kit was 34, with a positive concordance rate of 97.14%. The number of negative samples detected by both methods was 101, with a negative concordance rate of 99.02% and an overall concordance rate of 99.26%.

[0121] Table 2. Concordance rate analysis of magnetic particle chemiluminescence method

[0122]

[0123]

[0124] The present invention has been described in detail above. However, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, any modifications or improvements that do not depart from the spirit of the present invention are within the scope of protection of the present invention.

Claims

1. A hybridoma cell line that secretes a monoclonal antibody against Mycobacterium tuberculosis CFP10, characterized in that, The hybridoma cell line described was 2E7, which was deposited at the China Center for Type Culture Collection on May 14, 2025, with accession number CCTCC NO:C2025162.

2. A monoclonal antibody against Mycobacterium tuberculosis CFP10, characterized in that, Produced by the hybridoma cell line described in claim 1.

3. A monoclonal antibody 2E7 against Mycobacterium tuberculosis CFP10, characterized in that, The monoclonal antibody includes a heavy chain variable region and a light chain variable region. The light chain variable region includes CDR-L1, CDR-L2, and CDR-L3 with amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. The heavy chain variable region includes CDR-H1, CDR-H2, and CDR-H3 with amino acid sequences as shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively.

4. The anti-tuberculosis CFP10 monoclonal antibody 2E7 according to claim 3, characterized in that, The full-length amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.4, and the full-length amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

8.

5. The use of the anti-Mycobacterium tuberculosis CFP10 monoclonal antibody according to claim 2 and the anti-Mycobacterium tuberculosis CFP10 monoclonal antibody 2E7 according to any one of claims 3 to 4 in the preparation of a kit for detecting Mycobacterium tuberculosis.

6. The application according to claim 5, wherein the kit is a magnetic microparticle chemiluminescence detection kit.

7. A magnetic particle chemiluminescence assay kit for detecting pathogenic Mycobacterium tuberculosis, characterized in that, The kit comprises magnetically labeled Mycobacterium tuberculosis-specific protein antigen, monoclonal antibody 2E7 as described in any one of claims 3 to 4, and sample diluent.

8. The reagent kit according to claim 7, characterized in that, The Mycobacterium tuberculosis-specific protein antigen is the rHis-CFP10-ESAT6 fusion protein.

9. The reagent kit according to claim 7, characterized in that, The pathogenic tuberculosis mycobacteria include Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium bovis (M. bovis).

10. The kit according to claim 7, characterized in that, The sample diluent contains any one or more combinations of bovine serum albumin, bromonitol, surfactant brij35, chitosan, or hydrolyzed soybean protein.

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