CN101985473A - Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application thereof - Google Patents

Abstract

The present invention provides anti-mycobacterium tuberculosis early secreted antigen target protein monoclonal antibody TBEF3. The mice were immunized with specific antigen peptides, and the sensitized mouse B lymphocytes were collected for fusion with mouse myeloma cells. The positive clones were determined by enzyme-linked immunosorbent assay and Western blot detection, and the early secretion of anti-tuberculosis mycobacteria was established. The hybridoma cell line TBEF3 of the monoclonal antibody to the sex antigen target protein (ESAT-6), the positive cells were expanded and cultured and injected into the peritoneal cavity of mice of the same strain to induce ascites containing the antibody, after collection and antibody affinity purification, Obtaining monoclonal antibodies against target proteins of early secretory antigens of Mycobacterium tuberculosis. The monoclonal antibody provided by the invention is used in the detection of early secretory antigen target protein of Mycobacterium tuberculosis.

Description

Anti-tuberculosis ESAT-6 monoclonal antibody TBEF3 and its application

technical field

The invention belongs to the field of biotechnology, and relates to the preparation and application of a monoclonal antibody against mycobacterium tuberculosis early secretory antigen target protein (ESAT-6). Clone the hybridoma cell line of the antibody, induce ascites from the same strain of mice, prepare the anti-EAST-6 monoclonal antibody TBEF3, identify it as IgM, κ type, and then realize the antibody through affinity purification, electrophoresis, immunization and other technologies Applications.

Background technique

Early diagnosis of tuberculosis is an important problem that has been plaguing the field of medical diagnosis. In the 20th century, due to the development and wide application of antibiotic research, humans successfully controlled tuberculosis. However, due to the continuous emergence and accumulation of drug resistance, Mycobacterium tuberculosis is still a real threat to human beings in the 21st century. More seriously, in recent years, the prevalence of human immunodeficiency virus (HIV) and AIDS (AIDS) has become the main reason for the third rise in the incidence and mortality of tuberculosis in the world, and tuberculosis is also the most common opportunistic infection in AIDS patients. and cause of death. The tendency for TB and AIDS to co-exist makes treatment even more difficult. Diagnosis is the premise of treatment. If it can be diagnosed early, the harm of tuberculosis will be greatly reduced. As an area with a high rate of tuberculosis infection and a rapidly growing area of HIV infection in China, it is urgent to improve the early and accurate diagnosis of tuberculosis and HIV/AIDS combined tuberculosis infection.

The traditional means of early diagnosis is the tuberculin skin test. This is a skin test based on the principle of delayed-type hypersensitivity. But two flaws make this diagnostic approach very limited. One: This method cannot distinguish between healthy people who have ever been infected with Mycobacterium tuberculosis and those who have active Mycobacterium tuberculosis; Second: This method cannot distinguish between people who have been infected with Mycobacterium tuberculosis and those who have injected BCG. Therefore, traditional early diagnosis methods can only provide very limited reference information, and the diagnosis often has to wait until the patient has tuberculosis lesions. And it is too late to do treatment at this time, especially for drug-resistant Mycobacterium tuberculosis, the prognosis of patients is very poor.

ELISPOT technology has brought about a revolution in the early diagnosis of tuberculosis. First, because the ELISPOT method uses Mycobacterium tuberculosis-specific peptides, it can avoid the cross-reaction of BCG injectors; second, because ELISPOT detects the level of cellular immunity of the patient at that time, it can effectively distinguish healthy TB carriers from Onset of active TB patients. However, ELISPOT detection steps are cumbersome, require special equipment and expensive reagents, and are difficult to promote in most low-income countries and regions with a high burden of tuberculosis.

It is imminent to develop a rapid and sensitive method for early diagnosis of tuberculosis. Based on the above background, this project selects the currently recognized Mycobacterium tuberculosis specific antigen protein ESAT-6 as the target antigen, and uses modern bioinformatics and molecular biology techniques to design and synthesize several candidate ESAT-6 specific antigenic peptides Sequence, using fusion hybridoma technology to establish a hybridoma cell line that stably secretes monoclonal antibodies against specific antigenic proteins of Mycobacterium tuberculosis, and prepare, purify and identify these monoclonal antibodies in large quantities. The successful acquisition of the monoclonal antibody lays a material foundation for the establishment of a new type of diagnosis method for tuberculosis—diagnosis based on immunological techniques. At the same time, it plays an important role in the research of disease pathogenesis, diagnosis, prognosis and efficacy judgment.

The present invention uses hybridoma cell technology. This technology fuses the B lymphocytes of immunized mice with myeloma cells to establish a hybridoma cell line that secretes homogeneous antibodies, also known as monoclonal antibody technology. This technology involves a series of methods such as animal immunization, cell culture, cell fusion, cell clone culture and immunoassay.

Contents of the invention

The object of the present invention is to provide a monoclonal antibody against Mycobacterium tuberculosis early secretory antigen target protein (ESAT-6), which has the amino acid sequence of SEQ No.1: Met Thr Glu Gln GlnTrp Asn Phe Ala Gly Ile Glu Ala Ala. The subtype of the monoclonal antibody is IgM and κ, named TBEF3, and can specifically recognize the aa 1-14MTEQQWNFAGIEAA antigenic peptide sequence of Mycobacterium tuberculosis early secretory antigen target protein (ESAT-6). The anti-mycobacterium tuberculosis early secreted antigen target protein (ESAT-6) monoclonal antibody hybridoma cell has been preserved by the China Center for Type Culture Collection; address: Wuhan University, Wuhan, China; preservation date: August 17, 2010; Culture name, strain number and symbol (including genotype): Mycobacterium tuberculosis early secreted antigen target protein (ESAT-6) monoclonal antibody hybridoma cell TBEF3 (IgM, κ type), deposit number: CCTCC NO: C201080.

The second object of the present invention is to provide a preparation method of anti-ESAT-6 monoclonal antibody, which is achieved through the following steps and technical solutions:

(1) Animal immunization: 6-week-old BALB/C mice were selected, and the mice were immunized with antigenic peptides (1-14 amino acids of ESAT-6, 14 peptides MTEQQWNFAGIEAA). 14 Peptide MTEOQWNFAGIEAA was synthesized by solid-phase method on the peptide synthesizer (431A) of ABI Company in the United States, using the Fmoc (9-fluorenylmethoxycarbonyl) scheme, and the synthesis steps were carried out according to the peptide synthesis operation manual of ABI Company. Purified by high-performance liquid chromatography, the purity is >95%, and the sequence is identified by mass spectrometry.

(2) Culture of mouse myeloma cells: culture mouse myeloma cells SP2/0 and keep them in a good growth state for cell fusion.

(3) Cell fusion: the polyethylene glycol fusion method was used. Use BALB/C mouse peritoneal macrophages as feeder cells, inoculate BALB/C mouse peritoneal macrophages in a 96-well culture plate one day before fusion, culture in HAT medium containing 20% FCS for one day, take (1) The splenic lymphocytes of the middle mouse and the myeloma cells of the middle mouse were mixed and centrifuged, and then polyethylene glycol (PEG 6000) was used to mediate cell fusion. The fused cells were properly diluted and inoculated into the feeding Cell culture plates, cultured under appropriate conditions.

(4) Screening of hybridoma cells: the above cultures were cultured in HAT selective medium. When the cell colony grows to a suitable size, absorb the cell culture supernatant for antibody identification and screen positive clones.

(5) Cloning of hybridoma cells: The hybridoma cells were cloned by the limiting dilution method, and the cells diluted to a certain density were seeded into 96-well plates so that only one cell grew in each well. The culture supernatant was taken from the wells forming cell colonies for enzyme-linked immunosorbent assay (ELISA) to identify positive clones. Repeat limiting dilution cloning several times until the positive porosity of hybridoma cells reaches 100%. The cloned hybridoma cells were expanded and cultured for antibody identification and physical and chemical properties analysis.

(6) Induction of monoclonal antibody ascites: One week before inoculation of hybridoma cells, BALB/C mice were intraperitoneally injected with pristane 0.5ml each, and then each mouse was inoculated with 5×10 ⁶ positive hybridoma cells, and ascites was collected 10 days later Centrifuge, determine antibody titer, and purify monoclonal antibody.

(7) Purification of monoclonal antibodies: The monoclonal antibodies in ascites were purified by Protein L affinity purification.

(8) The present invention obtains a hybridoma cell line producing anti-ESAT-6 monoclonal antibody, ie TBEF3. The TBEF3 hybridoma cell line is cloned three times and cultured continuously for more than six months, and the secreted antibody is stable. The cell line was cryopreserved in liquid nitrogen and grew well after resuscitation, with no decline in antibody secretion. The titers of TBEF3 culture supernatant and ascites were 1:256 and 1:8192, respectively, measured by ELISA indirect method. The immunoglobulin subtype analysis of the monoclonal antibody showed that the antibody types produced by the hybridoma cells were all IgM. TBEF3 has been preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C201080.

The present invention provides hybridoma cells producing monoclonal antibodies, which are obtained after fusion, screening, cloning, subculture, and repeated freezing and recovery of immunized BALB/c mouse splenocytes and mouse myeloma cells SP2/0 The mouse hybridoma cell line TBEF3 can stably secrete the anti-ESAT-6 monoclonal antibody TBEF3. The hybridoma cell line has been preserved in the China Center for Type Culture Collection, and the preservation number is: CCTCC No.C201080.

Another object of the present invention is to provide the application of the monoclonal antibody TBEF3 in the detection of Mycobacterium tuberculosis early secretory antigen target protein and the expression level of the ESAT-6 protein in the sample for qualitative and quantitative application, achieved by the following approach:

①Using monoclonal antibody TBEF3 as a probe, use SDS-PAGE electrophoresis to detect ESAT-6 protein in Mycobacterium tuberculosis and its culture, various body fluid samples, genetically recombined and artificially synthesized samples by Western Blot method situation is identified.

②Using the monoclonal antibody TBEF3 as the binding antibody, immunoprecipitation method was used to identify the expression of ESAT-6 in the test sample mentioned in the above ①.

③Using monoclonal antibody TBEF3 as the coating antibody or detection antibody, the expression level of ESAT-6 protein in Mycobacterium tuberculosis culture, various body fluid samples, genetically recombined and artificially synthesized samples was tested by enzyme-linked immunosorbent assay (ELISA). Qualitative and quantitative analysis.

The advantage of the present invention is that it provides a monoclonal antibody against Mycobacterium tuberculosis specific antigen ESAT-6, which has not been reported in literature yet. The preparation method is simple and easy, and more importantly, the monoclonal antibody prepared by this method can have multiple uses, such as bacterial culture of Mycobacterium tuberculosis in clinical and laboratory, Mycobacterium tuberculosis, clinical body fluid samples, gene Qualitative and quantitative analysis of the expression of ESAT-6 molecules in recombinant proteins, as well as various research purposes.

Instructions attached

Figure 1 shows the immunoglobulin subtype analysis of the monoclonal antibody TBEF3.

Figure 2 The expression of recombinant ESAT-6/CFP-10 fusion protein, Mycobacterium tuberculosis and other mycobacteria and their culture supernatants were detected by immunoprecipitation and western-blot methods.

Fig. 3 ELISA standard reaction curve using monoclonal antibody as coating antibody and recombinant ESAT-6/CFP-10 fusion protein as standard antigen.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1. Preparation method of monoclonal antibody against ESAT-6

(1) Immunization of mice: For the first immunization, emulsify the 14 peptides (sequence aa1-14MTEQQWNFAGIEAA) of cross-linked KLH ESAT-6 with Freund's complete adjuvant, and emulsify with incomplete adjuvant for the second and third times, each BALB /C mouse 0.2ml (containing ESAT-6 antigen peptide 200μg), multi-point intradermal injection on the back, once every two weeks. A booster immunization was given 6 weeks later, and cell fusion was carried out 3 days later.

(2) Cultivation of mouse myeloma cell SP2/0: the SP2/0 myeloma cell line from BALB/C mouse was cultured and passaged with 10% FBS-DMEM medium, and contained 5% CO in _{a saturated} humidity chamber Cultured in a 37°C incubator. Subculture the day before fusion to ensure that the cells enter the logarithmic growth phase at the time of fusion.

(3) Cell fusion: use BALB/C mouse peritoneal macrophages as feeder cells, inoculate BALB/C mouse peritoneal macrophages in 96-well culture plates one day before fusion, and culture in HAT medium containing 20% FCS One day, the next day, take the spleen, put it in a 200-mesh stainless steel mesh, remove the fat and connective tissue, tear off the capsule, separate the spleen cells, wash the cells by centrifugation for 1 or 2 times, and then resuspend in culture medium. Mouse SP2/0 myeloma cells were collected, centrifuged, washed twice and then resuspended in culture medium to be used as SP2/0 cells to be fused. Mix 1.0×10 ⁸ splenic lymphocytes of immunized mice with 1.0×10 ⁷ mouse myeloma cells SP2/0, and fuse under the action of 50% PEG6000. Mix the two cells and wash once, centrifuge to discard the supernatant, flick the tube wall to suspend the cells (do not add liquid), add 0.8ml of 50% PEG6000 (pH 8.0) pre-warmed at 37°C to the cells dropwise within 60-90 seconds During the precipitation, gently shake the centrifuge tube, but do not blow it, let it stand for 1 minute, then add 1ml of serum-free RPMI 1640 within the first minute, and add 2ml within the second minute according to the principle of slow first and then fast For serum-free RPMI 1640, add 7ml of serum-free RPMI 1640 within 3 minutes, then gradually add 30-40ml of serum-free RPMI1640 medium pre-warmed at 37°C within 1 minute. Centrifuge at 800 rpm for 5-10 minutes at low speed. Then add 20% FCS HAT medium and inoculate them into 96-well culture plates with feeder cells. Generally, 4 plates are spread for each fused cell, and they are cultured in a 5% CO ₂ incubator at 37°C.

(4) Screening of hybridoma cells: Change 1/2 culture solution (HAT) every 4 days, and change to HT-containing culture solution after 10 days. The fused hybridoma cells were cultured for about two weeks in the selective medium containing HT. When the cell colony grows to an appropriate size (observed under a 10x objective lens, the size of the cell clone should occupy a field of view), absorb the supernatant of the culture medium, and perform ELISA after appropriate dilution to screen positive clones. Positive hybridoma clones were screened by ELISA indirect method. Main steps: ① Dilute ESAT-6/CFP-10 recombinant fusion protein with 0.01M pH9.6 carbonate buffer solution, and synthesized ESAT-6 specific polypeptide at a concentration of 200ng/ml, and add 0.1ml to 96-well ELISA plate respectively ②Wash the plate three times with 0.01M pH7.4 PBS-Tween 20; ③Seal with 2% BSA-0.01M PBS with pH 7.2 for 1 hour; Hybridoma culture supernatant, 0.1ml/well, set positive control (immunized mouse serum), negative control (SP2/0 culture supernatant) and blank control at the same time, react at room temperature for 2 hours; ⑥ wash the plate; ⑦ add 1 : 5000 diluted goat anti-mouse Ig(G+M)-HRP, 0.1ml/well, react at room temperature for 1 hour; ⑧wash the plate; ⑨add substrate (TMB) and react in the dark for 5-10 minutes at room temperature; ⑩2M H ₂ The reaction was terminated by SO4; the OD value was measured at 450nm, and P/N ≥ 2.1 was positive.

(5) Cloning of hybridoma cells: The cloning and culturing of hybridoma cells was carried out by the limiting dilution method, and the positive antibody detection (both ESAT-6/CFP-10 recombinant fusion protein and synthetic ESAT-6 specific polypeptide were positive) were selected. After the hybridoma well cells were properly proliferated, the cells were accurately counted. Dilute the cell suspension to 10 cells/ml with complete 1640 medium and inoculate it into a 96-well culture plate with feeder cells, 0.1ml per well, observe the cell growth after 7-10 days, and detect the antibody level in the supernatant , select the 5 culture wells with the highest antibody titer and the growth of single clonal cells for secondary cloning culture. This method can be repeated many times until the positive rate of antibody detection in monoclonal wells is 100%.

(6) Induction of ascites: one week before inoculation of hybridoma cells, BALB/C mice were injected intraperitoneally with 0.5ml of pristane, and then each mouse was inoculated with 5.0×106 positive hybridoma cells, and ascites was collected 10 days later to determine the antibody effect. price.

(7) Purification of monoclonal antibodies: The monoclonal antibodies in ascites were purified by affinity purification (Protein L cross-linked Sepharose). ① Dilute the ascites 3 times with cold Binding Buffer, and then centrifuge at 10,000 rpm at 4°C for 15 minutes to remove the precipitate. ② Fully wash the affinity purification column prepacked with Sepharose-Protein L with 10 times the column bed volume of Binding Buffer. ③Put the diluted ascitic fluid on the column, and control the flow rate to 8-10 drops/min. ④ Repeat the ascites flowing through the column once. ⑤ Fully wash with 20 times the column bed volume of Binding Buffer until the A280 absorbance of the flow-through solution is less than 0.01. ⑥Use Elution Buffer (elution buffer) to elute the bound monoclonal antibody, control the flow rate to 8-10 drops/min, collect the eluate in pre-added 0.1ml potassium phosphate buffer (PH7.9, 0.5M) Collect 0.5ml of antibody-containing eluate in each collection tube, and collect more than 20 tubes in total. ⑦ Measure the absorbance of the eluate in each tube at A280nm, and collect the eluate with an absorbance value greater than 0.2. ⑧Place the collected eluate in a dialysis card and dialyze against 0.1M PBS at pH 7.0. The medium was changed every 6 hours for a total of 24 hours of dialysis. ⑨ After diluting the dialyzed antibody solution (1:100), measure the protein content at 280nm. ⑩ Dispense the purified antibody into small tubes and store in a low-temperature refrigerator for later use.

(8) Subtype identification of the monoclonal antibody: the mouse monoclonal antibody immunoglobulin typing kit of Serotec Company was used for analysis. The purified monoclonal antibody was properly diluted for detection, and the operation was performed strictly according to the kit instructions. The test results showed that the monoclonal antibodies secreted by TBEF3 hybridoma cells were IgM and κ types.

The results are shown in Figure 1.

Embodiment 2. carry out the identification (qualitative) detection of ESAT-6 with this monoclonal antibody

The anti-ESAT-6 monoclonal antibody prepared by the present invention can be used for identification (qualitative detection), and identification method can be realized by following two methods:

1. Western Blotting by Western Blotting: The recombinant ESAT-6 and ESAT-6/CFP-10 fusion protein and Mycobacterium tuberculosis whole protein lysate were detected by Western Blotting with this monoclonal antibody, and the target band appeared at the corresponding position, indicating that the detection to the expression of ESAT-6 protein. Specific steps:

(1) SDS polyacrylamide gel electrophoresis: For the method, refer to F. Osper et al., "Refined Molecular Biology Experiment Guide" (Science Press, 1998). Using 15% separating gel and 5% stacking gel, the electrophoresis condition is a voltage of 150V, and the bromophenol blue dye band is about 1.5 cm away from the bottom edge of the gel to stop the electrophoresis.

(2) Electrotransfer: For the method, refer to F. Osper et al., "Refined Molecular Biology Experiment Guide" (Science Press, 1998). It was transferred to PVDF (0.22 μm) membrane by electrotransfer.

(3) Immunoblotting: After electrotransfer to the membrane, the membrane was blocked in 5% skimmed milk powder blocking solution at room temperature for 1 hour, and the monoclonal antibody TBEF3 prepared by the present invention was used as the primary antibody, incubated at room temperature for 2 hours or reacted overnight at 4°C, and then reacted with TBST (TBS added 0.5% Tween-20) washed 3 times, 10min each time. Horseradish peroxidase-labeled goat anti-mouse IgM was used as the secondary antibody, reacted at room temperature for 2 hours, washed with the above method, and reacted with ECL for 1 minute, and then exposed and imaged on a fully automatic chemiluminescent imager (Bio-Rad VersaDoc 5000MP).

2. Immunoprecipitation (IP): Combine recombinant ESAT-6 and ESAT-6/CFP-10 fusion protein, whole protein lysate of Mycobacterium tuberculosis, culture supernatant of Mycobacterium tuberculosis, tuberculous pleural effusion of clinical tuberculosis patients and The monoclonal antibody was subjected to immunoprecipitation reaction, and then detected by western-blot, and the target band appeared at the corresponding position, indicating that the expression of ESAT-6 protein was detected. Specific steps:

① Take 1ug of recombinant protein or 100ug of Mycobacterium tuberculosis lysate protein, 500ul of Mycobacterium tuberculosis culture supernatant or tuberculosis pleural effusion of clinical tuberculosis patients plus purified 1ug of anti-ESAT-6 monoclonal antibody TBEF3, shake slowly at 4°C for 2 hours ; Add 20 microliters of fully resuspended Protein L Agarose and shake slowly overnight at 4°C.

②Centrifuge at 2500rpm for 5 minutes, carefully aspirate the supernatant, and be careful not to aspirate Protein L Agarose.

③ Wash the precipitate 5 times with 0.5-1 ml TBST.

④ After the last wash, remove the supernatant, add 40 microliters of 1X SDS-PAGE electrophoresis loading buffer to resuspend the pellet, and centrifuge the sample to the bottom of the tube by instantaneous high-speed centrifugation.

⑤Treat at 100°C or in a boiling water bath for 3-5 minutes, take some samples for SDS-PAGE electrophoresis, and store the temporarily unused samples at -20°C.

⑥ After electrophoresis, transfer the protein to PVDF membrane.

⑦ After the membrane transfer, place the membrane in 5% skimmed milk powder blocking solution and block at room temperature for 1 hour.

⑧ Add rabbit anti-ESAT-6 polyclonal antibody and react at room temperature for 2 hours.

⑨ After washing the membrane 4 times, add anti-rabbit IgG-HRP and react at room temperature for 1 hour.

⑩After washing the membrane 4 times, the ECL color was developed, and the VersaDoc 5000 automatic chemiluminescence imager was exposed for imaging.

The results are shown in Figure 2, where M: protein molecular weight Marker (15% SDS-PAGE), 1. recombinant ESAT-6/CFP-10 fusion protein, 2. recombinant ESAT-6 protein, 3. Mycobacterium tuberculosis culture supernatant Liquid, 4. Tuberculous pleural effusion, 5. Mycobacterium avium intracellulare lytic protein, 6. Mycobacterium kansasii lytic protein, 7. Mycobacterium tuberculosis lytic protein.

Embodiment 3, carry out the quantitative detection of ESAT-6 molecule with this monoclonal antibody

The anti-ESAT-6 monoclonal antibody prepared by the present invention can be used to quantitatively detect recombinant ESAT-6 and ESAT-6/CFP-10 fusion protein, Mycobacterium tuberculosis culture supernatant, and various clinical body fluid samples in tuberculosis-specific antigen ESAT -6 level, the detection method can be realized by the following two methods:

1. Indirect ELISA method:

① Dilute the test sample appropriately in the coating solution of 0.01M pH9.6 carbonate buffer solution, and at the same time dilute the recombinant ESAT-6 and ESAT-6/CFP-10 fusion protein in appropriate serial concentrations in the coating solution, as Quantitative standard control. Add 100ul of the above-mentioned samples to be tested and standard products of serial concentrations to the corresponding microplate wells, incubate at room temperature for 2h or coat overnight at 4°C.

②Empty the liquid and pat dry the residual liquid, wash the plate three times with 0.01M pH7.4 PBS-Tween 20 washing solution.

③ Block with 2% BSA-0.01M PBS pH 7.2 for 1 hour.

④ Wash the plate 3 times as above.

⑤Add appropriately diluted anti-ESAT-6 monoclonal antibody TBEF3, 0.1ml/well, and react at room temperature for 2 hours.

⑥After washing the plate 3 times as above, add goat anti-mouse IgM-HRP, 0.1ml/well, and react at room temperature for 1 hour.

⑦ After soaking the plate with washing solution for 5 minutes, wash the plate 4 times as above, then add the substrate (TMB) and react in the dark at room temperature for 5-10 minutes.

⑧2M H ₂ SO4 to terminate the reaction, measure the OD value at 450nm.

⑨ Draw a standard reaction curve, and calculate the expression level of EAST-6 in the sample to be tested according to the standard curve.

2. Sandwich ELISA method:

① Coating: Dilute another ESAT-6 monoclonal antibody TBEA8 to 1 μg/mL with coating buffer. Add 0.1mL/well to the reaction well of the microplate plate, overnight at 4°C. The next day, the solution in the well was discarded, and the plate was washed 3 times with 0.01M pH 7.4 PBS-Tween 20 washing buffer, 3 minutes each time.

② Blocking: Block with 2% BSA-0.01M PBS at pH 7.2 for 1 hour at room temperature.

③ Adding samples: add appropriately diluted samples to be tested and 0.1 mL/well of recombinant ESAT-6 standard diluted in serial concentrations to the above-mentioned coated reaction wells, and put them in a wet box for 2 hours at room temperature.

④Wash the plate: Wash the plate 3 times with washing buffer, 3min each time.

⑤ Add secondary antibody: add the anti-ESAT-6 monoclonal antibody TBEF3 (IgM type, targeting different EAST-6 targets) or rabbit anti-ESAT-6 polyclonal antibody at appropriate dilution, 0.1ml/well, and react at room temperature for 2 hours.

⑥Add enzyme conjugate: After washing the plate 3 times as above, add goat anti-mouse IgM-HRP or anti-rabbit IgG-HRP, 0.1ml/well, and react at room temperature for 1 hour.

⑦Color development: After washing the plate 4 times as above, add the substrate (TMB) and react in the dark for 5-10 minutes at room temperature.

⑧ Termination and plate reading: 2M H ₂ SO4 to terminate the reaction, and measure the OD value at 450nm.

⑨Standard curve and calculation: take the standard concentration as the abscissa and the absorbance as the ordinate to generate a standard curve and a linear regression equation, and calculate the expression content of EAST-6 in the sample to be tested according to the standard response curve.

The results are shown in Figure 3 and Table 1

Table 1 The positive rate of ESAT-6 in clinical body fluid samples detected by ELISA and its application in the diagnosis of tuberculosis

Sample classification Number of cases Number of EAST-6 positive detections Mycobacterium tuberculosis culture supernatant 38 38 Culture supernatant of nontuberculous mycobacteria 20 0 Clinically diagnosed tuberculous pleural effusion 32 29 Clinically diagnosed nontuberculous pleural effusion twenty four 0 Serum from healthy people 20 0

Using the ELISA method established by ESAT-6 monoclonal antibody to test clinical samples, its sensitivity in the diagnosis of tuberculosis is 92.4%, and its specificity is 100%.

It should be understood that the present invention is described in conjunction with the best embodiment, but after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the present application. The scope defined by the appended claims.

Claims (6)

1. An anti-Mycobacterium tuberculosis early secretory antigen target protein monoclonal antibody TBEF3, the monoclonal antibody subtype is IgM, κ type, and can specifically bind to the early secretory antigen target protein ESAT-6 of Mycobacterium tuberculosis , produced by the hybridoma whose accession number is CCTCC No.C201080. 2. the preparation method of the monoclonal antibody TBEF3 of anti-mycobacterium tuberculosis early secretory antigen target protein according to claim 1, it is characterized in that this monoclonal antibody obtains by the following steps: (1) Immunization of mice: select 6-week-old BALB/C mice, and use specific antigenic peptides, namely the 1-14th amino acids of the early secretory antigen target protein gene of Mycobacterium tuberculosis, 14 peptides MTEQQWNFAGIEAA to treat the mice immunity. Each mouse was immunized with 200ug of antigen peptide mixed with adjuvant subcutaneously in multiple points, once every 2 weeks, a total of 3 times; the fourth booster immunization was used for fusion 3 days later. (2) Culture of mouse myeloma cells: culture mouse myeloma cells SP2/0 and keep it in a good growth state for hybridoma cell fusion; (3) Cell fusion: the polyethylene glycol cell fusion method was used, and BALB/C mouse peritoneal macrophages were used as feeder cells. The day before fusion, BALB/C mouse peritoneal macrophages were inoculated in a 96-well culture plate. Cultivate in HAT medium containing 20% FCS for one day, mix and centrifuge the spleen lymphocytes of the immunized mouse in step (1) and the myeloma cells of the mouse in step (2), then mediate cell fusion with polyethylene glycol, dilute The fused cells were inoculated into culture plates for culture; (4) Screening of hybridoma cells: culture the above-mentioned culture in HAT-containing selective medium, and when the cell colony grows to a suitable size, draw the culture supernatant and use enzyme-linked immunosorbent assay for antibody identification, and screen positive clone; (5) Cloning culture of hybridoma cells: positive hybridoma cells were cloned by limiting dilution method, and the cells diluted to a certain density were inoculated into 96-well cell culture plates, so that only one cell grew in each well to form a well of cell colonies Take the supernatant for enzyme-linked immunosorbent assay, screen and identify positive clones, and repeat the cloning several times until the positive porosity of the hybridoma cells reaches 100%. Identification; (6) Induction of mouse ascites: Inoculate 5×10 ⁶ positive hybridoma cells into each BALB/C mouse, collect ascites after 10 days and centrifuge, measure antibody titer, and purify monoclonal antibody in ascites; (7) Purification of monoclonal antibodies: The monoclonal antibodies in ascites were purified by Protein L affinity purification. 3. The application of the anti-Mycobacterium tuberculosis early secretory antigen target protein monoclonal antibody TBEF3 according to claim 1 in the detection of Mycobacterium tuberculosis early secretory antigen target protein. 4. the application of the monoclonal antibody TBEF3 of anti-mycobacterium tuberculosis early secretory antigen target protein in the detection of mycobacterium tuberculosis early secretory antigen target protein according to claim 3 is characterized in that: Immunosorbent assay, immunoblotting and immunoprecipitation were used to detect the expression of Mycobacterium tuberculosis early secretory antigen target protein in bacterial cultures and various body fluid samples. 5. The application of the anti-Mycobacterium tuberculosis early secretory antigen target protein monoclonal antibody TBEF3 according to claim 3 in the quantitative analysis of the Mycobacterium tuberculosis early secretory antigen target protein. 6. the application of the monoclonal antibody TBEF3 of the anti-mycobacterium tuberculosis early secretory antigen target protein quantitative analysis in the early secretory antigen target protein of tuberculosis according to claim 3 is characterized in that: by ELISA The method of adsorption test was used to qualitatively and quantitatively express the target protein expression level of Mycobacterium tuberculosis early secretory antigen in bacterial culture and body fluid samples.

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