Antigen protein composition for detecting bovine tuberculosis gamma-interferon and detection kit
By combining MTB8.4, ESAT6, CFP10, and Rv3615c antigen proteins with direct chemiluminescence technology, the problems of false positives and cumbersome operation in bovine tuberculosis detection have been solved, achieving a highly efficient and sensitive detection solution.
Patent Information
- Application Number
- CN202511184829.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-22
- Publication Date
- 2025-11-21
AI Technical Summary
Existing bovine tuberculosis detection technologies lack sufficient antigen specificity, making them prone to false positives. Furthermore, detection methods such as ELISA are cumbersome, time-consuming, and have limited sensitivity.
We used a combination of four antigen proteins—MTB8.4, ESAT6, CFP10, and Rv3615c—and combined them with direct chemiluminescence technology to optimize the anti-interference system and design a high-efficiency reagent kit.
It significantly improves the sensitivity and specificity of the test, reduces the detection limit to 0.1 pg/mL, lowers the false positive rate, shortens the test time to 15 minutes, and increases the throughput to 200 samples/hour, making it suitable for large-scale screening.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biological detection, and particularly relates to an antigen protein composition for detecting bovine tuberculosis gamma-interferon and a detection kit. BACKGROUND
[0002] Bovine tuberculosis is a disease caused by Mycobacterium bovis, which occurs in cattle. It is characterized by tuberculous nodular granuloma and caseous, calcified necrotic lesions of tissues and organs. Bovine tuberculosis is a chronic infectious disease of human and animals, mainly transmitted through the respiratory tract and digestive tract, and can also be transmitted through the placenta or mating.
[0003] Gamma-interferon is a cytokine produced by T lymphocytes and NK cells under the stimulation of specific antigens. Therefore, the level of gamma-interferon expressed in the whole blood of cattle is usually used to determine whether the detected cattle has been infected with bovine tuberculosis.
[0004] However, the existing detection technology has the following problems:
[0005] 1. Insufficient antigen specificity: traditional bovine tuberculosis detection uses bovine tuberculin (PPD) as a stimulating antigen, but it is easy to cross-react with environmental mycobacteria such as avian mycobacterium, resulting in false positives.
[0006] 2. Limitations of existing detection technology:
[0007] (i) ELISA: requires multiple washes, long reaction time (more than 2 hours), and low throughput for manual operation.
[0008] (ii) Enzymatic chemiluminescence: relies on horseradish peroxidase (HRP) to catalyze the substrate, with high background interference and limited sensitivity.
[0009] Therefore, based on this, the technical solution of the present application is proposed. SUMMARY
[0010] In order to solve the problems existing in the prior art, the present application provides an antigen protein composition for detecting bovine tuberculosis gamma-interferon, characterized in that it comprises MTB8.4, ESAT6, CFP10 and Rv3615c.
[0011] In order to facilitate understanding of the present application, the antigen proteins described in the present application are explained:
[0012] MTB8.4: is a small molecular protein isolated and purified from the culture filtrate of Mycobacterium bovis, with a relative molecular mass of 8400, which can induce specific Th1 immune response and secrete high levels of IFN-γ.
[0013] ESAT6 and CFP10: These are secretory antigens encoded by the RD1 region of Mycobacterium tuberculosis. They are highly specific and can recognize natural infections (excluding interference from BCG vaccination).
[0014] Rv3615c: Although located outside RD1, it belongs to the Esx-1 secretory system substrate protein. It is similar in structure and function to ESAT6 / CFP10 and can activate T cells to produce gamma interferon, thereby enhancing detection sensitivity.
[0015] Complementary antigenic epitopes: ESAT6 / CFP10 primarily targets Th1 cell immune responses, while Rv3615c covers more variant infections (such as drug-resistant strains), and MTB8.4 supplements Mycobacterium bovis-specific antigens, forming a multidimensional detection network.
[0016] Therefore, under the stimulation of the quadruple antigen, the intensity of gamma-interferon release from peripheral blood mononuclear cells (PBMCs) is significantly higher than that of a single antigen, resulting in a significant improvement in detection sensitivity. Furthermore, the quadruple antigen combination exhibits anti-interference ability against the high lipid / hemoglobin matrix in bovine whole blood, enhancing detection stability.
[0017] Preferably, the mass ratio of MTB8.4, ESAT6, CFP10 and Rv3615c is 1:1:1:1;
[0018] And / or, the concentration of the antigen protein composition is 2 to 6 μg / mL.
[0019] Based on the same technical concept, the present invention further provides the use of the antigen protein composition in the preparation of a kit for detecting bovine tuberculosis gamma interferon.
[0020] Based on the same technical concept, the present invention provides a kit for detecting bovine tuberculosis gamma interferon, the kit comprising the following antigen protein composition: MTB8.4, ESAT6, CFP10 and Rv3615c.
[0021] Preferably, the mass ratio of MTB8.4, ESAT6, CFP10 and Rv3615c is 1:1:1:1;
[0022] And / or, the concentration of the antigen protein composition is 2 to 6 μg / mL.
[0023] Preferably, the kit for detecting bovine tuberculosis interferon-gamma further includes separation magnetic beads, lymphocyte preservation solution, bovine tuberculosis interferon-gamma monoclonal antibody 4E5 U-shaped reaction cup, bovine tuberculosis interferon-gamma ester conjugate 2G8, pH adjuster, and anti-interference agent.
[0024] Preferably, the method for preparing the separation magnetic beads is as follows: bovine lymphocyte CD4 monoclonal antibody and CD56 monoclonal antibody are coupled with carboxyl magnetic beads by EDC method to prepare CD4 monoclonal antibody-coupled magnetic beads and CD56 monoclonal antibody-coupled magnetic beads respectively.
[0025] Preferably, the lymphocyte preservation solution comprises the following ingredients: 10% fetal bovine serum, 1% glucose, 0.5% glutamine, 1% arginine, 0.1% biotin, 0.2% vitamin B12, 0.5% choline, 0.8% inositol, 1% sodium pyruvate, 1% trehalose, and 0.1M HEPES; the pH of the lymphocyte preservation solution is 7.2–7.5.
[0026] Preferably, the U-shaped reaction cup coated with bovine tuberculosis gamma-interferon monoclonal antibody 4E5 is made of polystyrene and has a capacity of 2 mL. The U-shaped reaction cup coated with bovine tuberculosis gamma-interferon monoclonal antibody 4E5 is subjected to the following treatment:
[0027] Bovine tuberculosis gamma-interferon monoclonal antibody 4E5 was diluted to 10 μg / mL with CB diluent. 2 mL of the solution was added and reacted at 2–8 °C for 24 h. The reaction solution was then poured out, and 2 mL of casein blocking solution was added. The reaction solution was then reacted at 2–8 °C for another 24 h. The solution was then poured out, and the mixture was vacuumed at 20 °C for 12 h. The mixture was then sealed with aluminum foil and plastic film in a drying chamber at 20–25 °C and humidity below 10% for later use.
[0028] Preferably, the anti-interference agent includes a surfactant, a heterophile antibody blocker HBR5, and protamine; the surfactant is S9, S17, or S21.
[0029] The beneficial effects of this invention are as follows:
[0030] This invention achieves a multi-dimensional breakthrough in the detection of gamma-interferon in bovine tuberculosis by scientifically designing antigen-protein combinations, integrating advanced detection technologies, and optimizing the anti-interference system. It comprehensively improves the sensitivity, specificity, and practicality of the detection, providing an efficient and reliable solution for large-scale screening of bovine tuberculosis. Specific beneficial effects are as follows:
[0031] (1) The combination of antigen and protein has a synergistic effect, significantly improving detection sensitivity and specificity.
[0032] This invention utilizes four antigen proteins—MTB8.4, ESAT6, CFP10, and Rv3615c—to construct a composite, achieving a synergistic improvement in detection performance through complementary antigenic epitopes. ESAT6 and CFP10 are secreted antigens specific to the RD1 region of Mycobacterium tuberculosis, accurately identifying natural infections (excluding BCG vaccination interference). Rv3615c, as a substrate protein of the Esx-1 secretion system, functions similarly to ESAT6 / CFP10 and covers more variant strains (such as drug-resistant strains). MTB8.4 supplements the Mycobacterium bovis-specific antigenic epitope. The synergistic effect of these four antigens comprehensively activates peripheral blood mononuclear cells (PBMCs) to release interferon-gamma, increasing detection sensitivity to 96%, significantly higher than traditional single antigens or non-specific combinations (such as the combination containing MPB70 in the comparative validation example). Simultaneously, this composition avoids cross-reactivity with environmental mycobacteria such as avian mycobacteria, achieving a specificity of 98% and significantly reducing the false positive rate.
[0033] (2) Integrating direct chemiluminescence technology to improve detection sensitivity and efficiency
[0034] This invention combines direct chemiluminescence technology (instead of traditional ELISA or enzyme-catalyzed chemiluminescence), significantly improving detection sensitivity and automation. Compared to ELISA (where the detection limit is typically 10 pg / mL), direct chemiluminescence technology lowers the detection limit to 0.1 pg / mL, increasing sensitivity by at least 100 times, and accurately capturing low concentrations of gamma-interferon signals. Furthermore, this technology eliminates the need for multiple washing steps, is compatible with fully automated instruments, reduces the detection time for a single sample to 15 minutes, and achieves a throughput of 200 samples / hour, five times that of traditional ELISA (40 samples / hour), meeting the high-efficiency requirements of large-scale screening.
[0035] (3) Optimize the anti-interference system to adapt to complex animal sample matrices.
[0036] To address the interference issues caused by high lipid and hemoglobin content in bovine whole blood samples, this invention effectively inhibits non-specific binding by adding an anti-interference agent (1% protamine) and optimizing the microsphere surface modification (streptavidin density 1.0 μg / mg). In a high-interference model (containing 2 mg / mL hemoglobin and 1 mg / mL lipid), the background signal decreased from 250 RLU to 50 RLU, a reduction of 80%, while the positive signal remained stable, ensuring the accuracy and repeatability of complex sample detection (batch detection CV < 5%).
[0037] (4) The reagent kit is designed to be practical, facilitating industrialization and large-scale application.
[0038] This invention's kit includes an antigen-protein composition, magnetic separation beads, lymphocyte preservation solution, and other supporting components, each with optimized parameters: the magnetic separation beads are coupled with CD4 / CD56 monoclonal antibodies via EDC, enabling efficient enrichment of target cells; the lymphocyte preservation solution contains fetal bovine serum, glucose, and other components, maintaining cell viability for over 16 hours; and the U-shaped reaction cups coated with monoclonal antibodies undergo standardization to ensure batch-to-batch consistency. The entire kit is easy to use, requires no complex equipment, and can be directly applied to grassroots veterinary stations or large-scale farms, providing a practical tool for early screening and epidemic monitoring of bovine tuberculosis. Detailed Implementation
[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0040] Example 1: Determination of the concentration of the antigen protein composition
[0041] (1) Antigen preparation: MTB8.4, ESAT6, CFP10 and Rv3615c recombinant proteins (laboratory prokaryotic expression, purity ≥90%) were diluted to 6 μg / mL, 6 μg / mL, 6 μg / mL and 6 μg / mL respectively (mass ratio 1:1:1:1).
[0042] (2) Take 1 ml of fresh bovine blood, add it to an anticoagulant glass tube, invert and mix well, then add 1 ml of magnetic beads diluted 1 mg / ml at a 1:50 ratio, and shake well for 5 minutes at 300 rpm. Place the mixed whole blood into a magnetic microparticle luminescence analyzer, set the program, first use a magnetic rod to pick up the magnetic beads, then wash 5 times with lymphocyte preservation solution, then pick up the magnetic beads and transfer them to a 2 ml U-shaped reaction cup, then add 200 μl of lymphocyte preservation solution, add 50 μl of mixed tuberculosis antigen stimulant, react for 1-3 hours, then wash three times with PBST, add 100 μl of bovine tuberculosis gamma interferon 2G8 acridinium ester conjugate, react for 10 minutes, and finally add excitation solution to read the luminescence value.
[0043] Example 2: Determination of the optimal pH and incubation time for direct chemiluminescence reaction
[0044] (1) The reaction system consisted of a U-shaped reaction cup (4E5 antibody labeled, 5.0 μg / ml) and a tracer (2G5 antibody labeled).
[0045] (2) Positive control: recombinant bovine gamma interferon (10 pg / mL).
[0046] (3) pH gradient test: Adjust the pH of the reaction buffer to 6.0, 6.5, 7.0, 7.5 and 8.0, add the positive control, incubate at 37°C for 15 minutes, and detect the RLU value.
[0047] (4) Time gradient test: Under pH 7.0 conditions, incubate for 5, 10, 15 and 20 minutes and detect the RLU value.
[0048] The test results are shown in Table 1.
[0049] Table 1
[0050] pH RLU value (10 pg / mL) Incubation time (min) RLU value (10 pg / mL) 6.0 12000±1500 5 8000±1000 6.5 14500±1200 10 15000±1300 7.0 16000±1000 15 18000±1500 7.5 15500±1100 20 17500±1400
[0051] Conclusion: As shown in Table 1, the optimal conditions are: pH = 7.0 and incubation time = 15 min.
[0052] Example 3: Determination of Anti-interference Agent
[0053] (1) Construction of interference model: Add hemoglobin (2 mg / mL) and lipid (1 mg / mL) to negative bovine whole blood, and add 1.0 μg / mL antigen solution for stimulation, centrifuge and take the supernatant.
[0054] (2) Anti-interference agent test: 1% heterophilic antibody blocking agent HBR5, 1% S9 and 1% protamine were added to the reaction system respectively, and the background RLU value and positive sample (10pg / mL) signal were detected.
[0055] The test results are shown in Table 2.
[0056] Table 2
[0057]
[0058]
[0059] Conclusion: As shown in Table 2, these three anti-interference agents have significant effects and can significantly reduce the background.
[0060] Example 4: Determination of the clinical sensitivity and specificity of the kit
[0061] (1) Sample source:
[0062] Positive group: 50 whole blood samples from bovine tuberculosis gamma interferon positive (confirmed by bovine tuberculosis gamma interferon ELISA kit).
[0063] Negative group: 50 whole blood samples from healthy cattle (confirmed negative by bovine tuberculosis gamma interferon ELISA kit).
[0064] (2) Testing process:
[0065] The samples were treated according to the conditions of Example 1 (6.0 μg / mL antigen stimulation).
[0066] Direct chemiluminescence detection was performed under the conditions of Example 2 (pH 7.0, incubation for 15 minutes).
[0067] The test results are shown in Table 3.
[0068] Table 3
[0069] Sample type Number of positives detected Sensitivity / Specificity Positive group (50) 48 96％ Negative group (50) 6.0 1,000 30 1,000 Serum 0 / 50 0 / 50 6.5 1,000 30 1,000 Serum 0 / 50 0 / 50 7.0 1,000 30 1, 49 98％
[0070] In summary, the optimal parameters for the embodiment are as follows:
[0071] Antigen stimulation conditions: 6.0 μg / mL (MTB8.4+ESAT6+CFP10+Rv3615c, 1:1:1:1).
[0072] Test conditions: pH 7.0, incubation for 15 minutes.
[0073] Anti-interference agents: 1% protamine sulfate, 1% heterophile antibody blocking agents HBR5 and S9.
[0074] Clinical performance: Sensitivity 96%, Specificity 98%.
[0075] Comparison and verification examples
[0076] The antigen protein composition used in this comparative validation example included MPB70, MPB83, ESAT6, and CFP10. Among them, MPB70 showed cross-reactivity with avian mycobacteria (false positive rate 15%).
[0077] The results of a comprehensive comparison between the examples and the comparative verification examples are shown in Table 4.
[0078] Table 4
[0079]
[0080] As can be seen from the foregoing content and Table 4:
[0081] (1) Among 50 whole blood samples of bovine blood infected with Mycobacterium avium, the false positive rate of the experimental case was only 2% (specificity 98%), while the false positive rate of the comparative validation case was 15%.
[0082] (2) The detection limit of the comparative verification example is 2 pg / mL; the direct chemiluminescence technology of the example has higher sensitivity. In addition, the example of this invention combines quadruple antigen (MTB8.4, ESAT6, CFP10, Rv3615c) with photo-induced chemiluminescence technology, and the detection limit reaches 0.1 pg / mL.
[0083] (3) Serial dilution of recombinant bovine gamma interferon (0.1-1000 pg / mL) This method can stably detect 0.1 pg / mL (signal-to-noise ratio ≥3), and the sensitivity is 20 times higher than that of ELISA.
[0084] (4) The ELISA of the comparative validation example requires manual operation, takes more than 2 hours, and has a throughput of 40 samples / hour, which is not fully automated; while the example uses direct chemiluminescence technology, which does not require a washing step, is compatible with fully automated instruments, shortens the reaction time to 15 minutes, and has a throughput of 200 samples / hour.
[0085] When testing 200 samples in batches, the repeatability CV was <5%, which is significantly better than ELISA (CV = 10-15%).
[0086] (5) The bovine whole blood sample matrix was complex (high lipids, hemoglobin), resulting in a high background signal (RLU = 250) compared to the traditional ELISA method used in the validation example. The optimized example added 1% protamine and 1% heterophile antibody blockers HBR5 and S9 to inhibit non-specific binding. In the high-interference model (2 mg / mL hemoglobin), the background signal decreased from 4500 RLU to 900 RLU, a reduction of 80%.
[0087] In summary, the embodiments of the present invention eliminate cross-reactivity by replacing MPB70 with Rv3615c, improve sensitivity and efficiency by combining photo-induced chemiluminescence technology, and optimize the anti-interference system for animal samples, achieving a comprehensive breakthrough in sensitivity (96%), specificity (98%), and detection efficiency (200 samples / hour), providing an efficient solution for large-scale screening of bovine tuberculosis.
[0088] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. An antigen-protein composition for detecting bovine tuberculosis gamma interferon, characterized in that, Including MTB8.4, ESAT6, CFP10, and Rv3615c.
2. The antigen protein composition for detecting bovine tuberculosis gamma interferon according to claim 1, characterized in that, The mass ratio of MTB8.4, ESAT6, CFP10 and Rv3615c is 1:1:1:1; And / or, the concentration of the antigen protein composition is 2 to 6 μg / mL.
3. Use of the antigen protein composition according to claim 1 or 2 in the preparation of a kit for detecting bovine tuberculosis gamma interferon.
4. A kit for detecting gamma interferon in bovine tuberculosis, characterized in that, The kit comprises the antigen protein composition of claim 1 or 2: MTB8.4, ESAT6, CFP10, and Rv3615c.
5. The kit for detecting bovine tuberculosis gamma interferon according to claim 4, characterized in that, The mass ratio of MTB8.4, ESAT6, CFP10 and Rv3615c is 1:1:1:1; And / or, the concentration of the antigen protein composition is 2 to 6 μg / mL.
6. The kit for detecting bovine tuberculosis gamma interferon according to claim 4, characterized in that, It also includes magnetic beads for separation, lymphocyte preservation solution, U-shaped reaction cups coated with bovine tuberculosis interferon-gamma monoclonal antibody 4E5, acridinium ester conjugate of bovine tuberculosis interferon-gamma, pH adjuster and anti-interference agent.
7. The kit for detecting bovine tuberculosis gamma interferon according to claim 6, characterized in that, The method for preparing the separation magnetic beads is as follows: bovine lymphocyte CD4 monoclonal antibody and CD56 monoclonal antibody are coupled with carboxyl magnetic beads by EDC method to prepare CD4 monoclonal antibody-coupled magnetic beads and CD56 monoclonal antibody-coupled magnetic beads respectively.
8. The kit for detecting bovine tuberculosis gamma interferon according to claim 6, characterized in that, The lymphocyte preservation solution comprises the following ingredients: 10% fetal bovine serum, 1% glucose, 0.5% glutamine, 1% arginine, 0.1% biotin, 0.2% vitamin B12, 0.5% choline, 0.8% inositol, 1% sodium pyruvate, 1% trehalose, and 0.1M HEPES; the pH of the lymphocyte preservation solution is 7.2–7.
5.
9. The kit for detecting bovine tuberculosis gamma interferon according to claim 6, characterized in that, The U-shaped reaction cup coated with bovine tuberculosis gamma-interferon monoclonal antibody 4E5 is made of polystyrene and has a capacity of 2 mL. The U-shaped reaction cup coated with bovine tuberculosis gamma-interferon monoclonal antibody 4E5 is subjected to the following treatment: Bovine tuberculosis gamma-interferon monoclonal antibody 4E5 was diluted to 10 μg / mL with CB diluent. 2 mL of the solution was added and reacted at 2–8 °C for 24 h. The reaction solution was then poured out, and 2 mL of casein blocking solution was added. The reaction solution was then reacted at 2–8 °C for another 24 h. The solution was then poured out, and the mixture was vacuumed at 20 °C for 12 h. The mixture was then sealed with aluminum foil and plastic film in a drying chamber at 20–25 °C and humidity below 10% for later use.
10. The kit for detecting bovine tuberculosis gamma interferon according to claim 6, characterized in that, The anti-interference agent includes a surfactant, the heterophile antibody blocking agent HBR5, and protamine; the surfactant is S9, S17, or S21.