Preparation method of extracellular vesicle carrying mycobacterium tuberculosis specific antigen

Abstract

The invention provides a preparation method of extracellular vesicles carrying mycobacterium tuberculosis specific antigens, and belongs to the technical field of extracellular vesicles. THP-1 mononuclear cells are induced to be differentiated into macrophages through phorbol ester, a mycobacterium tuberculosis H37Rv standard strain is treated through ultrasonication and needle blowing and beating to prepare a monodisperse bacterium solution, and the monodisperse bacterium solution is added into the mycobacterium tuberculosis specific antigens to prepare the extracellular vesicles carrying the mycobacterium tuberculosis specific antigens. Infecting according to the infection complex value of 5, monitoring the real-time infection rate every 12 hours in the culture process, dynamically adjusting the initial bacterial liquid concentration according to the value to maintain the infection rate in the range of 45-55%, purifying the extracellular vesicles through a differential centrifugation method, detecting the particle size and concentration of the vesicles and the content of lipoarabinomannan, and determining the content of lipoarabinomannan in the extracellular vesicles. Qualified vesicle products are divided into three concentration grades according to the antigen content, and dimethyl sulfoxide is added for cryopreservation, so that the technical problem of poor antigen loading stability among batches of extracellular vesicle products carrying mycobacterium tuberculosis specific antigens is solved.

Description

Technical Field

[0001] This invention belongs to the field of extracellular vesicle technology, and more specifically, relates to a method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens. Background Technology

[0002] In tuberculosis diagnosis, the detection of Mycobacterium tuberculosis-specific antigens as biomarkers is required. Traditional methods involve collecting extracellular vesicles secreted by infected macrophages to obtain vesicle products carrying antigens such as lipofaranomannan, which are then used for the development of quality control materials for immunodiagnostic reagents or vaccine vector research. However, existing preparation methods lack dynamic monitoring of the infection process, resulting in significant fluctuations in infection efficiency between different batches of cells, leading to instability in the number of Mycobacterium tuberculosis bacteria taken up by macrophages. Since the antigen load in extracellular vesicles directly depends on the number of intracellular bacteria, batch-to-batch differences in infection efficiency cause significant fluctuations in the lipofaranomannan content of the final vesicle products, affecting the accurate classification of quality control concentration levels. In other words, existing technologies suffer from poor batch-to-batch stability of antigen load in extracellular vesicle products carrying Mycobacterium tuberculosis-specific antigens. Summary of the Invention

[0003] In view of this, the present invention provides a method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens, which can solve the technical problem of poor batch-to-batch antigen loading stability of extracellular vesicle products carrying Mycobacterium tuberculosis-specific antigens in the prior art.

[0004] This invention is implemented as follows: This invention provides a method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens, comprising: culturing THP-1 mononuclear cells in RPMI-1640 medium containing fetal bovine serum until the cell density reaches a preset density; adding phorbol ester to induce differentiation; discarding the medium and washing with phosphate-buffered saline; replacing with fresh medium containing fetal bovine serum for continued culture; collecting the cultured adherent cells; using flow cytometry to detect the expression levels of cell surface markers CD11b and CD14, and determining the differentiated macrophages; placing a suspension of Mycobacterium tuberculosis H37Rv standard strain in an ultrasonic homogenizer for ultrasonic treatment and repeatedly pipetting the bacterial solution with a needle; adjusting the bacterial solution concentration to the initial concentration by optical density measurement; adding the treated bacterial solution to differentiated macrophages for infection and incubation; washing the cells with medium containing gentamicin to remove extracellular bacteria; and further... The culture was continued using a maintenance medium containing gentamicin. Flow cytometry was used at preset time intervals to monitor the positive rate of fluorescently labeled Mycobacterium tuberculosis in infected cells and recorded as the real-time infection rate. The initial bacterial concentration was dynamically adjusted based on the real-time infection rate to maintain the real-time infection rate within the target range between different batches. Cell supernatant was collected after culture, and extracellular vesicles were separated using differential centrifugation. Nanoparticle tracking analysis was used to detect the particle size distribution and concentration of extracellular vesicles and to determine qualified vesicle products. Enzyme-linked immunosorbent assay (ELISA) was used to quantitatively detect the lipoarabinomannan and LAM carrier protein content in qualified vesicle products. Based on the lipoarabinomannan content, qualified vesicle products were divided into three concentration levels: high-concentration positive control, medium-concentration positive control, and low-concentration positive control. Positive control products of different concentration levels were aliquoted into polypropylene cryovials, and dimethyl sulfoxide was added as a cryoprotectant for cryopreservation.

[0005] The process involved culturing THP-1 monocytes in RPMI-1640 medium containing fetal bovine serum until the cell density reached a predetermined level, followed by the addition of phorbol ester to induce differentiation. Specifically, THP-1 monocytes were cultured in RPMI-1640 medium containing 10% fetal bovine serum until the cell density reached a predetermined level. Differentiation was induced for 48 hours by adding phorbol ester at a concentration of 100 ng / mL.

[0006] The phorbol ester is phorbol ester-12-myristate-13-acetate, which induces THP-1 monocytes to differentiate into adherent macrophages by activating the protein kinase C signaling pathway.

[0007] The phosphate buffer solution is an aqueous solution prepared by disodium hydrogen phosphate and potassium dihydrogen phosphate at a mass ratio of 8:1, with a pH of 7.4 and an osmotic pressure of 300 mOsm / kg.

[0008] The step of using flow cytometry to detect the expression levels of cell surface markers CD11b and CD14 and to determine the qualified macrophages for differentiation is as follows: when the positive expression rate of cell surface marker CD11b is >85% and the positive expression rate of cell surface marker CD14 is >80%, the macrophages are determined to be qualified for differentiation. If the positive expression rate of cell surface marker CD11b is ≤85% or the positive expression rate of cell surface marker CD14 is ≤80%, a second induction differentiation treatment is performed.

[0009] The secondary induction differentiation treatment refers to the process where, 48 hours after the first induction differentiation, if the positive expression rate corresponding to the expression level of cell surface marker CD11b or the positive expression rate corresponding to the expression level of cell surface marker CD14 does not meet the standard, the culture medium is replaced with fresh medium and phorbol ester at a concentration of 50 ng / mL is added again to continue induction for 24 hours.

[0010] The step of placing the Mycobacterium tuberculosis H37Rv standard strain suspension in an ultrasonic homogenizer for ultrasonic treatment and repeatedly blowing the bacterial solution with a needle is as follows: the Mycobacterium tuberculosis H37Rv standard strain suspension is placed in an ultrasonic homogenizer with a power of 150W for ultrasonic treatment for 5 seconds, with a 10-second interval, and the treatment is repeated 3 times. Then, the bacterial solution is repeatedly blown 12 times with a No. 27 needle.

[0011] The step of adjusting the bacterial suspension concentration to the initial bacterial suspension concentration by measuring the optical density value specifically involves adjusting the bacterial suspension concentration to the initial bacterial suspension concentration by measuring the optical density value. CFU / mL.

[0012] The step of adding the treated bacterial solution to differentiated macrophages for infection and incubation specifically involves adding the bacterial solution corresponding to the initial concentration of the treated solution to the differentiated macrophages for infection, with the infection multiplicity set to 5, and incubating at 37°C and 5%. After incubating in an incubator for 4 hours, the cells were washed three times with a medium containing 100 μg / mL gentamicin to remove extracellular bacteria, and then the medium was replaced with a maintenance medium containing 10 μg / mL gentamicin and cultured for another 48 hours.

[0013] The step of dynamically adjusting the initial bacterial concentration based on the real-time infection rate to maintain the real-time infection rate within the target range across different batches specifically involves monitoring the positivity rate of fluorescently labeled Mycobacterium tuberculosis in infected cells using flow cytometry every 12 hours and recording it as the real-time infection rate. When the real-time infection rate is <40%, the initial bacterial concentration is increased to [a higher concentration]. CFU / mL was used to re-infect the next batch of cells. When the real-time infection rate was >60%, the initial bacterial concentration was reduced to [a lower value]. CFU / mL, resulting in real-time infection rates between different batches ∈ [45%, 55%].

[0014] The key feature is that the step of separating extracellular vesicles using differential centrifugation involves first centrifuging at 300×g for 10 minutes to remove cell debris, then centrifuging at 2000×g for 20 minutes to remove apoptotic bodies, followed by centrifugation at 10000×g for 30 minutes to remove large vesicles, and finally ultracentrifugation at 100000×g for 70 minutes to precipitate the target extracellular vesicles, and resuspending the vesicle precipitate with phosphate buffer.

[0015] The step of using nanoparticle tracking analysis technology to detect the particle size distribution and concentration of extracellular vesicles and determine qualified vesicle products specifically involves using nanoparticle tracking analysis technology to detect the particle size distribution and concentration of target extracellular vesicles. When the main peak particle size of the particle size distribution is ∈ [80nm, 150nm] and the vesicle concentration is > A product is considered a qualified vesicle product when the number of vesicles per mL is reached.

[0016] The step of classifying qualified vesicle products into three concentration levels—high-concentration positive control products, medium-concentration positive control products, and low-concentration positive control products—based on the lipoarabinomannan content is as follows: Specifically, the lipoarabinomannan content of qualified vesicle products is classified into three concentration levels based on the lipoarabinomannan content: high-concentration positive control products correspond to lipoarabinomannan content ∈ [200 ng / mL, 300 ng / mL], medium-concentration positive control products correspond to lipoarabinomannan content ∈ [80 ng / mL, 120 ng / mL], and low-concentration positive control products correspond to lipoarabinomannan content ∈ [20 ng / mL, 40 ng / mL].

[0017] The process includes, after classifying qualified vesicle products into three concentration grades based on their lipoarabinomannan content, a further step of requiring each concentration grade to have a mass ratio of LAM carrier protein content to lipoarabinomannan content ∈ [0.6, 1.2].

[0018] The LAM carrier protein is a lipoprotein LprG secreted by Mycobacterium tuberculosis, which acts as a chaperone protein for lipoarabinomannan within bacterial cells, assisting in the transport and localization of lipoarabinomannan.

[0019] The step of aliquoting positive control samples of different concentrations into polypropylene cryovials, adding dimethyl sulfoxide as a cryoprotectant, and freezing them involves aliquoting high-concentration, medium-concentration, and low-concentration positive control samples into polypropylene cryovials, each with a volume of 200 μL, adding dimethyl sulfoxide at a final concentration of 10% as a cryoprotectant, pre-freezing at -20°C for 2 hours, and then transferring to a -80°C freezer for long-term storage.

[0020] This invention establishes a real-time infection rate monitoring and feedback control system. During the infection process, flow cytometry is used to detect the positive rate of fluorescently labeled Mycobacterium tuberculosis every 12 hours. The initial bacterial concentration is dynamically adjusted based on the real-time infection rate. When the real-time infection rate deviates from the target range, the inoculated bacterial load is increased or decreased promptly. This strategy maintains the infection efficiency between different batches within a narrow range of 45% to 55%, ensuring consistency in the number of bacteria taken up by macrophages in each batch, thereby stabilizing the loading of lipoarabinomannan in extracellular vesicles. Simultaneously, combined with precise control of the multiplicity of infection (MLI) and differential centrifugation purification, the batch-to-batch reproducibility of antigen content in the final vesicle product is guaranteed, enabling the precise preparation of high-, medium-, and low-concentration quality control products. In summary, this invention solves the technical problem mentioned in the background art of poor batch-to-batch antigen loading stability in extracellular vesicle products carrying Mycobacterium tuberculosis-specific antigens. Attached Figure Description

[0021] Figure 1 This is a graph showing the changes in the expression levels of cell surface markers at different induction time points in the examples.

[0022] Figure 2 This is a graph showing the particle size distribution and concentration analysis of extracellular vesicles in the example. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0024] This invention provides a method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens, comprising the following steps:

[0025] S01. THP-1 mononuclear cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum until the cell density reached the target level. The cells were divided into 100 ng / mL of phorbol ester and induced to differentiate for 48 hours. After discarding the culture medium, the cells were washed three times with phosphate buffer and then replaced with fresh culture medium containing 5% fetal bovine serum for another 24 hours.

[0026] S02. Collect adherent cells cultured in step S01 and use flow cytometry to detect the expression levels of cell surface markers CD11b and CD14. When the positive expression rate of cell surface marker CD11b is greater than 85% and the positive expression rate of cell surface marker CD14 is greater than 80%, the cells are considered to be qualified for differentiation. If the positive expression rate of cell surface marker CD11b is less than or equal to 85% or the positive expression rate of cell surface marker CD14 is less than or equal to 80%, a second induction differentiation treatment is performed.

[0027] S03. The suspension of Mycobacterium tuberculosis H37Rv standard strain was placed in a 150W ultrasonic homogenizer and sonicated for 5 seconds, with a 10-second interval, and repeated 3 times. Then, the bacterial suspension was repeatedly pipetted 12 times using a 27-gauge needle. The bacterial concentration was adjusted to the initial concentration by measuring the optical density. CFU / mL;

[0028] S04. Add the bacterial solution corresponding to the initial bacterial concentration after step S03 to the differentiated qualified macrophages obtained in step S02 for infection. The infection multiplicity value is set to 5. After incubating in a 37°C, 5% CO2 incubator for 4 hours, wash the cells 3 times with medium containing 100 μg / mL gentamicin to remove extracellular bacteria. Replace with maintenance medium containing 10 μg / mL gentamicin and continue culturing for 48 hours.

[0029] S05. Monitor the positivity rate of fluorescently labeled Mycobacterium tuberculosis in infected cells by flow cytometry every 12 hours and record it as the real-time infection rate. When the real-time infection rate is less than 40%, increase the initial bacterial concentration in step S03 to [a higher value]. CFU / mL and re-infect the next batch of cells. When the real-time infection rate is greater than 60%, reduce the initial bacterial concentration in step S03 to [value missing]. CFU / mL ensures that the real-time infection rate between different batches remains within the range of [45%, 55%];

[0030] S06. Collect the cell supernatant after 48 hours of culture in step S04. Use differential centrifugation to separate extracellular vesicles. First, centrifuge at 300×g for 10 minutes to remove cell debris, then centrifuge at 2000×g for 20 minutes to remove apoptotic bodies, then centrifuge at 10000×g for 30 minutes to remove large vesicles, and finally centrifuge at 100000×g for 70 minutes to precipitate the target extracellular vesicles. Resuspend the vesicle precipitate with phosphate buffer.

[0031] S07. Use nanoparticle tracking analysis technology to detect the particle size distribution and vesicle concentration of the target extracellular vesicles obtained in step S06. When the main peak particle size distribution is within the range of [80nm, 150nm] and the vesicle concentration is greater than […], the vesicle concentration is determined by the following method: A product is considered a qualified vesicle product when the number of vesicles / mL is reached.

[0032] S08. The content of lipoarabinomannan and LAM carrier protein in the qualified vesicle products obtained in step S07 is quantitatively detected by enzyme-linked immunosorbent assay. According to the lipoarabinomannan content, the qualified vesicle products are divided into three concentration levels: high concentration positive control products with lipoarabinomannan content in the range of [200ng / mL, 300ng / mL], medium concentration positive control products with lipoarabinomannan content in the range of [80ng / mL, 120ng / mL], and low concentration positive control products with lipoarabinomannan content in the range of [20ng / mL, 40ng / mL]. For each concentration level, the mass ratio of LAM carrier protein content to lipoarabinomannan content is required to be in the range of [0.6, 1.2].

[0033] S09. The high-concentration positive control sample, medium-concentration positive control sample and low-concentration positive control sample obtained in step S08 are respectively dispensed into polypropylene cryopreservation tubes, each with a volume of 200 μL. Dimethyl sulfoxide with a final concentration of 10% is added as a cryoprotectant. The tubes are first pre-frozen at -20°C for 2 hours, and then transferred to a freezer at -80°C for long-term storage. The storage period shall not exceed 6 months.

[0034] The phorbol ester is phorbol ester-12-myristate-13-acetate, which induces THP-1 monocytes to differentiate into adherent macrophages by activating the protein kinase C signaling pathway. The combination of a concentration of 100 ng / mL and an action time of 48 hours is the optimal parameter to achieve full differentiation while maintaining cell viability.

[0035] The phosphate buffer solution is an aqueous solution prepared by disodium hydrogen phosphate and potassium dihydrogen phosphate at a mass ratio of 8:1, with a pH of 7.4 and an osmotic pressure of 300 mOsm / kg. It is used to remove residual phorbol esters and metabolic waste when washing cells without affecting the cell membrane integrity.

[0036] The cell surface marker CD11b is a member of the integrin family. Its expression level is significantly upregulated during the differentiation of monocytes into macrophages. A positive expression rate of more than 85% indicates that the cells have completed the transformation to the macrophage phenotype.

[0037] The cell surface marker CD14 is a lipopolysaccharide receptor helper molecule that is highly expressed on the surface of mature macrophages. A positive expression rate of more than 80% is a marker of macrophage functional maturity.

[0038] The secondary induction differentiation treatment refers to the process where, 48 hours after the first induction differentiation, if the positive expression rate corresponding to the cell surface marker CD11b or the positive expression rate corresponding to the cell surface marker CD14 does not meet the standard, the culture medium is replaced with fresh medium and phorbol ester at a concentration of 50 ng / mL is added again to continue induction for 24 hours. By reducing the concentration of phorbol ester and shortening the induction time, the decrease in cell viability caused by overstimulation is avoided.

[0039] The Mycobacterium tuberculosis H37Rv standard strain is an internationally recognized virulence reference strain for Mycobacterium tuberculosis. It has an intact cell wall structure and a stable level of lipoarabinomannan expression. The strain number is ATCC 27294.

[0040] The ultrasonic disruptor uses the mechanical shearing force generated by the cavitation effect to break down the hydrophobic interactions within the Mycobacterium tuberculosis colony, dispersing the aggregated bacteria into individual cells. A power of 150W is the optimal power to achieve sufficient dispersion while ensuring that the bacterial activity is not damaged.

[0041] The No. 27 needle has an inner diameter of 0.4 mm. Repeatedly blowing 12 times utilizes the high shear force generated by the inner diameter of the needle to further break up the residual bacterial clumps. The number of blowing times is set to 12 based on a balance between bacterial clump dispersion efficiency and operational feasibility.

[0042] The optical density value is the absorbance at a wavelength of 600 nm. The concentration of viable bacteria in the bacterial solution is calculated by comparing the measured optical density value with a standard curve of known colony formation unit concentration. An optical density value of 0.6 corresponds to an initial bacterial solution concentration of... CFU / mL.

[0043] The infection multiplicity value is the number of Mycobacterium tuberculosis corresponding to each macrophage. It is calculated by dividing the total number of viable bacteria in the bacterial solution by the total number of macrophages. An infection multiplicity value of 5 indicates that the inoculation is carried out at a ratio of 5 Mycobacterium tuberculosis infecting 1 macrophage.

[0044] The fluorescently labeled Mycobacterium tuberculosis is a recombinant Mycobacterium tuberculosis strain H37Rv transfected with the green fluorescent protein gene. It emits green fluorescence under ultraviolet light excitation, and the infection rate is quantitatively monitored by detecting the intensity of the intracellular fluorescence signal using flow cytometry.

[0045] The differential centrifugation method separates particles of different sizes based on the difference in sedimentation rate in the centrifugal force field. By gradually increasing the centrifugal force and extending the centrifugation time, large particle impurities are removed in sequence, and extracellular vesicles of the target size are finally enriched.

[0046] The nanoparticle tracking and analysis technology is based on the Brownian motion principle. It tracks the trajectory of individual nanoparticles through laser scattering and calculates the particle size distribution. At the same time, it obtains information on the particle size and concentration of vesicles. The main peak particle size range corresponds to the typical size characteristics of exosomes and microvesicles in extracellular vesicles.

[0047] The lipoarabinomannan is a lipopolysaccharide component of the cell wall of Mycobacterium tuberculosis. It consists of a polysaccharide backbone composed of mannose and arabinose units linked to a lipid anchoring structure. After Mycobacterium tuberculosis infects macrophages, it is loaded into secreted extracellular vesicles and becomes a diagnostic marker.

[0048] The LAM carrier protein is a lipoprotein LprG secreted by Mycobacterium tuberculosis. In bacterial cells, it acts as a chaperone protein for lipoarabinomannan, assisting in the transport and localization of lipoarabinomannan. During macrophage infection, it is co-packaged with lipoarabinomannan into extracellular vesicles.

[0049] The mass ratio is the value obtained by dividing the LAM carrier protein content by the lipoarabinomannan content. The mass ratio in the range [0.6, 1.2] reflects the natural stoichiometry between LAM carrier protein and lipoarabinomannan in Mycobacterium tuberculosis.

[0050] The enzyme-linked immunosorbent assay (ELISA) achieves quantitative detection by antibody-specific recognition of the target antigen and enzyme-catalyzed colorimetric reaction. The content of lipoarabinomannan and LAM carrier protein was detected by anti-lipoarabinomannan monoclonal antibody CS-35 and anti-LAM carrier protein monoclonal antibody A194-01, respectively.

[0051] The concentration levels are classified according to the range of lipoarabinomannan content. High-concentration positive control samples are used to verify the upper limit of the sensitivity of the detection system, medium-concentration positive control samples are used for routine quality control, and low-concentration positive control samples are used to verify the detection limit of the detection system.

[0052] The dimethyl sulfoxide is a permeable cryoprotectant whose molecules can penetrate the lipid bilayer membrane of vesicles and enter the interior. During freezing, it stabilizes the membrane structure through hydrogen bonding and reduces the damage to vesicle integrity caused by ice crystal formation. A final concentration of 10% is the optimal concentration between protective effect and potential cytotoxicity.

[0053] The specific implementation methods of the above steps are described in detail below.

[0054] The specific implementation of step S01 involves seeding THP-1 mononuclear cells in RPMI-1640 medium containing 10% fetal bovine serum and continuously culturing them until the cell density reaches a certain level. The cells were in the logarithmic growth phase (cells / mL), at which point their metabolism was active and their proliferation capacity was stable. Then, phorbol ester-12-myristate-13-acetate was added to the culture system at a concentration of 100 ng / mL, and the induction treatment was maintained for 48 hours. This combination of concentration and time activated the protein kinase C signaling pathway, promoting the differentiation of monocytes into adherent macrophages while avoiding cell viability loss due to overstimulation. After induction, the old culture medium containing residual phorbol ester and cell metabolites was discarded. The adherent cells were washed three times with phosphate-buffered saline (PBSS) at pH 7.4 and an osmotic pressure of 300 mOsm / kg to remove chemical residues that might interfere with subsequent infection experiments. The isotonic nature of PBSS ensured that the cell membrane integrity was not compromised during the washing process. Finally, the culture medium was replaced with fresh medium containing 5% fetal bovine serum and cultured for another 24 hours to allow the cells to fully recover and stabilize the macrophage phenotype.

[0055] The specific implementation of step S02 involves collecting adherent cells after differentiation induction treatment and quantitatively detecting the expression levels of cell surface markers using flow cytometry. This technique is based on the principle of specific binding between fluorescently labeled antibodies and certain surface antigens. Precise analysis at the single-cell level is achieved by laser-excited fluorescence signals and detection via an optical system. During the detection process, cells are labeled with anti-CD11b and anti-CD14 fluorescent antibodies, respectively. CD11b, as a member of the integrin family, shows significantly upregulated expression during the differentiation of monocytes into macrophages. A positive expression rate greater than 85% indicates that the cells have completed the differentiation process. The transformation of macrophage phenotype is important. CD14, as a lipopolysaccharide receptor helper molecule, is highly expressed on the surface of mature macrophages with a positive expression rate of more than 80%, which is an important marker of functional maturity. When the positive expression rates of two markers meet the above threshold requirements, the macrophages are judged to be qualified for differentiation and enter the subsequent infection step. If the positive expression rate of either marker does not meet the standard, a second induction differentiation treatment is required. This remedial measure is achieved by replacing the culture medium with fresh medium and adding phorbol ester at a concentration reduced to 50 ng / mL for another 24 hours of induction. The reduced concentration and shortened time are designed to avoid cell viability decline due to overstimulation.

[0056] The specific implementation of step S03 involves placing a suspension of Mycobacterium tuberculosis H37Rv standard strain in an ultrasonic disruptor with a power setting of 150W for ultrasonic treatment. The treatment parameters are continuous ultrasonication for 5 seconds followed by a 10-second interval, and this cycle is repeated 3 times. The cavitation effect generated during ultrasonication disrupts the hydrophobic interactions within the bacterial clumps through mechanical shear force, dispersing the aggregated bacteria into individual cells. A power of 150W is the optimal parameter to achieve sufficient dispersion while ensuring that the bacterial activity is not damaged. After ultrasonic treatment, the bacterial solution is repeatedly agitated 12 times using a 27-gauge needle with an inner diameter of 0.4mm. The high shear force generated by the needle's inner diameter further breaks down any remaining bacterial clumps. The agitation frequency of 12 times is based on a balance between bacterial dispersion efficiency and operational feasibility. Subsequently, the optical density value at a wavelength of 600nm is measured using spectrophotometry and compared with a standard curve of unit concentration formed by known colonies to calculate the viable bacterial concentration in the bacterial solution. When the optical density value is 0.6, the corresponding initial bacterial concentration is... The bacterial culture concentration was adjusted to the target level by adding culture medium for dilution or by centrifugation to meet the precise inoculation requirements of subsequent infection experiments.

[0057] The specific implementation of step S04 is to adjust the bacterial concentration to the initial concentration. CFU / mL bacterial suspension was added to a culture system of properly differentiated macrophages at a ratio of 5 (Multiple Occurrence Value). The Multiple Occurrence Value (MOV) was defined as the number of Mycobacterium tuberculosis per macrophage, calculated by dividing the total number of viable bacteria in the bacterial suspension by the total number of macrophages. This ratio was designed to ensure a sufficient number of bacteria contact and be phagocytosed by the macrophages while avoiding excessive bacterial load leading to cell overload. The infection system was maintained at 37°C and 5% (presumably referring to a specific temperature range). Incubation in a high-concentration incubator for 4 hours provides sufficient time for bacterial invasion and colonization. After incubation, the cells are washed three times with a medium containing 100 μg / mL gentamicin to thoroughly remove any unphagocytosed extracellular bacteria. As an aminoglycoside antibiotic, gentamicin cannot penetrate the intact eukaryotic cell membrane, thus killing only extracellular bacteria without affecting bacteria already endocytosed by macrophages. After washing, the medium is replaced with a maintenance medium containing 10 μg / mL gentamicin and cultured for another 48 hours. The maintenance dose of gentamicin continuously inhibits any bacteria that may escape while not interfering with the intracellular infection process or the normal secretion of extracellular vesicles.

[0058] The specific implementation of step S05 involves using flow cytometry to monitor the positive rate of fluorescently labeled Mycobacterium tuberculosis in infected cells every 12 hours as a quantitative indicator of the real-time infection rate. The strain used is recombinant Mycobacterium tuberculosis H37Rv transfected with the green fluorescent protein gene, which emits green fluorescence under ultraviolet light excitation that can be detected by flow cytometry. The infection rate is accurately quantified by analyzing the distribution of intracellular fluorescence signal intensity. When the real-time infection rate is below 40%, it indicates that the activity of the current batch of bacterial culture is insufficient or the bacterial cells are not sufficiently dispersed, resulting in a reduced number of effective infections. In this case, the initial bacterial culture concentration needs to be increased to [a higher concentration]. CFU / mL was used to re-infect the next batch of cells to compensate for the decreased infection efficiency. When the real-time infection rate was higher than 60%, it indicated that the infection load was too heavy and may cause excessive cell stress or premature death, affecting vesicle yield and quality. In this case, the initial bacterial concentration should be reduced to [a lower value]. The CFU / mL is used to reduce the cell burden. Through this dynamic regulation mechanism, the real-time infection rate between different batches is maintained within the optimal range of 45% to 55%. This range corresponds to a balance between macrophages being able to effectively load Mycobacterium tuberculosis-specific antigens and maintain normal vesicle secretion function.

[0059] The specific implementation of step S06 involves collecting the cell supernatant after 48 hours of culture and separating extracellular vesicles using differential centrifugation. This method is based on the physical principle of the difference in sedimentation rate of particles of different sizes in a centrifugal force field to achieve stepwise separation. First, centrifugation at 300×g for 10 minutes removes large cell debris. Then, centrifugation at 2000×g for 20 minutes precipitates and removes apoptotic bodies. Centrifugation at 10000×g for 30 minutes removes large vesicles and other medium-sized impurities. Finally, centrifugation at 100000×g for 70 minutes completely precipitates the target extracellular vesicles at the bottom of the centrifuge tube. This ultracentrifugation condition ensures that exosomes and microvesicles with a particle size range of 80nm to 150nm are effectively enriched. After centrifugation, the supernatant is discarded and the vesicle precipitate is resuspended in phosphate buffer to obtain a high-purity target extracellular vesicle product.

[0060] The specific implementation of step S07 involves using nanoparticle tracking analysis technology to detect the particle size distribution and concentration of target extracellular vesicles. This technology, based on the Brownian motion principle, tracks the trajectory of individual nanoparticles through laser scattering and calculates the particle size distribution according to the Stokes-Einstein equation. Simultaneously, it obtains vesicle concentration information by statistically analyzing the number of particles within the field of view. During the detection process, the vesicle sample is appropriately diluted and injected into the detection cell, then irradiated with a laser. The scattered light is captured by a high-sensitivity camera, and the diffusion coefficient of each vesicle is analyzed by software to calculate the particle size. When the main peak particle size range of the particle size distribution curve is within the range of 80 nm to 150 nm, it indicates that the main components of the sample are exosomes and microvesicles, conforming to the size characteristics of typical extracellular vesicles. Simultaneously, a vesicle concentration greater than [missing value] is required. The sample size is 1 / mL to ensure sufficient sample volume for subsequent analysis and application. Samples that meet the above two quality indicators are judged as qualified vesicle products and proceed to the next step of antigen content quantification.

[0061] The specific implementation of step S08 involves using enzyme-linked immunosorbent assay (ELISA) to quantitatively detect the content of lipoarabinomannan and LAM carrier protein in qualified vesicle products. This method achieves quantitative analysis by antibody-specific recognition of target antigens combined with enzyme-catalyzed substrate colorimetric reaction. Lipoarabinomannan detection uses the anti-lipoarabinomannan monoclonal antibody CS-35, and LAM carrier protein detection uses the anti-LAM carrier protein monoclonal antibody A194-01. Both antigens are specific components of the Mycobacterium tuberculosis cell wall, which are co-packaged into secreted extracellular vesicles during macrophage infection. Based on the lipoarabinomannan content, qualified vesicle products are divided into three concentration groups. The positive control samples are categorized into three levels: high-concentration positive control samples with lipoarabinomannan content ranging from 200 ng / mL to 300 ng / mL for validation of the upper limit of sensitivity of the detection system; medium-concentration positive control samples with content ranging from 80 ng / mL to 120 ng / mL for routine quality control; and low-concentration positive control samples with content ranging from 20 ng / mL to 40 ng / mL for validation of the detection limit of the detection system. For each concentration level, the mass ratio of LAM carrier protein content to lipoarabinomannan content must be between 0.6 and 1.2 to reflect the natural stoichiometry between the two molecules within Mycobacterium tuberculosis and to ensure the biological authenticity of the vesicle-loaded antigen.

[0062] The specific implementation of step S09 involves aliquoting high-concentration, medium-concentration, and low-concentration positive control samples into polypropylene cryovials, with each aliquot containing 200 μL to meet the requirements for multiple tests. A final concentration of 10% dimethyl sulfoxide (DMSO) is added to each tube as a cryoprotectant. DMSO, as a permeable cryoprotectant, can penetrate the lipid bilayer of vesicles and stabilize the membrane structure through hydrogen bonding during freezing, reducing the damage to vesicle integrity caused by ice crystal formation. The final concentration of 10% represents the optimal balance between protective effect and potential cytotoxicity. The aliquoted samples are first pre-frozen at -20°C for 2 hours to allow the sample temperature to slowly decrease and form initial ice crystals. They are then transferred to a -80°C freezer for long-term storage. This two-step cooling process avoids sudden changes in osmotic pressure and the formation of large ice crystals caused by rapid freezing. The storage period is set to no more than 6 months to ensure that the stability of the vesicle structure and antigen activity do not significantly decrease due to long-term cryopreservation.

[0063] It should be noted that the key technical ideas of this invention include a dynamic infection rate monitoring and bacterial concentration regulation mechanism, a dual verification system for vesicle quality based on differential centrifugation combined with nanoparticle tracking analysis, and a concentration grading standard based on antigen stoichiometry. Dynamic infection rate monitoring uses flow cytometry to track the infection process of fluorescently labeled strains in real time and adjusts the bacterial concentration based on threshold feedback. Compared with traditional methods using fixed infection parameters, this can adapt to differences in the activity of different batches of strains and fluctuations in cell state, ensuring the consistency of infection load for each batch of product, thereby improving the reproducibility and stability of antigen loading in vesicles. The dual verification system of differential centrifugation combined with nanoparticle tracking analysis removes impurities and enriches vesicles of the target size through physical separation methods, and confirms product purity through particle size distribution and concentration quantification. Compared with single purification or detection methods, this significantly improves the specificity and batch-to-batch consistency of the product. The concentration grading standard based on the protein-to-protein ratio of lipoarabinomannan and LAM carrier not only considers the content of a single antigen but also focuses on the stoichiometry between the two molecules. This strategy ensures that the quality control products more realistically simulate the composition of natural Mycobacterium tuberculosis antigens rather than artificially enriching a single component, improving the representativeness and reliability of the quality control products for actual clinical sample testing. The synergistic effect of the three technical approaches lies in constructing a full-process quality assurance system from cell infection to vesicle preparation and quality control grading by controlling the consistency of the infection process at the source, standardizing the intermediate purification process, and verifying the end-stage quality evaluation in multiple dimensions. Compared with traditional methods that rely on a single quality control indicator, this approach can more comprehensively control product quality variation and improve the comparability between different batches and the accuracy of diagnostic applications.

[0064] It should be noted that this invention also solves the following technical problems: First, the problem of uneven macrophage differentiation leading to large differences in extracellular vesicle secretion capacity. This invention addresses this by setting dual criteria of CD11b positive expression rate greater than 85% and CD14 positive expression rate greater than 80% on the cell surface, combined with a secondary induction differentiation mechanism, to ensure that the macrophage population used for infection reaches a functionally mature state, thus maintaining consistency in extracellular vesicle secretion efficiency and vesicle carrying capacity between batches. Second, the problem of Mycobacterium tuberculosis cluster aggregation affecting the accuracy of infection multiplicity values. This invention uses a combination of 150W ultrasonic disruption and repeated 12-times blowing with a 27-gauge needle, utilizing the cavitation effect... The high shear force synergistically breaks down bacterial clusters into individual cells, and the concentration of the bacterial solution is precisely adjusted by measuring the optical density value, so that the actual number of viable bacteria inoculated matches the theoretical infection multiple value, avoiding local high concentration infection and uncontrolled infection efficiency caused by bacterial cluster aggregation; Thirdly, to address the problem of the imbalance between lipoarabinomannan and LAM carrier protein in extracellular vesicles affecting antigen stability, this invention sets a quality control index of the mass ratio of LAM carrier protein content to lipoarabinomannan content within the range of 0.6 to 1.2, ensuring the natural stoichiometry of antigen complexes in vesicle products, maintaining the stable anchoring state of lipoarabinomannan in the vesicle membrane structure, and preventing antigen degradation or aggregation during long-term cryopreservation.

[0065] Specifically, the principle of this invention is as follows: The solution to this technical problem lies in transforming the static infection mode into a dynamic monitoring and control mode. By quantitatively monitoring the key process parameter of real-time infection rate, a feedback regulation mechanism is established between bacterial concentration and infection efficiency. Since the survival and reproduction of Mycobacterium tuberculosis within macrophages affects the loading efficiency of extracellular vesicles onto lipoarabinomannan, and the infection rate directly reflects the distribution of intracellular bacteria, controlling the real-time infection rate within a certain range ensures the uniformity of the number of infected bacteria in the cell population. The 45% to 55% range set in this invention avoids both insufficient antigen loading due to excessively low infection rates and cell damage and abnormal vesicle secretion caused by excessively high infection rates. This range is the optimal range determined based on the balance between macrophage phagocytic capacity and metabolic load. Through high-frequency monitoring every 12 hours and proactive adjustment of bacterial concentration, the system can predict and correct deviations before the next batch of production, thereby achieving stable batch-to-batch control of antigen loading at the process level, meeting the stringent reproducibility requirements of quality control product preparation.

[0066] The following provides a specific embodiment 1 of the present invention, and the specific implementation of each step in this embodiment 1 is described in detail below.

[0067] In this embodiment, the specific implementation of step S01 is the same as described above, and will not be repeated in detail here.

[0068] The specific implementation of step S02 involves collecting adherent cells after differentiation induction treatment and quantitatively detecting the expression levels of cell surface markers using flow cytometry. This technology is based on the binding principle of fluorescently labeled antibodies and antigens. It achieves precise analysis at the single-cell level by exciting fluorescence signals with a laser and detecting them through an optical system. During the detection process, anti-CD11b fluorescent antibodies and anti-CD14 fluorescent antibodies are used to label cells, respectively. The qualification of macrophage differentiation is determined using a dual-threshold criterion, as described in detail below:

[0069] ;

[0070] In the formula, To determine the pass / fail outcome, the value is true if the conditions are met, and false otherwise. The positive expression rate corresponding to the expression level of the cell surface marker CD11b is expressed in % (%). The positive expression rate corresponding to the expression level of the cell surface marker CD14 is expressed in % (%). For logical AND operator.

[0071] The parameter acquisition method is as follows: The sample was obtained experimentally, including step 1: preparing a solution with a concentration of 10. Anti-CD11b fluorescent antibody working solution; Step 2: Take Cells suspended in 200 In phosphate buffer; Step 3: Add 10 After mixing the anti-CD11b fluorescent antibody working solution, incubate at 4°C in the dark for 30 min; Step 4: Wash twice with phosphate buffer and resuspend at 300 mL. In buffer solution; Step 5: Detect fluorescence signal intensity using flow cytometry. Set the positive threshold to the mean fluorescence intensity of the control group plus two standard deviations. Calculate the percentage of cells with fluorescence intensity exceeding the threshold. . The sample was obtained experimentally, including step 1: preparing a solution with a concentration of 10. Anti-CD14 fluorescent antibody working solution; Step 2: Take Cells suspended in 200 In phosphate buffer; Step 3: Add 10 After mixing the anti-CD14 fluorescent antibody working solution, incubate at 4°C in the dark for 30 min; Step 4: Wash twice with phosphate buffer and resuspend at 300 mL. In buffer solution; Step 5: Detect fluorescence signal intensity using flow cytometry. Set the positive threshold to the mean fluorescence intensity of the control group plus two standard deviations. Calculate the percentage of cells with fluorescence intensity exceeding the threshold. .

[0072] The dual-threshold criterion formula is based on the biological characteristics of the synergistic expression of surface markers during macrophage differentiation and maturation. The formula includes a CD11b positivity rate determination part and a CD14 positivity rate determination part, which are connected by a logical AND operator. The connection ensures that both markers are met simultaneously. The CD11b positivity rate reflects the degree of cell phenotypic transformation, while the CD14 positivity rate reflects the level of cell functional maturity. This dual determination mechanism effectively avoids false positive or false negative results caused by single marker detection. This formula ensures that macrophages entering subsequent infection steps possess both morphological characteristics and functional properties.

[0073] The specific implementation of step S03 involves placing a suspension of Mycobacterium tuberculosis H37Rv standard strain in an ultrasonic disruptor with a power setting of 150W for ultrasonic treatment. The treatment parameters are continuous ultrasonication for 5 seconds followed by a 10-second interval, and this cycle is repeated 3 times. The cavitation effect generated during ultrasonication disrupts the hydrophobic interactions within the bacterial clumps through mechanical shear force, dispersing the aggregated bacteria into individual cells. A power of 150W is the optimal parameter to achieve sufficient dispersion while ensuring that the bacterial activity is not damaged. After ultrasonic treatment, the bacterial solution is repeatedly agitated 12 times using a 27-gauge needle with an inner diameter of 0.4mm. The high shear force generated by the inner diameter of the needle further breaks up the remaining bacterial clumps. Subsequently, the optical density value at a wavelength of 600nm is measured using spectrophotometry and compared with a standard curve of unit concentration of known colonies to calculate the concentration of viable bacteria in the bacterial solution. The relationship between the optical density value and the bacterial solution concentration is specifically described as follows:

[0074] ;

[0075] In the formula, The concentration of viable bacteria in the bacterial solution, in units of... ; The optical density value at a wavelength of 600 nm is dimensionless. The standard optical density value is dimensionless, and its empirical value is 0.6. This represents the bacterial concentration corresponding to the standard optical density value, in units of... experience value .

[0076] The parameter acquisition method is as follows: The sample was obtained experimentally, including step 1: take 1 Step 1: Add the bacterial culture to the cuvette; Step 2: Place the cuvette into the sample chamber of the spectrophotometer; Step 3: Set the wavelength to 600 nm; Step 4: Zero the sample using a blank culture medium as a reference; Step 5: Read the absorbance value of the bacterial culture sample. , The range is typically 0.1 to 1.0.

[0077] The formula relating optical density to bacterial concentration is based on Beer-Lambert's law and includes a normalized portion of the measured optical density. and concentration calibration section The normalized portion of the measured optical density value is obtained by dividing by the standard optical density value. To ensure comparability of measurement results across different batches, the concentration calibration section uses known concentrations of standard strains at standard optical density values. As a calibration benchmark, this formula enables the quantitative conversion of optical density values ​​to viable bacterial concentration, ensuring precise control of bacterial concentration in subsequent infection experiments. This formula operates within a specific range of bacterial concentrations. to The internal linear relationship is good.

[0078] The specific implementation of step S04 is to adjust the bacterial concentration to the initial concentration. The bacterial culture was added to the culture system of differentiated macrophages at a ratio of 5 (multiple infection value of 5). The calculation of the multiple infection value is described in detail below:

[0079] ;

[0080] In the formula, The infection complex number is dimensionless. The total number of viable bacteria in the bacterial solution, in units of ; This represents the total number of macrophages, expressed in cells.

[0081] The specific formula for calculating the required volume of bacterial culture based on the multiplicity of infection is as follows:

[0082] ;

[0083] In the formula, The volume of bacterial solution to be added, in units of ; The target infection multiplicity value is 5, based on experience. The total number of macrophages in the culture system, expressed in cells; The concentration of viable bacteria in the bacterial solution, in units of... .

[0084] The parameter acquisition method is as follows: The cells were obtained experimentally, including step 1: treating adherent macrophages with trypsin digestion solution for 5 min to detach the cells from the culture dish; step 2: collecting the cell suspension and washing once with phosphate buffer; step 3: taking 10 Cell suspension and 10 Step 4: Mix the trypan blue staining solution; Step 5: Add the mixture to a hemocytometer; Step 6: Count the number of unstained live cells under a microscope. Record the total number of cells in the four large squares of the hemocytometer as [data missing]. , The calculation formula is:

[0085] ;

[0086] In the formula, The total number of macrophages in the culture system, expressed in cells; The total number of cells in the four large squares of the counting chamber, expressed in cells. The dilution factor is dimensionless, with an empirical value of 2, corresponding to the volume ratio of cell suspension to trypan blue solution. This is the volume correction factor for the hemocytometer, in units of... experience value This indicates the conversion relationship between the number of cells per milliliter of solution corresponding to a single large square on the counting plate; The total volume of the culture system is expressed in units of... .

[0087] The range is usually to indivual.

[0088] Formula for calculating the multiplicity of infection Based on the ratio of bacteria to host cells, the formula quantitatively describes the infection load level by the ratio of the total number of viable bacteria to the total number of macrophages. This ratio reflects the average number of bacteria that each macrophage comes into contact with and engulfs. An infection multiplicity of 5 ensures a sufficient number of bacteria come into contact with and are engulfed by macrophages while avoiding excessive bacterial load leading to cell overload. The formula for calculating bacterial volume is... This achieves the conversion from the target infection multiplicity to the actual operational volume, ensuring the reproducibility and standardization of infection experiments. The formula for calculating the total number of macrophages is also provided. The formula is established based on the relationship between the standard volume and dilution factor of a hemocytometer. It reflects the degree of dilution of the cell suspension. For each large square on the hemocytometer, 1 corresponds to a cell count. Conversion factor for cell count in solution. The three formulas work together to convert cell concentration per unit volume into the total number of cells in the culture system, achieving a complete conversion of infection multiplicity value from theoretical design to practical operation, ensuring the consistency of infection conditions for each batch of cells.

[0089] The specific implementation of step S05 involves using flow cytometry to monitor the positive rate of fluorescently labeled Mycobacterium tuberculosis in infected cells every 12 hours as a quantitative indicator of the real-time infection rate. The calculation formula for dynamically adjusting the initial bacterial concentration is specifically expressed as follows:

[0090] .

[0091] In the formula, The adjusted initial bacterial concentration is expressed in units of... ; This represents the initial bacterial concentration for the current batch, in units of... , defaults to ; The infection rate is expressed in real-time percentages (%). This is a compensation coefficient for low infection rates, dimensionless, with an empirical value of 1.5; The high infection rate inhibition coefficient is dimensionless and has an empirical value of 0.7. This is the lower limit threshold for the real-time infection rate, expressed in %, with an empirical value of 40. This is the upper limit threshold for the real-time infection rate, expressed in %, with an empirical value of 60%.

[0092] The parameter acquisition method is as follows: The cells were obtained experimentally, including step 1: collecting infected macrophages and treating them with trypsin digestion solution for 3 min to remove them from the culture dish; step 2: washing the cells twice with phosphate buffer and resuspending them in 500 mL of water. Step 3: Transfer the cell suspension to a flow cytometer tube; Step 4: Set the excitation wavelength to 488 nm and the green fluorescence emission detection wavelength to 530 nm; Step 5: Analyze at least... For each cell, a fluorescence threshold is set as the mean fluorescence intensity of the uninfected control group plus three standard deviations; Step 6: Calculate the percentage of cells with fluorescence intensity exceeding the threshold. , The range is typically 20% to 80%.

[0093] Formula for dynamically adjusting the initial bacterial concentration Established based on a real-time infection rate feedback mechanism, in which the control coefficient Based on real-time infection rate With threshold and The relationship is determined, and the formula includes a low infection rate compensation component, a high infection rate inhibition component, and a normal infection rate maintenance component. The low infection rate compensation component is achieved by increasing the bacterial concentration to the original concentration. The reduced infection efficiency due to decreased activity or insufficient dispersion of the compensated strain can be mitigated by reducing the bacterial concentration to the original concentration. This piecewise function design effectively reduces the excessive stress load on cells while maintaining the current concentration of the normal infection rate. It enables automatic regulation of the real-time infection rate between different batches within the optimal range of 45% to 55%, ensuring that macrophages can effectively load Mycobacterium tuberculosis antigens while maintaining normal vesicle secretion function.

[0094] The specific implementation methods for steps S06-S07 are the same as those described above, and will not be repeated in detail here.

[0095] The specific implementation of step S08 involves using enzyme-linked immunosorbent assay (ELISA) to quantitatively detect the content of lipoarabinomannan and LAM carrier protein in qualified vesicle products. The formula for determining the mass ratio is as follows:

[0096] ;

[0097] In the formula, The mass ratio of LAM carrier protein content to lipoarabinomannan content is dimensionless. LAM carrier protein content, in units of ; This refers to the content of arabinomannan, in units of... .

[0098] The formula for determining whether a concentration level is acceptable is described in detail below:

[0099] ;

[0100] In the formula, This is the result of the concentration level determination; This indicates a high concentration of positive control material; This indicates a medium-concentration positive control sample; This indicates a low-concentration positive control sample; This indicates that the product is unqualified. This represents the lower limit of high-concentration grade arabinomannan content, in units of... The experience value is 200; This represents the upper limit of the content of high-concentration grade arabinomannan, in units of... The experience value is 300; This represents the lower limit of lipofarabinomannan content for medium concentration grades, in units of... The experience value is 80; This represents the upper limit of lipofarabinomannan content for medium concentration grades, in units of... The experience value is 120; This represents the lower limit of lipofarabinomannan content for low-concentration grades, in units of... The experience value is 20; This represents the upper limit of lipofarabinomannan content for low-concentration grades, in units of... The experience value is 40; This is the lower limit of the mass ratio, dimensionless, with an empirical value of 0.6; This represents the upper limit of the mass ratio, is dimensionless, and has an empirical value of 1.2. For logical AND operator.

[0101] The parameter acquisition method is as follows: The antibody CS-35 was obtained experimentally, including step 1: diluting the anti-lipoarabinomannan monoclonal antibody CS-35 to 5 μL with carbonate buffer. Step 2: Take 100 Add antibody solution to the wells of the ELISA plate and coat overnight at 4°C; Step 3: Discard the coating solution and add 200 ml of antibody solution. Step 4: After washing 3 times, add 100 ml of phosphate buffer containing 5% skim milk powder. Vesicle samples were incubated at 37°C for 1 hour; Step 5: After washing 3 times, 100 ml of water was added. Biotin-labeled secondary antibody, incubated at 37°C for 1 hour; Step 6: After washing 3 times, add 100... Horseradish peroxidase-labeled streptavidin, incubated at 37°C for 30 min; Step 7: After washing 3 times, add 100 Substrate solution, incubate at room temperature for 15 minutes for color development; Step 8: Add 50 The reaction was terminated with a stop solution; Step 9: The absorbance value was measured at 450 nm using an ELISA reader and recorded as follows. Step 10: Calculate the lipoarabinomannan content in the sample based on the standard curve. The standard curve is established using purified lipoarabinomannan with a known concentration gradient, ranging from 10 to 500 mg / L. The equation of the standard curve is:

[0102] ;

[0103] In the formula, This refers to the content of arabinomannan, in units of... ; The slope of the standard curve for the detection of lipoarabinomannan is given in units of 1000 mcg. The absorbance values ​​are obtained by linear fitting of the absorbance values ​​of known concentration standards, and are usually taken as 400 to 600. The absorbance value of the lipoarabinomannan sample measured by an enzyme-linked immunosorbent assay (ELISA) at a wavelength of 450 nm is dimensionless. The intercept of the standard curve for the detection of lipoarabinomannan is given in units of 1000 mcg. The absorbance is obtained by linear fitting of the absorbance values ​​of a known concentration standard, and the value is usually taken as -5 to 5.

[0104] The antibody was obtained experimentally, including step 1: diluting the anti-LAM carrier protein monoclonal antibody A194-01 to 5 μL with carbonate buffer. Step 2: Take 100 Add antibody solution to the wells of the ELISA plate and coat overnight at 4°C; Step 3: Discard the coating solution and add 200 ml of antibody solution. Step 4: After washing 3 times, add 100 ml of phosphate buffer containing 5% skim milk powder. Vesicle samples were incubated at 37°C for 1 hour; Step 5: After washing 3 times, 100 ml of water was added. Biotin-labeled secondary antibody, incubated at 37°C for 1 hour; Step 6: After washing 3 times, add 100... Horseradish peroxidase-labeled streptavidin, incubated at 37°C for 30 min; Step 7: After washing 3 times, add 100 Substrate solution, incubate at room temperature for 15 minutes for color development; Step 8: Add 50 The reaction was terminated with a stop solution; Step 9: The absorbance value was measured at 450 nm using an ELISA reader and recorded as follows. Step 10: Calculate the LAM carrier protein content in the sample based on the standard curve. The standard curve is established using purified LAM carrier protein at known concentration gradients, ranging from 10 to 500 mg / L. The equation of the standard curve is:

[0105] ;

[0106] In the formula, LAM carrier protein content, in units of ; The slope of the LAM carrier protein detection standard curve is given in units of... The absorbance values ​​are obtained by linear fitting of the absorbance values ​​of known concentration standards, and are usually taken as 350 to 550. The absorbance values ​​of LAM carrier protein samples measured by an ELISA reader at a wavelength of 450 nm are dimensionless. This is the intercept of the standard curve for LAM vector protein detection, in units of... The absorbance is obtained by linear fitting of the absorbance values ​​of a known concentration standard, and the value is usually taken as -5 to 5.

[0107] Mass ratio determination formula Based on the natural stoichiometry between LAM carrier protein and lipoarabinomannan in Mycobacterium tuberculosis, the formula reflects the biological authenticity of vesicle-loaded antigen through the ratio of the two proteins' contents. The concentration grade qualification formula includes high-concentration grade, medium-concentration grade, and low-concentration grade determination parts. Each grade determination part considers both the absolute content and mass ratio of lipoarabinomannan. The high-concentration grade is used for verification of the upper limit of sensitivity of the detection system, the medium-concentration grade is used for routine quality control, and the low-concentration grade is used for verification of the detection limit of the detection system. This grading system covers the entire concentration range of Mycobacterium tuberculosis antigen in clinical samples. The mass ratio range of 0.6 to 1.2 ensures that the ratio of the two molecules in the vesicles conforms to the natural state. Standard curve equation. and Based on the linear relationship between absorbance and antigen concentration in enzyme-linked immunosorbent assay (ELISA), the slope parameter was established. and Intercept parameter reflects the antibody's sensitivity to antigen binding. and It reflects the background signal level of the detection system.

[0108] The formula for dynamically regulating the initial bacterial concentration establishes a feedback regulation mechanism based on the real-time infection rate. Through piecewise function design, the bacterial concentration is adaptively adjusted. This formula increases the bacterial concentration to compensate for the decrease in activity when the infection rate is low, and decreases the bacterial concentration to avoid cell overload when the infection rate is high. It maintains the real-time infection rate within the optimal range of 45% to 55%, ensuring that macrophages maintain normal vesicle secretion function while effectively loading Mycobacterium tuberculosis antigen, which significantly improves the yield and quality stability of extracellular vesicle products.

[0109] The specific implementation method of step S09 is the same as described above, and will not be repeated in detail here.

[0110] It should be explained that the dual-threshold criterion formula connects two independent cell surface marker threshold judgment conditions through a logical AND operator, making full use of the synergistic expression characteristics of CD11b and CD14 in the process of macrophage differentiation and maturation. This formula avoids false positive results that may be produced by single marker detection, ensuring that cells entering the subsequent infection steps have both morphological characteristics and functional properties, achieving precise control of macrophage differentiation quality, and improving the loading efficiency and stability of Mycobacterium tuberculosis antigen in subsequent extracellular vesicle products.

[0111] The formula relating optical density to bacterial concentration is based on Beer-Lambert's law. The formula includes a normalization part for the measured optical density and a concentration calibration part. The normalization part for the measured optical density eliminates systematic errors in measurement systems of different batches by dividing by the standard optical density value. This formula enables a rapid and accurate conversion from optical density to viable bacterial concentration, providing a precise means of controlling bacterial concentration for subsequent infection experiments, ensuring the consistency of infection conditions between different batches, and improving the reproducibility of extracellular vesicle products.

[0112] The multiplicity of infection (MLI) calculation formula is based on the ratio of bacteria to host cells, which reflects the average number of bacteria that each macrophage comes into contact with and engulfs. The bacterial culture volume calculation formula realizes the conversion from the target MII to the actual operating volume, ensuring the reproducibility and standardization of infection experiments. The macrophage total number calculation formula is based on the relationship between the standard volume of a hemocytometer and the dilution factor. The accurate total number of cells is obtained by multiplying the hemocytometer count, dilution factor, volume correction factor, and culture system volume. The three formulas work together to realize the complete conversion of the MII from theoretical design to actual operation, ensuring the consistency of infection conditions for each batch of cells.

[0113] The mass ratio determination formula and the concentration grade qualification determination formula together constitute the quality evaluation system for extracellular vesicle products. The mass ratio determination reflects the biological authenticity of the antigen loaded in the vesicles by the ratio of LAM carrier protein to lipoarabinomannan content. The concentration grade determination divides the products into three concentration grades (high, medium, and low) through a piecewise function, covering the entire concentration range for clinical testing applications. This evaluation system ensures the reliability and applicability of extracellular vesicle products as quality control materials for the diagnosis of Mycobacterium tuberculosis, and provides a standardized quality control tool for the early diagnosis of tuberculosis. The standard curve equation realizes the quantitative conversion of absorbance signal to antigen concentration in enzyme-linked immunosorbent assay (ELISA). The slope and intercept parameters established by linear fitting ensure the comparability and accuracy of test results from different batches.

[0114] To better understand this invention, a specific application scenario of the invention is provided in Example 2 below: A technical team needs to prepare a batch of high-quality extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens for the construction of a clinical diagnostic quality control system. The team carried out the preparation work according to the technical solution of this invention. First, THP-1 mononuclear cells were prepared according to... Inoculated at a density of 10% fetal bovine serum (FBS) per mL in RPMI-1640 medium and incubated at 37°C with 5% fetal bovine serum. Cells were cultured in an incubator until the logarithmic growth phase, and then phorbol ester-12-myristate-13-acetate at a concentration of 100 ng / mL was added to induce differentiation. After 48 hours of induction, the medium containing phorbol ester was discarded, and the cells were washed three times with phosphate buffer (pH 7.4, osmotic pressure 300 mOsm / kg) to remove residual inducing agent and metabolic waste. The medium was then replaced with fresh medium containing 5% fetal bovine serum and cultured for another 24 hours to allow the cells to fully adhere and return to normal metabolic state.

[0115] After collecting adherent cells, the technical team used flow cytometry to detect the expression levels of cell surface markers. Cells were labeled with anti-CD11b and anti-CD14 fluorescent antibodies, respectively. Flow cytometry analysis showed that the positive expression rate of CD11b was 88.3%, and the positive expression rate of CD14 was 84.7%, both meeting the criteria for qualified differentiated macrophages. Figure 2 As shown, the expression levels of cell surface markers gradually increased with the extension of induction time, reaching a stable plateau at 48 hours. The technical team cultured the Mycobacterium tuberculosis H37Rv standard strain in 7H9 liquid medium to the logarithmic growth phase, collected the cells, and resuspended them in phosphate buffer to form a bacterial suspension. The bacterial suspension was subjected to ultrasonic treatment using a 150W ultrasonic homogenizer, with a 10-second interval between each 5-second treatment to prevent excessive temperature from reducing bacterial activity. This treatment was repeated 3 times, followed by repeated pipetting 12 times with a 27-gauge needle to further disperse the bacterial clusters. The bacterial concentration was adjusted to [specific value missing] by measuring the optical density at 600 nm and comparing it with a standard curve. CFU / mL was used as the initial infection solution.

[0116] The technical team added the processed bacterial solution to well-differentiated macrophages and inoculated them at a ratio of 5 (multiple infection value of 5), meaning each macrophage contained 5 Mycobacterium tuberculosis bacteria. The culture was then stored at 37°C and 5%... After incubation in an incubator for 4 hours, the cells were washed three times with medium containing 100 μg / mL gentamicin to kill any unphagocytosed extracellular bacteria. The medium was then replaced with maintenance medium containing 10 μg / mL gentamicin and cultured for another 48 hours. Since this experiment used a recombinant Mycobacterium tuberculosis H37Rv strain transfected with the green fluorescent protein gene, the team monitored the fluorescence signal intensity within infected cells every 12 hours using flow cytometry to calculate the real-time infection rate. As shown in Table 1, the monitoring data showed that the infection rate rose rapidly in the initial stage, stabilized after 24 hours, and ultimately remained at 49.2%, within the target range of 45% to 55%.

[0117] Table 1. Monitoring data of macrophage infection rate at different time points

[0118]

[0119] After 48 hours of culture, the team collected the cell supernatant for extracellular vesicle isolation. Differential centrifugation was used to remove particles of different sizes in stages: first, centrifugation at 300×g for 10 minutes removed cell debris and unlysed intact cells; then, centrifugation at 2000×g for 20 minutes removed apoptotic bodies; next, centrifugation at 10000×g for 30 minutes removed large vesicles and bacterial debris; finally, ultracentrifugation at 100000×g for 70 minutes precipitated the target extracellular vesicles. After resuspending the precipitate in phosphate buffer, the team used nanoparticle tracking analysis to determine the particle size distribution and concentration of the vesicles. Figure 2 As shown, the particle size distribution exhibits a single-peak pattern, with the main peak particle size located at 112 nm, falling within the target range of 80 nm to 150 nm. The measured vesicle concentration value is... The number of cells / mL meets the criteria for qualified vesicle products.

[0120] The technical team further used enzyme-linked immunosorbent assay (ELISA) to quantitatively detect the content of Mycobacterium tuberculosis-specific antigen in the vesicles. The content of lipoarabinomannan was detected using the anti-lipoarabinomannan monoclonal antibody CS-35, and the results showed that the lipoarabinomannan content in this batch of vesicles was 246 ng / mL, which falls within the range of high-concentration positive control products. The content of LAM carrier protein was detected using the anti-LAM carrier protein monoclonal antibody A194-01, and the measured value was 197 ng / mL. The calculated mass ratio of LAM carrier protein content to lipoarabinomannan content was 0.80, which is within the target range of 0.6 to 1.2, reflecting that the natural stoichiometric relationship between LAM carrier protein and lipoarabinomannan in Mycobacterium tuberculosis was well maintained. The technical team aliquoted this batch of vesicles into polypropylene cryovials, each with a volume of 200 μL, and added 10% dimethyl sulfoxide as a cryoprotectant. After dispensing, place the cryovials in a -20°C freezer for 2 hours to allow the temperature to drop slowly and avoid damage to the vesicle membrane structure from rapid freezing. Then transfer them to a -80°C freezer for long-term storage.

[0121] Through subsequent six months of stability monitoring, the technical team found that the vesicle products stored at -80°C maintained stability in terms of particle size distribution, vesicle concentration, and antigen content, indicating that the preparation method and storage conditions of this invention can ensure the long-term stability of the quality control products. This invention represents a significant technological advancement over traditional Mycobacterium tuberculosis antigen extraction methods. Traditional methods directly extract lipoarabinomannan from Mycobacterium tuberculosis cells, requiring large amounts of organic solvents and high-temperature treatment, which easily damages the native conformation and biological activity of lipoarabinomannan and makes it difficult to maintain its native binding state with chaperone proteins. This invention, by simulating the physiological process of Mycobacterium tuberculosis infecting macrophages, utilizes the host cell's own extracellular vesicle secretion mechanism to co-package lipoarabinomannan and its chaperone proteins into vesicles. The entire process is carried out under physiological temperature and pH conditions, maximizing the preservation of the antigen's native state and biological activity. Furthermore, the lipid bilayer membrane structure of the extracellular vesicles provides a natural protective barrier for the internal antigen, significantly improving the stability of the antigen during storage and transportation—an advantage that is difficult to achieve with traditional antigen purification methods.

[0122] 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 changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens, characterized in that, include: THP-1 mononuclear cells were cultured in RPMI-1640 medium containing fetal bovine serum until the cell density reached the preset level. Phorbol ester was added to induce differentiation. After discarding the medium, the cells were washed with phosphate-buffered saline (PBS) and cultured in fresh medium containing PBS. Adherent cells were collected, and flow cytometry was used to detect the expression levels of cell surface markers CD11b and CD14 to determine the number of macrophages that had differentiated sufficiently. A suspension of Mycobacterium tuberculosis H37Rv standard strain was sonicated and repeatedly pipetted with a needle. The bacterial concentration was adjusted to the initial concentration using optical density measurement. The treated bacterial suspension was added to the differentiated macrophages for infection and incubation. The cells were washed with medium containing gentamicin to remove extracellular bacteria and cultured in maintenance medium containing gentamicin. The culture was repeated every [period not specified]. The positive rate of fluorescently labeled Mycobacterium tuberculosis in infected cells was monitored by flow cytometry at preset time intervals and recorded as the real-time infection rate. The initial bacterial concentration was dynamically adjusted according to the real-time infection rate to maintain the real-time infection rate within the target range between different batches. The cell supernatant after culture was collected, and extracellular vesicles were separated by differential centrifugation. The particle size distribution and vesicle concentration of extracellular vesicles were detected by nanoparticle tracking analysis technology to determine qualified vesicle products. The content of lipoarabinomannan and LAM carrier protein in qualified vesicle products was quantitatively detected by enzyme-linked immunosorbent assay. Based on the lipoarabinomannan content, qualified vesicle products were divided into three concentration levels: high-concentration positive control, medium-concentration positive control, and low-concentration positive control. The positive control products of different concentration levels were aliquoted into polypropylene cryovials, and dimethyl sulfoxide was added as a cryoprotectant for cryopreservation.

2. The method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens according to claim 1, characterized in that, THP-1 monocytes were cultured in RPMI-1640 medium containing fetal bovine serum until the cell density reached the preset level. Then, phorbol ester was added to induce differentiation. Specifically, THP-1 monocytes were cultured in RPMI-1640 medium containing 10% fetal bovine serum until the cell density reached the preset level. Differentiation was induced for 48 hours by adding phorbol ester at a concentration of 100 ng / mL.

3. The method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens according to claim 2, characterized in that, The phorbol ester is phorbol ester-12-myristate-13-acetate, which induces THP-1 monocytes to differentiate into adherent macrophages by activating the protein kinase C signaling pathway.

4. The method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens according to claim 3, characterized in that, The phosphate buffer solution is an aqueous solution prepared by disodium hydrogen phosphate and potassium dihydrogen phosphate at a mass ratio of 8:1, with a pH of 7.4 and an osmotic pressure of 300 mOsm / kg.

5. The method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens according to claim 4, characterized in that, The procedure for detecting the expression levels of cell surface markers CD11b and CD14 using flow cytometry and determining the differentiation-qualified macrophages is as follows: macrophages are considered to be differentiation-qualified when the positive expression rate of cell surface marker CD11b is >85% and the positive expression rate of cell surface marker CD14 is >80%. If the positive expression rate of cell surface marker CD11b is ≤85% or the positive expression rate of cell surface marker CD14 is ≤80%, a second differentiation induction treatment is performed.

6. The method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens according to claim 5, characterized in that, The secondary induction differentiation treatment refers to replacing the culture medium with fresh phorbol ester at a concentration of 50 ng / mL and adding it again for another 24 hours after the first induction differentiation 48 hours later, if the positive expression rate corresponding to the expression level of cell surface marker CD11b or cell surface marker CD14 does not meet the standard.

7. The method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens according to claim 6, characterized in that, The procedure involves placing the Mycobacterium tuberculosis H37Rv standard strain suspension in an ultrasonic homogenizer for ultrasonic treatment and repeatedly blowing the bacterial solution with a needle. Specifically, the Mycobacterium tuberculosis H37Rv standard strain suspension is placed in an ultrasonic homogenizer with a power of 150W for ultrasonic treatment for 5 seconds, with a 10-second interval, and the treatment is repeated 3 times. Then, the bacterial solution is repeatedly blown 12 times with a No. 27 needle.

8. The method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens according to claim 7, characterized in that, The step of adjusting the bacterial suspension concentration to the initial bacterial suspension concentration by measuring the optical density value is specifically to adjust the bacterial suspension concentration to the initial bacterial suspension concentration by measuring the optical density value. CFU / mL.

9. The method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens according to claim 8, characterized in that, The step of adding the treated bacterial solution to differentiated macrophages for infection and incubation involves adding the bacterial solution corresponding to the initial concentration of the treated solution to the differentiated macrophages for infection, setting the multiplicity of infection (MOU) to 5, and incubating at 37°C and 5%. After incubating in an incubator for 4 hours, the cells were washed three times with a medium containing 100 μg / mL gentamicin to remove extracellular bacteria, and then the medium was replaced with a maintenance medium containing 10 μg / mL gentamicin and cultured for another 48 hours.

10. The method for preparing extracellular vesicles carrying Mycobacterium tuberculosis-specific antigens according to claim 9, characterized in that, The steps involve dynamically adjusting the initial bacterial concentration based on the real-time infection rate to maintain the real-time infection rate within the target range across different batches. Specifically, flow cytometry is used every 12 hours to monitor and record the positivity rate of fluorescently labeled Mycobacterium tuberculosis in infected cells as the real-time infection rate. When the real-time infection rate is <40%, the initial bacterial concentration is increased to [a higher concentration]. CFU / mL was used to re-infect the next batch of cells. When the real-time infection rate was >60%, the initial bacterial concentration was reduced to [a lower value]. CFU / mL, resulting in real-time infection rates between different batches ∈ [45%, 55%].

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