Method for efficient and rapid concentration of LPS or LAM, and apparatus

Abstract

The present invention relates to the field of biological testing. Disclosed are a method for efficient and rapid concentration of lipopolysaccharide (LPS) or lipoarabinomannan (LAM), and an apparatus. The inventor has found by researches that a non-polar macroporous resin within a specific pore size range exhibits adsorption efficiency for LPS or LAM comparable to affinity adsorption, has a large adsorption capacity and a high adsorption rate, and can efficiently elute LPS in combination with a specific eluent to obtain a concentrated LPS sample under test, thereby creating good conditions for the detection of LPS or LAM. By means of the method, efficient concentration of LAM is achieved, providing excellent conditions for the detection of mycobacterium tuberculosis.

Description

A highly efficient and rapid concentration method and apparatus for LPS or LAM Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a highly efficient and rapid concentration method and apparatus for LPS or LAM. Technical issues

[0002] In vitro diagnostics (IVD) refers to a testing method that analyzes human samples (such as blood, urine, cerebrospinal fluid, saliva, and tissues) outside the human body to obtain clinical diagnostic information. In practical applications, the analysis of human samples involves identifying the effective target analytes (including proteins, DNA / RNA, cells, and compounds) contained within the sample to determine the individual's health status or disease condition. However, the concentration of effective target analytes in samples is often very low. Due to limitations in the sensitivity of testing instruments or reagent cards, direct testing of bodily fluids often fails to detect effective target analytes, or the detected levels have significant errors, leading to inaccurate results. Therefore, enriching and concentrating effective target analytes from samples is a fundamental and primary task. Enriching and concentrating effective target analytes offers the following advantages:

[0003] First, it can improve detection sensitivity. In in vitro detection, the concentration of certain target substances may be very low, making accurate results difficult to obtain through direct detection. By concentrating the sample, the relative content of the target substance can be increased, making it easier to detect and thus significantly improving detection sensitivity. For example, in the detection of trace biomarkers, concentrated samples can bring substances that are originally below the detection limit to detectable levels, providing a more reliable basis for the early diagnosis of diseases.

[0004] Second, it can reduce the influence of interfering substances. In vitro testing samples often contain various interfering substances, which may affect the accuracy and specificity of the test. Concentrating the sample can reduce the relative proportion of interfering substances to a certain extent, thus reducing their interference with the detection. For example, in blood testing, concentration can reduce the influence of interfering substances such as proteins and salts in plasma, making the target analyte easier to identify and quantify.

[0005] Third, it saves on testing costs and time. Concentrated samples can reduce the volume of samples and reagents required for testing, thereby lowering costs. Furthermore, for some testing methods that require complex pretreatment, concentrated samples can simplify procedures and shorten testing time. For example, in nucleic acid testing, concentrated samples can reduce the workload of nucleic acid extraction and purification, improving testing efficiency.

[0006] Fourth, it facilitates sample storage and transportation. The concentrated sample has a smaller volume, making it easier to store and transport. This is highly advantageous for situations requiring remote testing or long-term sample preservation. For example, in clinical laboratories, concentrated samples can be cryopreserved and tested when needed, reducing sample waste and the risk of duplicate collection.

[0007] Macroporous resin, also known as fully porous resin, is prepared by polymerization of monomers and additives such as crosslinking agents, porogens, and dispersants. After polymer formation, the porogens are removed, leaving interconnected pores of various sizes and shapes within the resin. Therefore, macroporous resin has a high porosity and large pore size in its dry state, ranging from 5 to 1000 nm.

[0008] Macroporous adsorption resins are mainly made from styrene, α-methylstyrene, methyl methacrylate, propionitrile, etc., with a certain amount of vinylbenzene added as a crosslinking agent, and toluene and xylene as pore-forming agents. They are mostly spherical particles, generally with a diameter between 0.3 and 1.25 mm, and are usually classified into non-polar, weakly polar, and moderately polar macroporous adsorption resins. These resins crosslink and polymerize to form a porous framework structure. The resin is generally a white spherical particle with a particle size of 20-60 mesh. It is a type of crosslinked polymer containing ion exchange groups, and its physicochemical properties are stable. It is insoluble in acids, alkalis, and organic solvents, and is unaffected by inorganic salts and strong ionic low-molecular-weight compounds. The adsorption of the resin relies on the van der Waals attraction between it and the adsorbed molecules (adsorbate). It works through physical adsorption via its large specific surface area, allowing organic compounds to be separated by elution with a certain solvent according to their adsorption force and molecular weight, achieving different purposes such as separation, purification, impurity removal, and concentration.

[0009] Macroporous adsorption resins can be classified into three categories—non-polar, moderately polar, and polar—based on their polarity and the molecular structure of the monomers used.

[0010] (1) Non-polar macroporous adsorption resin

[0011] Nonpolar macroporous adsorption resins are polymerized from monomers with very small dipole moments and do not contain any functional groups. They have strong hydrophobicity on the pore surface and can adsorb organic matter in solution by interacting with the hydrophobic part of small molecules. They are most suitable for adsorbing nonpolar substances in polar solvents and are also called aromatic adsorbents, such as styrene and divinylbenzene polymers.

[0012] (2) Medium polarity macroporous adsorption resin

[0013] Medium-polarity macroporous adsorption resins are ester-containing adsorption resins with multifunctional methacrylate groups acting as crosslinking agents. Their surface possesses both hydrophobic and hydrophilic components. They can adsorb polar substances from both polar and non-polar solvents, and are also known as aliphatic adsorbents, such as polyacrylate polymers.

[0014] (3) Polar macroporous adsorption resin

[0015] Polar macroporous adsorption resins refer to adsorption resins containing nitrogen, oxygen, and sulfur polar functional groups such as amide, cyano, and phenolic hydroxyl groups. They adsorb polar substances, such as acrylamide, through electrostatic interactions.

[0016] While current technologies utilize various commercially available macroporous resins for the separation and purification of polysaccharides and active components of natural drugs, such as steviol glycosides, flavonoids, alkaloids, ginkgo flavonoids, ginsenosides, and tea polyphenols, macroporous resins have not been applied to the adsorption and concentration of lipopolysaccharides. The separation and concentration effects of macroporous resins are influenced by multiple factors, and the adsorption and elution times are relatively long, generally making it difficult to meet the needs of rapid detection.

[0017] Lipopolysaccharide (LPS) is a component of the outer wall of Gram-negative bacterial cell walls, and is a substance composed of lipids and polysaccharides (glycolipids). LPS consists of three parts: lipid A, core polysaccharide, and O-antigen. The lipid A portion of LPS enters the lipid bilayer as part of the outer layer, while the glycan portion remains exposed outside the cell, thus existing on the cell surface of Gram-negative bacteria in this form.

[0018] Lipopolysaccharide (LPS) concentration detection can be applied to: 1. Disease diagnosis: LPS is a major component of bacterial endotoxins, mainly found in the cell walls of Gram-negative bacteria. Its detection is of great significance for the diagnosis of digestive system diseases, bacterial infectious diseases, and fever of unknown origin. 2. Pathogen examination: LPS detection is a powerful supplement to bacterial culture and can be considered the "gold standard" for pathogen examination of bacterial infectious diseases. 3. Observing the effect of antibiotic use: By detecting LPS, the effect of antibiotic use can be observed and the treatment effect can be evaluated. IV. Immunological Research: LPS is a widely used inducer / stimulant in immunological research. It can trigger a cascade of immune stimulation and toxic pathophysiological activities in the body, as well as studies on inflammation and immune response mechanisms. LPS can activate immune cells carrying CD14 / TLR4 / MD2 receptor complexes, thus playing an important role in studying the mechanisms of inflammation and immune responses. V. Animal Model Establishment: LPS is used to establish various disease-related animal models, such as acute lung injury and acute pancreatitis, to study disease development and treatment. VI. Drug Development: LPS has received increasing attention in recent years as a target for the development of anti-Gram-negative bacterial drugs because it is an important barrier for bacteria to defend against the external environment and a significant factor in the inherent drug resistance of Gram-negative bacteria. VII. Biomarkers of Infection and Inflammation: The presence and concentration of LPS can serve as biomarkers for infection and inflammation, helping clinicians assess the condition and guide treatment.

[0019] Lipoarabinomannan (LAM) is mainly found on the cell walls of mycobacteria (such as Mycobacterium tuberculosis), which is the pathogen that causes tuberculosis. LAM antigen detection can provide direct evidence for the diagnosis of tuberculosis. Highly efficient and rapid concentration of lipoarabinomannan (LAM) is of great significance for the diagnosis of diseases, especially the detection of Mycobacterium tuberculosis.

[0020] Lipopolysaccharides (LPS) are generally extracted using processes similar to those for polysaccharides, such as phenol-water extraction, trichloroacetic acid extraction, water extraction with alcohol precipitation, and enzymatic hydrolysis. CN102898537A discloses a purification method for LPS, which yields highly purified LPS after ultrafiltration concentration, hydrophobic chromatography, and ion exchange chromatography. The purified LPS is suitable for vaccine preparation. However, this method is time-consuming and cannot meet the needs of rapid detection. Liu Yuchen. Research on the isolation and purification process of inactivated Mycobacterium tuberculosis LPS antigen [J]. Zhejiang University of Technology, 2012. The disclosed LPS extraction method is also complex and cannot meet the needs of rapid detection.

[0021] The technical solution disclosed in CN101974099A utilizes the structural difference between LAM antigen, which contains three mannose impurities (LM, PIM, etc.) and only contains one mannose, and utilizes the characteristic that lectins (such as lentil lectin) have a much higher affinity for the three mannose branch structure than for the two mannose and one mannose. Through affinity chromatography, lectins are used as affinity purification reagents to purify LAM antigen.

[0022] Specific steps:

[0023] Column packing: Pack the lectin, such as COA-Sepharose 4B or LCA-Sepharose 4B, into the centrifuge column and equilibrate it with an appropriate buffer.

[0024] Sample loading: Add the sample into the column, centrifuge after a certain time, and collect the column liquid.

[0025] Washing: Wash the column with the same buffer solution and collect the wash solution.

[0026] Dissociation: Add a buffer solution containing methyl glucoside to dissociate and collect the dissociated solution.

[0027] Electrophoretic separation and analysis: The purified products were separated by SDS-PAGE electrophoresis and analyzed by silver staining or other staining methods.

[0028] Other methods for purifying LAM include centrifugation or ultrasonication, but these involve numerous steps and complex processes.

[0029] For example, ultrasound methods include pretreatment: placing the LAM sample in an appropriate buffer to maintain its stability and activity.

[0030] Ultrasonic disruption: The sample is disrupted using an ultrasonic disruptor. Collection of disrupted products: After sonication, undisturbed cell debris and other large particulate impurities are removed by low-speed centrifugation. Post-ultrasonic processing: Further centrifugation may be required to collect the small particles and LAM (laminar endothelial growth factor) from the ultrasonic disruption.

[0031] The steps for purifying LAM by centrifugation include: Differential centrifugation: First, use low-speed centrifugation to remove large particles, then use high-speed centrifugation to separate smaller particles. Density gradient centrifugation: Pre-place media of different densities in centrifuge tubes to create a density gradient. During centrifugation, particles move along the gradient according to density until they form a band at a point of equal density. LAM collection: After centrifugation, LAM can be collected from specific locations in the gradient based on its density characteristics. Washing and resuspending: The collected LAM may need to be washed and resuspended with appropriate buffer to remove residual impurities.

[0032] There are also methods to separate and purify LAM using the specific affinity of antibodies (Yan ZH, Zhao B, Pang Y, et al. Generation of mycobacterial lipoarabinomannan-specific monoclonal antibodies and their ability to identify mycobacterium isolates[J]. Journal of Microbiology, Immunology and Infection, 2021, 54(3): 437-446., Hamasur B, Källenius G, Svenson S B. A new rapid and simple method for large-scale purification of mycobacterial lipoarabinomannan[J]. FEMS Immunology & Medical Microbiology, 1999, 24(1): 11-17.).

[0033] In summary, existing methods for separating and purifying lipopolysaccharides (LPS) or lipoarabinomannan (LAM) are relatively complex and costly, making them difficult to apply to the concentration and purification of LPS or LAM or the detection of Mycobacterium tuberculosis. Solution

[0034] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a highly efficient and rapid concentration method and apparatus for LPS or LAM.

[0035] The technical solution adopted in this invention is:

[0036] A first aspect of the present invention provides: a highly efficient and rapid method for concentrating lipopolysaccharides or lipoarabinomannan, comprising the following steps:

[0037] S1) Pack macroporous resin into a chromatography column, load the sample to be concentrated onto the chromatography column, and allow it to adsorb.

[0038] S2) After adsorption is complete, drain the liquid;

[0039] S3) Add elution buffer, elute and collect to obtain lipopolysaccharide or lipoarabinomannan concentrate;

[0040] Wherein: the macroporous resin has a particle size of 0.03 to 1.2 mm and a pore size range of 5 to 50 nm;

[0041] The eluent is selected from at least one of the following: aqueous ethanol solution, aqueous methanol solution, ethyl acetate solution, a mixture of ethanol and buffer solution, and a mixture of methanol and buffer solution.

[0042] In some examples of efficient and rapid concentration methods, the macroporous resin has a particle size of 0.03–0.4 mm. Experimental data show that the finer the particle size of the macroporous resin, the better the adsorption effect on lipopolysaccharides or lipoarabinomannan; however, excessively fine particle size leads to excessively long adsorption times, which is not conducive to rapid concentration.

[0043] In some examples of efficient and rapid concentration methods, the macroporous resin has a pore size range of 5–16 nm. Experimental data show that nonpolar macroporous resins with this pore size have better adsorption effects on lipopolysaccharides or lipoarabinomannan.

[0044] In some examples of efficient and rapid concentration methods, the macroporous resin has a particle size of 0.03–0.4 mm and a pore size range of 5–16 nm.

[0045] In some examples of efficient and rapid concentration methods, the macroporous resin is a modified nonpolar resin.

[0046] In some examples of efficient and rapid concentration methods, the nonpolar resin is prepared from a polymeric monomer, a crosslinking agent, a porogen, an initiator, and a stabilizer. The polymeric monomer is at least one selected from styrene, α-methylstyrene, methyl methacrylate, and propionitrile. The crosslinking agent is divinylbenzene, the porogen is toluene, a fatty alcohol, or a fatty acid, and the stabilizer is gelatin.

[0047] In some examples of efficient and rapid concentration methods, the nonpolar macroporous resin is selected from at least one of D101, H103, HPD100, ADS-8, X-5, D101-I, D50, D700, D910 and D4020.

[0048] In some examples of efficient and rapid concentration methods, the total content of methanol and / or ethanol in the eluent is 10 to 90 v / v.

[0049] There are no restrictions on the type of buffer used for elution, as long as it does not affect subsequent detection. PBS has virtually no impact on the detection of LPS or LAM and is a better choice. Of course, other buffers can also be used, including but not limited to Tris buffer (Tris-HCl), acetate buffer, carbonate buffer, citrate buffer, HEPES buffer (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid), MES buffer (2-(N-morpholino)ethanesulfonic acid), MOPS buffer (3-(N-morpholino)propanesulfonic acid), TAE and TBE buffers, Good's buffers, including Tricine, Bicine, EPPS, PIPES, etc., glycine buffer, succinate buffer, EDTA buffer, and Tricine buffer.

[0050] In some examples of efficient and rapid concentration methods, the eluent consists of 10–90 v / v% methanol or ethanol, with the balance being PBS solution. This eluent effectively elutes adsorbed lipopolysaccharides or lipoarabinomannan. Preferably, the eluent consists of 10–50 v / v%, 10–40 v / v%, 10–35 v / v%, 10–30 v / v%, 10–25 v / v% ethanol, with the balance being PBS solution. Ethanol is safer and provides better elution, making it a superior choice.

[0051] In some examples of efficient and rapid concentration methods, the diameter-to-height ratio (diameter:height) of the chromatography column is 1:1 to 5:1, preferably 1:1 to 3:1. Columns with this diameter-to-height ratio can adsorb lipopolysaccharides or lipoarabinomannan more quickly and effectively.

[0052] In some examples of efficient and rapid concentration methods, the column is activated using an activation solution before loading the sample to be concentrated onto the chromatography column. This activation solution is a buffer solution containing 30–95 v / v% ethanol or methanol. The activation solution activates the resin and simultaneously wets it, preventing dry resin from adversely affecting the adsorption of lipopolysaccharides (LPS) or lipoarabinomannan, thus solving the problem of resin inactivation during long-term storage. The buffer solution in the activation solution has no special requirements, as long as it does not affect resin activation or subsequent LAM or LPS adsorption. PBS has no adverse effect on resin activation and does not affect LAM or LPS adsorption, making it a better choice. Of course, other buffers can also be used, including but not limited to Tris buffer (Tris-HCl), acetate buffer, carbonate buffer, citrate buffer, HEPES buffer (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid), MES buffer (2-(N-morpholino)ethanesulfonic acid), MOPS buffer (3-(N-morpholino)propanesulfonic acid), TAE and TBE buffers, Good's buffers, including Tricine, Bicine, EPPS, PIPES, etc., glycine buffer, succinate buffer, EDTA buffer, and Tricine buffer.

[0053] In some examples of efficient and rapid concentration methods, during elution, the concentration of lipopolysaccharide (LPS) or lipoarabinomannan in different sections of the eluent is measured, and the fraction with the higher LPS or lipoarabinomannan concentration is selected for detection. For ease of collection, multiple elutions can be performed, and the eluent is collected after each elution. Data shows that when the eluent-to-resin volume ratio (mL:g) is not higher than 2, the amount of LPS or lipoarabinomannan eluted is relatively small. With further increases in the eluent volume, the amount of LPS or lipoarabinomannan eluted increases significantly. When the eluent-to-resin volume ratio is between 2 and 4, the concentration of LPS or lipoarabinomannan in the eluent is higher. After the eluent-to-resin volume ratio reaches 5, almost all LPS or lipoarabinomannan has been eluted. If necessary, all the eluent can be collected to obtain more of the LPS or lipoarabinomannan. When the testing requirements are met, only the segments with higher concentrations of lipopolysaccharide or lipoarabinomannan can be collected.

[0054] In some examples of efficient and rapid concentration methods, the mixing ratio of nonpolar macroporous resin to the sample to be concentrated is 0.1 g:10 mL to 0.5 g:10 mL. This generally meets the needs for concentrating lipopolysaccharides or lipoarabinomannan while requiring relatively less resin.

[0055] In some examples of efficient and rapid concentration methods, the ratio of nonpolar macroporous resin to eluent is 1 g: (1–5) mL. This effectively elutes lipopolysaccharides or lipoarabinomannan.

[0056] In some examples of efficient and rapid concentration methods, the sample to be concentrated is selected from common samples such as blood, urine, cerebrospinal fluid, saliva, or tissue. Tissue samples are liquefied if necessary.

[0057] In some examples of efficient and rapid concentration methods, the sample to be concentrated is urine.

[0058] In some examples of efficient and rapid concentration methods, the non-polar macroporous resin has a particle size of 0.03–0.4 mm and a pore size range of 5–16 nm, and the eluent consists of 10–90 v / v% methanol or ethanol, with the balance being PBS solution.

[0059] These features can be combined arbitrarily as long as they do not conflict with each other.

[0060] A second aspect of the present invention provides: a method for rapidly determining the content of lipopolysaccharide or lipoarabinomannan (LAM), comprising:

[0061] A concentrated solution of lipopolysaccharide or lipoarabinomannan (LAM) is obtained by the efficient and rapid concentration method described in the first aspect of the present invention;

[0062] Take a concentrated solution of lipopolysaccharide or lipoarabinomannan (LAM) and determine its content by testing.

[0063] In some instances of this method, the sample to be concentrated is urine, and the LAM content is measured.

[0064] A third aspect of the present invention provides: an apparatus for implementing the efficient and rapid concentration method described in the first aspect of the present invention, comprising:

[0065] A chromatography tube includes an upper inlet and a lower outlet. An upper sieve plate and a lower sieve plate are provided between the inlet and the outlet, and the space between the upper sieve plate and the lower sieve plate is used to fill adsorption particles.

[0066] The mounting base includes an upper cover plate and a base, wherein the upper cover plate is provided with a plurality of placement holes;

[0067] A collector for collecting the effluent from the outlet.

[0068] In some examples of the apparatus, an inner filter tube is also included, which is placed inside the chromatography tube through the through-hole.

[0069] In some examples of the apparatus, there is a gap between the outer wall of the filter inner tube and the inner wall of the chromatography through tube.

[0070] In some examples of the device, the upper end of the filter inner tube is provided with a tube beam section for easy gripping.

[0071] In some examples of the apparatus, the inner filter tube is placed on the chromatography tube via the lower end face of the tube beam, and the portion of the inner filter tube at the lower end face of the tube beam is inserted into the chromatography tube.

[0072] In some examples of the device, the head opening of the tube beam gradually narrows downwards.

[0073] In some examples of the device, the lower end face of the tube beam is provided with several support columns.

[0074] In some examples of the device, the base is provided with a waste liquid tank.

[0075] In some examples of the device, at least a portion of the placement hole is provided with a limiting groove.

[0076] In some examples of the apparatus, the placement holes are divided into two rows, with the rear row housing the chromatography tubes and the front row housing the collectors, and the waste liquid tank is located below the rear row of placement holes.

[0077] In some examples of the apparatus, the diameter of the chromatography tube gradually decreases near the outlet.

[0078] In some examples of the devices, the adsorbed particles are macroporous resins.

[0079] In some examples of the apparatus, the macroporous resin has a particle size of 0.03 to 0.4 mm.

[0080] In some examples of the apparatus, the pore size of the macroporous resin ranges from 5 to 16 nm.

[0081] In some examples of the apparatus, the macroporous resin is a nonpolar resin or a modified nonpolar resin.

[0082] In some examples of the apparatus, the nonpolar resin is prepared from a polymeric monomer, a crosslinking agent, a porogen, an initiator, and a stabilizer. The polymeric monomer is selected from at least one of styrene, α-methylstyrene, methyl methacrylate, and propionitrile. The crosslinking agent is divinylbenzene, the porogen is toluene, a fatty alcohol, or a fatty acid, and the stabilizer is gelatin.

[0083] In some examples of the apparatus, the nonpolar macroporous resin is selected from at least one of D101, H103, HPD100, ADS-8, X-5, D101-I, D50, D700, D910 and D4020.

[0084] These features can be combined arbitrarily as long as they do not conflict with each other.

[0085] A fourth aspect of the invention provides a rapid test kit for lipoarabinomannan, comprising the apparatus described in the third aspect of the invention. Beneficial effects

[0086] The inventors discovered that nonpolar macroporous resins with a specific pore size range (5–50 nm) exhibit similar affinity adsorption efficiency for lipoarabinomannan, with a large adsorption capacity and fast adsorption rate. When combined with an eluent of a specific composition, lipoarabinomannan can be eluted efficiently, yielding concentrated lipoarabinomannan samples for testing, thus creating favorable conditions for the detection of lipoarabinomannan.

[0087] The present invention provides a highly efficient and rapid method for concentrating arabinomannan in some examples. By using a non-polar macroporous resin with a specific particle size, the adsorption rate can be significantly improved.

[0088] The present invention provides a highly efficient and rapid concentration method for lipoarabinomannan in several examples, which is applicable to the processing of various samples and provides the possibility for rapid detection of lipoarabinomannan.

[0089] The apparatus and method described in some examples of the present invention can effectively realize the efficient and rapid concentration method of lipoarabinomannan in some examples of the present invention. Attached Figure Description

[0090] Figure 1 is a schematic diagram of the overall structure of a concentration device according to some examples of the present invention.

[0091] Figure 2 is a schematic diagram of the overall structure of the concentration device of some examples of the present invention, including a cross-section.

[0092] Figure 3 is a cross-sectional structural diagram of the combination of the chromatography tube and the filter inner tube in some examples of the present invention.

[0093] Figure 4 is a schematic cross-sectional view of the chromatography tube in some examples of the present invention.

[0094] Figure 5 is a schematic diagram of the structure of some examples of the collector of the present invention.

[0095] Figure label:

[0096] Chromatography tube-1, tube inlet-11, outlet-12, upper sieve plate-13, lower sieve plate-14

[0097] Fixing base-2, upper cover plate-21, base-22, placement hole-23, waste liquid tank-24, limiting groove-25

[0098] Collector-3, tube body-31, tube cap-32, connector-33

[0099] Filter inner tube-4, tube beam section-41.

[0100] Figure 6 shows the standard curves of LAM samples at different concentrations before and after concentration. Embodiments of the present invention

[0101] Referring to Figures 1 to 5, an apparatus for implementing the efficient and rapid concentration method described in the first aspect of the present invention includes:

[0102] The chromatography tube 1 includes an upper inlet 11 and a lower outlet 12. An upper sieve plate 13 and a lower sieve plate 14 are provided between the inlet 11 and the outlet 12. The space between the upper sieve plate 13 and the lower sieve plate 14 is used to fill adsorption particles.

[0103] The fixing base 2 includes an upper cover plate 21 and a base 22, wherein the upper cover plate 21 is provided with a plurality of placement holes 23;

[0104] Collector 3 is used to collect the effluent from the outlet 12.

[0105] When using it, add the sample solution to the chromatography tube, and allow it to be adsorbed and eluted to obtain a concentrated solution.

[0106] The upper sieve plate 13 and the lower sieve plate 14 can prevent adsorbed particles from falling off.

[0107] In some examples of the apparatus, an inner filter tube 4, an upper sieve plate 13, and a lower sieve plate 14 are also included. The inner filter tube 4 is placed inside the chromatography tube 1 through the through-port 11.

[0108] In some examples of the apparatus, there is a gap between the outer wall of the inner filter tube 4 and the inner wall of the chromatography tube 1. The presence of this gap facilitates the flow of the filtered sample solution into the chromatography tube 1.

[0109] When using, add the sample solution to be concentrated into the filter tube for preliminary filtration to prevent impurities in the sample solution from clogging the chromatography tube. Then, add the sample solution further into the chromatography tube for adsorption.

[0110] In some examples of the device, the upper end of the inner filter tube 4 is provided with a tube beam 41 for easy gripping.

[0111] In some examples of the apparatus, the inner filter tube 4 is placed on the chromatography tube 1 via the lower end face of the tube beam portion 41, and the portion of the inner filter tube 4 at the lower end face of the tube beam portion 41 is inserted into the chromatography tube 1.

[0112] In some embodiments of the device, the head opening of the tube section 41 gradually tapers downwards. This facilitates the addition of liquid.

[0113] In some examples of the apparatus, the lower end face of the tube beam 41 is provided with several support columns. This creates a gap between the inner filter tube 4 and the chromatography through tube 1, facilitating gravity chromatography.

[0114] In some examples of the device, the base 22 is provided with a waste liquid tank 24.

[0115] In some embodiments of the device, at least part of the placement hole 23 is provided with a limiting groove 25. This better restricts the movement of the collector 3 and facilitates operation.

[0116] In some examples of the device, the placement holes 23 are divided into two rows, with the chromatography tube 1 placed in the rear row and the collector 3 placed in the front row. The waste liquid tank 24 is located below the rear row of placement holes 23.

[0117] In some examples of the apparatus, the diameter of the chromatography tube 1 gradually narrows near the outlet 12. This allows for the maximum possible drainage of the liquid.

[0118] In some examples of the devices, the filling adsorbent particles are macroporous resins.

[0119] In some examples of the apparatus, the macroporous resin has a particle size of 0.03 to 0.4 mm.

[0120] In some examples of the apparatus, the pore size of the macroporous resin ranges from 5 to 16 nm.

[0121] In some examples of the apparatus, the macroporous resin is a nonpolar resin or a modified nonpolar resin.

[0122] In some examples of the apparatus, the nonpolar resin is prepared from a polymeric monomer, a crosslinking agent, a porogen, an initiator, and a stabilizer, wherein the polymeric monomer is styrene, the crosslinking agent is divinylbenzene, the porogen is toluene, a fatty alcohol, or a fatty acid, and the stabilizer is gelatin.

[0123] In some examples of the apparatus, the nonpolar macroporous resin is selected from at least one of D101, H103, HPD100, ADS-8, X-5, D101-I, D50, D700, D910 and D4020.

[0124] In some examples of devices, different macroporous resins can be used either alone or in combination.

[0125] In some examples of the apparatus, the diameter-to-height ratio of the filled macroporous resin is 1:1 to 5:1, preferably 1:1 to 3:1.

[0126] These features can be combined arbitrarily as long as they do not conflict with each other. Specific examples of concentration devices:

[0127] A concentration apparatus, comprising:

[0128] The chromatography tube 1 includes an upper inlet 11 and a lower outlet 12, and the diameter of the chromatography tube 1 gradually decreases at the end near the outlet 12.

[0129] An upper sieve plate 13 and a lower sieve plate 14 are provided between the inlet 11 and the outlet 12. The space between the upper sieve plate 13 and the lower sieve plate 14 is filled with macroporous resin. The particle size of the macroporous resin is 0.03 to 1.2 mm and the pore size range is 5 to 50 nm.

[0130] The filter inner tube 4 is placed inside the chromatography tube 1 through the through-hole 11 at its lower part. There is a gap between the outer wall of the lower part of the filter inner tube 4 and the inner wall of the chromatography tube 1. The filter inner tube 4 has an upper sieve plate 13 and a lower sieve plate 14. The upper end of the filter inner tube 4 is provided with a tube beam part 41 that is easy to hold. The head opening of the tube beam part 41 gradually narrows downward. The lower end face of the tube beam part 41 is provided with several support columns. After the filter inner tube 4 is inserted into the chromatography tube 1, the support columns contact the upper tube wall of the chromatography tube 1.

[0131] The fixed base 2 includes an upper cover plate 21 and a base 22. The upper cover plate 21 is provided with a plurality of placement holes 23, which are divided into two rows, front and back. The rear row is used to place the chromatography tube 1, and a waste liquid tank 24 is provided on the base below the rear row of placement holes 23. The front row of placement holes 23 is used to place the collector 3, and the front row of placement holes 23 is also provided with a limiting groove 25.

[0132] Collector 3, specifically a reagent tube, includes a cap 32 and a tube body 31, with a connecting part 33 between the cap 32 and the tube body 31, used to collect the outflow liquid from the outlet 12.

[0133] When using, first insert the filter inner tube into the chromatography tube, add the sample and then discharge the waste liquid to allow LPS or LAM to be adsorbed onto the macroporous resin. Then elute and collect the eluent using a collector for later use.

[0134] The technical solution of the present invention will be further illustrated below with examples.

[0135] Pretreatment of adsorption resin:

[0136] The purchased commercial non-polar macroporous resin was dried to remove moisture, then crushed and sieved to obtain resin powder with different particle size ranges.

[0137] Before use, clean the resin powder with an ethanol solution or other solution to remove unpolymerized monomers, porogens, dispersants, preservatives, and other organic residues remaining in the resin pores, thereby improving resin cleanliness and safety. After cleaning, dilute the adsorption resin with a 75% ethanol aqueous solution to 0.3 g / ml for later use.

[0138] Adsorption rate = (AB) / A × 100%

[0139] Elution rate = C / (AB) × 100%

[0140] In the formula, A is the total LAM content in the sample to be concentrated, B is the total residual LAM in the effluent after the sample is concentrated, and C is the total LAM content in the eluent. The total content can be obtained by multiplying the concentration of LAM in the liquid by the volume of the liquid.

[0141] Recovery rate = Adsorption rate × Elution rate × 100%.

[0142] Effective concentration factor = concentration of lipopolysaccharide or lipoarabinomannan after concentration / concentration of lipopolysaccharide or lipoarabinomannan in the sample to be concentrated.

[0143] Example 1: The effect of different macroporous resins on LAM adsorption

[0144] S1) Place the chromatography tube on the fixed base, insert the lower sieve plate, and press the lower sieve plate firmly and securely.

[0145] S2) Place the pretreated adsorption resin on a magnetic stirrer and stir. While stirring, add 0.5 ml of adsorption resin powder (0.3 g / ml, particle size 0.03-1.2 mm) into the chromatography tube, place the upper sieve plate horizontally and press it down. The adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1.

[0146] S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until the addition is complete;

[0147] S4) Add 5 ml of LAM solution with a concentration of 100 pg / mL as the sample to be concentrated, and let it stand until the dripping is complete;

[0148] S5) Add elution buffer (15% ethanol / PBS) to the chromatography column twice, 0.4 ml for the first time and 0.2 ml for the second time. Let it stand until the elution is complete, and collect the concentrated sample after the second wash.

[0149] The collected samples were tested using a lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Red Biotechnology Co., Ltd. The test results are shown in Table 1.

[0150] Table 1. Effect of different types of macroporous resins on LAM adsorption

[0151] 0.15g of different resin powders + 10ml of lipoarabinomannan solution with a concentration of 100 pg / mL were concentrated by gravity according to steps S1) to S5) above and then eluted. The lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Red Biotechnology Co., Ltd. was used for detection. The detection results are shown in Table 2.

[0152] Table 2. Effect of different types of macroporous resins on LAM adsorption

[0153] Group Resin Model Luminescence Value LAM Concentration Value (pg / mL) Total Adsorption Rate Elution Rate Recovery Rate Effective Concentration Factor Before Concentration / 20 41 61 00 / / / / Elution 1 ADS-8 76 85 20 38 82 90% 86% 77% 39 Elution 2 D 50 74 270 83 75 190% 83% 75% 38 Elution 3 D 10 175 66 20 38 22 92% 83% 76% 38 Elution 4 D 70 0 64 78 94 32 72 79% 83% 66% 33 Elution 5 D 91 0 66 84 69 33 76 79% 86% 68% 34 Elution 6 D 40 20 74 80 55 37 78 93% 81% 75% 38

[0154] Conclusion: After being processed into powder, D50, D101, D4020 and ADS-8 have comparable adsorption rates and concentration effects, while D700 and D910 have slightly lower adsorption rates.

[0155] Comparison of adsorption effects of macroporous adsorption resins with different polarities and pore sizes on LAM

[0156] S1) Place the chromatography tube on the fixed base, insert the lower sieve plate, and press the lower sieve plate firmly and securely.

[0157] S2) Place the pretreated adsorption resin on a magnetic stirrer and stir. While stirring, add 0.3g of different types of adsorption resin into the chromatography tube, place it horizontally on the upper sieve plate and press it down.

[0158] S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until the addition is complete;

[0159] S4) Add 10 ml of LAM solution with a concentration of 100 pg / mL as the sample to be concentrated, let it stand until the dripping is complete, detect the concentration of residual LAM in the supernatant of the effluent, and calculate the adsorption rate.

[0160] The lipoarabinomannan (LAM) assay kit (chemiluminescence method) produced by Guangzhou Red Biotechnology Co., Ltd. was used for detection, and the results are shown in Table 3.

[0161] Table 3. Comparison of adsorption effects of macroporous adsorption resins with different polarities and pore sizes on LAM

[0162] Resin Polarity Average Pore Size (nm) Supernatant LAM Concentration (pg / mL) Adsorption Rate D101 Nonpolar 9~10 21.79% HPD100 Nonpolar 8.5~9.0 10.90% ADS-8 Nonpolar 12~16 8.92% D50 Nonpolar 5.0~7.5 22.78% D700 Nonpolar 3.0~5.0 41.54% XAD1180N Nonpolar 30~40 27.73% XAD7HP Nonpolar 40~50 30.70% X-5 Nonpolar 2.9~3.0 56.44% AB-8 Weakly Polar 13~14 35.65% DM130 Medium Polar 9~10 60.40% HPD600 Polar 86.535%

[0163] Conclusion: The above experimental data show that the adsorption effect of non-polar resins is better than that of weakly polar, moderately polar and polar resins. Among the non-polar resins, those with pore sizes of 5-50 nm have higher adsorption rates, and those with pore sizes of 5-16 nm have even better adsorption rates.

[0164] Adsorption effect of nonpolar resins on LPS or LAM:

[0165] S1) Place the chromatography tube on the fixed base, insert the lower sieve plate, and press the lower sieve plate firmly and securely.

[0166] S2) Place the pretreated adsorption resin on a magnetic stirrer and stir. While stirring, add 1 ml of adsorption resin (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) into the chromatography tube, place the upper sieve plate horizontally and press it down. The adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1.

[0167] S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until the addition is complete;

[0168] S4) Add 10 ml of LAM standard solution and endotoxin standard solution as samples to be concentrated, and let stand until the dripping is complete;

[0169] S5) Detect the concentration of residual LAM and endotoxin in the supernatant after adsorption, and calculate the adsorption rate.

[0170] Endotoxin concentration was detected using the Limulus Amebocyte Lysate (LIL) endotoxin detection kit (microplate quantitative chromogenic matrix method).

[0171] The concentration of LAM was detected using a lipoarabinomannan (LAM) detection kit (chemiluminescence method) manufactured by Guangzhou Rede Biotechnology Co., Ltd. The results are shown in Table 4.

[0172] Table 4

[0173] Conclusion: The above experimental results show that nonpolar resins can effectively adsorb lipopolysaccharides or lipoarabinomannan, and the adsorption rate is high.

[0174] Experiments with different resin dosages and sample volume ratios:

[0175] Different amounts of adsorption resin (0.3 g / ml, particle size 0.03–0.4 mm, ADS-8) were added to chromatography tubes, followed by 10 ml of 10 pg / mL LAM solution. After incubation at 30 rpm, the supernatant and resin were separated. The supernatant was centrifuged at 1500 g for 5 min and then detected using a lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Rede Biotechnology Co., Ltd. The results are shown in Table 5.

[0176] Table 5

[0177] Conclusion: Increasing the amount of resin and extending the adsorption time can improve the adsorption rate, but the increase in adsorption rate is slow as the amount of resin increases. Therefore, the preferred ratio of resin amount to sample volume is 0.1:10 to 0.5:10.

[0178] Example 2: Comparison of the elution effects of different eluents on lipid LAM

[0179] Comparison of LAM adsorption rates of the same resin with different particle sizes (mixing method: 0.3g resin + 10ml lipoarabinomannan solution with a concentration of 5 pg / mL, incubated at 30rpm for 30min, then the supernatant and resin were separated, the supernatant was centrifuged at 1500g for 5min and then detected using the lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Rede Biotechnology Co., Ltd. The results are shown in Table 6.

[0180] Table 6

[0181] The results showed that the smaller the resin particle size, the higher the adsorption rate and the shorter the adsorption time, with 0.03 to 0.4 mm being the preferred size.

[0182] The effect of resin particle size on the adsorption of arabinomannan:

[0183] Experiment 1:

[0184] S1) Place the chromatography tube on the fixed base, insert the lower sieve plate, and press the lower sieve plate firmly and securely.

[0185] S2) Place the pretreated adsorption resin on a magnetic stirrer and stir. While stirring, add 0.5 ml of adsorption resin powder (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) into the chromatography tube, place the upper sieve plate horizontally and press it down. The adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1.

[0186] S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until the addition is complete;

[0187] S4) Add 5 ml of LAM solution with a concentration of 100 pg / mL as the sample to be concentrated, and let it stand until the dripping is complete;

[0188] S5) Add 0.2 ml of elution buffer with different compositions to the chromatography column, let it stand until the eluent is completely added, and collect the concentrated sample.

[0189] The collected samples were analyzed using a lipoarabinomannan (LAM) detection kit (chemiluminescence method) manufactured by Guangzhou Rede Biotechnology Co., Ltd.

[0190] Repeat step S5, take the elution fraction with higher concentration, and the test results are shown in Table 7.

[0191] Table 7

[0192] The results showed that different eluents had significant differences in their elution effects on depolysaccharides. Eluents containing ethanol could effectively elute LAM adsorbed by the adsorption resin, while 0.5% Tween-20 / PBS, 0.2M glycine-hydrochloric acid (pH 1.5), and 0.2M NaOH (pH 12) could not effectively elute the LAM.

[0193] Experiment 2:

[0194] S1) Place the chromatography tube on the fixed base, insert the lower sieve plate, and press the lower sieve plate firmly and securely.

[0195] S2) Place the pretreated adsorption resin on a magnetic stirrer and stir. While stirring, add 0.5 ml of adsorption resin (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) into the chromatography tube, place the upper sieve plate horizontally and press it down. The adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1.

[0196] S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until the addition is complete;

[0197] S4) Add 10 ml of LAM solution with a concentration of 100 pg / mL as the sample to be concentrated, let it stand until the dripping is complete, detect the concentration of residual LAM in the supernatant of the effluent, and calculate the adsorption rate;

[0198] S5) Add elution buffer with different compositions to the chromatography column twice, adding 0.4 ml the first time and 0.2 ml the second time. Let it stand until the elution is complete, and collect the concentrated sample after the second wash.

[0199] The collected samples were analyzed using a lipoarabinomannan (LAM) assay kit (chemiluminescence method) produced by Guangzhou Rede Biotechnology Co., Ltd. The elution volume was obtained after elution, and the results are shown in Table 8. Samples eluted with ethanol or methanol concentrations exceeding 20% ​​required dilution before analysis.

[0200] Table 8

[0201] Note: * indicates the luminescence value of a 100 pg / mL LAM solution.

[0202] The results in the table show that methanol, ethanol, and ethyl acetate have good elution efficiency for LAM.

[0203] Example 3: Effects of different ethanol content eluents and elution cycles on elution efficiency

[0204] S1) Place the chromatography tube on the fixed base, insert the lower sieve plate, and press the lower sieve plate firmly and securely.

[0205] S2) Place the pretreated adsorption resin on a magnetic stirrer and stir. While stirring, add 0.5 ml of adsorption resin powder (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) into the chromatography tube, place the upper sieve plate horizontally and press it down. The adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1.

[0206] S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until the addition is complete;

[0207] S4) Add 5 ml of LAM solution with a concentration of 100 pg / mL as the sample to be concentrated, and let it stand until the dripping is complete;

[0208] S5) Add 0.1 ml of eluent to the chromatography column multiple times; let it stand until the eluent is completely added, and then collect the concentrated samples.

[0209] The eluents collected from the 3rd to the 6th collections were analyzed using a lipoarabinomannan (LAM) detection kit (immunochromatography) manufactured by Guangzhou Rede Biotechnology Co., Ltd. For ease of comparison, the total LAM elution volume from the 4th to the 7th collections was considered 100%. The results are shown in Table 9.

[0210] Table 9

[0211] Note: T value is the detected value, C value is the quality control value, and T / C value is the relative concentration of LAM in the eluent.

[0212] The experimental results show that eluents with different ethanol concentrations have different elution effects. Overall, the amount of LAM eluted was lower in the first three elutions, which may be related to the ethanol concentration in the eluent. The preferred ethanol concentration in the eluent is 15%; if the ethanol concentration is reduced, the elution peak shifts later, the elution rate decreases, the operation time is prolonged, and the operation cost increases. Therefore, the ethanol concentration in the eluent should not be lower than 15%.

[0213] Example 4: Effect of Resin Dosage on LAM Concentration

[0214] S1) Place the chromatography tube on the fixed base, insert the lower sieve plate, and press the lower sieve plate firmly and securely.

[0215] S2) Place the pretreated adsorption resin on a magnetic stirrer and stir. While stirring, add different amounts of adsorption resin powder (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) into the chromatography tube, place the upper sieve plate horizontally and press it down. The adsorption resin forms the chromatography column.

[0216] S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until the addition is complete;

[0217] S4) Add 5 ml of LAM solution with a concentration of 100 pg / mL as the sample to be concentrated, and let it stand until the dripping is complete;

[0218] S5) Add elution buffer to the chromatography column, 100 μL each time, and elute multiple times. After each elution, allow the column to stand until the eluent has completely dissolved, and then collect the concentrated sample.

[0219] The collected samples were tested using a lipoarabinomannan (LAM) detection kit (immunochromatographic method) produced by Guangzhou Red Biotechnology Co., Ltd. The test results are shown in Table 10.

[0220] Table 10

[0221] As shown in Table 10, the optimal ratio of resin dosage to sample volume is 0.15–0.3 g: 5 ml, with 0.15: 5 ml being the best. Further increasing the resin dosage will cause the elution peak to shift significantly to the later stage, thus increasing the concentration and elution time.

[0222] Example 5: Effect of column diameter-to-height ratio on the concentration of arabinomannan

[0223] S1) Place the chromatography tube on the fixed base, insert the lower sieve plate, and press the lower sieve plate firmly and securely.

[0224] S2) Place the pretreated adsorption resin on a magnetic stirrer and stir. While stirring, add 0.15 g of adsorption resin powder (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) into the chromatography tube, place the upper sieve plate horizontally and press it down to prepare chromatography columns with different diameter-to-height ratios.

[0225] S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until the addition is complete;

[0226] S4) Add 5 ml of LAM sample with a concentration of 100 pg / mL to the specific chromatography column, let it stand until the drip is complete, record the drip time, collect the eluent, detect the amount of residual LAM, and determine the adsorption rate.

[0227] S5) Add elution buffer (15% ethanol / PBS) to the chromatography column twice, adding 0.4 ml the first time and 0.2 ml the second time. Let it stand until the elution is complete, and collect the concentrated sample obtained after the second wash for detection to determine the elution rate.

[0228] The collected samples were tested using a lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Red Biotechnology Co., Ltd., and the results are shown in Table 11.

[0229] Table 11

[0230] As shown in Table 11, there are significant differences in the time required to complete the sample drop when the diameter-to-height ratio (diameter:height) is 1:1 to 3:1. The time is significantly shorter and the adsorption rate is higher.

[0231] Example 6: Establishing the LAM Standard Curve

[0232] S1) Place the chromatography tube on the fixed base, insert the lower sieve plate, and press the lower sieve plate firmly and securely.

[0233] S2) Place the pretreated adsorption resin on a magnetic stirrer and stir. While stirring, add 0.5 ml of adsorption resin powder (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) into the chromatography tube, place the upper sieve plate horizontally and press it down. The adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1.

[0234] S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until the addition is complete;

[0235] S4) Add 5 ml of samples with different LAM concentrations as the samples to be concentrated, and let stand until the dripping is complete;

[0236] S5) Add 0.3 mL of elution buffer (PBS solution of 15 v / v ethanol) to the chromatography column and let it stand until the elution is complete;

[0237] S6) Add 0.2 ml of elution buffer to the chromatography column, let it stand until the elution is complete, and collect the concentrated sample.

[0238] The collected samples were tested using a lipoarabinomannan (LAM) detection kit (immunochromatographic method) produced by Guangzhou Red Biotechnology Co., Ltd. The test results are shown in Table 12 and Figure 6.

[0239] Table 12

[0240]

[0241] The experimental results show that the concentration method greatly improves the detection sensitivity, and the standard curve has excellent linearity with a correlation coefficient of 0.9986.

[0242] Example 7: Detection of experimental urine samples

[0243] Sixteen positive samples and four negative samples from clinical samples were validated, and the specific steps are as follows:

[0244] S1) Place the chromatography tube on the fixed base, insert the lower sieve plate, and press the lower sieve plate firmly and securely.

[0245] S2) Place the pretreated adsorption resin on a magnetic stirrer and stir. While stirring, add 0.15 g of adsorption resin powder (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) into the chromatography tube, place the upper sieve plate horizontally and press it down to prepare chromatography columns with different diameter-to-height ratios.

[0246] S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until the addition is complete;

[0247] S4) Add 5 ml of urine sample to the chromatography column, let it stand until the drip is complete, record the drip time, collect the eluent, and detect the amount of residual LAM to determine the adsorption rate;

[0248] S5) Add elution buffer (15% ethanol / PBS) to the chromatography column twice, 0.4 ml for the first time and 0.2 ml for the second time. Let it stand until the elution is complete, and collect the concentrated sample obtained after the second wash for detection.

[0249] The collected samples were tested using a lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Red Biotechnology Co., Ltd., and the results are shown in Table 13.

[0250] Table 13

[0251] Sample No. Luminescence Value Before Concentration Luminescence Value After Concentration Sample No. Luminescence Value Before Concentration Luminescence Value After Concentration TB1 785 2458 TB1 11 12 237 079 TB2 797 2632 TB1 21 2298 261 TB3 847 4417 TB1 31 285 7867 TB4 876 2146 TB1 41 30 210 123 TB5 895 2048 TB1 51 340 8266 TB6 898 5593 TB1 61 35 215 237 TB7 96 12 470 N0 19 12 1404 TB8 10 135 370 N0 290 51 682 TB9 10 26 25 25 N0 385 81 142 TB1 10 10 22 601 N0 487 61 048

[0252] The experimental results show that the concentration method greatly improves the detection sensitivity. Samples that were not detected before concentration can be detected as positive after concentration, and negative samples are not affected in their detection results. The results indicate that this method can improve detection sensitivity and ensure detection accuracy.

[0253] Comparative Example 1: Direct adsorption of Mycobacterium tuberculosis by macroporous resin

[0254] Referring to Embodiment 4 of Chinese Patent Application CN110129241A, specifically:

[0255] (1) Use 95% alcohol to repeatedly clean and remove dust from the surface of the macroporous resin, so that the surface of the macroporous resin is clean and dry.

[0256] (2) Add 10 ml of bacterial solution containing Mycobacterium tuberculosis to a beaker, then add 2 g of macroporous resin D301 treated in step (1) to the beaker, mix thoroughly and let stand for 24 h for adsorption.

[0257] (3) After sufficient adsorption, the adsorbed bacterial solution is discharged through the outlet of the beaker. After the bacterial solution is fully discharged, the outlet of the beaker is closed, and the eluent is added to the beaker. After the macroporous resin and the eluent are fully mixed, the eluent is discharged, and the content of Mycobacterium tuberculosis is detected in the eluent.

[0258] The bacterial culture used was 10 ml of pleural effusion sample containing Mycobacterium tuberculosis. The Mycobacterium tuberculosis content in the sample was adjusted to 10^7 CFU / mL using PBS solution. The elution buffer was PBS containing 15 v / v% ethanol. The Mycobacterium tuberculosis content was determined by solid culture method.

[0259] The above operations were set up in 3 experimental groups, and the test results are shown in Table 14.

[0260] Table 14

[0261] Group comparison 1, comparison 2, comparison 3: Test results: No obvious bacterial species detected.

[0262] Experimental results show that the adsorption effect of Mycobacterium tuberculosis in Comparative Example 1 is not ideal. Compared with Experiment 1 in Example 2 of this invention, the amount of resin required to concentrate 10 ml of sample solution in this invention is only 0.15 g, and the adsorption time is much less than 24 h. The time in Experiment 1 is only about 30 minutes. Therefore, in comparison, this invention has an advantage in concentrating large sample volumes.

[0263] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

A highly efficient and rapid method for concentrating lipopolysaccharides or lipoarabinomannan includes the following steps: S1) A non-polar macroporous resin is packed into a chromatography column, and the sample to be concentrated is loaded onto the chromatography column so that lipopolysaccharide or lipoarabinomannan is adsorbed onto the resin. S2) After adsorption is complete, drain the liquid; S3) Add elution buffer, elute and collect to obtain lipopolysaccharide or lipoarabinomannan concentrate; in: The macroporous resin has a particle size of 0.03–1.2 mm and a pore size range of 5–50 nm. The eluent is selected from at least one of the following: aqueous ethanol solution, aqueous methanol solution, ethyl acetate solution, a mixture of ethanol and buffer solution, methanol, and a mixture of isopropanol and buffer solution. According to the efficient and rapid concentration method of claim 1, the non-polar resin is prepared from a polymeric monomer, a crosslinking agent, a pore-forming agent, an initiator, and a stabilizer. The polymeric monomer is selected from at least one of styrene, α-methylstyrene, methyl methacrylate, and propionitrile. The crosslinking agent is divinylbenzene, the pore-forming agent is toluene, a fatty alcohol, or a fatty acid, and the stabilizer is gelatin. And / or the nonpolar macroporous resin is selected from at least one of D910, D4020, D101, H103, HPD100, ADS-8, X-5, D101-I, D50, and D700. The efficient and rapid concentration method according to claim 1 is characterized in that, The total content of methanol and / or ethanol in the eluent is 10-90 v / v. The efficient and rapid concentration method according to claim 1 is characterized in that, The diameter-to-height ratio of the chromatography column is 1:1 to 5:

1. The efficient and rapid concentration method according to claim 1 is characterized in that, Before loading the sample to be concentrated onto the chromatography column, the chromatography column is activated with an activation solution, which is a buffer solution containing 30–95 v / v% ethanol or methanol. The efficient and rapid concentration method according to any one of claims 1 to 5 is characterized in that, It satisfies at least one of the following properties: 1) The mixing ratio of macroporous resin to the sample to be concentrated is 0.1 g: 10 mL to 0.5 g: 10 mL; 2) The ratio of macroporous resin to eluent is 0.1 g to 1 g : (1 to 5) mL; 3) The volume range of the eluent is 50uL to 1ml; 4) The volume of the sample to be concentrated is 0.5ml to 20ml; 5) The macroporous resin is a modified nonpolar resin; 6) The sample to be concentrated is selected from one of serum, urine, cerebrospinal fluid, saliva or tissue homogenate. A method for rapidly determining the content of lipopolysaccharide or lipoarabinomannan, characterized in that, include: S1) Obtain a lipopolysaccharide concentrate or a lipoarabinomannan concentrate using the efficient and rapid concentration method according to any one of claims 1 to 6; S2) Take a concentrated solution of lipopolysaccharide or lipoarabinomannan and test it to determine the content of lipopolysaccharide or lipoarabinomannan. An apparatus for implementing the efficient and rapid concentration method according to any one of claims 1 to 6, characterized in that, include: A chromatography tube includes an upper inlet and a lower outlet. An upper sieve plate and a lower sieve plate are provided between the inlet and the outlet, and the space between the upper sieve plate and the lower sieve plate is used to fill adsorption particles. A mounting base for supporting the chromatography tube includes an upper cover plate and a base, wherein the upper cover plate is provided with several placement holes; A collector for collecting the effluent from the outlet. The apparatus according to claim 8, characterized in that, It also includes an inner filter tube, which is placed inside the chromatography tube through the through-hole. The apparatus according to claim 9 is characterized in that, The upper end of the filter inner tube is provided with a tube beam section that is easy to grip. The apparatus according to any one of claims 8 to 10 is characterized in that, The base is provided with a waste liquid tank; and / or at least some of the placement holes are provided with limiting grooves. A rapid test kit for lipoarabinomannan, characterized in that, Includes the apparatus described in any one of claims 8 to 11.

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