Method and device for efficiently and quickly concentrating LPS (Lipopolysaccharide) or LAM (Lipopolysaccharide)

Abstract

The invention belongs to the field of biological detection, and discloses an efficient and rapid concentration method and device for LPS or LAM. The inventor finds out that the non-polar macroporous resin with a specific aperture range has similar affinity adsorption efficiency to lipopolysaccharide or lipoarabinomannan, the adsorption capacity is large, the adsorption speed is high, the lipopolysaccharide can be efficiently eluted in cooperation with a specific eluent, a concentrated lipopolysaccharide to-be-detected sample is obtained, and the non-polar macroporous resin with the specific aperture range can be used for detecting the lipopolysaccharide or lipoarabinomannan in the lipopolysaccharide or lipoarabinomannan in the lipopolysaccharide or lipoarabinomannan. And a good condition is created for the detection of lipopolysaccharide or lipoarabinomannan. Efficient concentration of LAM is realized by using the method, and excellent conditions are provided for detection of mycobacterium tuberculosis.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular relates to a method and a device for efficiently and quickly concentrating LPS or LAM. Background Art

[0002] In vitro testing, or in vitro diagnosis, refers to a testing method that obtains clinical diagnostic information by testing and analyzing human samples (such as blood, urine, cerebrospinal fluid, saliva, tissue, etc.) outside the human body. In practical applications, the analysis of human samples is to detect the effective target detectable substances (including proteins, DNA / RNA, cells, compounds, etc.) contained in the samples to detect the health status of the human body or whether the human body is sick. However, the content of effective target detectable substances in the samples is often very low. Due to the sensitivity of the detection instrument or the detection reagent card, if the body fluid is directly tested, the effective target detectable substances are often not detected, or the content of the detected effective target detectable substances has a large error, resulting in inaccurate judgment results. Therefore, enriching and concentrating effective target detectable substances from the specimen is a basic and primary task. Enriching and concentrating effective target detectable substances can bring the following advantages: First, it can improve the detection sensitivity. In in vitro detection, the concentration of some target substances may be very low, and direct detection may be difficult to obtain accurate results. By concentrating the sample, the relative content of the target substance can be increased, making it easier to detect, thereby significantly improving the sensitivity of the detection. For example, in the detection of trace biomarkers, concentrated samples can make substances that were originally below the detection limit reach a detectable level, providing a more reliable basis for the early diagnosis of the disease.

[0003] Second, it can reduce the impact of interfering substances. Various interfering substances are often present in in vitro test samples, which may affect the accuracy and specificity of the test. Concentrated samples can reduce the relative proportion of interfering substances to a certain extent and reduce their interference with the test. For example, in blood testing, concentration can reduce the impact of interfering substances such as proteins and salts in plasma, making the target analyte easier to identify and quantify.

[0004] 3. Save testing costs and time. Concentrated samples can reduce the sample volume and reagent dosage required for testing, thereby reducing testing costs. At the same time, for some testing methods that require complex pre-treatment, concentrated samples can simplify the operation steps and shorten the testing time. For example, in nucleic acid testing, concentrated samples can reduce the workload of nucleic acid extraction and purification and improve detection efficiency.

[0005] Fourth, it is convenient for sample storage and transportation. The concentrated sample is smaller in volume and easier to store and transport. This is very beneficial for situations where remote testing is required or samples need to be stored for a long time. For example, in a clinical laboratory, the concentrated sample can be frozen and stored, and then tested when needed, reducing the waste of samples and the risk of repeated collection.

[0006] Macroporous resin is also called fully porous resin. Macroporous resin is prepared by polymerization reaction of polymer monomers and additives such as crosslinking agents, porogens, and dispersants. After the polymer is formed, the porogen is removed, leaving holes of different sizes, shapes, and interconnected in the resin. Therefore, macroporous resin has a high porosity inside in a dry state, and the pore size is large, which can be between 5 and 1000nm.

[0007] Macroporous adsorption resins are mainly made of styrene, α-methylstyrene, methyl methacrylate, propionitrile, etc. as raw materials, with a certain amount of vinylbenzene added as a crosslinking agent, toluene and xylene as porogens. They are mostly spherical particles with a diameter generally between 0.3 and 1.25 mm. They are usually divided into non-polar, weakly polar and medium-polar macroporous adsorption resins, which are crosslinked and polymerized to form a porous skeleton structure. The resin is generally white spherical particles with a particle size of 20 to 60 meshes. It is a type of cross-linked polymer containing ion exchange groups. Its physical and chemical properties are stable, insoluble in acids, alkalis and organic solvents, and not affected by inorganic salts and strong ionic low-molecular compounds. The adsorption of the resin relies on the van der Waals attraction between it and the adsorbed molecules (adsorbates). It works through its huge specific surface area for physical adsorption, so that organic compounds can be separated by elution with a certain solvent according to their adsorption capacity and molecular weight to achieve different purposes such as separation, purification, impurity removal, and concentration.

[0008] Macroporous adsorption resins can be divided into three categories: non-polar, neutral polar and polar according to their polarity and the selected monomer molecular structure.

[0009] (1) Non-polar macroporous adsorption resin Non-polar macroporous adsorption resins are made by polymerizing monomers with very small dipole moments and do not carry any functional groups. The pore surface is highly hydrophobic and can adsorb organic matter in the solution by interacting with the hydrophobic parts of small molecules. They are most suitable for adsorbing non-polar substances in polar solvents and are also called aromatic adsorbents, such as styrene and divinylbenzene polymers.

[0010] (2) Medium polarity macroporous adsorption resin Medium polar macroporous adsorption resin is an adsorption resin containing ester groups, and multifunctional methacrylate is used as a crosslinking agent. Its surface has both hydrophobic and hydrophilic parts. It can adsorb polar substances from polar solvents and polar substances from non-polar solvents. It is also called aliphatic adsorbent, such as polyacrylate polymer.

[0011] (3) Polar macroporous adsorption resin Polar macroporous adsorption resins refer to adsorption resins containing polar functional groups such as amide, cyano, and phenolic hydroxyl groups containing nitrogen, oxygen, and sulfur. They adsorb polar substances such as acrylamide through electrostatic interactions.

[0012] Although the existing technology currently uses a variety of commercial macroporous resins to separate and purify polysaccharides and active parts of natural medicines such as stevioside, flavonoids, alkaloids, flavonoids, ginkgo flavonoids, ginsenosides, tea polyphenols, etc., macroporous resins have not been used for adsorption and concentration of lipopolysaccharides. The separation and concentration effects of macroporous resins are affected by many factors, and the adsorption and elution time is relatively long, which generally cannot meet the needs of rapid detection.

[0013] Lipopolysaccharide is a component of the outer cell wall of Gram-negative bacteria. It is a substance composed of lipids and polysaccharides (glycolipids). Lipopolysaccharide consists of three parts: lipid A, core polysaccharide, and O-antigen. The lipid A part of lipopolysaccharide enters the lipid layer as a component of the outer layer of the lipid bilayer, and the sugar chain part is exposed outside the cell, and then exists in this form on the cell surface of Gram-negative bacteria.

[0014] Lipopolysaccharide (LPS) concentration detection can be applied to: 1. Disease diagnosis: LPS is the main component of bacterial endotoxins and is mainly present in the cell wall of Gram-negative bacteria. Its detection is of great significance for the diagnosis of digestive system diseases, bacterial infectious diseases, fever and other diseases. 2. Pathogenic examination: LPS detection is a powerful supplement to bacterial culture and can be used as the "gold standard" for pathogenic examination of bacterial infectious diseases. 3. Observation of the effect of antibiotic application: By detecting LPS, the effect of antibiotic application can be observed and the treatment effect can be evaluated. 4. Immunological research: LPS is a widely used inducer / stimulator in immunological research, which can cause cascade reactions of immune stimulation and toxic pathophysiological activities of the body, inflammation and immune response mechanism research: LPS can activate immune cells carrying CD14 / TLR4 / MD2 receptor complexes, so it plays an important role in studying inflammation and immune response mechanisms. 5. Animal model establishment: LPS is used to establish animal models related to various diseases, such as acute lung injury, acute pancreatitis, etc., to study the development and treatment of diseases; 6. Drug development: LPS, as a target for the development of anti-Gram-negative drugs, has received increasing attention in recent years because it is an important barrier for bacteria to resist the external environment and an important factor in the inherent drug resistance of Gram-negative bacteria. 7. Biomarkers of infection and inflammation: The presence and concentration of LPS can be used as a biomarker of infection and inflammatory status, which helps clinicians assess the condition and guide treatment.

[0015] Lipoarabinomannan (LAM) mainly exists on the cell wall of mycobacteria (such as Mycobacterium tuberculosis). Mycobacterium tuberculosis is the pathogen that causes tuberculosis. The lipopolysaccharide in its structure is an effective antigen that is currently widely used for the production of diagnostic antibodies. Its main component is lipoarabinomannan (LAM). Efficient and rapid concentration of lipoarabinomannan (LAM) is of great significance for the diagnosis of diseases, especially the detection of Mycobacterium tuberculosis.

[0016] Lipopolysaccharide is generally extracted by referring to the process of polysaccharide, such as phenol-water extraction, trichloroacetic acid method, water extraction and alcohol precipitation method, enzymatic hydrolysis method, etc. CN102898537A discloses a method for purifying lipopolysaccharide, which obtains lipopolysaccharide with high purity after ultrafiltration concentration, hydrophobic chromatography, and ion exchange chromatography purification. The purified lipopolysaccharide is suitable for preparing vaccines. This method is time-consuming and cannot meet the needs of rapid detection. Liu Yuchen. Study on the separation and purification process of inactivated tuberculosis lipopolysaccharide antigen [J]. Zhejiang University of Technology, 2012. The disclosed lipopolysaccharide extraction method is also complicated to operate and cannot meet the needs of rapid detection.

[0017] In the technical solution disclosed in CN101974099A, the structural difference that LAM antigen contains three mannoses while other impurities (LM, PIM, etc.) contain only one mannose is utilized, and the affinity of lectin (such as Lentillum lentil agglutinin) to the three mannose branch structure is much higher than the affinity of 2 mannose and 1 mannose is utilized. Through affinity chromatography technology, lectin is used as an affinity purification reagent to purify LAM antigen.

[0018] Specific steps: Column loading: Load the lectin such as COA-Sepharose 4B or LCA-Sepharose 4B into a spin column and equilibrate with an appropriate buffer.

[0019] Loading: Add the sample to the column, centrifuge after a certain period of time, and collect the column liquid.

[0020] Wash: Wash the column with the same buffer and collect the wash solution.

[0021] Dissociation: Add buffer containing methyl glucoside for dissociation and collect the dissociation solution.

[0022] Electrophoretic separation and analysis: Separate the purified product using SDS-PAGE electrophoresis and analyze it using silver staining or other staining methods.

[0023] There are also methods for purifying LAM by centrifugation or ultrasound, which have many steps and complex processes. For example, the sonication method includes pretreatment: placing the LAM sample in an appropriate buffer to maintain its stability and activity.

[0024] Ultrasonic disruption: Use an ultrasonic disruptor to disrupt the sample. Collect the disrupted product: After ultrasonication, remove unbroken cell debris and other large particles by low-speed centrifugation. Post-ultrasonication treatment: Further centrifugation steps may be required to collect small particles and LAM after ultrasonic disruption.

[0025] The steps for centrifugation purification of LAM include: Differential centrifugation: First, use low-speed centrifugation to remove large particles, and then use high-speed centrifugation to separate smaller particles. Density gradient centrifugation: Pre-place media of different densities in the centrifuge tube to form a density gradient. During the centrifugation process, the particles will move in the gradient according to the density until a zone is formed at the position with equal density. Collecting LAM: After the centrifugation is completed, LAM can be collected from a specific position in the gradient according to the density characteristics of LAM. Washing and resuspension: The collected LAM may need to be washed and resuspended with an appropriate buffer to remove residual impurities.

[0026] LAM is also separated and purified by 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.).

[0027] In summary, the existing methods for separation and purification of lipopolysaccharide or LAM are relatively complex and costly, and are difficult to apply to the concentration and purification of lipopolysaccharide or lipoarabinomannan (LAM) or the detection of Mycobacterium tuberculosis. Summary of the invention

[0028] The object of the present invention is to overcome at least one disadvantage of the prior art and to provide a method for efficiently and quickly concentrating lipopolysaccharide or lipoarabinomannan (LAM).

[0029] The technical solution adopted by the present invention is: The first aspect of the present invention provides: A method for efficiently and quickly concentrating lipopolysaccharide or lipoarabinomannan comprises the following steps: The sample to be concentrated is contacted with the macroporous resin and adsorbed; After adsorption is completed, the liquid is discharged; adding an eluent, eluting and collecting to obtain a lipopolysaccharide or lipoarabinomannan concentrate; Wherein: the particle size of the macroporous resin is 0.03-1.2 mm, and the pore size range is 5-50 nm; The eluent is selected from at least one of an ethanol aqueous solution, a methanol aqueous solution, an ethyl acetate solution, a mixture of ethanol and a buffer solution, and a mixture of methanol and a buffer solution.

[0030] In some examples of efficient and rapid concentration methods, the particle size of the macroporous resin is 0.03-0.4 mm.

[0031] In some examples of efficient and rapid concentration methods, the pore size of the macroporous resin ranges from 5 to 16 nm.

[0032] In some examples of the efficient and rapid concentration method, the macroporous resin is a non-polar resin or a modified non-polar resin.

[0033] In some examples of efficient and rapid concentration methods, the non-polar resin is prepared from a polymerized monomer, a cross-linking agent, a porogen, an initiator, and a stabilizer. The polymerized monomer is at least one selected from styrene, а-methylstyrene, methyl methacrylate, and propionitrile. The cross-linking agent is divinylbenzene, the porogen is toluene, a fatty alcohol or a fatty acid, and the stabilizer is gelatin.

[0034] In some examples of the efficient and rapid concentration method, the non-polar macroporous resin is selected from at least one of D101, H103, HPD100, ADS-8, X-5, D101-I, D50, D700, D910 and D4020.

[0035] In some examples of the efficient and rapid concentration method, the total content of methanol and / or ethanol in the eluent is 10-90 v / v%.

[0036] Preferably, the composition of the eluent is: 10-50 v / v%, 10-40 v / v%, 10-35 v / v%, 10-30 v / v%, 10-25 v / v% ethanol, and the remainder is PBS solution.

[0037] In some examples of efficient and rapid concentration methods, the macroporous resin is packed into a chromatography column, and the diameter-to-height ratio (diameter: height) of the chromatography column is 1:1-5:1, preferably 1:1-3:1.

[0038] In some examples of efficient and rapid concentration methods, before loading the sample to be concentrated onto the chromatography column, the chromatography column is activated with an activation solution, wherein the activation solution is composed of a buffer solution containing 30 to 95 v / v% ethanol or methanol.

[0039] In some examples of efficient and rapid concentration methods, during elution, the concentration of lipopolysaccharide or lipoarabinomannan in the eluate of different sections is detected, and the portion with a higher concentration of lipopolysaccharide or lipoarabinomannan is selected for detection.

[0040] In some examples of efficient and rapid concentration methods, the mixing ratio of the macroporous resin to the sample to be concentrated is 0.1 g:10 mL to 0.5 g:10 mL.

[0041] In some examples of efficient and rapid concentration methods, the amount of eluent used ranges from 50uL to 1ml.

[0042] In some examples of efficient and rapid concentration methods, the volume of the sample to be concentrated is 0.5 ml-20 ml.

[0043] In some examples of efficient and rapid concentration methods, the ratio of macroporous resin to eluent is 0.1-1 g: (1-5) mL.

[0044] In some examples of efficient and rapid concentration methods, the sample to be concentrated is selected from blood, urine, cerebrospinal fluid, saliva or tissue.

[0045] In some examples of the efficient and rapid concentration method, the sample to be concentrated is urine.

[0046] In some examples of efficient and rapid concentration methods, the particle size of the non-polar macroporous resin is 0.03-0.4 mm, the pore size range is 5-16 nm, and the composition of the eluent is: 10-90 v / v% methanol or ethanol, and the remainder is PBS solution.

[0047] The above features can be combined arbitrarily unless they conflict with each other.

[0048] The second aspect of the present invention provides: A method for rapidly determining the content of lipopolysaccharide or lipoarabinomannan (LAM), comprising: Obtaining a lipopolysaccharide or lipoarabinomannan (LAM) concentrate according to the efficient and rapid concentration method described in the first aspect of the present invention; Take the lipopolysaccharide or lipoarabinomannan (LAM) concentrate and test to determine the lipopolysaccharide or lipoarabinomannan (LAM) content.

[0049] In some examples of the methods, the sample to be concentrated is urine, and the LAM content is detected.

[0050] The third aspect of the present invention provides: A device for implementing the efficient and rapid concentration method according to the first aspect of the present invention comprises: The chromatography tube comprises a tube opening at the upper end and a liquid outlet at the lower end, an upper sieve plate and a lower sieve plate are arranged between the tube opening and the liquid outlet, and the space between the upper sieve plate and the lower sieve plate is used to fill adsorption particles; The fixing seat comprises an upper cover plate and a base, wherein the upper cover plate is provided with a plurality of placement holes; A collector is used to collect the outflowing liquid from the liquid outlet.

[0051] In some examples of the device, it also includes a filtering inner tube, which is placed in the chromatography through-tube through the through-tube opening.

[0052] In some examples of the device, there is a gap between the outer wall of the inner filtration tube and the inner wall of the chromatography tube.

[0053] In some device examples, the upper end of the inner filter tube is provided with a tube beam portion for easy gripping.

[0054] In some device examples, the inner filter tube is placed on the chromatography tube through the lower end surface of the tube beam portion, and the portion of the inner filter tube at the lower end surface of the tube beam portion is inserted into the chromatography tube.

[0055] In some device examples, the head opening of the tube beam portion gradually narrows downward.

[0056] In some device examples, a plurality of support columns are provided on the lower end surface of the tube beam portion.

[0057] In some device examples, the base is provided with a waste liquid tank.

[0058] In some device examples, at least some of the placement holes are provided with limiting grooves.

[0059] In some device examples, the placement holes are divided into two rows, front and back rows, wherein the rear row is for placing chromatography tubes, the front row is for placing collectors, and the waste liquid tank is arranged below the rear row placement holes.

[0060] In some device examples, the diameter of the chromatography tube at one end close to the liquid outlet gradually shrinks.

[0061] In some embodiments of the device, the adsorbent particles are macroporous resins.

[0062] In some embodiments of the device, the particle size of the macroporous resin is 0.03-0.4 mm.

[0063] In some device examples, the pore size of the macroporous resin ranges from 5 to 16 nm.

[0064] In some device examples, the macroporous resin is a non-polar resin or a modified non-polar resin.

[0065] In some device examples, the non-polar resin is prepared from a polymerizable monomer, a cross-linking agent, a porogen, an initiator, and a stabilizer, the polymerizable monomer being selected from at least one of styrene, а-methylstyrene, methyl methacrylate, and propionitrile, the cross-linking agent being divinylbenzene, the porogen being toluene, a fatty alcohol or a fatty acid, and the stabilizer being gelatin.

[0066] In some device examples, the non-polar macroporous resin is selected from at least one of D101, H103, HPD100, ADS-8, X-5, D101-I, D50, D700, D910 and D4020.

[0067] The above features can be combined arbitrarily unless they conflict with each other.

[0068] A fourth aspect of the present invention provides: A rapid test kit for lipoarabinomannan, comprising the device described in the third aspect of the present invention.

[0069] The beneficial effects of the present invention are: The inventors have found that non-polar macroporous resins in a specific pore size range (5 to 50 nm) have similar affinity adsorption efficiency for lipopolysaccharide or lipoarabinomannan, with a large adsorption capacity and a fast adsorption rate. When used with an eluent of a specific composition, lipopolysaccharide or lipoarabinomannan can be efficiently eluted to obtain concentrated lipopolysaccharide or lipoarabinomannan test samples, creating good conditions for the detection of lipopolysaccharide or lipoarabinomannan.

[0070] In some embodiments of the present invention, the high-efficiency and rapid concentration method of lipopolysaccharide or lipoarabinomannan can significantly increase the adsorption rate by using a non-polar macroporous resin with a specific particle size.

[0071] The efficient and rapid concentration methods of lipopolysaccharide or lipoarabinomannan in some examples of the present invention are suitable for processing a variety of samples, and provide a possibility for rapid detection of lipopolysaccharide or lipoarabinomannan.

[0072] The devices and use methods of some examples of the present invention can well implement the efficient and rapid concentration methods of lipopolysaccharide or lipoarabinomannan of some examples of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Schematic diagram of the overall structure of some example concentration devices of the present invention.

[0074] Figure 2 It is a schematic diagram of the overall structure including cross-section of some example concentration devices of the present invention.

[0075] Figure 3 It is a schematic diagram of the cross-sectional structure of some examples of the chromatography tube and the filtering inner tube when they are matched.

[0076] Figure 4 Schematic diagram of the cross-sectional structure of some examples of chromatography tubes of the present invention.

[0077] Figure 5 Schematic diagram of the structure of some collectors of the present invention.

[0078] Reference numerals: Chromatography tube-1, tube port-11, liquid outlet-12, upper sieve plate-13, lower sieve plate-14 Fixed seat-2, upper cover-21, base-22, placement hole-23, waste liquid tank-24, limit slot-25 Collector-3, tube body-31, tube cover-32, connection part-33 Filter inner tube-4, tube beam part-41.

[0079] Figure 6 This is the standard curve of LAM samples with different concentrations before and after concentration. DETAILED DESCRIPTION

[0080] The first aspect of the present invention provides: A method for efficiently and quickly concentrating lipopolysaccharide or lipoarabinomannan comprises the following steps: The sample to be concentrated is contacted with the macroporous resin and adsorbed; After adsorption is completed, the liquid is discharged; adding an eluent, eluting and collecting to obtain a lipopolysaccharide or lipoarabinomannan concentrate; Wherein: the particle size of the macroporous resin is 0.03-1.2 mm, and the pore size range is 5-50 nm; The eluent is selected from at least one of an ethanol aqueous solution, a methanol aqueous solution, an ethyl acetate solution, a mixture of ethanol and a buffer solution, and a mixture of methanol and a buffer solution.

[0081] Experimental data show that macroporous resins with a pore size range of 5 to 50 nm, especially non-polar macroporous resins, have unexpected adsorption effects on lipopolysaccharide or lipoarabinomannan, which can meet the needs of efficient and rapid concentration of lipopolysaccharide or lipoarabinomannan. Different macroporous resins can be used alone or in combination. Macroporous resins with different particle sizes can be mixed and used.

[0082] In some examples of efficient and rapid concentration methods, the particle size of the macroporous resin is 0.03-0.4 mm. Experimental data show that the finer the particle size of the macroporous resin, the better the adsorption effect on lipopolysaccharide or lipoarabinomannan, but too fine a particle size will result in too long an adsorption time, which is not conducive to rapid concentration.

[0083] In some examples of efficient and rapid concentration methods, the pore size of the macroporous resin is in the range of 5 to 16 nm. Experimental data show that the non-polar macroporous resin with this pore size has a better adsorption effect on lipopolysaccharide or lipoarabinomannan.

[0084] In some examples of efficient and rapid concentration methods, the particle size of the macroporous resin is 0.03-0.4 mm, and the pore size range is 5-16 nm.

[0085] In some examples of the efficient and rapid concentration method, the total content of methanol and / or ethanol in the eluent is 10-90 v / v%.

[0086] There is no restriction on the type of buffer used for the elution solution, as long as it does not affect the subsequent detection. PBS has little effect 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 (Acetate Buffer), carbonate buffer (Carbonates Buffer), citrate buffer (Citrate Buffer), HEPES buffer (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonicacid), MES buffer (2-(N-morpholino)ethanesulfonic acid), MOPS buffer (3-(N-morpholino) propanesulfonic acid), TAE and TBE buffer, Good's buffer, including Tricine, Bicine, EPPS, PIPES, etc., glycine buffer (Glycine Buffer), succinate buffer (Succinate Buffer), EDTA buffer, Tricine buffer.

[0087] In some examples of efficient and rapid concentration methods, the composition of the eluent is: 10-90 v / v% methanol or ethanol, and the balance is PBS solution. This eluent can elute the adsorbed lipopolysaccharide or lipoarabinomannan well. Preferably, the composition of the eluent is: 10-50 v / v%, 10-40 v / v%, 10-35 v / v%, 10-30 v / v%, 10-25 v / v% ethanol, and the balance is PBS solution. Ethanol is safer and has a better elution effect, making it a better choice.

[0088] In some examples of efficient and rapid concentration methods, the macroporous resin is packed into a chromatography column, and the diameter-to-height ratio of the chromatography column is 1:1-5:1, preferably 1:1-3:1. The chromatography column with such a diameter-to-height ratio can adsorb lipopolysaccharide or lipoarabinomannan faster and better.

[0089] In some examples of efficient and rapid concentration methods, before loading the sample to be concentrated onto the chromatography column, the chromatography column is activated with an activation solution, wherein the activation solution is composed of a buffer solution containing 30 to 95 v / v% ethanol or methanol. The activation solution can activate the resin and wet the resin at the same time, thereby preventing the dry resin from adversely affecting the adsorption of lipopolysaccharide or lipoarabinomannan, and solving the problem of resin inactivation during long-term storage. There are no special requirements for the buffer solution in the activation solution, as long as it does not affect the activation of the resin and the subsequent adsorption of LAM or LPS. PBS has no adverse effect on the activation of the resin, nor does it affect the adsorption of LAM or LPS, and is a better choice. Of course, other buffers can also be used, including but not limited to Tris buffer (Tris-HCl), acetate buffer (Acetate Buffer), carbonate buffer (Carbonates Buffer), citrate buffer (Citrate Buffer), HEPES buffer (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid), MES buffer (2-(N-morpholino)ethanesulfonic acid), MOPS buffer (3-(N-morpholino) propanesulfonicacid), TAE and TBE buffers, Good's buffer, including Tricine, Bicine, EPPS, PIPES, etc., glycine buffer (Glycine Buffer), succinate buffer (Succinate Buffer), EDTA buffer, Tricine buffer.

[0090] In some examples of efficient and rapid concentration methods, during elution, the concentration of lipopolysaccharide or lipoarabinomannan in the eluate of different sections is detected, and the part with higher concentration of lipopolysaccharide or lipoarabinomannan is selected for detection. For the convenience of collection, multiple elutions can be performed and the eluates after elution are collected separately. The data show that when the volume mass ratio of eluent: resin (mL: g) is not higher than 2, the amount of eluted lipopolysaccharide or lipoarabinomannan is small. With the further increase of eluent, the elution amount of lipopolysaccharide or lipoarabinomannan has a significant increase. When the volume mass ratio of eluent: resin is 2 to 4, the concentration of lipopolysaccharide or lipoarabinomannan in the eluate is higher. After the volume mass ratio of eluent: resin reaches 5, almost all lipopolysaccharide or lipoarabinomannan has been eluted. If necessary, all eluents can be collected to make it more likely to obtain all lipopolysaccharide or lipoarabinomannan. When the detection needs are met, only the segments with higher lipopolysaccharide or lipoarabinomannan concentrations can be collected.

[0091] In some examples of efficient and rapid concentration methods, the mixing ratio of non-polar macroporous resin to the sample to be concentrated is 0.1 g: 10 mL to 0.5 g: 10 mL, which can basically meet the needs of lipopolysaccharide or lipoarabinomannan concentration and the amount of resin used is relatively less.

[0092] In some examples of efficient and rapid concentration methods, the ratio of non-polar macroporous resin to eluent is 1 g: (1-5) mL, which can effectively elute lipopolysaccharide or lipoarabinomannan.

[0093] 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. The tissue sample is liquefied when necessary.

[0094] In some examples of efficient and rapid concentration methods, the non-polar resin is prepared from a polymerized monomer, a cross-linking agent, a porogen, an initiator, and a stabilizer. The polymerized monomer is at least one selected from styrene, а-methylstyrene, methyl methacrylate, and propionitrile. The cross-linking agent is divinylbenzene, the porogen is toluene, a fatty alcohol or a fatty acid, and the stabilizer is gelatin.

[0095] In some examples of efficient and rapid concentration methods, the non-polar macroporous resin is selected from at least one of D101, H103, HPD100, ADS-8, X-5, D101-I, D50, D700, D910 and D4020. These are mature commercial non-polar macroporous resins.

[0096] In some examples of efficient and rapid concentration methods, the sample to be concentrated is urine, and the rapid concentration of lipoarabinomannan can be better used for the detection of Mycobacterium tuberculosis.

[0097] In some examples of efficient and rapid concentration methods, the particle size of the macroporous resin is 0.03-0.4 mm, the pore size range is 5-16 nm, and the composition of the eluent is: 10-90 v / v% methanol or ethanol, and the remainder is PBS solution.

[0098] The above features can be combined arbitrarily unless they conflict with each other.

[0099] The second aspect of the present invention provides: A method for rapidly determining the content of lipopolysaccharide or lipoarabinomannan, comprising: Obtaining lipopolysaccharide or lipoarabinomannan concentrate according to the efficient and rapid concentration method described in the first aspect of the present invention; Take the lipopolysaccharide or lipoarabinomannan concentrate and test to determine the lipopolysaccharide or lipoarabinomannan content.

[0100] In some examples of the methods, the sample to be concentrated is urine, and the concentration target is lipoarabinomannan.

[0101] The third aspect of the present invention provides: Reference Figure 1 to Figure 5 , a device for implementing the efficient and rapid concentration method according to the first aspect of the present invention, comprising: The chromatography tube 1 comprises a tube opening 11 at the upper end and a liquid outlet 12 at the lower end, an upper sieve plate 13 and a lower sieve plate 14 are provided between the tube opening 11 and the liquid outlet 12, and adsorption particles are filled between the upper sieve plate 13 and the lower sieve plate 14; The fixing seat 2 comprises an upper cover plate 21 and a base 22, wherein the upper cover plate 21 is provided with a plurality of placement holes 23; The collector 3 is used to collect the outflowing liquid from the liquid outlet 12 .

[0102] When in use, add the sample solution into the chromatography tube, adsorb and elute to obtain a concentrated solution.

[0103] The upper sieve plate 13 and the lower sieve plate 14 can prevent the adsorbed particles from falling.

[0104] In some examples of the device, it further includes a filtering inner tube 4 , an upper sieve plate 13 and a lower sieve plate 14 , wherein the filtering inner tube 4 is placed in the chromatography through-tube 1 through the through-tube opening 11 .

[0105] In some device examples, there is a gap between the outer wall of the inner filter tube 4 and the inner wall of the chromatography tube 1. The existence of the gap is more conducive to the filtered sample liquid flowing into the chromatography tube 1.

[0106] When in use, the sample liquid to be concentrated is added to the filter inner tube for preliminary filtration to prevent impurities in the sample liquid from clogging the chromatography tube, and then the sample liquid is further added to the chromatography tube for adsorption.

[0107] In some device examples, the upper end of the filtering inner tube 4 is provided with a tube beam portion 41 for easy gripping.

[0108] In some device examples, the inner filter tube 4 is placed on the chromatography tube 1 through the lower end surface of the tube beam portion 41 , and the inner filter tube 4 is inserted into the chromatography tube 1 at the lower end surface of the tube beam portion 41 .

[0109] In some device examples, the head opening of the tube beam portion 41 gradually contracts downwards, which makes it easier to add liquid.

[0110] In some device examples, a plurality of support columns are provided on the lower end surface of the tube beam portion 41. This can form a gap between the inner filter tube 4 and the chromatography through tube 1 to facilitate gravity chromatography.

[0111] In some examples of the device, the base 22 is provided with a waste liquid tank 24 .

[0112] In some device examples, at least part of the placement holes 23 are provided with limiting grooves 25. This can better limit the movement of the collector 3 and facilitate operation.

[0113] In some device examples, the placement holes 23 are divided into two rows, front and back rows, wherein the chromatography tubes 1 are placed in the back row, the collectors 3 are placed in the front row, and the waste liquid tank 24 is arranged below the placement holes 23 in the back row.

[0114] In some device examples, the diameter of the chromatography tube 1 near the liquid outlet 12 is gradually reduced. This can guide the liquid out as much as possible. In some embodiments of the device, the adsorbent particles filled are macroporous resins.

[0115] In some embodiments of the device, the particle size of the macroporous resin is 0.03-0.4 mm.

[0116] In some device examples, the pore size of the macroporous resin ranges from 5 to 16 nm.

[0117] In some device examples, the macroporous resin is a non-polar resin or a modified non-polar resin.

[0118] In some device examples, the non-polar resin is prepared from a polymerizable monomer, a cross-linking agent, a porogen, an initiator, and a stabilizer. The polymerizable monomer is styrene, the cross-linking agent is divinylbenzene, the porogen is toluene, a fatty alcohol or a fatty acid, and the stabilizer is gelatin.

[0119] In some device examples, the non-polar macroporous resin is selected from at least one of D101, H103, HPD100, ADS-8, X-5, D101-I, D50, D700, D910 and D4020.

[0120] In some device embodiments, different macroporous resins can be used alone or in combination.

[0121] In some device examples, the diameter-to-height ratio of the filled macroporous resin is 1:1-5:1, preferably 1:1-3:1.

[0122] The above features can be combined arbitrarily unless they conflict with each other.

[0123] Specific examples of concentrating devices: A concentrating device, comprising: The chromatography tube 1 comprises a tube opening 11 at the upper end and a liquid outlet 12 at the lower end, and the diameter of the chromatography tube 1 at one end close to the liquid outlet 12 gradually shrinks; An upper sieve plate 13 and a lower sieve plate 14 are provided between the pipe opening 11 and the liquid outlet 12. A macroporous resin is filled between the upper sieve plate 13 and the lower sieve plate 14. The particle size of the macroporous resin is 0.03 to 1.2 mm, and the pore size range is 5 to 50 nm. The filter inner tube 4, the lower part of the filter inner tube 4 is placed in the chromatography through tube 1 through the through tube opening 11, 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 through tube 1, the upper sieve plate 13 and the lower sieve plate 14 are provided with a tube beam part 41 for easy gripping at the upper end of the filter inner tube 4, the head opening of the tube beam part 41 gradually shrinks downward, and the lower end surface of the tube beam part 41 is provided with a plurality of support columns, after the filter inner tube 4 is inserted into the chromatography through tube 1, the support columns are in contact with the upper tube wall of the chromatography through tube 1; The fixing seat 2 comprises an upper cover plate 21 and a base 22, wherein the upper cover plate 21 is provided with a plurality of placement holes 23, and the placement holes 23 are divided into two rows, front and rear rows, wherein the chromatography through tube 1 is placed in the rear row, and a waste liquid tank 24 is provided on the seat below the rear row placement holes 23; the front row placement holes 23 are provided with collectors 3, and the front row placement holes 23 are also provided with limit grooves 25; The collector 3 , specifically a reagent tube, comprises a tube cover 32 and a tube body 31 . A connecting portion 33 is provided between the tube cover 32 and the tube body 31 for collecting the outflowing liquid from the liquid outlet 12 .

[0124] When in use, first insert the inner filter tube into the chromatography tube, discharge the waste liquid after adding the sample, allow LPS or LAM to be adsorbed on the macroporous resin, and then elute, and use a collector to collect the eluate for later use.

[0125] The technical solution of the present invention is further illustrated below in conjunction with examples.

[0126] Pretreatment of adsorption resin The commercial non-polar macroporous resin purchased is dried to remove moisture, then crushed and sieved to obtain resin powders with different particle size ranges.

[0127] Before use, use ethanol solution or other solution to clean the resin powder, remove the unpolymerized monomers and organic residues such as porogens, dispersants, and preservatives remaining in the pores of the resin, improve the cleanliness of the resin, and improve the safety of the resin. After cleaning, dilute the adsorption resin with 75% ethanol aqueous solution to 0.3g / ml and set aside.

[0128] Adsorption rate = (AB) / A×100% Elution rate = C / (AB) × 100% Wherein, A is the total LAM content in the sample to be concentrated, B is the total residual amount of LAM in the effluent after the sample to be concentrated is loaded and concentrated, and C is the total content of LAM in the eluate. The total content can be obtained by multiplying the concentration of LAM in the liquid by the volume of the liquid.

[0129] Recovery rate = adsorption rate × elution rate × 100%.

[0130] Effective concentration multiple = LPS or LAR concentration after concentration / LPS or LAR concentration in the sample to be concentrated.

[0131] Example 1: Effects of different macroporous resins on the adsorption of lipoarabinomannan S1) Place the chromatography tube on the fixed seat, put in the lower sieve plate, and press the lower sieve plate firmly; S2) placing the pretreated adsorption resin on a magnetic stirrer and stirring, while stirring, 0.5 ml of adsorption resin powder (0.3 g / ml, particle size 0.03-1.2 mm) is pipetted into the chromatography tube, and the upper sieve plate is horizontally placed and pressed, so that the adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1; S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until it is completely added; S4) adding 5 ml of LAM solution with a concentration of 100 pg / mL as the sample to be concentrated, and letting it stand until the solution is completely dripped; S5) Add eluent (15% ethanol / PBS) to the column twice, 0.4 ml for the first time and 0.2 ml for the second time, let stand until the eluent is finished, collect the concentrated sample after the second wash.

[0132] The collected samples were tested using the lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Rede Biotechnology Co., Ltd. The test results are shown in Table 1.

[0133] Table 1

[0134] 0.15 g of different resin powders + 10 ml of 100 pg / mL lipoarabinomannan solution was concentrated by gravity and 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.

[0135] Table 2

[0136] Conclusion: After different resins are processed into powder, D50, D101, D4020 and ADS-8 have comparable adsorption rates and concentration effects, while D700 and D910 are slightly lower.

[0137] Comparison of adsorption effects of macroporous adsorption resins with different polarities on lipoarabinomannan S1) Place the chromatography tube on the fixed seat, put in the lower sieve plate, and press the lower sieve plate firmly; S2) placing the pretreated adsorption resin on a magnetic stirrer and stirring, while stirring, 0.3 g of different types of adsorption resins are pipetted into the chromatography tube, placed horizontally on the upper sieve plate and pressed tightly; S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until it is completely added; S4) Add 10 ml of 100 pg / mL LAM solution as the sample to be concentrated, let it stand until the solution is completely dripped, detect the concentration of residual LAM in the supernatant of the effluent, and calculate the adsorption rate.

[0138] The lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Rede Biotechnology Co., Ltd. was used for detection. The results are shown in Table 3.

[0139] Table 3

[0140] Conclusion: From the above experimental data, it can be seen that the adsorption effect of non-polar resin is better than that of weak polar, medium polar and polar resins, and the adsorption rate of non-polar resin with a pore size of 5-50nm is higher, and the adsorption rate of pore size of 5-16nm is better.

[0141] Adsorption effect of non-polar resin on lipopolysaccharide or lipoarabinomannan: S1) Place the chromatography tube on the fixed seat, put in the lower sieve plate, and press the lower sieve plate firmly; S2) placing the pretreated adsorption resin on a magnetic stirrer and stirring, while stirring, 1 ml of adsorption resin (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) is drawn into the chromatography tube, and the upper sieve plate is horizontally placed and pressed, so that the adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1; S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until it is completely added; S4) adding 10 ml of a concentration standard LAM solution and a concentration standard endotoxin solution as the sample to be concentrated, and letting it stand until it is completely dripped; S5) Detect the concentration of residual LAM and endotoxin in the supernatant after adsorption, and calculate the adsorption rate.

[0142] Endotoxin concentration was detected according to the endotoxin detection horseshoe crab kit (microplate quantitative colorimetric matrix method).

[0143] Detection method of LAM concentration: 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 4.

[0144] Table 4

[0145] Conclusion: From the above experimental results, it can be seen that non-polar resin can effectively adsorb lipopolysaccharide or lipoarabinomannan with a high adsorption rate.

[0146] Test of different resin dosage and sample volume ratio: Different amounts of adsorption resin (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) were added to the chromatography tube, and 10 ml of LAM solution with a concentration of 10 pg / mL was added. After incubation on a rotating disk at 30 rpm, the supernatant and the resin were separated. The supernatant was centrifuged at 1500 g for 5 min and then detected using the lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Red Biotechnology Co., Ltd. The results are shown in Table 5.

[0147] Table 5

[0148] Conclusion: Increasing the amount of resin and prolonging the adsorption time can improve the adsorption rate, but with the increase of the amount of resin, the adsorption rate increases slowly. Therefore, the ratio of resin amount to sample volume is preferably 0.1:10-0.5:10.

[0149] Effect of resin particle size on adsorption of lipoarabinomannan: Comparison of LAM adsorption rates of the same resin with different particle sizes (mixing method: 0.3 g resin + 10 ml 5 pg / mL lipoarabinomannan solution, incubated at 30 rpm for 30 min, separated the supernatant and resin, centrifuged at 1500 g for 5 min, and detected using the lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Red Biotechnology Co., Ltd. The results are shown in Table 6.

[0150] Table 6

[0151] The results show that the smaller the resin particle size, the higher the adsorption rate and the shorter the adsorption time, preferably 0..03~0.4mm.

[0152] Example 2: Comparison of the elution effects of different eluents on lipoarabinomannan Experiment 1: S1) Place the chromatography tube on the fixed seat, put in the lower sieve plate, and press the lower sieve plate firmly; S2) placing the pretreated adsorption resin on a magnetic stirrer and stirring, while stirring, 0.5 ml of adsorption resin powder (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) is pipetted into the chromatography tube, and the upper sieve plate is horizontally placed and pressed, so that the adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1; S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until it is completely added; S4) adding 5 ml of LAM solution with a concentration of 100 pg / mL as the sample to be concentrated, and letting it stand until the solution is completely dripped; S5) Add 0.2 ml of eluent of different compositions to the chromatography column, let it stand until it is completely dripped, and collect the concentrated sample.

[0153] The collected samples were tested using the lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Red Biotechnology Co., Ltd. Repeat step S5 and take the elution segment with higher concentration. The test results are shown in Table 7.

[0154] Table 7

[0155] The results showed that there were significant differences in the elution effects of different eluents on polysaccharide removal. The eluent containing ethanol could effectively elute LAM adsorbed by the adsorption resin, while 0.5% Tween-20 / PBS, 0.2M glycine-hydrochloric acid (pH1.5), and 0.2M NaOH (pH12) could not effectively elute.

[0156] Experiment 2: S1) Place the chromatography tube on the fixed seat, put in the lower sieve plate, and press the lower sieve plate firmly; S2) placing the pretreated adsorption resin on a magnetic stirrer and stirring, while stirring, 0.5 ml of adsorption resin (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) is pipetted into the chromatography tube, and the upper sieve plate is horizontally placed and pressed, so that the adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1; S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until it is completely added; S4) adding 10 ml of a LAM solution with a concentration of 100 pg / mL as the sample to be concentrated, leaving it to stand until it is completely dripped, detecting the concentration of residual LAM in the supernatant of the effluent, and calculating the adsorption rate; S5) Add different eluent compositions to the chromatography column twice, 0.4 ml for the first time and 0.2 ml for the second time, let stand until the eluent is finished, collect the second wash to obtain a concentrated sample.

[0157] The collected samples were tested using the lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Red Biotechnology Co., Ltd. to obtain the elution volume after elution. The test results are shown in Table 8. Samples eluted with ethanol or methanol concentrations exceeding 20% ​​need to be diluted before testing.

[0158] Table 8

[0159] Note: The luminescence value marked with * is the luminescence value of LAM solution with a concentration of 100 pg / mL.

[0160] From the results in the table, it can be seen that methanol, ethanol and ethyl acetate have better elution efficiency for lipopolysaccharide.

[0161] Example 3: Effects of different ethanol content eluents and elution times on elution effect S6) placing the chromatography tube on the fixed seat, inserting the lower sieve plate, and pressing the lower sieve plate firmly; S7) placing the pretreated adsorption resin on a magnetic stirrer and stirring, while stirring, 0.5 ml of adsorption resin powder (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) is pipetted into the chromatography tube, and the upper sieve plate is horizontally placed and pressed, so that the adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1; S8) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until it is completely added; S9) adding 5 ml of LAM solution with a concentration of 100 pg / mL as the sample to be concentrated, and letting it stand until it is completely dripped; S10) adding 0.1 ml of eluent to the chromatography column multiple times, each time; letting it stand until it is completely added, and collecting the concentrated samples.

[0162] The eluates collected from the 3rd to 6th times were tested using the lipoarabinomannan (LAM) detection kit (immunochromatography) produced by Guangzhou Red Biotechnology Co., Ltd. For the sake of comparison, the total LAM elution amount of the eluates collected from the 4th to 7th times was counted as 100%. The test results are shown in Table 9.

[0163] Table 9

[0164] Note: T value is the detection value, C value is the quality control value, and T / C value is the relative concentration of LAM in the eluent.

[0165] The experimental results show that different ethanol concentrations of eluents have different elution effects. In general, the elution amount of LAM was low in the first three elutions, which may be related to the ethanol concentration in the eluent. The ethanol concentration in the eluent is preferably 15%; if the ethanol concentration is reduced, the elution peak will shift backward, the elution rate will decrease, the operation time will be extended, and the operation cost will increase. Therefore, the ethanol concentration of the eluent should not be less than 15%.

[0166] Example 4: Effect of resin dosage on lipoarabinomannan concentration S1) Place the chromatography tube on the fixed seat, put in the lower sieve plate, and press the lower sieve plate firmly; S2) placing the pretreated adsorption resin on a magnetic stirrer and stirring, while stirring, taking different amounts of adsorption resin powder (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) and adding them into the chromatography tube, horizontally placing the upper sieve plate and pressing it, the adsorption resin forms a chromatography column; S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until it is completely added; S4) adding 5 ml of LAM solution with a concentration of 100 pg / mL as the sample to be concentrated, and letting it stand until the solution is completely dripped; S5) Add elution solution to the chromatography column, 100 μL each time, and perform multiple elutions. Allow the solution to stand for a while until it is completely eluted, and collect the concentrated sample.

[0167] The collected samples were tested using the lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Rede Biotechnology Co., Ltd. The test results are shown in Table 10.

[0168] Table 10

[0169] From the results in Table 10, it can be seen that the ratio of resin dosage to sample volume is 0.15-0.3 g:5 ml, which is the best ratio, and 0.15:5 ml is the best. Further increasing the resin dosage will significantly shift the elution peak to the back, increasing the time of concentrated elution.

[0170] Example 5: Effect of column diameter-height ratio on lipoarabinomannan concentration S1) Place the chromatography tube on the fixed seat, put in the lower sieve plate, and press the lower sieve plate firmly; S2) placing the pretreated adsorption resin on a magnetic stirrer and stirring, while stirring, 0.15 g of adsorption resin powder (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) was pipetted into the chromatography tube, and horizontally placed on the upper sieve plate and pressed tightly to prepare chromatography columns with different diameter-to-height ratios; S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until it is completely added; S4) adding 5 ml of LAM sample with a concentration of 100 pg / mL to the chromatography column, leaving it to drip completely, recording the dripping completion time, collecting the effluent, detecting the amount of residual LAM therein, and determining the adsorption rate; S5) Add eluent (15% ethanol / PBS) to the column twice, 0.4 ml for the first time and 0.2 ml for the second time, let stand until the eluent is complete, collect the concentrated sample obtained after the second wash for testing, and determine the elution rate.

[0171] The collected samples were tested using the lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Rede Biotechnology Co., Ltd. The results are shown in Table 11.

[0172] Table 11

[0173] It can be seen from Table 11 that the time it takes for the sample to drip completely varies significantly for different diameter-to-height ratios (diameter: height). When the diameter-to-height ratio is 1:1 to 3:1, the time is significantly shorter and has a higher adsorption rate.

[0174] Example 6: Establishment of LAM standard curve S1) Place the chromatography tube on the fixed seat, put in the lower sieve plate, and press the lower sieve plate firmly; S2) placing the pretreated adsorption resin on a magnetic stirrer and stirring, while stirring, 0.5 ml of adsorption resin powder (0.3 g / ml, particle size 0.03-0.4 mm, ADS-8) is pipetted into the chromatography tube, and the upper sieve plate is horizontally placed and pressed, so that the adsorption resin forms a chromatography column with a diameter-to-height ratio of 2:1; S3) Add 1 ml of activation solution (40% ethanol PBS solution) to the chromatography column and let it stand until it is completely added; S4) adding 5 ml of samples with different LAM concentrations as samples to be concentrated, and letting them stand until they are completely dripped; S5) Add 0.3 mL of eluent (15 v / v% ethanol in PBS) to the column and let it stand until it is completely added; S6) Add 0.2 ml of eluent to the chromatography column, let it stand until it is completely added, and collect the concentrated sample.

[0175] The collected samples were tested using the lipoarabinomannan (LAM) detection kit (chemiluminescence method) produced by Guangzhou Red Biotechnology Co., Ltd. The test results are shown in Table 12 and Figure 6 shown.

[0176] Table 12

[0177] The experimental results show that this concentration method greatly improves the sensitivity of detection, and the linearity of its standard curve is excellent, with a correlation coefficient of 0.9986.

[0178] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A method for efficiently and rapidly concentrating lipopolysaccharide or lipoarabinomannan, comprising the following steps: S1) contacting and adsorbing the sample to be concentrated with the macroporous resin; S2) After adsorption is completed, the liquid is discharged; S3) adding an eluent, eluting and collecting to obtain a lipopolysaccharide or lipoarabinomannan concentrate; in: The particle size of the macroporous resin is 0.03 to 1.2 mm, and the pore size range is 5 to 50 nm; The eluent is selected from at least one of an ethanol aqueous solution, a methanol aqueous solution, an ethyl acetate solution, a mixture of ethanol and a buffer solution, and a mixture of methanol and a buffer solution.

2. The efficient and rapid concentration method according to claim 1, characterized in that: The macroporous resin has at least one of the following characteristics: 1) The particle size of the macroporous resin is 0.03-0.4 mm; 2) The pore size of the macroporous resin is in the range of 5 to 16 nm; 3) The macroporous resin is a non-polar resin.

3. The efficient and rapid concentration method according to claim 1, characterized in that: The total content of methanol and / or ethanol in the eluent is 10 to 90 v / v%.

4. The efficient and rapid concentration method according to claim 1, characterized in that: The macroporous resin is packed into a chromatography column, and the diameter-to-height ratio of the chromatography column is 1:1--5:

1.

5. The efficient and rapid concentration method according to claim 4, characterized in that: Before loading the sample to be concentrated onto the chromatography column, the chromatography column is activated using an activation solution, wherein the activation solution is composed of a buffer solution containing 30 to 95 v / v% ethanol or methanol.

6. The high-efficiency and rapid concentration method according to claim 2, wherein the non-polar resin is prepared from a polymerizable monomer, a cross-linking agent, a porogen, an initiator, and a stabilizer, wherein the polymerizable monomer is selected from at least one of styrene, α-methylstyrene, methyl methacrylate, and propionitrile, the cross-linking agent is divinylbenzene, the porogen is toluene, a fatty alcohol or a fatty acid, and the stabilizer is gelatin; And / or the non-polar macroporous resin is selected from at least one of D101, H103, HPD100, ADS-8, X-5, D101-I, D50, D700, D910 and D4020.

7. The efficient and rapid concentration method according to any one of claims 1 to 6, characterized in that: It satisfies at least one of the following characteristics: 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-1 g: (1-5) mL; 3) The dosage range of the eluent is: 50uL-1ml; 4) The volume of the sample to be concentrated is 0.5ml-20ml; 5) The macroporous resin is a non-polar resin or a modified non-polar resin; 6) The sample to be concentrated is selected from serum, urine, cerebrospinal fluid, saliva or tissue.

8. A method for rapidly determining the content of lipopolysaccharide or lipoarabinomannan, characterized in that: include: S1) obtaining a lipopolysaccharide concentrate or a lipoarabinomannan concentrate according to the efficient and rapid concentration method according to any one of claims 1 to 7; S2) taking the lipopolysaccharide concentrate or the lipoarabinomannan concentrate, and testing to determine the lipopolysaccharide or lipoarabinomannan content.

9. A device for implementing the efficient and rapid concentration method according to any one of claims 1 to 7, characterized in that: include: The chromatography tube comprises a tube opening at the upper end and a liquid outlet at the lower end, an upper sieve plate and a lower sieve plate are arranged between the tube opening and the liquid outlet, and the space between the upper sieve plate and the lower sieve plate is used to fill adsorption particles; The fixing seat comprises an upper cover plate and a base, wherein the upper cover plate is provided with a plurality of placement holes; A collector is used to collect the outflowing liquid from the liquid outlet.

10. The device according to claim 9, characterized in that It also includes a filtering inner tube, which is placed in the chromatography through-tube through the through-tube opening.

11. The device according to claim 10, characterized in that The upper end of the filtering inner tube is provided with a tube beam portion which is convenient for holding.

12. The device according to any one of claims 9 to 11, characterized in that: The base is provided with a waste liquid tank; and / or at least part of the placement holes are provided with limiting grooves.

13. A rapid test kit for lipoarabinomannan, characterized in that: The invention comprises the device according to any one of claims 9 to 11.

Images