JP6595215B2 - Immunochromatographic analyzer, manufacturing method thereof, and immunochromatographic analysis method - Google Patents

Description

  The present invention relates to an immunochromatography analyzer, a method for producing the same, and an immunochromatography analysis method.

  In recent years, immunochromatographic immunoassays that do not require pretreatment of specimens have been used as simple in-vitro diagnostic kits or portable diagnostic devices that detect antigens in sample solutions using the specific reactivity of antibodies. The importance is increasing. In particular, pathogen testing kits such as viruses and bacteria are familiar immunochromatographic analyzers widely used in general hospitals and clinics.

  The simplest structure of a conventional immunochromatography analyzer is that a sample addition unit, a labeling substance holding unit, a chromatographic medium unit carrying a detection unit, and an absorption unit that absorbs liquid that has passed through the detection unit are interconnected. Structure.

For such an immunochromatography analyzer, high sensitivity is currently required in order to detect a trace amount of antigen such as influenza virus.
For example, in order to prevent non-specific antigen-antibody reaction, a method of adding a protein such as BSA, casein, gelatin, or mammalian IgG to a sample diluent or a developing solution, or a sample with a surfactant solution having a steroid skeleton A method of developing and then amplifying a labeled substance (Patent Document 1), or a method of developing a solution containing a test substance and a labeled substance modified with a first antibody against the test substance on a porous carrier at a specific rate (Patent Document 2) and the like have been proposed.
However, the conventional immunochromatography analyzer cannot increase the detection sensitivity of the substance to be detected to the required level.

JP 2012-211849 A JP 2008-197038 A

  Accordingly, an object of the present invention is to provide an immunochromatography analyzer, a method for producing the same, and an immunochromatography method that can significantly increase the detection sensitivity of a substance to be detected.

The present invention is as follows.
1. A sample addition unit for adding a sample, a labeling substance holding unit for holding a labeling substance for recognizing a detection substance contained in the sample, and a chromatographic medium part on which a detection unit for detecting the detection substance is supported In the immunochromatography analyzer including sequentially,
The detection part in the chromatographic medium part is formed by applying a coating liquid containing at least an aprotic polar solvent and an antibody that specifically reacts with the substance to be detected on the chromatographic medium part. A characteristic immunochromatographic analyzer.
2. 2. The immunochromatographic analyzer according to 1 above, wherein the aprotic polar solvent is dimethyl sulfoxide (DMSO).
3. 3. The immunochromatographic analyzer according to 1 or 2 above, wherein the concentration of the aprotic polar solvent in the coating solution is 0.02 to 0.4% by volume.
4). 4. The immunochromatographic analyzer according to any one of 1 to 3, wherein the chromatographic medium part includes nitrocellulose or cellulose acetate.
5). A sample addition unit for adding a sample, a labeling substance holding unit for holding a labeling substance for recognizing a detection substance contained in the sample, and a chromatographic medium part on which a detection unit for detecting the detection substance is supported In the manufacturing method of the immunochromatography analyzer including sequentially,
The detection part in the chromatographic medium part is formed by applying a coating liquid containing at least an aprotic polar solvent and an antibody that specifically reacts with the substance to be detected on the chromatographic medium part. A method for producing an immunochromatographic analyzer.
6). 6. The method for producing an immunochromatographic analyzer according to 5 above, wherein the aprotic polar solvent is dimethyl sulfoxide (DMSO).
7). 7. The method for producing an immunochromatographic analyzer according to 5 or 6, wherein the concentration of the aprotic polar solvent in the coating solution is 0.02 to 0.4% by volume.
8). 8. The method for producing an immunochromatographic analyzer according to any one of 5 to 7, wherein the chromatographic medium part contains nitrocellulose or cellulose acetate.
9. The immunochromatography analysis method according to any one of 1 to 4 above, wherein the following steps (1) to (4) are sequentially performed.
(1) A step of adding the sample to the sample addition unit (2) A step of recognizing a substance to be detected contained in the sample by a labeling substance held in the labeling substance holding unit (3) A sample and a labeling substance (4) A step of detecting a substance to be detected in the developed mobile phase by a detection unit.

  According to the present invention, the aprotic polar solvent interacts with the hydrophobic portion (for example, Fc region) of the antibody and suppresses the aggregation of the antibody on the chromatographic medium portion, thereby being based on the presence of the substance to be detected. The signal-to-noise ratio, which is the ratio of the signal and noise emitted, is improved, and the detection sensitivity of the substance to be detected is significantly increased.

It is sectional drawing for demonstrating the structure of an immunochromatography analyzer.

  Below, the form for implementing this invention is demonstrated.

The immunochromatography analyzer of the present invention and the manufacturing method thereof include a sample addition unit for adding a sample, a labeling substance holding unit for holding a labeling substance that recognizes a target substance contained in the sample, and detecting the target substance A chromatographic medium part on which the detection part is supported, and an absorption part that absorbs the liquid that has passed through the detection part if necessary, and the detection part in the chromatographic medium part is an aprotic polar solvent And a coating solution containing at least an antibody that specifically reacts with the substance to be detected is formed on the chromatographic medium part.
Hereinafter, an embodiment of an immunochromatography analyzer of the present invention and a manufacturing method thereof will be described with reference to the drawings.

  As shown in FIG. 1, the immunochromatographic analyzer of the present invention comprises a sample adding part (also called a sample pad) (1), a labeling substance holding part (also called a conjugate pad) (2), and a chromatographic medium part (3). , A detection unit (4), an absorption unit (5), and a backing sheet (6).

  A sample addition part (1) is a site | part which dripped a test substance (sample) in an immunochromatography analyzer. The sample may be any material as long as it is a material used in a normal immunochromatography analyzer. As the sample addition part (1), a glass fiber or cellulose film for absorbing and holding the sample is usually used.

  The labeling substance holding part (2) contains a labeling substance (marker substance) to be described later that is bound to an antibody that binds to the substance to be detected in advance. When the substance to be detected moves in the labeling substance holding part, it binds to the antibody and is labeled. The labeling substance holding part (2) is made of, for example, a glass fiber nonwoven fabric or a cellulose film.

  The chromatographic medium part (3) is a developed part of the chromatograph. The chromatographic medium part (3) is an inert film made of a microporous material that exhibits capillary action. From the viewpoint of not having reactivity with a detection reagent, an immobilization reagent or a substance to be detected used in a chromatograph, and from the viewpoint of improving the effect of the present invention, for example, a membrane made of nitrocellulose (hereinafter referred to as “ A nitrocellulose membrane ”or a cellulose acetate membrane (hereinafter sometimes referred to as“ cellulose acetate membrane ”) is preferable, and a nitrocellulose membrane is more preferable. Cellulose membranes, nylon membranes and porous plastic cloth (polyethylene, polypropylene) can also be used.

  As the nitrocellulose membrane, it is sufficient if nitrocellulose is mainly contained, and a membrane mainly composed of nitrocellulose such as a pure product or a nitrocellulose mixed product can be used.

  The nitrocellulose membrane can further contain a substance that promotes capillary action. As the substance, a substance that lowers the surface tension of the film surface and brings about hydrophilicity is preferable. For example, a substance having an amphipathic action such as saccharides, amino acid derivatives, fatty acid esters, various synthetic surfactants or alcohols, which has no effect on the movement of a substance to be detected on an immunochromatograph and is a marker substance Substances that do not affect the color development (eg, gold colloid) are preferred.

  Nitrocellulose membranes are porous and exhibit capillary action. The index of the capillary phenomenon can be confirmed by measuring the water absorption speed (water absorption time: capillary flow time). The water absorption rate affects detection sensitivity and inspection time.

  The form and size of the chromatographic medium part (3) represented by the above nitrocellulose membrane and cellulose acetate membrane are not particularly limited, and are appropriate in terms of actual operation and reaction results. If it is.

  Furthermore, in order to make the operation easier, it is preferable to provide a support made of plastic or the like on the back surface of the chromatographic medium part (3). The properties of the support are not particularly limited, but when the measurement result is observed by visual judgment, the support preferably has a color that is not similar to the color caused by the labeling substance. Usually, it is preferably colorless or white.

The detection part (4) is formed on the chromatographic medium part (3). That is, an antibody that specifically binds to the substance to be detected is immobilized at an arbitrary position.
In the present invention, the detection part (4) is formed by applying a coating liquid containing at least an aprotic polar solvent and an antibody that specifically reacts with the substance to be detected on the chromatographic medium part (3). It is characterized by that.

  Examples of the aprotic polar solvent include dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, acetonitrile, propanenitrile, butyronitrile, sulfolane and the like. Dimethyl sulfoxide (DMSO) is particularly preferable from the viewpoint of suppressing aggregation of the antibody on the medium part and improving the SN ratio.

  The concentration of the aprotic polar solvent in the coating solution is preferably in the range of 0.01 to 1.0% by volume. When the concentration of the aprotic polar solvent is less than 0.01% by volume, the added amount is too small to achieve the effects of the present invention. On the contrary, if it exceeds 1.0% by volume, the chromatographic medium part such as nitrocellulose membrane or cellulose acetate membrane is dissolved, which is not preferable. The concentration of the aprotic polar solvent in the coating solution is more preferably 0.02 to 0.4% by volume, and further preferably 0.02 to 0.3% by volume.

Examples of antibodies include polyclonal antibodies and monoclonal antibodies. Monoclonal antibodies and polyclonal antibodies or fragments thereof are known and available, and can be prepared by known methods.
The concentration of the antibody in the coating solution is, for example, 0.1 mg / mL to 2 mg / mL, preferably 0.3 mg / mL to 1.5 mg / mL, and further 0.4 mg / mL to 1.2 mg / mL. preferable.

  In the coating solution, components that are usually used in addition to the above components include various buffer solutions, alcohols, sugars, amino acids, proteins, water-soluble synthetic polymers, and the like.

  Examples of a method for applying the coating solution on the chromatographic medium part (3) include known methods such as an ink jet printing method and a spot method. Moreover, the shape of the applied detection part is not particularly limited, but examples include a circular spot, a line extending perpendicularly to the developing direction of the chromatographic medium part (3), a number, a character, or a symbol such as +,-. It is done. Also, the coating amount can be equivalent to a known one.

  In addition, after applying the coating liquid on the chromatographic medium part (3), in order to prevent the accuracy of analysis from being reduced due to non-specific adsorption, The blocking process can be performed by a known method. In general, proteins such as bovine serum albumin, skim milk, casein or gelatin are preferably used for the blocking treatment. After such blocking treatment, if necessary, for example, one or a combination of two or more surfactants such as Tween 20, Triton X-100, or SDS may be washed.

  The absorption section (5) is installed at the end of the chromatographic medium section (3) as necessary to absorb the liquid such as the specimen and the developing solution that has passed through the detection section (4). In the immunochromatography analyzer of the present invention, the absorption part (5) can be made of glass fiber, for example. When the absorption part (5) is made of glass fiber, the return of the sample liquid can be greatly reduced.

  The backing sheet (6) is a base material. By applying an adhesive on one side or sticking an adhesive tape, one side has adhesiveness, and the sample addition part (1), labeling substance holding part (2), chromatographic medium part ( 3) A part or all of the detection unit (4) and the absorption unit (5) are provided in close contact with each other. The backing sheet (6) is not particularly limited as long as the backing sheet (6) becomes impermeable and impermeable to the sample solution by the adhesive.

  The immunochromatographic analyzer produced as described above is usually subjected to a drying treatment before commercialization. The drying temperature is, for example, 20 to 50 ° C., and the drying time is 0.5 to 1 hour. An aprotic polar solvent, such as dimethyl sulfoxide (DMSO), remains in the detection part (4), for example, 0.01 to 0.5 μg / kit mass% per kit under the above drying conditions.

The immunochromatographic analysis method of the present invention includes the following steps (1) to (4), and detects a substance to be detected contained in a specimen using the above immunochromatographic analyzer.
(1) A step of adding the sample to the sample addition unit (2) A step of recognizing a substance to be detected contained in the sample by a labeling substance held in the labeling substance holding unit (3) A sample and a labeling substance Step of developing the chromatographic medium portion as a mobile phase (4) Step of detecting a substance to be detected in the developed mobile phase by the detection portion Each step will be described below.

(1) Step of adding sample to sample adding portion In step (1), first, the sample diluent is adjusted to a concentration that allows the sample to move smoothly through the immunochromatographic medium without reducing measurement accuracy. It is preferable to prepare or dilute the sample-containing solution by adjusting or dilution with Secondly, a predetermined amount (usually 0.1 to 2 ml) of the specimen-containing liquid is dropped onto the sample addition part (1). When the specimen-containing liquid is dropped, the specimen-containing liquid starts to move in the sample addition part (1).

  The sample diluent can also be used as a developing solution, but usually water is used as a solvent, and a buffer solution, a salt, and a nonionic surfactant, and further, for example, an antigen-antibody reaction is promoted. Or you may add 1 type, or 2 or more types, such as a protein for suppressing a nonspecific reaction, high molecular compounds (PVP etc.), an ionic surfactant, or a polyanion, or an antibacterial agent, a chelating agent. When used as a developing solution, a sample and developing solution mixed in advance can be supplied and dropped onto the sample addition unit, or developed, or after the sample is first supplied and dropped onto the sample addition unit. Alternatively, the developing solution may be supplied and dropped on the sample addition unit to be developed.

  Samples containing the substance to be detected include, for example, biological samples, that is, whole blood, serum, plasma, urine, saliva, sputum, nasal cavity or throat wipe, cerebrospinal fluid, amniotic fluid, nipple discharge, tears, sweat, skin As well as extracts from milk, eggs, wheat, beans, beef, pork, chicken, and foods containing them, and the like.

  Specific examples of the substance to be detected include carcinoembryonic antigen (CEA), HER2 protein, prostate specific antigen (PSA), CA19-9, α-fetoprotein (AFP), immunosuppressive acidic protein (IPA), CA15- 3, CA125, estrogen receptor, progesterone receptor, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), syphilis antibody, Examples include, but are not limited to, influenza virus, human hemoglobin, chlamydia antigen, group A β-streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin or glycated albumin.

(2) The step of recognizing the substance to be detected contained in the specimen by the labeling substance held in the labeling substance holding part In the process (2), the specimen-containing liquid added to the sample addition part in the step (1) is labeled In this step, the substance to be detected is detected in the specimen by the labeling substance held in the labeling substance holding part by moving to the substance holding part (2).

  The labeling substance labels the antibody. Generally, an enzyme or the like is used for labeling the detection reagent in the immunochromatography method. However, since it is suitable for visually determining the presence of the substance to be detected, it is preferable to use an insoluble carrier as the labeling substance. . A labeled detection reagent can be prepared by sensitizing the antibody to an insoluble carrier. The means for sensitizing the antibody to the insoluble carrier may be in accordance with a known method.

  Insoluble carriers as labeling substances include colloidal metal particles such as gold, silver or platinum, colloidal metal oxide particles such as iron oxide, colloidal nonmetal particles such as sulfur, and latex particles composed of synthetic polymers. Or others can be used. In particular, colloidal gold is preferable because it is easy to detect. The average particle diameter of the colloidal gold particles is, for example, 10 nm to 250 nm, preferably 35 nm to 120 nm. The average particle diameter was measured with a transmission electron microscope (TEM: manufactured by JEOL Ltd., JEM-2010), and the projected area circle equivalent diameter of 100 particles was measured randomly using the photographed projection photograph. It can be calculated from the average value.

  The insoluble carrier is a labeling substance suitable for visually determining the presence of the substance to be detected, and is preferably colored in order to facilitate the visual determination. The colloidal metal particles and the colloidal metal oxide particles themselves exhibit a specific natural color corresponding to the particle diameter, and the color can be used as a label.

(3) Step of developing a sample and a labeling substance as a mobile phase in the chromatographic medium part Step (3) is a step in which the substance to be detected is recognized after the substance to be detected is recognized by the labeling substance in the labeling substance holding part in step (2). In this step, the substance is passed through the chromatographic medium as a mobile phase.

(4) A step of detecting a substance to be detected in the developed mobile phase by the detection unit Step (4) is a step in which the substance to be detected in the specimen that has passed as a mobile phase on the chromatographic medium part is a specific antigen / antibody. This is a step in which the detection part is colored by specific reaction and binding so as to be sandwiched between the antibody and the labeling reagent that are held or immobilized on the detection part by the target binding reaction.

When the substance to be detected does not exist, the labeling reagent dissolved in the moisture of the sample does not cause a specific binding reaction even when it passes through the detection part on the chromatographic medium part, so that the detection part is not colored.
In the present invention, the aprotic polar solvent contained in the coating solution interacts with the hydrophobic part (for example, Fc region) of the antibody, and the antibody aggregates on a chromatographic medium part such as a nitrocellulose membrane or a cellulose acetate membrane. Is suppressed, the signal-to-noise ratio, which is a ratio of a signal and noise generated based on the presence of the substance to be detected, is improved, and the detection sensitivity of the substance to be detected is significantly increased.

  Finally, the moisture in the specimen-containing liquid moves to the absorption part (5).

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not restrict | limited to the following example.

(1) Preparation of sample addition part The nonwoven fabric (Millipore company_made: 300 mm x 30 mm) which consists of glass fiber was used as a sample addition part.
(2) Preparation of labeling substance holding part 0.5 ml of colloidal gold suspension (Tanaka Kikinzoku Kogyo Co., Ltd .: LC 40 nm) diluted with phosphate buffer (pH 7.4) to a concentration of 0.05 mg / ml 0.1 ml of the mouse-derived anti-influenza A monoclonal antibody (second antibody) was added and allowed to stand at room temperature for 10 minutes.
Next, 0.1 ml of a phosphate buffer (pH 7.4) containing 1% by weight of bovine serum albumin (BSA) was added, and the mixture was allowed to stand at room temperature for 10 minutes. Then, after sufficiently stirring, centrifugation was performed at 8000 × g for 15 minutes to remove the supernatant, and 0.1 ml of a phosphate buffer solution (pH 7.4) containing 1% by mass of BSA was added. The labeling substance solution was prepared by the above procedure.
After adding 300 μL of a 10% by mass trehalose aqueous solution and 1.8 mL of distilled water to 300 μL of the prepared labeling substance solution to a 15 mm × 300 mm glass fiber pad (Millipore), vacuum is added. The labeling substance holding part was produced by drying with a dryer.

(3) Preparation of chromatographic medium part and detection part As a membrane, a sheet made of nitrocellulose (manufactured by Millipore, trade name: HF120, 300 mm × 25 mm) was used.
Next, 150 μL of a solution obtained by diluting a mouse-derived anti-influenza A monoclonal antibody (first antibody) with a phosphate buffer solution (pH 7.4) containing 5% by mass of isopropyl alcohol so as to have a concentration of 1.0 mg / ml was obtained. Then, a detection site (detection line) on the dried membrane was applied in a line shape with an amount of 1 μL / mm (25 μL per sheet) using an immunochromatographic dispenser “XYZ3050” (manufactured by BIODOT) with a width of 1 mm. As the solution, a plurality of solutions having dimethyl sulfoxide (DMSO) concentrations shown in Table 1 were prepared.
In addition, in order to confirm the presence / absence and development speed of the gold nanoparticle labeling reagent, the goat-derived antiserum having a wide affinity for the gold nanoparticle labeling substance or animal meat protein as the detection substance downstream of the detection site Was diluted with a phosphate buffer (pH 7.4) and applied to a control site (control line). Then, it was dried at 50 ° C. for 30 minutes and dried overnight at room temperature to prepare a chromatographic medium part and a detection part.

(4) Preparation of immunochromatographic analyzer Next, in order to absorb the sample addition part, the labeling substance holding part, the chromatographic medium part carrying the detection part, the developed sample and the labeling substance on the base material composed of a backing sheet Glass fiber non-woven fabrics were sequentially bonded together as an absorbent part. And it cut | judged so that a width | variety might be set to 5 mm with the cutter, and it was set as the immunochromatography analyzer. The length of the labeling substance holding part and the incubation part in the sample development direction was 8 mm.

(5) Sample diluent 1% by mass of nonionic surfactant (manufactured by NOF Corporation, trade name: MN811 and Nacalai Tesque, trade name NP-40, 1: 1 mixture), 80 mM potassium chloride, 20 mM A 50 mM HEPES buffer solution (pH 7.5) containing 0.4% by weight of guanidine hydrochloride and 0.4% by weight of polyvinylpyrrolidone (average molecular weight of 360,000) was prepared as a reagent for subjecting the specimen to dilution treatment.

(6) Measurement The presence or absence of influenza A virus in the sample was measured by the following method using the prepared immunochromatography analyzer. That is, one tube of the suction trap was inserted into the suction pump, and the other tube was inserted into the back of the nasal cavity of a person who was not infected with influenza, and the suction pump was set to negative pressure to collect nasal discharge. The collected nasal discharge was diluted 20 times with the above-mentioned sample diluent, and this was used as a negative sample sample. Moreover, what added the commercially available inactivated influenza A virus so that protein concentration might be set to 50 ng / mL to the negative sample sample was made into the positive sample sample.

  150 μL of both the negative sample sample and the positive sample sample are placed on the sample addition part of the immunochromatography analyzer and developed, and after each elapsed time shown in Table 1, the degree of coloring (color intensity) of the detection part is measured with a densitometer (The unit in the table is mAbs). The results are shown in Table 1. The number of samples was 3 (No. 1 to 3).

In Table 1, first, referring to the result of the negative specimen sample, in the sample to which DMSO was not added, colored noise was observed after 15 minutes and 30 minutes from the start of the test. In the sample added in the range of ˜0.40% by volume, the generation of noise was greatly reduced.
In addition, when the S / N ratio is calculated from the results of the positive specimen sample and the negative specimen sample by the following formula, the S / N ratio is improved in the sample to which DMSO is added in the range of 0.02 to 0.40% by volume. It was suggested that the detection sensitivity of the substance was remarkably increased. Furthermore, the measurement variation (SD: standard deviation) after the lapse of 15 minutes for the positive sample is also greatly reduced in the sample to which DMSO was added in the range of 0.02 to 0.40 vol%. These effects are obtained by adding an aprotic polar solvent when the antibody is applied to the detection unit, so that the orientation of the detection unit antibody immobilized on the solid phase is improved, thereby suppressing nonspecific reactions and measurement variations. It is considered that an increase in specific reaction has been obtained.
SN ratio = color intensity after 15 minutes of positive sample / color intensity after 30 minutes of negative sample

1 Sample addition part (sample pad)
2 Labeling substance holding part 3 Chromatographic medium part 4 Detection part 5 Absorption part 6 Backing sheet

Claims (5)

A sample addition unit for adding a sample, a labeling substance holding unit for holding a labeling substance for recognizing a detection substance contained in the sample, and a chromatographic medium part on which a detection unit for detecting the detection substance is supported In the immunochromatography analyzer including sequentially,
The detection part in the chromatographic medium part is formed by applying a coating liquid containing at least an antibody that specifically reacts with dimethyl sulfoxide (DMSO) and the substance to be detected on the chromatographic medium part,
The immunochromatographic analyzer, wherein the chromatographic medium part contains nitrocellulose or cellulose acetate. The immunochromatography analyzer according to claim 1, wherein the concentration of the dimethyl sulfoxide (DMSO) in the coating solution is 0.02 to 0.4% by volume. A sample addition unit for adding a sample, a labeling substance holding unit for holding a labeling substance for recognizing a detection substance contained in the sample, and a chromatographic medium part on which a detection unit for detecting the detection substance is supported In the manufacturing method of the immunochromatography analyzer including sequentially,
The detection part in the chromatographic medium part is formed by applying a coating liquid containing at least an antibody that specifically reacts with dimethyl sulfoxide (DMSO) and the substance to be detected on the chromatographic medium part, and the chromatograph The method for producing an immunochromatographic analyzer, wherein the medium part contains nitrocellulose or cellulose acetate. The method for producing an immunochromatographic analyzer according to claim 3, wherein the concentration of the dimethyl sulfoxide (DMSO) in the coating solution is 0.02 to 0.4% by volume. An immunochromatography analysis method, wherein the following steps (1) to (4) are sequentially performed using the immunochromatography analyzer according to claim 1 or 2.
(1) A step of adding the sample to the sample addition unit (2) A step of recognizing a substance to be detected contained in the sample by a labeling substance held in the labeling substance holding unit (3) A sample and a labeling substance (4) A step of detecting a substance to be detected in the developed mobile phase by a detection unit.

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