WO2022092312A1 - 尿検体を保存するための試薬、キット及び方法 - Google Patents
尿検体を保存するための試薬、キット及び方法 Download PDFInfo
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- WO2022092312A1 WO2022092312A1 PCT/JP2021/040248 JP2021040248W WO2022092312A1 WO 2022092312 A1 WO2022092312 A1 WO 2022092312A1 JP 2021040248 W JP2021040248 W JP 2021040248W WO 2022092312 A1 WO2022092312 A1 WO 2022092312A1
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- urine
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/405—Concentrating samples by adsorption or absorption
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B10/0045—Devices for taking samples of body liquids
- A61B10/007—Devices for taking samples of body liquids for taking urine samples
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
- A61B10/0096—Casings for storing test samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/493—Physical analysis of biological material of liquid biological material urine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/521—Single-layer analytical elements
- G01N33/523—Single-layer analytical elements the element being adapted for a specific analyte
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/70—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving creatine or creatinine
Definitions
- the present invention relates to a reagent and a kit for storing a urine sample, which is used for a test for measuring a urinary component.
- the present invention relates to reagents, kits and methods for suppressing changes in the concentration of urinary components during storage and transportation of urine specimens.
- Human or animal-derived urine specimens like blood, contain many biomarkers that reflect the health status of the target human or animal and the presence or absence of disease. It is a sample that can be collected non-invasively as compared with blood, and when the subject is a human (subject), the subject himself can collect it by himself. Therefore, general-purpose test agents (OTC test agents) such as urine sugar / urine protein test agents, pregnancy test agents, and ovulation test agents for the subject himself / herself to perform urinalysis at home or the like are widely used. In addition to this, there is a mail inspection in which the subject collects a urine sample, mails it to a medical institution, a hygiene laboratory, etc., and inspects various items in urine at the mailing destination.
- OTC test agents general-purpose test agents
- the subject When performing a urine test by mail inspection, the subject can either put the urine sample in a container such as a polypropylene tube as it is and mail it, or mail the urine soaked in filter paper etc. and dried (filter paper urine). is assumed. If the urine sample is put in the container as it is and mailed, there is a risk of leakage due to cracking of the container or malfunction of the sealing plug, but in order to reduce this, measures using filter paper urine (for example, Non-Patent Document 1) are taken. obtain.
- the present invention provides a urine sample used for a test for measuring urinary components, which is easy to transport and can obtain a test result equivalent to that of fresh urine even after the sample is transported.
- reagents, kits and methods for storage are provided.
- a kit comprising the reagent according to any one of (1) to (5).
- a method for storing a urine sample comprising a step of contacting the urine sample with a filter paper containing an aromatic alcohol, a phenol derivative or a polyvalent aliphatic alcohol.
- a urine sample used for a test for measuring a urinary component can be easily transported, and a test result equivalent to that of fresh urine can be obtained even after the sample is transported. It is possible to provide reagents, kits and methods for storage.
- A shows the rate of change in the measured values of the filter paper urine sample without phenoxyethanol added and (B) with phenoxyethanol added. It is a graph which shows the relationship between the change rate of the vitamin B1 measured value, and the incubation time.
- (A) shows the rate of change in the measured values of the filter paper urine sample without phenoxyethanol added and (B) with phenoxyethanol added. It is a graph which shows the relationship between the change rate of the vitamin B2 measured value, and the incubation time.
- (A) shows the rate of change in the measured values of the filter paper urine sample without phenoxyethanol added and (B) with phenoxyethanol added. It is a graph which shows the relationship between the change rate of a magnesium measurement value and an incubation time.
- (A) shows the rate of change in the measured values of the filter paper urine sample without phenoxyethanol added and (B) with phenoxyethanol added. It is a graph which shows the relationship between the change rate of the creatinine measurement value of a normal sample, and the incubation time.
- (A) shows the rate of change in the measured values of the filter paper urine sample without phenoxyethanol added and (B) with phenoxyethanol added.
- a to B (A and B are numerical values) shall represent “A or more and B or less” unless otherwise specified.
- % refers to% by weight unless otherwise specified.
- Reagent> A reagent comprising a filter paper, wherein the filter paper contains an aromatic alcohol, a phenol derivative or a polyhydric fatty alcohol, for storing a urine sample.
- the term "reagent” as used herein includes not only compound compounds but also filter papers containing the same.
- the "urine sample" to be stored with the reagent of the present invention is a urine sample collected from a human or an animal to be tested for urinalysis.
- the test target is not particularly limited as long as it is a human or animal that excretes urine, for example, humans, primates including chimpanzees, pet animals such as dogs and cats, and domestic animals such as cows, pigs, horses, sheep and goats. , Mice, rodents such as rats, animals kept in zoos, etc. are included. It is preferably human.
- the pH and salt concentration of urine samples differ greatly from one to another, and there is a problem that bacteria and the like are easily mixed. Since deterioration is likely to occur, the measurement of urinary components is usually performed in a short period after urination, such as fresh urine or 24-hour urine storage. Alternatively, if long-term storage is unavoidable, freezing is performed.
- the present inventors have found that the numerical values of urine samples that have been allowed to stand at room temperature for several days or more, especially filter paper urine, change in some urinalysis items as compared with fresh urine.
- filter paper urine left at room temperature a phenomenon was observed in which the measured value of 8-hydroxydeoxyguanosine (8-OHdG), which is an oxidative stress marker, was higher than that of fresh urine.
- the urine sample in the present invention is filter paper urine.
- filter paper urine refers to a piece of filter paper impregnated with urine by contact (dropping, impregnation, etc.) and dried.
- the difference in weight between the filter paper urine sample and the filter paper before urine contact is due to the difference in weight between the filter paper urine sample and urine immediately after urine contact.
- the weight difference of the filter paper before contact 100%, it can be dried until the weight of the filter paper is 10.0% or less, that is, the weight of the urine sample is 10.0% or less immediately after contact with urine.
- the means for preparing such filter paper urine is not particularly limited, but for example, (1) the filter paper piece is brought into contact with urine and then air-dried at room temperature for several hours to overnight, and (2) the filter paper piece is brought into contact with urine. After that, put it in a plastic bag with a chuck containing a desiccant such as silica gel and leave it at room temperature. (3) After contacting the filter paper piece with urine, put it in a plastic bag with a chuck and put it at room temperature. be able to.
- the drying of the filter paper urine usually proceeds in the order of (2), (1), and (3).
- the term "filter paper” can be any of the same as those used for conventional filter paper blood.
- the material is not particularly limited, but cellulose can be preferably used.
- the thickness, shape and size are also not particularly limited, but for example, the thickness is 0.10 to 4.0 mm, the shape is circular, elliptical, square or rectangular, and the major axis is 3.0 to 150 mm.
- the filter paper preferably has a water absorption of a certain level or higher, and for example, a filter paper having a water absorption degree (JIS L 1907: 2010 water absorption test by the Birec method) of 4.0 cm or more, particularly 6.0 cm, and further 8.0 cm or more. Can be suitably used.
- the amount of the urine sample used in the present invention is not particularly limited, but is preferably 20 to 500 ⁇ L, particularly preferably 50 to 200 ⁇ L, in order to make the filter paper urine in a dry state more easily. It is preferable that the filter paper urine is prepared so that the amount of urine soaked in the filter paper is constant.
- the means for impregnating the filter paper is not particularly limited, but for example, a means for dropping urine into the filter paper using a dropper or the like, a means for immersing the filter paper in the urine in the urine cup for a predetermined time (for example, 3 seconds), or the like can be taken. I can.
- the reagent of the present invention requires that the filter paper for preparing the filter paper urine contains an aromatic alcohol, a phenol derivative or a polyhydric fatty alcohol (hereinafter, also referred to as "aromatic alcohol or the like"). It is prepared by soaking a filter paper with a solution of aromatic alcohol or the like and drying it.
- the means for impregnating the filter paper with a solution such as aromatic alcohol is not particularly limited, and any means such as dropping, spraying, and impregnating can be used.
- any means for drying the filter paper impregnated with the solution any means that does not cause deterioration or decrease in the content of aromatic alcohol or the like can be used, and for example, means such as air drying and vacuum drying can be used.
- the "aromatic alcohol” contained in the reagent of the present invention is not particularly limited as long as it is an alcohol having an aromatic ring, and is, for example, benzyl alcohol, salicyl alcohol, phenethyl alcohol, phenoxyethanol, 3-phenyl-1-propanol and the like, and You can choose from these derivatives.
- phenoxyethanol (formula I) and phenethyl alcohol (formula II) can be preferably used.
- the "phenol derivative" contained in the reagent of the present invention is not particularly limited as long as it is a compound having a phenol structure, and for example, as a substituent, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert.
- -It can be selected from a phenol derivative having a butyl group, a methyl carboxylate group, an ethyl carboxylate group, a propyl carboxylate group, a butyl carboxylate group and the like.
- isopropylmethylphenol (formula III) and propylparaben (formula IV) can be preferably used.
- the "polyhydric aliphatic alcohol” contained in the reagent of the present invention is not particularly limited as long as it is an aliphatic alcohol having two or more hydroxyl groups, and is, for example, ethylene glycol, glycerin, propanediol (propylene glycol), butanediol ( Butylene Glycol), Butane Triol, Pentan Diol, Pentan Triol, Hexan Diol, Hexa Triol, Heptan Diol, Heptan Triol, Octane Diol, Octane Triol, Cyclopentan Diol, Cyclopentan Triol, Methyl Pentan Diol, Cyclo Xentan Diol, Cyclo Xenthan Triol, Methyl It can be selected from cyclohexanediol, cycloheptanediol, cycloheptantriol, cyclooxanediol, cyclooxan
- the reagent of the present invention preferably contains an aromatic alcohol. Above all, it is more preferable to contain phenoxyethanol because it is possible to stabilize the sample for a long period of time at a low concentration.
- the content of aromatic alcohol or the like contained in the reagent of the present invention is preferably 0.5 to 100 mg, particularly 1.0 to 50 mg per 1 mL of the urine sample when the urine sample is applied.
- the content thereof is preferably 1.0 to 50 mg, particularly 7.5 to 20 mg per 1 mL of the urine sample when the urine sample is applied.
- a urine sample stored using the reagent of the present invention is used for measuring urinary components. Before measurement, it is necessary to extract the components to be measured from the filter paper using a liquid medium. Specifically, for example, a means for immersing a filter paper piece in a liquid medium, shaking it for a certain period of time, and collecting the supernatant can be used, but the present invention is not limited thereto.
- a liquid medium water, physiological saline, PBS and the like can be preferably used.
- the urinary component (item) to be measured may be any component measured by a normal urine sample test, for example, sugar, protein, occult blood, ketone body, urobilinogen, bilirubin, albumin, creatin, creatinine, etc.
- the reagent of the present invention includes 8-OHdG, various vitamins (for example, vitamin B1, vitamin B2), various metal ions (for example, magnesium), etc., in which the oxidation of the sample has a great influence on the measured value. It is useful in the examination of urinary components of.
- 8-OHdG is a molecule generated by OH modification of the carbon at the 8-position of the guanidine base when DNA is oxidatively damaged by active oxygen, as shown in the formula VI.
- 8-OHdG in DNA is excreted from DNA by the action of a DNA repair enzyme, excreted extracellularly, and excreted in urine. This 8-OHdG is known as a biomarker that reflects oxidative stress in the living body.
- the measuring means of 8-OHdG is not particularly limited, but can be measured by, for example, an immunoassay method using an anti-8-OHdG monoclonal antibody.
- Competitive methods can preferably be used to measure small molecule substances such as 8-OHdG.
- Antigen 12 (8-OHdG) is immobilized on the surface of a microwell plate, magnetic beads, or the like (microwell plate 11 in the figure) (A).
- A a sample containing antigen 13 (8-OHdG) is added (B).
- the labeled anti-8-OHdG antibody 14 is added thereto to cause an antigen-antibody reaction (C). Unbound molecules are removed by washing (D).
- the label is an enzyme
- take measures such as adding an enzyme substrate to detect the signal derived from the label.
- the greater the amount of 8-OHdG (antigen) in the sample the smaller the signal obtained because most of the labeled antibody binds to the antigen of the sample and is removed by washing.
- the less antigen in the sample the larger the signal obtained.
- a calibration curve can be prepared by using a plurality of 8-OHdG solutions having different concentrations and known concentrations as standard solutions and acquiring each signal in the same manner as in the sample. It is possible to calculate the amount of 8-OHdG in the sample by comparing the signal of the sample with the calibration curve.
- the labeling method for the antibody is not particularly limited as long as it is a labeling method that can be used for immunoassay, such as enzyme labeling (Horseradish peroxidase (HRP), alkaline phosphatase (ALP), etc.), fluorescent labeling, isotope labeling, etc. Any known method can be used, but an enzyme label that does not require special equipment or the like can be preferably used.
- enzyme labeling Haseradish peroxidase (HRP), alkaline phosphatase (ALP), etc.
- fluorescent labeling fluorescent labeling
- isotope labeling etc. Any known method can be used, but an enzyme label that does not require special equipment or the like can be preferably used.
- any known method usually used can be used.
- a method for measuring vitamin B1 for example, measurement by high performance liquid chromatography (HPLC), measurement by LC / MS / MS, and the like are known.
- a method for measuring vitamin B2 for example, measurement by HPLC, measurement by LC / MS / MS, and the like are known.
- any known method commonly used for example, a colorimetric method (xylidyl blue method) or an enzyme method
- a colorimetric method xylidyl blue method
- an enzyme method for example, a colorimetric method (xylidyl blue method) or an enzyme method
- metal ion measuring methods any known method usually used can be used.
- the change with time of the 8-OHdG measured value of the filter paper urine left at 25 ° C. and 37 ° C. is reduced as compared with the case of using a normal filter paper. Demonstrated in.
- Kit > The kit of the present invention is ⁇ 1.
- the kit of the present invention may include an instruction manual explaining the procedure for preparing a urine sample for a subject of a mail inspection.
- a urine collection container such as a urine cup, a plastic bag with a zipper for storing the prepared urine sample, a mailing envelope, a target person information entry form, and the like may be provided as needed.
- the kit of the present invention may include a reagent for measuring a target urinary component together with a reagent for storing a urine sample.
- the urinary component intended here is not particularly limited as long as it is a component measured by a urinalysis, but the kit of the present invention is suitable for measuring a component susceptible to oxidation, for example. Suitable for measuring 8-OHdG. Therefore, preferably, a reagent for measuring 8-OHdG is provided. Unless otherwise explained, the types of reagents for measuring 8-OHdG, measurement conditions, etc. are described in ⁇ 1. As explained in the section of Reagents>.
- the method of the present invention is a method for storing a urine sample, comprising contacting the urine sample with a filter paper containing an aromatic alcohol, a phenol derivative or a polyhydric fatty alcohol. Detailed conditions and the like relating to the method of the present invention are described in ⁇ 1. The conditions are the same as those described in the section of Reagents>.
- the method of the present invention requires that the filter paper urine contains an aromatic alcohol or the like.
- the filter paper is impregnated with aromatic alcohol or the like in advance, dried if necessary, and contacted with urine (dropping, impregnation, etc.) to obtain aromatic alcohol or the like in the obtained filter paper urine. Can be included.
- the amount of aromatic alcohol or the like contained in the filter paper is preferably 0.5 to 100 mg, particularly 1.0 to 50 mg per 1 mL of the urine sample.
- the method of the present invention may include a step of extracting a target urinary component in a stored urine sample.
- Filter paper urine requires a step of extracting the component to be measured from the filter paper using a liquid medium.
- the step of extracting the urinary component can be, for example, a step of immersing a filter paper piece in a liquid medium, shaking it for a certain period of time, and then collecting the supernatant, but the step is not limited to this.
- the liquid medium water, physiological saline, PBS and the like can be preferably used.
- the method of the present invention may further include a step of measuring a target urinary component of a sample extracted from filter paper urine.
- the target urinary component referred to here is usually not particularly limited as long as it is a component measured by urinalysis, but the method of the present invention is suitable for measuring a component susceptible to oxidation, for example.
- Suitable for measuring 8-OHdG. Therefore, preferably, a step of measuring 8-OHdG is provided. Unless otherwise explained, the procedure, principle, etc. for measuring 8-OHdG are described in ⁇ 1. As explained in the section of Reagents>.
- Example 1 Storage stability test of filter paper urine using a filter paper containing phenoxyethanol> (1) Sample preparation A filter paper (cellulose, thickness 1.4 mm) was cut to prepare a 7 mm ⁇ 14 mm filter paper piece. Each filter paper piece was placed in a 1.5 mL polypropylene tube and 45 ⁇ L of 2.0% phenoxyethanol / ethanol solution was added. It was dried in a desiccator with the lid open. 90 ⁇ L of a urine sample containing 200 ng / mL of 8-OHdG was added to the dried filter paper (the final concentration of phenoxyethanol was 1.0% / urine). The tubes were incubated at 25 ° C and 37 ° C for 0, 7, 14 and 21 days.
- Specimens that had been incubated for a predetermined period were stored at ⁇ 20 ° C., returned to room temperature after all incubations were completed, and dried in a vacuum dryer to prepare filter paper urine samples.
- 900 ⁇ L of PBS was added to the tube of each filter paper urine sample, and the mixture was shaken and stirred at room temperature for 1 hour. Each supernatant after stirring was collected to obtain a sample for measurement. The test was performed using the urine of two subjects (subjects A and B).
- the outline of the measurement principle of 8-OHdG is as shown in the above and FIG.
- the 8-OHdG-BSA complex was diluted with PBS, 50 ⁇ L each was dispensed into a 96-well microplate, and the mixture was shaken at room temperature for 1 hour. After washing 4 times with 0.01% Tween20-PBS (TPBS), 250 ⁇ L of 1% BSA / PBS was added and incubated at room temperature for 1 hour. After washing 4 times with TBSB, 50 ⁇ L of each sample prepared in (1), positive control and 8-OHdG standard solution having a known concentration were added. 50 ⁇ L of each HRP-labeled anti-8-OHdG antibody solution was dispensed.
- TMB tetramethylbenzidine
- Tables 1 and 2 The numerical values in the table were calculated with the 8-OHdG measured value on the 0th day of the sample under the same conditions as 100.
- Table 1 shows the measurement results of each filter paper urine when the filter paper urine was incubated at 25 ° C.
- Table 2 shows the measurement results of each filter paper urine when the filter paper urine was incubated at 37 ° C.
- the underlined values indicate the values at which the measured values were higher than 10% and lower than 10% compared to the 0th day. It was shown that the measured values were stabilized in the filter paper urine supplemented with phenoxyethanol at both 25 ° C. and 37 ° C. conditions.
- Example 2 Storage stability test of filter paper urine using filter paper containing various components (8-OHdG)> Filter paper urine was prepared in the same manner as in Example 1 except that the solutions having the concentrations shown in Table 3 were added instead of the 1.0% phenoxyethanol / ethanol solution. Tubes containing each filter paper urine were incubated at 25 ° C and 37 ° C for 0,7,14 days. Specimens that had been incubated for a predetermined period were stored at ⁇ 20 ° C., returned to room temperature after all incubations were completed, and dried in a vacuum dryer to prepare filter paper urine samples. 900 ⁇ L of PBS was added to the tube of each filter paper urine sample, and the mixture was shaken and stirred at room temperature for 1 hour. Each supernatant after stirring was collected to obtain a sample for measurement. The test was performed using the urine of one subject. 8-OHdG was measured by the same method as in Example 1.
- Tables 4, 5 and 6 show the measurement results of 8-OHdG in filter paper urine supplemented with aromatic alcohols, phenol derivatives and polyhydric fatty alcohols, respectively.
- the numerical values in the table were calculated with the 8-OHdG measured value on the 0th day of the sample under the same conditions as 100.
- the underlined values indicate the values at which the measured values were higher than 10% and lower than 10% compared to the 0th day.
- the stability of the filter paper urine having a final concentration of 1.0% was superior to that of the filter paper urine without the addition.
- Phenethyl alcohol had excellent filter paper urine stability at final concentrations of 1.0% and 5.0%.
- Example 3 Measurement value stability test of various measurement items of filter paper urine> The stability of the measured values of 8-OHdG, vitamin B1, vitamin B2 and magnesium in the filter paper urine sample was investigated.
- Filter paper urine was prepared in the same manner as in Example 1 except that urine to which 8-OHdG was not added was used.
- As a control filter paper urine was prepared using filter paper containing no phenoxyethanol. Tubes containing each filter paper urine were incubated at 25 ° C and 37 ° C for a predetermined period of 0-21 days. Specimens that had been incubated for a predetermined period were stored at ⁇ 20 ° C., returned to room temperature after all incubations were completed, and dried in a vacuum dryer to prepare filter paper urine samples. 900 ⁇ L of PBS was added to the tube of each filter paper urine sample, and the mixture was shaken and stirred at room temperature for 1 hour. Each supernatant after stirring was collected to obtain a sample for measurement.
- FIG. 2 is a graph showing the relationship between the rate of change in 8-OHdG measured values and the incubation time.
- FIG. 2A shows the rate of change in the measured values of the filter paper urine sample without phenoxyethanol added and FIG. 2B shows with phenoxyethanol added.
- FIG. 3 is a graph showing the relationship between the rate of change in vitamin B1 measured values and the incubation time.
- FIG. 3A shows the rate of change in the measured values of the filter paper urine sample without phenoxyethanol added and
- FIG. 3B shows the filter paper urine sample with phenoxyethanol added.
- FIG. 4 is a graph showing the relationship between the rate of change in vitamin B2 measured values and the incubation time.
- FIG. 4A shows the rate of change in the measured values of the filter paper urine sample without phenoxyethanol added and FIG. 4B shows the filter paper urine sample with phenoxyethanol added.
- FIG. 5 is a graph showing the relationship between the rate of change in magnesium measurement values and the incubation time.
- FIG. 5A shows the rate of change in the measured values of the filter paper urine sample without phenoxyethanol added and FIG. 5B shows with phenoxyethanol added.
- Example 4 Change in creatinine measurement value depending on the degree of dryness of filter paper urine> The stability of creatinine measurements in filter paper urine samples was investigated.
- Filter paper urine was prepared in the same manner as in Example 3.
- As a control filter paper urine was prepared using filter paper containing no phenoxyethanol (normal sample). Tubes containing each filter paper urine were incubated at 25 ° C and 37 ° C for a predetermined period of 0-21 days. At this time, it was confirmed that all the filter papers were in a dry state in appearance after 7 days had passed.
- FIG. 6 is a graph showing the relationship between the rate of change in creatinine measured values and the incubation time in a normal sample of filter paper urine.
- FIG. 6A shows the rate of change in the measured values of the filter paper urine sample without phenoxyethanol added
- FIG. 6B shows the filter paper urine sample with phenoxyethanol added.
- the measured value of creatinine gradually decreased in a high temperature state of 37 ° C., and in particular, after the 7th day, there was a tendency that the measured value greatly decreased by more than 20%.
- the measured values were relatively stable under the condition of 25 ° C. From these facts, it was found that it is desirable to properly control the temperature or to process the sample earlier in order to measure the filter paper urine sample more accurately.
- FIG. 7 is a graph showing the relationship between the rate of change in creatinine measured values and the incubation time in a dried sample of filter paper urine.
- FIG. 7A shows the rate of change in the measured values of the filter paper urine sample without phenoxyethanol added
- FIG. 7B shows with phenoxyethanol added.
- the measured creatinine value did not tend to decrease.
- Example 5 Dry state by means for storing filter paper urine> A filter paper (cellulose, thickness 1.4 mm) having a width of 7 mm and a length of 14 mm was attached to one end of a strip-shaped plastic slice having a width of 7 mm and a length of 85 mm in the longitudinal direction to prepare a filter paper stick. The part of the plastic flakes without filter paper was used as the handheld part. The urine sample was placed in a urine cup, and the filter paper portion of the filter paper stick was immersed in the urine sample to impregnate the filter paper with urine. When removing the filter paper stick from the urine sample, the filter paper portion was pressed against the wall of the urine cup to remove excess liquid.
- a filter paper cellulose, thickness 1.4 mm
- a plastic bag with a chuck (Horiaki Co., Ltd., product name "wrap-in", PE-4) with and without a desiccant (manufactured by Toyota Kako Co., Ltd., silica gel 10 g) was prepared.
- a filter paper stick impregnated with urine was placed in a plastic bag containing and without a desiccant.
- Each plastic bag was allowed to stand at room temperature for 6 days, and the weight change of the filter paper stick during this period was observed.
- each plastic bag was allowed to stand in a desiccator for 6 days, and the weight change of the filter paper stick was observed.
- the room temperature was 27 ° C. and the relative humidity was 33 to 55%.
- the temperature in the desiccator was 27 ° C. and the relative humidity was 24-33%.
- Table 7 shows the change in the weight of the filter paper stick in the plastic bag with and without the desiccant left at room temperature.
- the difference between the weight immediately after urine impregnation and the weight before urine impregnation of the filter paper stick is 100%, the time until the difference from the weight before urine impregnation becomes 10% or less is required as the "drying time". The time was measured. Under the condition of containing a desiccant, the drying time was 19.5 to 24 hours. On the other hand, under the condition without a desiccant, it took 115.5 hours to reach the same state.
- Table 8 shows the change in the weight of the filter paper in the plastic bag with and without the desiccant left in the desiccator.
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Abstract
Description
(1)ろ紙を備え、前記ろ紙が芳香族アルコール、フェノール誘導体又は多価脂肪族アルコールを含む、尿検体を保存するための試薬。
(2)ろ紙の芳香族アルコール、フェノール誘導体又は多価脂肪族アルコールの含有量が、適用される尿検体1mLあたり0.5~100mgである、(1)の試薬。
(3)前記ろ紙がフェノキシエタノールを含む、(1)又は(2)に記載の尿検体を保存するための試薬。
(4)ろ紙のフェノキシエタノール含有量が、適用される尿検体1mLあたり1.0~50mgである、(3)に記載の試薬。
(5)前記尿検体が、8-ヒドロキシデオキシグアノシン、ビタミン類、金属イオンからなる群より選択される、少なくとも1つの含有量を測定するための検体である、(1)~(4)のいずれかに記載の試薬。
(6)(1)~(5)のいずれかに記載の試薬を備える、キット。
(7)尿検体を、芳香族アルコール、フェノール誘導体又は多価脂肪族アルコールを含むろ紙に接触させる工程を備える、尿検体を保存するための方法。
(8)ろ紙の芳香族アルコール、フェノール誘導体又は多価脂肪族アルコールの含有量が、適用される尿検体1mLあたり0.5~100mgである、(7)に記載の方法。
ろ紙を備え、前記ろ紙が芳香族アルコール、フェノール誘導体又は多価脂肪族アルコールを含む、尿検体を保存するための試薬である。ここでいう「試薬」は、各化合物成のみならず、これを含むろ紙をも包含するものとする。
本発明のキットは、<1.試薬>の項で説明した試薬を備える。上記の通り、「試薬」は、各化合物のみならず、これを含むろ紙を包含する。本発明のキットに係る詳細な条件等は、別途の説明のない、<1.試薬>の項に記載した条件と同様である。
本発明の方法は、尿検体を、芳香族アルコール、フェノール誘導体又は多価脂肪族アルコールを含むろ紙に接触させる工程を備える、尿検体を保存するための方法である。本発明の方法に係る詳細な条件等は、別途の説明のない限り、<1.試薬>の項に記載した条件と同様である。
(1)試料調製
ろ紙(セルロース、厚さ1.4mm)をカットして7mm×14mmのろ紙片を作製した。各ろ紙片を1.5mLポリプロピレンチューブに入れ、2.0%フェノキシエタノール/エタノール溶液45μLを加えた。フタを開けたまま減圧乾燥器で乾燥させた。乾燥後のろ紙に、8-OHdGを200ng/mL含む尿検体を90μL添加した(フェノキシエタノール終濃度は1.0%/尿)。チューブを、25℃及び37℃で、0、7、14及び21日間インキュベートした。所定期間のインキュベートが終了した検体は、-20℃で保管し、全てのインキュベートが終了した後に常温に戻し、減圧乾燥器で乾燥させ、ろ紙尿検体を調製した。各ろ紙尿検体のチューブにPBSを900μL添加して、室温で1時間振とう攪拌を行った。攪拌後の各上清を回収して、測定用の試料を得た。試験は2名の被験者(被験者A、B)の尿を用いて行った。
8-OHdGの測定原理の概略は、前記及び図1に示す通りである。8-OHdG-BSA複合体をPBSで希釈して、96ウェルマイクロプレートに50μLずつ分注し、室温で1時間振とうした。0.01%Tween20-PBS(TPBS)で4回洗浄した後、1%BSA/PBS 250μLを添加して室温で1時間インキュベートした。TPBSで4回洗浄した後、(1)で調製した各試料、ポジティブコントロール及び濃度既知の8-OHdG標準液をそれぞれ50μL添加した。HRP標識抗8-OHdG抗体溶液を各50μL分注した。10分間振とう後、室温で50分間インキュベートした。TPBSで4回洗浄した後、テトラメチルベンジジン(TMB)100μLを分注して10分間静置して発色させて、1Mリン酸50μLを分注して停止させた。450nm/570nmの吸光度を測定した。
各試料の測定結果を表1及び表2に示す。表中の数値は、同じ条件の検体の0日目の8-OHdG測定値を100として算出した。表1はろ紙尿を25℃でインキュベートした際の、各ろ紙尿の測定結果を示す。表2は、ろ紙尿を37℃でインキュベートした際の、各ろ紙尿の測定結果を示す。下線は、測定値が、0日目と比較して10%を超えて高くなった数値、及び10%を超えて低くなった数値を示す。25℃、37℃の両方の条件において、フェノキシエタノールを添加したろ紙尿では、測定値が安定化することが示された。
1.0%フェノキシエタノール/エタノール溶液に代えて、表3に示す溶液の濃度の溶液をそれぞれ添加した以外は、実施例1と同様の方法でろ紙尿を調製した。各ろ紙尿の入ったチューブを、25℃及び37℃で、0、7、14日間インキュベートした。所定期間のインキュベートが終了した検体は、-20℃で保管し、全てのインキュベートが終了した後に常温に戻し、減圧乾燥器で乾燥させ、ろ紙尿検体を調製した。各ろ紙尿検体のチューブにPBSを900μL添加して、室温で1時間振とう攪拌を行った。攪拌後の各上清を回収して、測定用の試料を得た。試験は1名の被験者の尿を用いて行った。実施例1と同様の方法にて、8-OHdGの測定を行った。
ろ紙尿検体における、8-OHdG、ビタミンB1、ビタミンB2及びマグネシウムの測定値の安定性を調査した。8-OHdGを添加しない尿を使用した以外は、実施例1と同様の方法でろ紙尿を調製した。対照として、フェノキシエタノールを含まないろ紙を用いてろ紙尿を調製した。各ろ紙尿の入ったチューブを、25℃及び37℃で、0~21日間の所定期間インキュベートした。所定期間のインキュベートが終了した検体は、-20℃で保管し、全てのインキュベートが終了した後に常温に戻し、減圧乾燥器で乾燥させ、ろ紙尿検体を調製した。各ろ紙尿検体のチューブにPBSを900μL添加して、室温で1時間振とう攪拌を行った。攪拌後の各上清を回収して、測定用の試料を得た。
ろ紙尿検体における、クレアチニン測定値の安定性を調査した。実施例3と同様の方法でろ紙尿を調製した。対照として、フェノキシエタノールを含まないろ紙を用いてろ紙尿を調製した(通常検体)。各ろ紙尿の入ったチューブを、25℃及び37℃で、0~21日間の所定期間インキュベートした。この際、7日間経過時点で、いずれのろ紙も外観上乾燥した状態であることが確認された。一方、フェノキシエタノール含有/非含有ろ紙片に、尿検体を90μL添加した後、室温で6時間風乾し、次いで25℃及び37℃の条件下で、所定時間開放状態で静置した(乾燥検体)。所定期間のインキュベートが終了した検体は、-20℃で保管し、全てのインキュベートが終了した後に常温に戻し、減圧乾燥器で乾燥させ、ろ紙尿検体を調製した。各ろ紙尿検体のチューブにPBSを900μL添加して、室温で1時間振とう攪拌を行った。攪拌後の各上清を回収して、測定用の試料を得た。各ろ紙尿検体のクレアチニンの測定は、通常の酵素法を用いて実施した(n=7)。
幅7mm×長さ85mmの帯状のプラスチック薄片の長手方向一方端部に、幅7mm×長さ14mmのろ紙(セルロース、厚さ1.4mm)を取り付け、ろ紙スティックを作製した。プラスチック薄片のろ紙のない部分を手持ち部分とした。尿検体を尿カップに入れ、ろ紙スティックのろ紙部分を尿検体に浸漬してろ紙に尿を含浸させた。ろ紙スティックを尿検体から取り出す際に、ろ紙部分を尿カップの壁部に押し当てて、余分な液体を除去した。チャック付きポリ袋(ホリアキ株式会社、製品名「ラップイン」、PE-4)に乾燥剤(豊田化工株式会社製、シリカゲル10g)を入れたものと入れていないものを準備した。乾燥剤入り、乾燥剤なしのポリ袋に、それぞれ、尿を含浸させたろ紙スティックを入れた。各ポリ袋を、室温で6日間静置し、この間のろ紙スティックの重量変化を観察した。同様に、各ポリ袋をデシケーター内に6日間静置し、ろ紙スティックの重量変化を観察した。観察期間の室温は27℃、相対湿度は33~55%であった。デシケーター内の温度は27℃、相対湿度は24~33%であった。各条件のポリ袋及びろ紙スティック尿検体をn=3で準備した。
Claims (8)
- ろ紙を備え、前記ろ紙が芳香族アルコール、フェノール誘導体又は多価脂肪族アルコールを含む、尿検体を保存するための試薬。
- ろ紙の芳香族アルコール、フェノール誘導体又は多価脂肪族アルコールの含有量が、適用される尿検体1mLあたり0.5~100mgである、請求項1に記載の試薬。
- 前記ろ紙がフェノキシエタノールを含む、請求項1又は2に記載の尿検体を保存するための試薬。
- ろ紙のフェノキシエタノール含有量が、適用される尿検体1mLあたり1.0~50mgである、請求項3に記載の試薬。
- 前記尿検体が、8-ヒドロキシデオキシグアノシン、ビタミン類、金属イオンからなる群より選択される、少なくとも1つの含有量を測定するための検体である、請求項1~4のいずれか1項に記載の試薬。
- 請求項1~5のいずれか1項に記載の試薬を備える、キット。
- 尿検体を、芳香族アルコール、フェノール誘導体又は多価脂肪族アルコールを含むろ紙に接触させる工程を備える、尿検体を保存するための方法。
- ろ紙の芳香族アルコール、フェノール誘導体又は多価脂肪族アルコールの含有量が、適用される尿検体1mLあたり0.5~100mgである、請求項7に記載の方法。
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