WO2005012557A1 - 赤血球採取方法及びソルビトール測定方法 - Google Patents
赤血球採取方法及びソルビトール測定方法 Download PDFInfo
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- WO2005012557A1 WO2005012557A1 PCT/JP2004/011045 JP2004011045W WO2005012557A1 WO 2005012557 A1 WO2005012557 A1 WO 2005012557A1 JP 2004011045 W JP2004011045 W JP 2004011045W WO 2005012557 A1 WO2005012557 A1 WO 2005012557A1
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- sorbitol
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
Definitions
- the present invention relates to a method for measuring sorbitol, a method for collecting red blood cells, a method for measuring sorbitol in red blood cells using the same, and a kit or device thereof.
- Serum, plasma or whole blood is mainly used for blood tests in clinical tests.
- whole blood is a measurement target
- whole blood is used as it is
- serum or plasma is a measurement target
- centrifuged supernatant is used for simple test reagents.
- a method of effectively using a separation membrane to collect serum or plasma has been implemented.
- erythrocytes are used as test subjects
- erythrocytes are collected after separating erythrocytes from serum or plasma components by centrifugation to remove serum or plasma components in the supernatant.
- a step of washing several times with a physiological saline solution is also performed in order to completely remove serum or plasma components. This requires several additional centrifugation operations, which is complicated.
- Sorbitol is a representative polyol of a polyol metabolite produced from gnorecose by the action of aldose reductase. Sorbitol is said to accumulate in cells due to enhanced polyol metabolism in diabetic patients, which is one of the factors that cause diabetic complications. Therefore, the measurement of sorbitol in tissues has been used as an index reflecting the metabolism of polyols in tissues in diabetic patients.
- the amount of sorbitol in tissues is correlated with the amount of sorbitol in erythrocytes, which indicates that the measurement of sorbitol in erythrocytes can be used to determine diabetic neuropathy, diabetic retinopathy, diabetic nephropathy. It is considered to be useful for diagnosis and management of diabetic complications such as diabetes.
- sorbitol in erythrocytes For the measurement of sorbitol in erythrocytes, a method of measuring NADH or NADPH generated by the action of sorbitol dehydrogenase is generally used.
- the measurement of the produced NADH or NADPH is carried out by a method of measuring the fluorescence of NADH or NADPH itself (Diabetes 29: 861-864, 1980, Medicine and Pharmacy 43 (3), 555, 2000), a formazan coloring method ( 6-189790), a method of generating hydrogen peroxide from NADH or NADPH and measuring by color development (JP-A-6-209793) or fluorescence (JP-A-6-109726) has been proposed.
- the method of measuring the fluorescence of NADH or NADPH itself from the surface is mainly used.
- Measuring sorbitol in erythrocytes which directly measures the fluorescence emitted by NADH or NADPH, requires a large amount of erythrocytes, and the measurement device is implemented using a general-purpose fluorometer and a square cell. Since this measurement operation is very complicated, it is difficult to perform daily measurement in a laboratory or the like.
- some instruments such as a fluorescent plate reader, can measure a large number of samples at the same time.Power is low for a fluorescent plate reader that has lower detection sensitivity than a general fluorometer because the fluorescence intensity of the NADH or NADPH to be measured is low. In reality, it is impossible to measure with high accuracy.
- Patent Document 1 JP-A-6-189790
- Patent Document 2 JP-A-6-209793
- Patent Document 3 JP-A-6-109726
- Non-Patent Document 1 Diabetes 29: 861-864, 1980
- Non-patent Document 2 Medicine and Pharmacy 43 (3), 555, 2000
- red blood cells In clinical tests, the measurement of components in red blood cells has hardly been performed.
- the method of collecting red blood cells as a measurement sample is complicated. Since red blood cells are collected by centrifugation, expensive centrifugation equipment is required. Clear serum or plasma components must be removed. Further, when washing red blood cells, it is necessary to repeat the centrifugation operation and the operation of removing the supernatant several times, which is not suitable for general examination. Under these circumstances, the measurement of sorbitol in red blood cells, which is considered to be useful for the diagnosis and management of diabetic complications, is very complicated because the collection of red blood cells is complicated, and the measurement method is complicated. As it is not suitable for general use, it has not yet reached general widespread use.
- the present inventors diligently studied to solve the above-mentioned problems, and first devised a simple method for collecting red blood cells. That is, after blood is added to an erythrocyte holding layer capable of holding erythrocytes in blood and a serum or plasma component is penetrated into an absorbing layer capable of absorbing serum or plasma existing in contact with the erythrocyte holding layer, According to the method for collecting red blood cells from the retained red blood cell holding layer, it has been found that red blood cells can be easily collected from whole blood.
- the present invention provides a method in which blood is added to an erythrocyte holding layer capable of holding erythrocytes in blood, and a serum or plasma component is added to an absorption layer capable of absorbing serum or plasma existing in contact with the erythrocyte holding layer. After the permeation of the red blood cells, the red blood cell-retaining layer holding the red blood cells is immersed in a red blood cell component extract to extract red blood cell components, and the extract is subjected to a protein removal operation.
- An object of the present invention is to provide a method for measuring sorbitol in erythrocytes, wherein DP and diaphorase are added and reacted to measure the fluorescence of a generated phosphor.
- the present invention is also characterized in that sorbitol dehydrogenase, a fluorescent substance, NAD or NADP, and diaphorase are added to a sample to be measured for sorbitol and reacted, and the resulting phosphor is measured for fluorescence.
- the present invention further comprises adding blood to an erythrocyte holding layer capable of holding erythrocytes in blood, and adding serum or plasma components to an absorption layer capable of absorbing serum or plasma existing in contact with the erythrocyte holding layer.
- a method for collecting erythrocytes or erythrocyte components characterized by immersing the erythrocyte holding layer holding erythrocytes after permeation into a solution to collect erythrocytes or erythrocyte components.
- the present invention provides an apparatus and a kit for performing the method. That is, it has an erythrocyte holding layer capable of holding erythrocytes in blood, and an absorbing layer which is in contact therewith and can absorb serum or plasma, wherein the erythrocyte holding layer is removable. And a sorbitol measurement kit comprising sorbitol dehydrogenase, NAD or NADP, diaphorase, and a fluorescent substrate, as well as an erythrocyte collection device for collecting erythrocytes from blood.
- the present invention has made it possible to easily carry out erythrocyte collection, which has been complicated in a clinical test. Further, the present invention has conventionally required several centrifugation operations to obtain washed red blood cells, whereas another centrifugation operation is required to obtain the washed red blood cells by a very simple operation without any centrifugation. An advantageous advantage has also resulted. In addition, the sorbitol measurement method of the present invention has extremely high sensitivity, so that sorbitol can be measured even in a small amount of red blood cells.
- the measurement of sorbitol in erythrocytes becomes extremely easy, resulting in diabetic neuropathy, diabetic retinopathy, and diabetes. Diagnosis and management of diabetic complications such as sexual nephropathy can be easily implemented.
- FIG. 1 is a cross-sectional view of an erythrocyte collection device type A.
- FIG. 2 is a cross-sectional view of the erythrocyte collection device type A in a state where a unit including a erythrocyte holding layer is removed.
- FIG. 3 is a cross-sectional view of a red blood cell collection device type B.
- FIG. 4 is a cross-sectional view of a device provided with a mechanism for facilitating removal of a red blood cell holding layer and an auxiliary tool in a red blood cell collection device type B.
- FIG. 5 is a graph showing the relationship between the blood volume and the red blood cell collection amount in Example 1.
- FIG. 6 is a graph showing the relationship (calibration curve) between sorbitol concentration and fluorescence intensity in Example 2.
- FIG. 7 is a graph showing the correlation between the method of the present invention and the conventional method in Example 3.
- blood is added to an erythrocyte holding layer capable of holding erythrocytes in blood, and serum or plasma components are penetrated into an absorption layer capable of absorbing serum or plasma existing in contact with the erythrocyte holding layer.
- the erythrocyte holding layer holding the erythrocytes is immersed in the erythrocyte component extract to extract the erythrocyte component, the extract is subjected to a protein removal operation, and the extract after the deproteinization is performed. Sorbitol dehydrogenase, a fluorescent substrate, NAD or NADP, and diaphorase, and reacting the resulting mixture to measure the fluorescence of the resulting phosphor.
- the method for measuring sorbitol in erythrocytes will be described in detail.
- the red blood cell holding layer may be any layer that can hold red blood cells.
- a plasma separation membrane which is a sheet-like membrane, is used.
- commercially available plasma separation membranes made of glass fiber, polyester, polyethersulfone, and the like can be used.
- an anti-red blood cell antibody, a lectin, and the like can be contained in the red blood cell holding layer in order to easily hold the red blood cells.
- the absorbent layer may be any one that can absorb the aqueous solution, such as filter paper, nonwoven fabric, and cotton.
- the size is preferably the same or larger than the erythrocyte retention layer.
- filter paper is preferred.
- filter paper in a form such as holder type B, filter paper, nonwoven fabric, cotton, etc. can be used.
- the amount of blood added to the red blood cell holding layer depends on the volume of the red blood cell holding layer. Although the optimum amount of added calorie may be determined in advance by an experiment, it is preferable to add a slightly larger amount of blood than the extent that the entire surface of the red blood cell holding layer becomes red. Usually, it is suitable to provide a red blood cell holding layer of a size capable of holding red blood cells, with a blood force of 10 ⁇ L-500 ⁇ L, preferably 50 ⁇ L-200 ⁇ L.
- the blood added to the erythrocyte holding layer allows its serum or plasma components to penetrate into the absorbing layer due to the contact between the erythrocyte holding layer and the absorbing layer.
- the erythrocyte holding layer and the absorption layer can be brought into contact with each other as separate devices at the time of use. However, it is preferable to use the erythrocyte holding layer and the absorption layer that have been brought into contact from the beginning to form one device. That is, it is optimal to use the red blood cell collection device of the present invention as described below.
- red blood cells may reach the absorption layer.
- the maximum amount of red blood cells retained depends on the volume of the red blood cell holding layer, the collection of red blood cells is performed. Has no effect. Further, by adding an isotonic solution such as physiological saline to the erythrocyte holding layer, the washed erythrocytes can be collected.
- the red blood cell component is collected from the red blood cell holding layer by placing the red blood cell holding layer in a container such as a test tube containing the red blood cell component extract.
- the main purpose is to extract sorbitol in erythrocytes, and as the erythrocyte component extract, a liquid having a hemolytic action may be used.
- a liquid having a hemolytic action purified water is preferable in consideration of the influence on the measurement system and the like, but an aqueous solution containing salted ammonium, saponin, a surfactant and the like may be used.
- red blood cell holding layer If it is desired to immediately extract the components in the red blood cells from the red blood cell holding layer, it may be stirred. If not used immediately, for example, if left in a refrigerator, the components are completely extracted from the red blood cell holding layer in a few hours. If you want to collect red blood cells from the red blood cell holding layer in the form of cells, use an isotonic saline solution instead of the red blood cell component extract. A solution may be used.
- the extracted liquid is subjected to a protein removal operation in order to remove hemoglobin and the like contained in red blood cells.
- a perchloric acid method is particularly preferable if an existing protein removing method such as a perchloric acid method, a trichloroacetic acid method, or a zinc hydroxide method is used.
- the perchloric acid method is to remove a precipitate formed by mixing a sample and a solution containing perchloric acid, and then add potassium carbonate or the like to the supernatant to remove excess perchloric acid. Is precipitated as an insoluble potassium salt and removed.
- a suspension of an ion exchanger is used as the above-described component extract in red blood cells, hemolysis and deproteinization can be performed simultaneously with red blood cell collection. It is very easy to collect a sample from which protein has been removed.
- the ion exchanger used may be a cation exchanger or an anion exchanger, but it is preferable to use an anion exchanger.
- the pH of the suspension may be set so long as hemoglobin and protein adsorb to the ion exchanger and sorbitol does not adsorb.
- optimal conditions may be determined according to the performance of the anion exchanger to be used, where weak alkalinity of about neutral pHIO is preferred.
- pH 8-9 for DEAE Sephadettas manufactured by Pharmacia and purified water can be used for the anion-exchange cellulose DE53 manufactured by Whatman without pH adjustment.
- the optimal concentration of the ion exchanger in the suspension should be determined according to the amount of red blood cells.
- the sorbitol measurement method of the present invention can measure serum or plasma, tissues, and trace samples of sorbitol contained in cells, which can be measured not only by sorbitol in erythrocytes, but also can be obtained from animals such as mice, rats, etc., not only human samples. The sample can also be measured with high accuracy.
- the sorbitol dehydrogenase used in the present invention is a sorbitol dehydrogenase in the presence of NAD or NADP.
- NAD or NADP sorbitol dehydrogenase in the presence of NAD or NADP.
- sorbitol dehydrogenase derived from the genus Flavimonas or the genus Pseudomonas is preferable because of its excellent specificity.
- the fluorescent substrate used in the present invention is preferably resazurin or alama blue, which has a strong and stable fluorescent intensity, as long as it is reduced to a fluorescent substance in the presence of NADH or NADPH and diaphorase.
- Masire, The origin and the like of the diaphorase used in the present invention are not particularly limited as long as it acts on NADH or NADPH to change a fluorescent substrate into a fluorescent substance.
- the erythrocyte collection device of the present invention has an erythrocyte holding layer capable of holding erythrocytes in blood, and an absorption layer that is in contact with and absorbs serum or plasma, and the erythrocyte holding layer is removable.
- This is a red blood cell collection device that collects red blood cells from blood. Erythrocyte collection power
- This device is suitable for performing the process up to the extraction of red blood cell components.
- the red blood cell collecting apparatus is also applied to the part of the process up to the red blood cell collection described above as it is.
- the substantial parts will be described with reference to the drawings, taking Type A and Type B as examples. These are examples of the red blood cell collection device of the present invention.
- Type A is composed of unit B1 and unit B2 as shown in FIG.
- the unit B2 has a structure in which the absorbing layer A4 is bonded to the supporting part A1, and the unit B1 is composed of the supporting part A2 and the red blood cell holding layer A3.
- the unit B1 and the unit B2 are in a state where the red blood cell holding layer A3 and the absorption layer A4 overlap each other without bonding.
- the unit B1 and the unit B2 are physically bonded only at a part where they are in contact with each other, and have a structure that can be easily separated.
- a double-sided tape, an adhesive or the like can be used for each bonding.
- the material of the support portions A1 and A2 is preferably a plastic strip and a thin piece of polystyrene or the like.
- red blood cells In order to collect red blood cells by type A, first, blood is added to the red blood cell holding layer A3 of the unit B1. In the added whole blood, red blood cells are retained in the blood cell retention layer A3, and the serum or plasma components move to the absorption layer A4 of the unit B2. Then, remove unit B1 from unit B2. Remove.
- the unit B1 is placed in a container such as a test tube containing the red blood cell component extract, the red blood cell component can be collected in the extract. Also, if it is put into an isotonic solution, it can be collected as red blood cells.
- Type B has a configuration in which the red blood cell holding layer A3 and the absorption layer A4 are accommodated in holders A5 and A6.
- the erythrocyte holding layer A3 and the absorption layer A4 overlap and are in close contact, and the absorption layer A4 is sandwiched and fixed between the holder A5 and the holder A6.
- the erythrocyte holding layer A3 is not sandwiched between the holder A5 and the holder A6, and can be easily removed. As shown in FIG.
- the holder A6 can be provided with a hole A7 at the center so that an auxiliary tool B5 for removing the red blood cell holding layer A3 can be used.
- the auxiliary tool B5 is a stick having a thickness enough to fit into the hole A7 of the holder B6, which penetrates the absorption layer A4 and pushes and removes the red blood cell holding layer A3.
- red blood cells in type B it is basically the same as in type A. However, in the case of type B, after the serum or plasma components have moved to the absorption layer A4, set the device upside down in a container such as a test tube containing the extract of red blood cell components, and place the red blood cells in the container. Retaining layer A3 can be removed. If it is difficult to remove the red blood cell holding layer A3, the auxiliary tool B5 can be inserted through the hole A7 and removed.
- the sorbitol measurement according to the present invention can also be carried out using the following sorbitol measurement kit of the present invention.
- the sorbitol measurement kit of the present invention contains at least sorbitol dehydrogenase, NAD or NADP, diaphorase, and a fluorescent substrate. These components may be mixed together to form one reagent, or the components may be appropriately combined and divided. Specifically, the stability is better when the reagent (a) is divided into an enzyme reagent containing sorbitol dehydrogenase and NAD or NADP and diaphorase, and the reagent (b) is a fluorescent substrate. Is preferred. Each of these components or reagents may be in the form of a solution or a lyophilized product, and may contain, in addition to the above components, a buffering agent for pH adjustment, a stabilizer, and the like.
- the above reagents are impregnated into test paper or the like to form a dry test piece.
- the sorbitol measurement kit of the present invention can add a reagent (c): a deproteinizing agent, if necessary. Further, in order to measure sorbitol in erythrocytes, reagent (d): an extract of erythrocyte components can be further added. All of these components have already been described.
- the sorbitol measurement kit of the present invention further comprises an erythrocyte holding layer capable of holding erythrocytes in blood, which is the device of the present invention, and an absorption layer present in contact therewith and capable of absorbing serum or plasma.
- an erythrocyte collection device from which the erythrocyte holding layer is detachable and having the same package the kit is extremely convenient for measuring sorbitol in erythrocytes.
- the sorbitol measurement according to the present invention can be carried out by a general-purpose fluorometer using each cell by reacting in a test tube, but a large number of samples can be measured simultaneously with a small amount of a sample using a 96-well microplate.
- a fluorescent plate reader is used.
- a red blood cell collecting apparatus was prepared in which GA100 (Advantech) cut into a circular shape of 6 mm was used as an erythrocyte holding layer and an AP10 absorption pad (Millipore) was used as an absorption layer. To this, 100 ⁇ L of whole blood and 200 ⁇ L were added, and the mixture was left until completely absorbed. Thereafter, three drops of physiological saline were added to completely absorb the erythrocytes, and the erythrocyte holding layer was taken out and placed in a test tube containing 1 mL of purified water to collect components in erythrocytes. As a control, 100 ⁇ L / 200 ⁇ L of whole blood was put in 1 mL of purified water, and the hemoglobin concentration in this solution was measured to determine the relationship between blood volume and red blood cell recovery.
- GA100 Advancedtech
- FIG. 5 shows the results.
- the erythrocyte collection device prepared this time showed a maximum and constant hemoglobin concentration at 150 ⁇ L or more of whole blood, indicating that red blood cells could be collected without using a centrifuge. . That is, when the hematocrit value was 50%, it was considered that up to 75 ⁇ L of red blood cells could be collected.
- Reagent A 0.3 mg of resazurin (manufactured by Wako Pure Chemical Industries) was dissolved in 60 mL of purified water.
- Test Solution B 260 mg of -NAD (manufactured by Oriental), 2000 units of diaphorase (manufactured by Unitika), and 100 units of sorbitol dehydrogenase (manufactured by Unitika) were dissolved in 100 mL of Tris buffer (pH 8.2).
- FIG. 6 shows the results. As is clear from the figure, since good linearity was observed between the sorbitol concentration and the fluorescence intensity, it was obtained that the sorbitol can be measured by the method of the present invention.
- the sorbitol concentration of 30 human erythrocyte samples was measured using the conventional method and the method of the present invention in Example 2.
- Blood was collected from 30 diabetic patients, and red blood cells were obtained by centrifugation.
- a mixture of 20 ⁇ L of red blood cells and 180 ⁇ L of purified water was used as a measurement sample.
- a mixture of 500 ⁇ L of red blood cells and 2 mL of purified water was used as a measurement sample.
- the measurement of sorbitol according to the method of the present invention was carried out according to Example 2.
- the conventional method of measuring sorbitol is a method of measuring the fluorescence intensity of NADH generated by the enzymatic reaction of sorbitol dehydrogenase, and the measurement was performed using a Hitachi F-2500 fluorometer using a square cell.
- FIG. 7 shows the results. As is clear from the figure, there is a high correlation between the method of the present invention and the conventional method. Thus, the method of the present invention was confirmed to be an effective method that can replace the conventional method. Further, the method according to the present invention can use a fluorescent plate reader, so that significant savings can be achieved.
- red blood cell separation method of the present invention the following operation was performed. Blood collected from two healthy subjects was added to the erythrocyte collection device prepared in Example 1 at 150 zL. After the blood was completely absorbed, the erythrocyte-retaining layer was taken out, placed in a test tube containing 1 mL of purified water, and the components in the erythrocyte were collected to be used as a measurement sample of the method of the present invention. On the other hand, red blood cell separation according to the conventional method was performed by centrifuging 1 mL of whole blood and removing the supernatant, and a mixture of 500 ⁇ L of the red blood cells and 2 mL of purified water was used as a measurement sample.
- Sorbitol was measured in the same manner as in Example 3 for both the method of the present invention and the conventional method.
- the sorbitol concentration was calculated by measuring the hemoglobin concentration of the measurement sample and converting it to the concentration per lg of hemoglobin.
- Table 1 shows the results. According to the erythrocyte separation method and the sorbitol measurement method, several combinations of the present invention method and the conventional method were set, and the measured values were compared. The value was shown. On the other hand, when the method of collecting erythrocytes was carried out by the method of the present invention and the method of measuring sorbitol was carried out by the conventional method, the measurement was near the lower limit of measurement due to the small amount of the sample, and an accurate measured value could not be obtained.
- Example 2 150 ⁇ L of blood collected from two healthy subjects was added to the red blood cell collection device prepared in Example 1. I got calo. After the blood was completely absorbed, the erythrocyte retention layer was removed, 5 g of ion exchanger DE53 (manufactured by Whatman) was suspended in 10 mL of purified water, and the mixture was stirred in a test tube containing ImL. The supernatant was used as a measurement sample. The measurement of sorbitol was performed by the method of Example 2. At the time of measurement, the step of adding the perchloric acid solution and the carbonated lithium solution during the measurement operation in Example 2 was omitted.
- the erythrocyte holding layer was taken out, placed in a test tube containing ImL of purified water, and the components in the erythrocytes were collected as the measurement sample.
- the measurement of sorbitol was performed according to the method of 2.
- Table 2 shows the results. As shown in Table 2, when the ion-exchange suspension was used, the supernatant was already colorless and transparent without performing the slow protein operation using the perchloric acid solution and the potassium carbonate solution. . For this reason, sorbitol in erythrocytes could be measured without performing a protein removal operation at the time of measurement. In addition, almost the same measured values were obtained in both methods, indicating that there was no adsorption of sorbitol to the ion exchanger. From this, it was confirmed that the method of the present invention is an effective method that can replace the conventional method.
- Ion exchanger Colorless and transparent None 0.31 nmoL / mL 0.35 nmoL / mL Purified water Red Yes 0.29 nmoL / mL 0.34 nmoL / mL
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