JPH041303B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for measuring blood components. More specifically, when measuring using a blood filter paper used for mass screening of children, a composition consisting of the blood filter paper and ether:water:acetone:alcohol or a composition consisting of methylene chloride:water:acetone:alcohol is used. This method of measuring blood components is characterized by using blood filter paper in which hemoglobin has been denatured and fixed by contacting it with an object. Blood components measured in the method of the present invention include glucose, galactose,
Galactose-1-phosphate, uridine-diphosphate galactose, ornithine, phenylalanine,
These are sugars and amino acids such as histidine, leucine, methionine, and tyrosine. In pediatric mass screening, in order to detect metabolic abnormalities in newborns early, the footpad is punctured with a lancet on the 5th to 7th day after birth, blood is absorbed into a designated filter paper, and the dried blood filter paper is sent to a testing center. A method of testing is the so-called Guthrie test. This test measures sugar, amino acids, etc. as mentioned above, which can lead to galactosemia, phenylketonuria, histidinemia, maple syrup urine disease, homocystinuria, etc.
Hypertyrosinemia is diagnosed. This test method is
First, blood filter paper is punched out into disks with a diameter of 3 to 10 mm, and this disk is reacted with the measurement reagent, or the bacterial suppression test method (Bacterial
The target substance is measured by inhibition assay (abbreviated as BIA method). Examples of the former include, for example, a galactose microquantification method in which galactose dehydrogenase and NAD are allowed to act on the blood filter paper disk and the fluorescence or visible absorption of the generated NADH is measured. In addition, examples of the BIA law include, for example:
In the measurement of phenylalanine, antimetabolite β
There is a method in which Bacillus subtilis spores are inoculated into a minimal nutrient plate medium containing -2-thienylalanine, blood filter paper disks are arranged there, and semi-quantification is carried out from the growth zone of the bacteria. In the above measurements, hemoglobin and the like were eluted from the blood filter paper disk used, which interfered with photometer measurements or made the growth zone unclear. As a known technique related to the above problem, a method of denaturing and fixing hemoglobin using formic acid, acetic acid, acetone, water vapor, etc. has been reported (Nagoya City Institute of Health Bulletin Vol. 25, p. 17, 1978, Tohoku Journal of Extract). Perimental Medicine (Tohoku J.Exp.Med.) Volume 133, Page 371,
(e.g. 1981). Disadvantages of these prior art techniques include that the denaturation and fixation operation takes a long time, that the analyte is degraded due to the high temperature treatment, and that the chemicals used are irritating. Furthermore, after denaturation and fixation, it may take a long time or special equipment to remove and dry the used drug. That is, when formic acid, acetic acid, etc. are used, it is necessary to operate under reduced pressure in a desiccator in order to remove excess acid adhering to the filter paper, and it is also necessary to have activated carbon present. Furthermore, when a mixed system of organic solvent and water was used, it took overnight to dry at a low temperature. The present inventors are conducting research to improve the above drawbacks: Water: Acetone:
Although it was found that using a composition made of alcohol was easier to operate, it still had drawbacks such as requiring a long time. The present invention was born from the above knowledge, and
The gist is that the hemoglobin on the blood filter paper is denatured and fixed by contacting the blood filter paper with a composition consisting of ether:water:acetone:alcohol or a composition consisting of methylene chloride:water:acetone:alcohol, and the hemoglobin is eluted. This is a method for measuring blood components using blood filter paper that completely prevents blood loss. In the method of the present invention, blood components can be measured by known methods using the modified fixed blood filter paper obtained as described above. That is, examples include the following method. The first is NAD conjugated to a measuring substance, such as the method for microquantitating galactose mentioned above.
This method involves an enzymatic reaction involving redox of (P) or NAD(P)H. In this method, the change in fluorescence or visible absorption caused by the reaction is measured using a fluorometer or spectrophotometer. The second method is to use the above-mentioned bacterial suppression test method. These methods measure the aforementioned sugars, amino acids, etc.
The effects of the present invention are particularly remarkable in trace measurement methods that utilize fluorescence or visible absorption. As mentioned above, the hemoglobin denaturation fixation reagent used in the method of the present invention is a composition consisting of ether:water:acetone:alcohol or methylene chloride:water:acetone:alcohol. The alcohol preferably has a low boiling point, such as methanol or ethanol. The mixing ratio of reagents effective for denatured immobilization can be selected within a wide range as described below. The denaturation fixation operation can be performed, for example, as follows. A few drops of blood is soaked into a Guthrie paper filter (manufactured by Daiichi Chemical), which is used for mass screening of newborns with congenital metabolic abnormalities, and air-dried. The blood filter paper thus obtained contains approximately 20-30 μl of blood per 1 cm diameter. Cut out a disk of an appropriate size (usually 3 mm diameter) from this blood filter paper, place it in a suitable container, such as a test tube used for measurement, add the denatured fixation reagent there, and let it cool at room temperature or around 37°C. Approximately 30 minutes in a constant temperature water tank
Process for minutes to 1 hour. The amount of denatured fixation reagent to be added is sufficient to soak into the disk, and is approximately 20 μl for one 3 mm diameter disk. In the method of the present invention, denatured fixation and drying are completed in the above-mentioned time, that is, about 30 minutes to 1 hour, so there is no need to perform a separate drying operation such as heating or reduced pressure. Once the disk is dry,
You can also move on to the next measurement operation using the same test tube. The blood filter paper disk obtained as described above does not elute hemoglobin at all and does not cause any hindrance to the reaction system. Furthermore, the method of the present invention has made it possible to carry out denaturation and immobilization very quickly and easily. The composition of the reagent for denaturation and immobilization will be explained below. That is, the aforementioned blood filter paper was punched out into a disk with a diameter of 3 mm, placed in a test tube, and 20 μl of a reagent having the composition shown in Table 1 was added to examine the ability to denature and fix hemoglobin. Table 1 shows the time required for denaturation fixation and drying, and the results of measuring the degree of elution of hemoglobin by soaking the obtained disk in water. In the same table,
There are four levels of time required: + means less than 1 hour;
indicates within 2 hours, within 4 hours, 〓 indicates over 4 hours, and cases where hemoglobin elution was observed were indicated as (x), and cases where no hemoglobin was observed were indicated as (○). Furthermore, comprehensive evaluation was performed, and C represents one that can be used for denaturation and immobilization, B represents a more suitable one, and A represents a most suitable one.

【table】

[Table] As can be seen from the results shown in Table 1, the reagent composition suitable for denatured immobilization is ether: water: acetone:
The ratio of methanol is 20-83:3-32:2-48:3-48, preferably 20-83:3-16:2-48:7-40. Table 2 shows the case of methylene chloride:water:acetone:methanol system. How to write a table is the first
Same as table.

[Table] As can be seen from the results shown in Table 2, in the case of methylene chloride:water:acetone:methanol, the suitable ratio is 25 to 83:3 to 20:2 to 30:2 to 45, and preferably 50 The ratio is ~83:3~10:2~30:2~30. In addition to the above-mentioned methods, the method of the present invention can also be used to prevent tailing in filter paper chromatography, thin layer chromatography, starch zone electrophoresis, etc., and to prevent clogging of packing materials in gas chromatography, liquid chromatography, etc. Useful. Test Example 1. Several drops of blood were impregnated into Guthrie filter paper (manufactured by Daiichi Kagaku) and air-dried to obtain blood filter paper. This is diameter 3
Punch out 1 mm disks, place them one by one in a test tube, and add denatured immobilization reagent with the composition shown in Table 3.
20 μl was added and left at room temperature for 1 hour. In order to check whether the hemoglobin in the obtained blood filter paper disk was completely immobilized, water was placed in a test tube and the elution of the hemoglobin was observed. i.e., the fluorescence intensity of the aqueous eluate of the denatured fixed blood disk is determined by the excitation wavelength.
The measurement was performed at 381 nm and the fluorescence wavelength was 382 nm. The standard solution used was 0.29 μM quinine sulfate (fluorescence intensity 0.1), and the control solution used water (also 0). The results are shown in Table 3. As can be seen from the table, no elution of hemoglobin was observed from the blood disks denatured and fixed by the method of the present invention.

[Table] Example 1. Measurement of galactose Various blood samples containing a known amount of galactose were impregnated into the aforementioned Guthrie filter paper, air-dried, and then punched out to a diameter of 3 mm to obtain a blood filter paper disk. Place the disks one by one in a test tube and add ether:water:acetone:methanol in a ratio of 50:
20 μl of a 10:10:30 composition was added and left at room temperature for 1 hour to perform denaturation and fixation treatment of hemoglobin. Then, in a test tube, add 13mM NDA, 5 x 10 ^-2 mg/ml galactose dehydrogenase (Boehringer Mann High),
10 μl of 1M Tris-HCl buffer (pH 8.0) was added to each, and the mixture was reacted at 37° C. for 1 hour. 3 ml of water was added, and the fluorescence of the generated NADH was measured at an excitation wavelength of 340 nm and a fluorescence wavelength of 450 nm. As shown in FIG. 1, good linearity was obtained between the galactose concentration and the fluorescence intensity. Example 2. Measurement of uridine-diphosphoric acid (UDP) galactose A blood sample was impregnated into a Guthrie paper filter, air-dried, and then punched out to a diameter of 3 mm to obtain a blood filter paper disk. Place the disk in a test tube and add the ratio of methylene chloride:water:acetone:methanol.
20 μl of a 70:10:5:15 composition was added and left at room temperature for 1 hour to perform denaturation and fixation treatment of hemoglobin. Then add 13mM NDA and 0.2u/ml UDP to the test tube.
Galactose-4-epimerase (manufactured by Sigma),
10 μl each of 0.03 u/ml UDP glucose dehydrogenase (manufactured by Boehringer Mannheim) and 1M Tris-HCl buffer (ph 8.0) were added and reacted at 37° C. for 1 hour. The following was generated in the same manner as in Example 1.
The fluorescence of NADH was measured, and the amount of UDP galactose was determined from the calibration curve. As a result, the concentration in this blood sample was 0.1mM.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the relationship between galactose concentration and fluorescence intensity in the measurement of galactose by the method of the present invention.

Claims (1)

[Claims] 1 Ether in measurements using blood filter paper:
A method for obtaining blood components characterized by using a blood filter paper in which hemoglobin contained in the blood filter paper has been denatured and fixed by the action of a composition consisting of water:acetone:alcohol or a composition consisting of methylene chloride:water:acetone:alcohol. Measurement method.