JP4306116B2 - Glycated hemoglobin measuring device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a glycated hemoglobin measuring apparatus capable of measuring glycated hemoglobin in blood of various kinds of animals such as domestic animals, pets, and experimental research animals.
[0002]
[Prior art]
Glycated hemoglobin in blood reflects the average blood glucose level over the past 1 to 2 months, and has already been widely measured in humans as an indicator for early detection by determining the progress of diabetes treatment and screening for diabetes. Human glycated hemoglobin is measured using a cation exchange column, a high-performance liquid chromatography apparatus using an affinity column using an aminophenylboronic acid group, a measuring reagent using a specific antibody against glycated hemoglobin, etc. As the measurement reagent, a reagent that specifically employs an immunoturbidimetric method or latex agglutination method is commercially available.
[0003]
[Problems to be solved by the invention]
The above-described conventional glycated hemoglobin measuring device and the like have been developed to measure glycated hemoglobin present in human blood, but today, they exist in the blood of many types of vertebrates other than humans. There is an increasing demand for devices for measuring glycated hemoglobin. In other words, compounds that are effective in treating diabetes in humans are screened using vertebrates that have developed diabetes, the efficacy and side effects of candidate compounds obtained in this way are confirmed, and are further raised as pet animals. In order to diagnose, prevent, and treat diabetes in various animals, a device capable of measuring glycated hemoglobin in many kinds of animals is required.
[0004]
However, hemoglobin has different amino acid sequences depending on the animal species, and as a result, the isoelectric point is different. Therefore, the high performance liquid chromatography apparatus using the cation exchange column described above is used for measuring glycated hemoglobin of various animals. Has a problem that measurement conditions for each animal species must be set. The salt concentration and gradient pattern of the eluent may be adjusted each time so that the hemoglobin and glycated hemoglobin of the animal species to be measured can be separated and measured, but all types of eluents expected in advance are prepared and expected. It is practically impossible to store gradient patterns and the like for all animal species in the apparatus. In addition, even if all types of eluents expected in advance are prepared and gradient patterns for all animal species are stored in the apparatus, blood to be actually measured in a situation where blood samples of various animals are mixed. Unless the apparatus automatically recognizes what animal the sample is derived from, it is necessary for the operator to input animal species information, and an automatic measurement apparatus cannot be provided. And even if all of the above problems are solved, the glycated hemoglobin measurement is still carried out under different measurement conditions.
[0005]
With the measurement reagent using the specific antibody described above, it is impossible to measure glycated hemoglobin of various animals depending on the recognition site of the antibody used, and even if the measurement is possible, the result is low reproducibility. There is a problem that it cannot be obtained.
[0006]
On the other hand, a high performance liquid chromatography apparatus using an affinity column using an aminophenylboronic acid group is affected by the difference in isoelectric point because it uses the affinity between the aminophenylboronic acid group and the cisdiol bond of the glycated protein. First, it has been reported that glycated proteins from various animals can be separated from non-glycated proteins (see, for example, US Pat. No. 4,269,605, V. Bouriotis et.al., Diabetogia 21, 579 (1981), TS). Reid et.al., J. of High Resolution Chromatography 12,249 (1989)). However, there has been no report that glycated hemoglobin was measured in various animals using the device, and the influence of various glycated proteins present in the sample on various animal blood samples was excluded under the same measurement conditions. Whether or not glycated hemoglobin can be measured is unknown. Furthermore, even if it is possible to measure glycated hemoglobin in various animals under the same conditions, it is completely unknown what the conditions are.
[0007]
Therefore, an object of the present invention is to provide an apparatus for measuring glycated hemoglobin present in the blood of various animal species under the same conditions.
[0008]
[Means for Solving the Problems]
The invention of claim 1 completed to achieve the above object is a liquid chromatography apparatus for measuring glycated hemoglobin in the blood of many kinds of animals, wherein an aminophenylboronic acid group is used as a measurement column. An apparatus for measuring glycated hemoglobin in blood of an animal, equipped with a column filled with a filler. The invention of claim 2 relates to the invention of claim 1, characterized in that the glycated hemoglobin to be measured is hemoglobin A1c. Hereinafter, the present invention will be described in detail.
[0009]
The apparatus of the present invention is characterized in that it is equipped with a column packed with a packing material having aminophenylboronic acid groups as a measurement column. In addition to the column, there are parts equipped with a general liquid chromatography apparatus. Is included. Examples of such parts include a liquid feed pump, a sample injection valve, a measurement column, a detector, a column oven that keeps the measurement column at a constant temperature, and an autosampler that automatically dilutes and hemolyzes the sample blood. It is not limited to these. Among them, a column oven that keeps the measurement column at a constant temperature and an autosampler that automatically dilutes and hemolyzes the test sample blood are preferably employed to provide a highly accurate automatic measurement device. In addition, what is necessary is just to employ | adopt the component which has the pressure | voltage resistance requested | required, respectively, when providing the so-called high performance liquid chromatography apparatus which enables shortening of measurement time, for example.
[0010]
The apparatus of the present invention may be configured such that a diluent is added to blood collected from an animal, diluted and hemolyzed, and set. However, the apparatus automatically samples animal blood collected in a blood collection tube or the like, May be added to automatically dilute hemolysis, and then glycated hemoglobin may be measured automatically. The diluent used at this time is, for example, a nonionic surfactant solution.
[0011]
If the measurement column is a column packed with a filler having an aminophenylboronic acid group, a commercially available column or the like can be used. An example of such a commercially available column is TSKgel Boronate-5PW (4.6 mm ID × 35 mm) (trade name, manufactured by Tosoh Corporation). As the detector, a general UV-visible detector for liquid chromatography, for example, a detector capable of detecting absorption at 415 nm suitable for hemoglobin detection can be employed.
[0012]
Next, measurement of glycated hemoglobin of various animals using the apparatus of the present invention will be described. Two kinds of eluents (hereinafter referred to as the first liquid and the second liquid) are used for the measurement. The first solution is a buffer solution whose salt concentration, pH, etc. are adjusted so as to allow the binding of glycated hemoglobin to the aminophenylboronic acid group and not allow the adsorption of non-glycated hemoglobin to the aminophenylboronic acid group. The second solution is a buffer solution in which the salt concentration, pH, and the like are adjusted so as to cause rapid detachment of the adsorbed glycated hemoglobin. The type of these buffer solutions (type of buffer), salt concentration, pH, and the like can be appropriately prepared based on the description in the above-mentioned literature, but according to the knowledge of the present inventors, 150 mM ammonium acetate, A buffer solution of pH 8.8 containing 50 mM magnesium chloride and 5% acetonitrile and a buffer solution of pH 8.0 containing 50 mM Tris, 100 mM sorbitol, 50 mM EDTA, 5% acetonitrile are used as the first solution and the second solution, respectively. It is possible to perform glycated hemoglobin measurement of various animals. In addition to the above, the first liquid specifically includes a liquid containing a buffer such as HEPES, ammonium acetate or glycine, 10 to 100 mM magnesium chloride, and 1 to 15% acetonitrile, ethanol or methanol. it can. As the second liquid, in addition to the above, specifically, a buffer such as trishydroxyaminomethane (Tris), 10 to 100 mM sorbitol, mannitol, xylitol or glucitol, etc., 10 to 100 mM EDTA, and 1 to A liquid containing 15% acetonitrile, ethanol, methanol or the like can be exemplified.
[0013]
A certain amount of blood sample from the diluted hemolyzed animal is sampled, the sample is injected into the liquid chromatography liquid supply line by the sample injection valve, and the first liquid and the second liquid are alternately switched and sent. It is preferable that the timing of switching the liquid supply between the first liquid and the second liquid is interlocked with the blood sample injection interval (T) in order to quickly measure and improve the number of measurements per fixed time. For example, a blood sample is first injected into a measurement column equilibrated with the first liquid, and after the blood sample is injected, the first liquid is supplied for a certain period of time (T1), and then the second liquid is injected for a certain period of time (T2). Then, the first liquid is again fed for a certain time (T3). Here, T = T1 + T2 + T3. Since the non-glycated hemoglobin component in the sample does not adsorb to the measurement column, it elutes together with the first liquid. On the other hand, the glycated hemoglobin component is eluted from the column when the second liquid is fed. The elution of the hemoglobin component from the column can be monitored by continuously measuring the absorbance at 415 nm, and can be recorded by a data processor or a chart recorder.
[0014]
The% glycated hemoglobin serving as an index of diabetes can be calculated from the integrated value of the absorbance of the glycated hemoglobin fraction with respect to the sum of the integrated value of the absorbance of the eluted non-glycated hemoglobin fraction and the integrated value of the absorbance of the glycated hemoglobin fraction. Further, when the obtained glycated hemoglobin% is converted into hemoglobin A1c%, it can be obtained by measuring and calibrating a calibrator whose hemoglobin A1c% value is known in advance.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLES Examples will be described below to describe the present invention in more detail, but the present invention is not limited to these examples.
[0016]
FIG. 1 is a diagram showing an outline of an example of a liquid chromatography apparatus for measuring glycated hemoglobin in blood of various kinds of animals. The apparatus includes a switching valve 4 for switching an eluent, and a sample blood. A liquid feed pump 5 for feeding liquid, an injection valve 6 for injecting a sample, a measurement column 7, a detector 8, and a printer 9 are included. The illustrated apparatus is an apparatus of the present invention obtained by modifying a commercially available apparatus (HLC-723GHbV, trade name, manufactured by Tosoh Corporation) for automatically measuring human glycated hemoglobin. The type and number, the time (timing) for switching the delivery of the eluent, and the measurement column are changed. The measurement column was changed from an ion exchange column to a commercially available column (TSKgel Boronate-5PW, 4.6 mm ID × 35 mm, trade name, manufactured by Tosoh Corporation) packed with a filler having an aminophenylboronic acid group. .
[0017]
Measurements were performed on blood samples from various animals using the apparatus shown. In the measurement, the hemolysis dilution of the blood sample and the hemolysis washing solution commercially available as the washing solution for the sampler portion including the injection valve (the above-mentioned commercially available HSi hemolysis washing solution for automatic glycohemoglobin analyzer, manufactured by Tosoh Corporation), the first solution (Fig. Medium 1 ) (pH 8.8 buffer containing 150 mM ammonium acetate, 50 mM magnesium chloride and 5% acetonitrile) and second liquid ( 2 in the figure) (pH 8 containing 50 mM Tris, 100 mM sorbitol, 50 mM EDTA, and 5% acetonitrile) 0.0 buffer) was used. In addition, although the eluent 3 is described in the figure, this is an eluent when using the commercially available apparatus, and is not used in this embodiment.
[0018]
Blood samples were collected from rats and rabbits and measurements were performed under the above conditions. 10 μl of each blood sample was taken in an Eppendorf tube, 1 ml of the hemolysis washing solution was added , and hemolysis dilution was performed using a vortex mixer. The device simply sets the hemolyzed sample in the autosampler of the device and gives a start instruction, and automatically aspirates the hemolyzed sample at regular intervals and executes the measurement. In this example, T was set to 2.5 minutes, and T1, T2, and T3 were set to 50 seconds, 30 seconds, and 70 seconds, respectively.
[0019]
Changes in 415 nm absorption when measurement is performed as described above are shown in FIGS. FIG. 2 shows the results for a rat blood sample, and FIG. 3 shows the results for a rabbit blood sample.
[0020]
As is clear from the figure, glycated hemoglobin in the blood of different vertebrate animals, rat and rabbit, could be measured under the same measurement conditions.
[0021]
【The invention's effect】
As described above, according to the apparatus of the present invention, it is possible to measure glycated hemoglobin in the blood of various animals under the same conditions. As a result, it is not necessary to change the measurement conditions depending on the animal species to be measured and adjust the salt concentration and gradient pattern of the eluent. Therefore, for example, even in a situation where blood samples of various animals are mixed, measurement can be automatically performed without an operator inputting animal species information.
[0022]
Therefore, the apparatus of the present invention screens a compound effective for human diabetes treatment using a vertebrate that has developed diabetes, confirms the efficacy and side effects of the candidate compound thus obtained, and It is useful for diagnosis, prevention, and treatment of diabetes in various animals raised as pet animals.
[Brief description of the drawings]
FIG. 1 is a diagram showing an outline of an embodiment of an apparatus of the present invention.
FIG. 2 shows a chromatogram obtained in an example.
FIG. 3 shows a chromatogram obtained in an example.
[Explanation of symbols]
1, 2 Eluent 4 Switching valve 5 Pump 6 Injection valve 7 Measurement column 8 Detector 9 Printer

Claims (2)

A liquid chromatography device for measuring glycated hemoglobin in the blood of mammals other than humans under the same conditions, equipped with a column packed with a filler having an aminophenylboronic acid group as a measurement column ,
When measuring glycated hemoglobin using the column,
It consists of a buffer containing 10 to 100 mM magnesium chloride and 1 to 15% acetonitrile that allows adsorption of glycated hemoglobin to the aminophenylboronic acid group of the filler and does not allow adsorption of non-glycated hemoglobin. A first eluent, and
Using a second eluent consisting of a buffer containing 10 to 100 mM sorbitol, 10 to 100 mM EDTA, and 1 to 15% acetonitrile, causing rapid release of the glycated hemoglobin adsorbed on the filler. A device for measuring glycated hemoglobin , which is characterized . The measuring apparatus according to claim 1, wherein the glycated hemoglobin to be measured is hemoglobin A1c.

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