JP2020146622A - Method and apparatus for measuring glycosylated hemoglobin by affinity method - Google Patents

Abstract

To provide an apparatus capable of measuring hemoglobin by an affinity method in which a base line does not vary and calculation processing is simple.SOLUTION: A liquid chromatograph apparatus is provided that comprises: an eluant feed pump 3 for feeding eluent 1; a sample injection mechanism 8 connected to a position downstream of the eluant feed pump; an analysis column 10 filled with an affinity gel capable of selectively adsorbing glycosylated hemoglobin; a visible light detector 11; and a switching valve 9, the switching valve can switch a state of the liquid chromatograph apparatus between a first state in which the sample injection mechanism, the visible light detector, and the analysis column are fluidly connected to each other in this order and a second state in which the sample injection mechanism, the analysis column, and the visible light detector are fluidly connected to each other in this order.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method and an apparatus for measuring glycated hemoglobin by an affinity method.

Diagnosis is often made based on the proportion of glycated hemoglobin (SA1c) in the blood as an index for determining diabetes. As a method for measuring SA1c%, a measuring method based on the principle of affinity chromatography is widely used. Specifically, after hemolyzing and diluting blood, the blood is introduced into a column packed with a gel containing an aminophenylboronic acid group, and glycated hemoglobin is adsorbed on the gel with the first eluent to elute non-glycated hemoglobin. After an hour, it is switched to a second eluent that desorbs glycated hemoglobin to elute glycated hemoglobin.

Various buffer solutions and concentrations have been proposed for the above two types of eluents, but in general, the compositions of the two solutions are significantly different, and the background of the detector is also different, so that the baseline fluctuates stepwise. Therefore, it is difficult to calculate each peak area accurately.

An object of the present invention is to provide an apparatus capable of measuring glycated hemoglobin by an affinity method, which has no baseline fluctuation and is easy to calculate.

In order to solve the above problems, the present inventors have reached the present invention as a result of repeated diligent studies.

That is, one aspect of the present invention is
A liquid feed pump for feeding the eluent and
A sample injection mechanism connected downstream of the liquid feed pump,
An analytical column filled with an affinity gel capable of specifically adsorbing glycated hemoglobin,
Visible light detector and
Switching valve and
It is a liquid chromatograph device equipped with
The switching valve is a first state in which the sample injection mechanism, the visible photodetector, and the analysis column are fluidly connected in this order.
It is characterized in that the sample injection mechanism, the analysis column, and the visible photodetector can be switched to the second fluid-connected state in this order.

Hereinafter, the present invention will be described in detail.

FIG. 1 shows a configuration of an apparatus according to an aspect of the present invention, and is a diagram showing a state of a flow path switching valve. The thick solid line in the figure indicates the connected flow paths.

At the start of measurement, the switching valve is set to the first state, and the eluent for adsorbing glycated hemoglobin is sent. In this state, the sample is injected from the sample injection mechanism. As a result, the sample flows in the order of the visible light detector and the analysis column, and is discharged. Since the sample is directly introduced into the visible light detector, the detector can obtain a signal proportional to the total amount of the sample. The sample exiting the detector is introduced into the column, but the glycated hemoglobin component is adsorbed on the gel, and the other components move within the column (see FIG. 1a).

Next, the switching valve is switched to the second state. As a result, the eluent flows in the order of the analysis column and the visible photodetector, and is discharged. The components other than glycated hemoglobin that were moving in the analysis column are guided to the detector. That is, the detector can obtain a signal proportional to the amount of components other than glycated hemoglobin (see FIG. 1b).

If the valve switching timing is too early, the detector will not be able to obtain an accurate signal for the total amount of the sample, and if the timing is too late, components other than glycated hemoglobin that were moving in the analysis column will be discharged. In the first state, it is preferable to set the mode so that the output signal of the detector automatically switches when the output signal falls below an appropriate threshold value. The appropriate threshold value may be appropriately determined by a test run using a sample or the like.

From the detector signal (A) obtained in the first state and the detector signal (B) obtained in the second state of the switching valve, the ratio of glycated hemoglobin (SA1c%) to the total amount can be calculated. That is, the amount of glycated hemoglobin corresponds to the difference between the total amount (A) and the amount of non-glycated hemoglobin (B). The ratio of the difference (A)-(B) to the total amount (A) is the glycated hemoglobin ratio (SA1c%). The amounts of (A) and (B) correspond to the integrated value or peak area of the peaks obtained in each step.

In the conventional affinity method, it is necessary to sequentially switch between two eluents (for glycated hemoglobin adsorption and glycated hemoglobin desorption) in one analysis, whereas the apparatus of the present invention is a valve as described above. Since the total amount of the sample and components other than glycated hemoglobin can be detected by the detector, the glycated hemoglobin ratio can be measured by using only the eluent for adsorbing glycated hemoglobin. Therefore, the present invention enables measurement in a state where the composition of the eluent does not change and the background does not change (see FIG. 2).

Further, when the sample is continuously analyzed by the apparatus which is one aspect of the present invention, the saccharified hemoglobin adsorbed on the analysis column is accumulated, but there is no problem until the adsorption limit of the gel used is exceeded. Continuous analysis is possible. FIG. 3 schematically shows the state of the switching valve and the change in the obtained detector signal.

If the limit of gel adsorption is exceeded, or if the number of measurements exceeds a preset number of measurements, the analysis column may be replaced, but for example, a switching valve (see FIG. 4a) on the upstream side of the liquid feed pump. By providing an on-off valve (see FIG. 4b) or by providing two liquid feed pumps (see FIG. 4c), it becomes possible to distribute the glycated hemoglobin adsorption eluent and the glycated hemoglobin desorption eluent a certain number of times. After continuous measurement, the glycated hemoglobin desorption eluent may be flushed to wash away the accumulated glycated hemoglobin component, and the regeneration operation may be performed.

By using the apparatus according to one aspect of the present invention, the blood sample is flushed from the sample injection mechanism with the eluent for adsorbing glycated hemoglobin, the change in blood concentration is measured by the visible light detector, and the measured blood sample is measured. Was guided to an analysis column filled with an affinity gel capable of specifically adsorbing glycated hemoglobin, and then the concentration change of components other than glycated hemoglobin was measured with a visible light detector, and the amount of blood concentration change and other than glycated hemoglobin were measured. The amount of glycated hemoglobin can be easily calculated from the difference from the amount of change in the concentration of the components of the blood sample, but the blood sample is pressed with the eluent for adsorbing glycated hemoglobin in the order of the visible light detector, the analysis column, and the visible light detector. If it can flow, the device used is not limited to the above-mentioned device.

It has become possible to measure glycated hemoglobin by the affinity method, which has no baseline fluctuation and is easy to calculate.

It is a figure which showed the structure of the apparatus which is one aspect of this invention. FIG. 1a shows the switching valve in the first state, and FIG. 1b shows the switching valve in the second state. It is a figure which showed the difference of the variation of the baseline by the conventional affinity chromatography and the method of this invention schematically. It is a figure which showed typically the pattern of separation of glycated hemoglobin by the method of this invention, and the mode of a flow path switching valve. It is a figure which showed the other aspect of this invention. FIG. 4a shows a mode in which only one liquid feed pump is used and one switching valve switches between two liquids, and FIG. 4b shows a mode in which only one liquid feed pump is used and two liquids are switched by one on-off valve, FIG. 4c. Is a mode in which two liquid feeding pumps are used and two liquids are switched. FIG. 5 is a diagram in which the results of measuring the calibrator three times in succession and the results of measuring the hemolytic lavage fluid in Example 1 are superimposed. FIG. 5a is a sample of Level_1 (Low), and FIG. 5b is a sample of Level_1 (High). FIG. 5 is a diagram in which the results of measuring the control three times in succession and the results of measuring the hemolytic lavage fluid in Example 1 are superimposed. FIG. 6a is a sample of Level_1 (Low), and FIG. 6b is a sample of Level_1 (High). It is a figure which showed the influence on the area% of SA1c by the sample concentration change. The horizontal axis is the logarithm of the dilution rate, and the vertical axis is the calculated SA1c area%. It is a figure which showed the measurement result of Example 3. In the figure, the solid line is the result of sample measurement, and the broken line is the result obtained by passing the glycated hemoglobin desorption eluent through the column after measuring the sample multiple times in succession. FIG. 8a shows the results of one sample measurement, FIG. 8b shows the results of two sample measurements, and FIG. 8c shows the results of five sample measurements. Same as FIG. FIG. 9a shows the results of sample measurement 10 times, FIG. 9b shows the results of sample measurement 20 times, and FIG. 9c shows the results of sample measurement 50 times. It is a figure which showed the measurement result of Example 3. The horizontal axis is the logarithm of the number of consecutive measurements, and the vertical axis is the logarithm of the peak area of glycated hemoglobin at the time of desorption.

In order to clarify the effect of the present invention, the system of FIG. 4a was constructed and verified.

The analytical column (10) / visible photodetector (11) was connected via a liquid feed pump (3), a sample injection mechanism (8) and a 6-port 2-position switching valve (9). In addition, a switching valve (7) was arranged on the upstream side of the liquid feed pump (3) so that the SA1c adsorption eluent (1) and the SA1c desorption eluent (2) could be selected.
The 6-port 2-position switching bulb (9) can take two states (flow paths). In the first state, the eluent to be sent is the visible light detector, the analysis column, and the drain. In the second state, the eluent to be sent can flow in the order of the analysis column, the visible photodetector, and the drain.
All components use 8020 series HPLC manufactured by Tosoh Corporation, and the analysis column uses TSKgel Boronate-5PW (manufactured by Tosoh Corporation, particle size 10 μm) using m-aminophenylboronic acid as a ligand. A column tube having an inner diameter of 4.6 mm and a length of 10 mm was filled.

As the SA1c adsorption eluent (pH 8.8), one prepared with the following composition was used.
Glycine 50 mM
Magnesium chloride hexahydrate 5 mM
Sodium chloride 50 mM
As the SA1c desorption eluent (pH 8.8), one prepared with the following composition was used.
Glycine 50 mM
Sodium chloride 50 mM
D-sorbitol 100 mM
Other measurement conditions are as follows.
Column temperature: 45 ° C
Flow velocity: 1.0 mL / min
Detection wavelength: 415 nm (single wavelength)
Detector response: 0.15s
Data collection sampling pitch: 50ms
As the cleaning solution of the sample injection mechanism (8), a hemolysis / cleaning solution dedicated to a glycohemoglobin analyzer (HLC-723 series) manufactured by Tosoh Corporation was used. When blood was used as a sample, hemolysis / dilution was performed with the above-mentioned hemolysis / washing solution.

(Example 1)
It was verified whether continuous measurement was possible with only the SA1c adsorption eluent.
As the sample, the calibrator sample and the control sample prepared as follows were used.
Freeze-dried A1c calibrator (manufactured by Toso Co., Ltd.) Level_1 (HbA1c 5.87% [NGSP]) and Level_1 (HbA1c 10.87% [NGSP]) are dissolved in 1.3 mL of purified water at room temperature. Level_1 (HbA1c 4.9 ± 0.3% [NGSP]) and Level_2 (HbA1c 9.9 ± 0.5% [NGSP]) of A1c control (manufactured by Toso Co., Ltd.) ) Was first dissolved in 0.5 mL of purified water, left at room temperature for 30 minutes, and 0.5 mL of purified water was added to 30 μL of the first dissolved product. Calibrator Level_1 was added three times in a row. The calibrator Level_2 was measured three times in a row. Similarly, the control Level_1 was measured three times in a row, and then the Control Level_1 was measured three times in a row. As for the liquid flow, only the SA1c adsorption eluent was continuously fed.
In the measurement sequence, the valve was in the first state from the time of sample injection to 0.25 minutes, and the valve was in the second state from 0.25 minutes to 2.80 minutes.

FIG. 5 is a diagram in which the results of the calibrators Level_1 and 2 are superimposed, and FIG. 6 is a diagram in which the results of the controls Level_1 and 2 are superimposed. The broken line in the figure is a background showing the variation of the chromatogram, that is, the baseline when the hemolytic washing solution is injected as a sample.
In both FIGS. 5 and 6, the sharp peak appearing around 0.1 minute is the change in the absorbance of the entire injected sample, and the component other than glycated hemoglobin that was moving in the affinity column as a broad peak around 0.45 minute. The change in absorbance can be confirmed.

Table 1 shows the quantification results of the calibrator sample.

Since the area of glycated hemoglobin (SA1c) corresponds to the difference between the area of peak 1 and the area of peak 2, the area% of SA1c is
{(Peak 1 area)-(Peak 2 area)} * 100 / (Peak 1 area)
It is calculated by.
When a calibration curve is created with SA1c area% (4) as X and reference value (5) as Y, the regression equation is y = 0.7873x − 4.6712.
It became.

Next, Table 2 shows the quantitative results of the control sample. In addition, column (6) in the table is a value calculated by SA1c% by the above calibration curve.

The average SA1c% of control Level_1 was 5.3%, and that of control Level_1 was 10.4%.

(Example 2)
Next, the effects of the concentration of the sample on the conventional affinity method and the method of the present invention were compared.
A step gradient was carried out in which the SA1c adsorption eluent (1) and the SA1c desorption eluent (2) were switched by a switching valve (7) arranged upstream of the liquid feed pump (3). Since the bulb is fixed in the second state, it flows in the order of the analysis column, the visible photodetector, and the drain, so that a general chromatography flow path can be taken.
In the measurement sequence, the SA1c adsorption eluent was flowed from the time of sample injection to 0.15 minutes, and the SA1c desorption eluent was flowed from 0.15 minutes to 0.65 minutes. Further, the chromatogram (background) obtained by injecting the hemolytic washing solution was subtracted from the chromatogram obtained by the measurement, and the peak was detected by the difference chromatogram.

Samples prepared by adjusting whole blood to 5 different concentrations from 1/25 to 1/150 were subjected to the method of the present invention and the conventional affinity method, and the effect on SA1c area% was confirmed. Table 3 shows the quantification results by the method of the present invention, and Table 4 shows the quantification results by the conventional affinity method. Since the concentration range is wide, the concentration is logarithmic.

In the conventional affinity method, the SA1c area% tends to increase as the concentration decreases, but in the continuous affinity method of the present invention, it can be seen that the influence of the concentration is small (see FIG. 7).
In the conventional affinity method, there is a background fluctuation due to switching between two types of eluents, and the fluctuation amount is included in the SA1c area. However, since the method of the present invention uses only one type of eluent, This is because there is no change in the background and it is not easily affected by the sample concentration.

(Example 3)
It was verified whether the glycated hemoglobin component was adsorbed even when the method of the present invention was continuously carried out. The basic measurement conditions are the same as in Example 1. The glycated hemoglobin adsorption eluent (1) is fed, the sample is measured multiple times, and then the glycated hemoglobin desorption eluent (2) is fed and adsorbed. The components were detected.
For the measurement, the sample was injected once, twice, five times, ten times, 20 times, and 50 times, respectively, and then the eluent was switched. As the sample, a control sample (Level_1) was used. The results are shown in FIGS. 8 and 9. In the figure, the solid line shows the result of sample measurement (overlaying of the measurement result n times), and the broken line shows the result obtained at the time of attachment / detachment.

At the time of sample measurement, a sharp peak showing the total amount appears around 0.1 minutes, a broad peak showing the amount of components other than glycated hemoglobin appears around about 0.3 minutes, and at the time of desorption, around 1.6 minutes. A broad peak indicating the amount of glycated hemoglobin appears in, and it can be seen that the peak intensity of the peak increases as the number of measurements increases.
FIG. 10 shows a plot of the logarithm of the number of consecutive measurements on the horizontal axis and the logarithm of the peak area of glycated hemoglobin at the time of desorption on the vertical axis. It can be seen that a linear relationship is established between the number of injections and the peak area of glycated hemoglobin at the time of desorption, and it can be seen that only the glycated hemoglobin component is quantitatively adsorbed on the affinity gel.

1 SA1c adsorption eluent 2 SA1c desorption eluent 3, 4 Liquid transfer pump 5, 6 On-off valve 7 Switching valve 8 Sample injection mechanism 9 6 Port 2 Position switching valve 10 Analytical column 11 Visible photodetector 12 Column oven

Claims (3)

A liquid feed pump for feeding the eluent and
A sample injection mechanism connected downstream of the liquid feed pump,
An analytical column filled with an affinity gel capable of specifically adsorbing glycated hemoglobin,
Visible light detector and
Switching valve and
It is a liquid chromatograph device equipped with
The switching valve is a first state in which the sample injection mechanism, the visible photodetector, and the analysis column are fluidly connected in this order.
The apparatus, wherein the sample injection mechanism, the analysis column, and the visible photodetector can be switched to a second state fluidly connected in this order. The apparatus according to claim 1, wherein an on-off valve or a switching valve is provided upstream of the liquid feed pump, or two or more liquid feed pumps are provided so that two or more kinds of eluents can be fed. A method of calculating the amount of glycated hemoglobin in a blood sample using a liquid chromatograph device.
The blood sample was flushed from the sample injection mechanism with an eluent for adsorbing glycated hemoglobin, and the change in blood concentration was measured with a visible light detector.
After the measurement, the blood sample was guided to an analysis column filled with an affinity gel capable of specifically adsorbing glycated hemoglobin, and then the concentration change of components other than glycated hemoglobin was measured with a visible photodetector.
A method for calculating the amount of glycated hemoglobin from the difference between the amount of change in blood concentration and the amount of change in concentration of components other than glycated hemoglobin.