JP2000065839A - Human hemoglobin detector - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily carry out blood sugar examination and the like at home by arranging sensor parts detecting a human hemoglobin quantity and a human glycosylated hemoglobin quantity and supplying body fluid to the sensor parts via a supply path. SOLUTION: Firstly, cross-linked glycosylated human hemoglobin and cross- linked glycosylated human hemoglobin A1c stored in a standard liquid storage part 11 and provided with known concentrations are mixed with a buffer solution stored in a buffer 14 via a valve 16 and a mixing chamber 17 so as to be supplied an SPR sensor 18 using an anti-human hemoglobin antibody and to an SPR sensor 19 using an anti-stabilized human glycosylated hemoglobin antibody. Detection signals for a human hemoglobin concentration and a human hemoglobin A1c concentration from the sensors 18, 19 are processed in a processing unit 20 so as to be stored as initial values. Subsequently, urine stored in a sample storage part 12 is mixed with a buffer solution in a hemolytic buffer 13 so as to be hemolyzed, and a detection signal is calibrated on the basis of the initial values in the processing unit 20 via the sensors 18, 19, and consequently, a human hemoglobin concentration, a human hemoglobin A1c rate are displayed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an apparatus for detecting human hemoglobin.

[0002]

2. Description of the Related Art As the name implies, diabetes has a condition in which sugar (glucose) is excreted in urine. However, an increase in blood glucose concentration is observed even when the level does not reach the level excreted in urine, or a transiently high blood glucose level is exhibited after eating. Frequent blood glucose testing is also required at home to monitor such fluctuations in blood glucose levels.

[0003] However, a plurality of blood glucose measurements every day is a burden on the patient, and the clinical significance is lost unless the measurement is performed at an appropriate timing. It was difficult to do.

[0004] Therefore, glycated hemoglobin (Hb), which is a non-enzymatic conjugate of hemoglobin and glucose in blood.
It is widely used to measure the ratio of A1c, HbA1). Glycated hemoglobin is a product in which glucose is bonded to the amino group of valine at the end of the hemoglobin β chain,
It has been shown that the ratio of the glycated form to the whole hemoglobin is useful for grasping the blood sugar control status in the past month. In this method, all short-period blood sugar level fluctuations associated with daily meals can be comprehensively determined.

[0005]

As described above, although the method of measuring both hemoglobin and glycated hemoglobin is a relatively simple method, it is still difficult to perform a home-based test by a patient.

[0006] It is an object of the present invention to provide an apparatus capable of easily performing a blood glucose test or the like at home.

[0007]

According to the present invention, there is provided a sensor unit having a function of detecting the amount of human hemoglobin, a function of detecting the amount of human glycated hemoglobin, and a liquid for the sensor unit. Provided is a human hemoglobin detection device including a supply path for supplying.

According to the present invention, the amount of human hemoglobin and the amount of human glycated hemoglobin can be detected only by supplying a liquid such as urine to the sensor unit via the supply channel, so that a blood glucose test or the like can be performed by a simple method. .

[0009] In a preferred embodiment, the sensor section can be provided with a sensor for detecting human hemoglobin and a sensor for detecting human glycated hemoglobin separately.
In another embodiment, one sensor may be switched between using it as a sensor for detecting human hemoglobin and using it as a sensor for detecting human glycated hemoglobin.

[0010] Specifically, first, an anti-human hemoglobin antibody is disposed on the sensor to provide a sensor for detecting human hemoglobin, then the anti-human hemoglobin antibody is removed from the sensor, and then the anti-human glycated hemoglobin antibody is removed from the sensor. A method of arranging it on the top to form a sensor for detecting human glycated hemoglobin may be considered.

Further, as a form of the sensor, a sensor utilizing surface plasmon resonance is preferable.

Next, as an embodiment of the supply path, the supply path to the sensor for detecting human hemoglobin and the second supply path to the sensor for detecting human glycated hemoglobin may be branched. Preferably, the sensor for detecting human hemoglobin and the sensor for detecting human glycated hemoglobin may be arranged in one supply path.

Further, the detection value of human hemoglobin,
By providing a calculating means for calculating the ratio between the amount of human hemoglobin and the amount of human glycated hemoglobin based on the detected value of glycated hemoglobin, it is not necessary to calculate the ratio manually.

The measurement of glycated hemoglobin by such an immunoassay requires calibration with hemoglobin and a standard solution of glycated hemoglobin.

Therefore, according to the present invention, the storage means for storing the detection calibration value of the amount of human hemoglobin, the detection correction value of the amount of human glycated hemoglobin, and the storage contents of the storage means when notifying the detection result of the sensor unit And a notifying unit for notifying in consideration of the above, the detection error can be reduced as much as possible.

The contents of the notification include the concentration of human hemoglobin and the ratio of the concentration of human glycated hemoglobin to the concentration of human hemoglobin.

The storage timing is such that when a solution containing a predetermined standard substance is supplied to the sensor unit, the detected value of human hemoglobin and the detected value of human glycated hemoglobin are compared with the amount of human hemoglobin. Detection calibration value,
It is desirable to store each as a detection calibration value of the amount of human glycated hemoglobin.

Further, as a standard substance, human hemoglobin (human glycated hemoglobin) itself may be used.
A long-term preservation of a standard substance is possible by using a known amount of a substitute for human hemoglobin (human glycated hemoglobin) that is less likely to be denatured over time than human hemoglobin (human glycated hemoglobin).

In particular, a substance in which the molecular structure of human hemoglobin (human glycated hemoglobin) has been changed, a substance in which the subunits of human hemoglobin (human glycated hemoglobin) are linked, and β of human hemoglobin (human glycated hemoglobin) Substances with interchain bonds are preferred.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS [Preparation of Crosslinked Hemoglobin] The basic method of preparing crosslinked hemoglobin is described in White and Ols.
en (Archives of B)
iochemistry and biophysics
s 1987 258 51-57). The reaction scheme is shown in FIG. Fresh 1.7 mM (as monomer) human hemoglobin and 3.0 mM bis (3,5-dib
romosalicyl fumarate and 10 mM
1 ml of 50 mM phosphate buffer (pH 7.2) containing sodium cyanate was allowed to stand at 37 ° C. for 2 hours to carry out a crosslinking reaction. After completion of the reaction, the sample is passed through a desalting column to be a reaction by-product (3,5-dibro
(mosalicyl) fumarate was removed to obtain crosslinked hemoglobin. The crosslinked hemoglobin was confirmed by SDS-polyacrylamide gel electrophoresis.

[Measurement with SPR sensor] Commercially available hemoglobin (Sigma) and the crosslinked hemoglobin shown in FIG. 1 were converted to 50 mM NaCl, 0.05% Triton X-1.
00 mM HEPES buffer (pH
As the standard solution of 10 μg / ml adjusted in 7.4),
The solution was left to stand at 4 ° C., 25 ° C., and 35 ° C., and surface plasmon resonance (SPR) was measured at regular intervals using an anti-hemoglobin monoclonal antibody, and the residual ratio from the start of use was determined.
It was used as an indicator of the stabilizing effect of hemoglobin. The result is shown in FIG. As shown in FIG. 2, it was found that the one using the crosslinked product as the standard solution retained 90% or more antigenicity even after one month at 35 ° C., and had a large stabilizing effect.

[Measurement by latex agglutination method] The measurement by the latex agglutination method is basically performed by a reagent H for an automatic analyzer.
For bA0 measurement (Eiken Science) was used. The standard hemoglobin (Eiken hemoglobin A0 standard) prepared with the standard diluent automatic analysis reagent HbA0 and the crosslinked hemoglobin shown in FIG. 1 were adjusted to a concentration of 500 ng / ml, and allowed to stand at 37 ° C. at constant temperature. 200 μl was taken every hour, mixed with 150 μl of hemoglobin latex emulsion, and left to stand at 37 ° C. for 15 minutes, and then quantified at a wavelength of 660 nm with an ultraviolet-visible absorptiometer. The residual rate from the start was determined and used as an index of the stabilizing effect of hemoglobin. The result is shown in FIG.
As shown in FIG. 3, when cross-linked hemoglobin was used as the standard solution, it maintained about 90% antigenicity even after 10 days at 37 ° C., indicating that the stabilizing effect was large.

[Application to Toilet Apparatus] FIG. 4 shows a configuration of a home test apparatus 1 capable of measuring occult blood in the urine. The home test apparatus 1 includes a urine collecting mechanism 3 in a toilet bowl 2 and a measuring apparatus. It has a main body 4. Inside the measuring device main body 4, a urine storage unit (not shown), a β-chain crosslinked hemoglobin storage unit (not shown) shown in FIG. 1, and an SPR sensor shown in [Measurement by SPR sensor] (FIG. (Not shown), and a display unit 5 for displaying the detection value (detected value of hemoglobin concentration) of the SPR sensor is provided on the surface of the measuring device main body 4.

A urine sample was collected in the urine reservoir by the urine collecting mechanism 3, and red blood cells in the urine were mixed with a surfactant to lyse hemolysin, and the released hemoglobin was detected by an immunosensor equipped in the main body.

The experiment was performed under the same conditions as in [Measurement by SPR sensor], except that the temperature was not controlled, and the sample was left at room temperature. In this case, the above β
The use of chain-crosslinked hemoglobin confirmed that the antigenicity of the standard hemoglobin was stable for several months even at room temperature (FIG. 5), indicating that it was not necessary to frequently exchange the standard solution.

The conventional hemoglobin standard requires an operation of dissolving the freeze-dried hemoglobin standard in a fixed amount of water (preferably distilled water or ultrapure water) homogeneously immediately before calibration. Users without knowledge of the measurement could not expect the reliability of the measurement. In addition, due to the complexity, the desire for continuous testing was reduced.

FIG. 6 shows a control flow of the home inspection equipment 1 shown in FIG. First, an SPR sensor value is detected from the β-chain crosslinked hemoglobin stored in a storage unit (not shown), and the detected value is stored as an initial value (step S1).

When the urine hemoglobin detection timing comes (step S2), the SPR sensor value is detected by the urine hemoglobin stored in the storage unit (not shown) (step S3). Calibration is performed based on the stored initial values, and the calibrated values are displayed on the display unit 5.

Here, the detection timing of urinary hemoglobin may be automatically performed each time urination is performed, or may be performed by a user of the toilet 2.

Thereafter, the process returns to step S2 to repeat the process until the timing for updating the initial value comes. Therefore, the initial value is used at a plurality of times of detecting urine hemoglobin, and it is not necessary to update the initial value every time urine hemoglobin is detected.

When it is time to update the initial value (step S5), the process returns to step S1 to repeat the processing. At this time, since the β-chain crosslinked hemoglobin is not exchanged, the β-chain crosslinked hemoglobin is used at a plurality of initial value update timings, and it is necessary to exchange the β-chain crosslinked hemoglobin every time the initial value is updated. Absent.

Here, the update timing of the initial value may be automatically performed every time the water temperature or the air temperature fluctuates due to the season or the like, but the point is that it corresponds to the fluctuation of the disturbance factor of the SPR sensor detection value. Just do it.

In the above embodiment, the amount of hemoglobin in urine is detected. However, human hemoglobin (HbA,
By measuring the ratio of human glycated hemoglobin (HbA1c, HbA1) to HbA1, HbA2, HbF), the amount of sugar in urine can be estimated. This is shown below.

As the name implies, diabetes has a condition in which sugar (glucose) is excreted in urine. However, an increase in blood glucose concentration is observed even when the level does not reach the level excreted in urine, or a transiently high blood glucose level is exhibited after eating. Frequent blood glucose testing is also required at home to monitor such fluctuations in blood glucose levels.

However, the blood glucose measurement performed a plurality of times every day is a burden on the patient, and the clinical significance is lost if the measurement is not performed at an appropriate timing. It was difficult to do.

Thus, glycated hemoglobin (Hb), which is a non-enzymatic conjugate of hemoglobin and glucose in blood,
It has become widely used to measure the ratio of A1c, HbA1). This indicates that glucose is bound to the amino group of valine at the end of hemoglobin β chain, and it has been shown that the ratio of the glycated form to the whole hemoglobin is useful for grasping the blood sugar control situation in the past month or so. In this method, all short-period blood sugar level fluctuations associated with daily meals can be comprehensively determined.

As shown in FIG. 7, saccharification of hemoglobin proceeds through a two-step reaction. At this time, the state in which glucose is bound to valine at the end of the hemoglobin β chain is a reversible formation of Schiff bases, and generates unstable aldimine (unstable HbA1c). Further, irreversible Amadori rearrangement generates a stable ketoamine type (stable HbA1c). It is known that the presence of such an unstable type greatly affects HbA1c measurement values particularly in cases where blood sugar levels fluctuate rapidly and in severe diabetes with ketoacidosis. Therefore, as a dissociation substance or a method for removing an unstable form, a phosphoric acid condensate (Japanese Patent No. 1978894), a short-time heat treatment with high-concentration boric acid (Japanese Patent Application Laid-Open No. 4-109163), and an organic acid mixture such as maleic acid and malonic acid (JP-A-6-34634) and the like have been developed.

In order to measure glycated hemoglobin after removing the unstable form in this way, an ion exchange chromatography method is used.
There are an electrophoresis method, a colorimetric method, and the like, and as a clinical test, an HPLC method applying an ion exchange chromatography method is used as a standard. However, in the case of the HPLC method, a dedicated device requires a high-precision pump, a spectrophotometer, a high-precision flow path system, and the like, which has a disadvantage that the device becomes expensive and its maintenance becomes complicated. .

Here, glycated hemoglobin has glucose bound to the end of the β chain, and can be measured using a monoclonal antibody having the binding site as an epitope. Outside Japan, assay kits based on enzyme immunoassay are also commercially available. In this case, the ratio of the glycated hemoglobin concentration is determined from the total hemoglobin concentration measured using antibodies that bind to all hemoglobins and the concentration measured using antibodies that bind to glycated hemoglobin.

The use of a monoclonal antibody capable of binding only to stable HbA1c may have the advantage that the removal of unstable HbA1c as described above becomes unnecessary. Since such an enzyme immunoassay can share measurement items with measurement items of other enzyme immunoassays frequently used in clinical tests, it has the advantage of not requiring a special device for measuring glycated hemoglobin. is there. Further, a simple measurement device can be constructed as an immunosensor in combination with an appropriate sensor such as a surface plasmon resonance (SPR) sensor.

In the measurement of glycated hemoglobin by such an immunoassay, calibration with hemoglobin and a standard solution of glycated hemoglobin is required. Hemoglobin is also used as a standard substance in measurements such as fecal occult blood, but hemoglobin is easily formed due to environmental factors such as temperature because hemoglobin is formed by associating two heme-containing polypeptides consisting of α-chain and β-chain. It is known that dissociation occurs and denaturation occurs. It is also known that even during freeze-drying, heme iron is oxidized and denatured, and antigen activity is reduced. Such a situation can occur in glycated hemoglobin as well.

The details will be described below.

[Purification of hemoglobin A1c] 20 mM
2- (N-morpholino) ethanesulfonic acid (MES)
In a buffer, human whole blood was diluted 15-fold with a hemolysis reagent containing 0.33% (V / V) of octylphenoxy-polyethoxy-ethanol and 0.6M borate, and mixed 5 times.
The lysed blood was prepared by leaving it for more than a minute. The lysed blood was added to a column (Hemoglobin A1c Column Test Micro; manufactured by Bio-Rad) and the first buffer solution (50 mM boric acid 5
After washing with 0 mM phosphate buffer (pH 7.0) and completely draining, a second buffer (50 mM phosphate buffer, pH 6.7) was added, and the eluate was washed with hemoglobin A1.
c.

[Preparation of Crosslinked Hemoglobin A1c] The crosslinked hemoglobin can be prepared in the same manner as described above.

[Stability of Crosslinked Glycated Hemoglobin A1c—Measurement with SPR Sensor] Commercially available hemoglobin A1c
(BioRad) and crosslinked hemoglobin A1c
10 mM HEPESbuffer containing mM NaCl, 0.05% Triton X-100 (pH 7.
4) Prepare the standard solution of 10 μg / ml prepared in 4), leave it at room temperature, and measure surface plasmon resonance (SPR) at regular intervals using a sensor chip on which anti-human hemoglobin A1c antibody (Exocell) was immobilized. The residual rate from the start of use was determined and used as an index of the stabilizing effect of hemoglobin.
The result is shown in FIG. As shown in the figure, the one using the crosslinked product as the standard solution retained 90% or more of the antigenicity even after one month, indicating that the stabilizing effect was large.

[Ratio of Glycated Hemoglobin to Total Hemoglobin in Blood—Measurement by SPR Sensor] Human blood is diluted with a buffer containing a surfactant such as octylphenoxy-polyethoxy-ethanol and hemolyzed. This diluted solution was used to prepare crosslinked glycated human hemoglobin A1
The concentrations were calculated with an SPR sensor using an anti-stable human glycated hemoglobin antibody and an SPR sensor using an anti-human hemoglobin antibody (see FIG. 9), which were calibrated with c and the cross-linked human hemoglobin standard solution, and the respective concentrations were calculated. The ratio (%) of the stable human glycated hemoglobin to the hemoglobin concentration in blood was calculated. When the measurement was performed on the blood of three healthy subjects, the ratio of stable human glycated hemoglobin was 4.3%, 4.5%, and 4.8%, respectively.

FIG. 9 shows the measuring device main unit 4 shown in FIG.
Is a detailed example of the standard liquid storage unit 11 and the urine collecting mechanism 3 of FIG.
Reservoir 12 that communicates with the buffer 1 for hemolysis
3, buffer 14, regenerating solution storage unit 15, valve 16,
A mixing chamber 17, an SPR sensor 18 using an anti-human hemoglobin antibody, an SPR sensor 19 using an anti-stable human glycated hemoglobin antibody, and a processing device 20 are provided. The use state will be described below.

[Calibration of SPR Sensors 18 and 19] A known concentration of crosslinked glycated human hemoglobin and crosslinked glycated human hemoglobin A1c stored in the standard solution storage section 11 are supplied to the buffer 14 via the valve 16 and the mixing chamber 17 via the buffer 16. Mixed with the buffer stored in the SPR sensor 18,
Supply to 19.

The detection signals of the human hemoglobin concentration and the human hemoglobin A1c concentration are transmitted from the SPR sensors 18 and 19 to the processing unit 20, respectively, and when a predetermined operation is performed, the processing unit 20 stores the detection signals as initial values. (Corresponding to step S1 in FIG. 6)

Subsequently, the cleaning liquid stored in the regenerating liquid storage section 15 is supplied to the SPR sensors 18 and 19 via the valve 16 and the mixing chamber 17, and the SPR sensor 1
8 and 19 are washed.

[Detection of SPR sensor value] The urine stored in the sample storage section 12 is mixed with the buffer stored in the hemolysis buffer 13 via the valve 16 and the mixing chamber 17 to cause hemolysis. Supply to 19.

The detection signals of the human hemoglobin concentration and the human hemoglobin A1c concentration are transmitted from the SPR sensors 18 and 19 to the processing device 20 (corresponding to step S3 in FIG. 6), and the processing device 20 performs the processing based on the stored initial values. ,
The detection signals of the human hemoglobin concentration and the human hemoglobin A1c concentration are calibrated.

Then, the processing device 20 digitizes the human hemoglobin concentration and the ratio of human hemoglobin A1c to human hemoglobin based on the calibrated detection signals, and causes the display unit 5 to display the numerical values. (Step S4 in FIG. 6)
Corresponding to)

Subsequently, the cleaning liquid stored in the regenerating liquid storage section 15 is supplied to the SPR sensors 18 and 19 via the valve 16 and the mixing chamber 17, and the SPR sensor 1
8 and 19 are washed.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of preparation of crosslinked hemoglobin

FIG. 2 SPR of commercially available hemoglobin and cross-linked hemoglobin
Sensor measurement result

FIG. 3 shows the results of measurement of commercially available hemoglobin and crosslinked hemoglobin by latex agglutination method

FIG. 4 is a configuration diagram of a home test device 1 capable of measuring urinary occult blood.

FIG. 5 shows the results of SPR sensor measurement of cross-linked hemoglobin in the usage environment of home test equipment 1

FIG. 6 is a control flow of the home inspection device 1;

FIG. 7 shows glycation of hemoglobin.

FIG. 8 shows the stabilizing effect of cross-linked hemoglobin.

FIG. 9 is a diagram showing details of a measuring device main body 4.

[Description of Signs] 1 ... Home inspection equipment, 2 ... Toilet bowl, 3 ... Urine collection mechanism, 4 ... Measurement device main body, 5 ... Display unit, 11 ... Standard solution storage unit, 12 ...
Sample reservoir, 13: hemolysis buffer, 14: buffer, 15: regenerating solution reservoir, 16: valve, 17: mixing chamber, 18: SPR sensor using anti-human hemoglobin antibody, 19: anti-stable human saccharified type SPR sensor using hemoglobin antibody, 20 ... processing unit

Continued on front page F term (reference) 2G045 AA01 AA15 AA36 BA11 BA13 BB01 BB14 BB29 BB31 BB39 BB41 BB50 BB51 CA02 CA25 CB03 DA30 DA31 DA44 DA45 DA46 DA47 DA48 DA49 DA51 FA11 FA29 FB03 GC10 HA01 HA03 HA06 HA09 JA04

Claims (6)

[Claims] A function of detecting the amount of human hemoglobin;
A human hemoglobin detection device comprising: a sensor unit having a function of detecting the amount of human glycated hemoglobin; and a supply path for supplying a liquid to the sensor unit. 2. The sensor unit, comprising: a sensor for detecting human hemoglobin; a sensor for detecting human glycated hemoglobin;
The human hemoglobin detection device according to claim 1, which comprises: 3. The supply path branched into a first supply path to the human hemoglobin detection sensor and a second supply path to the human glycated hemoglobin detection sensor. Human hemoglobin detector. 4. The method according to claim 1, further comprising calculating means for calculating the ratio between the amount of human hemoglobin and the amount of human glycated hemoglobin based on the detected value of human hemoglobin and the detected value of glycated hemoglobin. The human hemoglobin detecting device according to any one of the above. 5. A storage unit for storing a detection calibration value of a human hemoglobin amount and a detection calibration value of a human glycated hemoglobin amount, and when notifying the detection result of the sensor unit, the storage contents of the storage unit are taken into account. The human hemoglobin detection device according to claim 1, further comprising: a notification unit configured to perform notification. 6. The storage unit stores the detected value of human hemoglobin and the detected value of human glycated hemoglobin when the solution containing a predetermined standard substance is supplied to the sensor unit. 6. The human hemoglobin detection device according to claim 5, wherein the detection calibration value is stored as a detection calibration value of the human glycated hemoglobin amount.