JPH11264824A - Method for detecting human hemoglobin, manufacture of substance for detecting human hemoglobin, human hemoglobin detector and tray device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring a human hemoglobin utilizing an accurate human hemoglobin calibrator solution with high sensitivity by utilizing the stability of hemoglobin for suppressing a decrease in antigen properties due to an alteration in a solution of the hemoglobin. SOLUTION: Crosslinked α-chains or β-chains of a human hemoglobin by a specific crosslinking reagent are used as a hemoglobin measuring calibrator. The crosslinked hemoglobin does not dissociate to a sub-unit as compared with an uncrosslinked hemoglobin. An oxidizing speed of a heme is decelerated, the alteration speed brought about by an oxidation of the heme is decelerated, and a stable antigen-antibody reaction is continued for a long period. Accordingly, an accurate antigen-antibody reaction with high sensitivity is stably obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting human hemoglobin, a method for producing a substance for detecting human hemoglobin,
The present invention relates to a human hemoglobin detection device.

[0002]

2. Description of the Related Art Occult blood test is used to diagnose various diseases,
It is used in the determination of therapeutic effects and is performed on bodily fluids such as feces and urine, or blood and gastric juice.
As a test method for lower gastrointestinal diseases such as colorectal cancer, detection of occult blood components in feces caused by gastrointestinal bleeding, particularly human hemoglobin, is widely performed.

Conventionally, in the measurement of occult blood, a method of detecting hemoglobin in stool using a reagent to which an anti-hemoglobin antibody is bound and utilizing an immunological reaction has been widely used because of its excellent speed and simplicity. I was Then, a target reagent containing human hemoglobin is attached to the reagent used for this detection, and used for confirming the activity of the antibody binding reagent.

[0004] Here, human hemoglobin is composed of α-chain and β-chain.
It is known that since two heme-containing polypeptides comprising chains are associated with each other, dissociation easily occurs due to environmental factors such as temperature and denaturation. It is also known that even during freeze-drying, heme iron is oxidized and denatured, and antigen activity is reduced.

In particular, when hemoglobin is formulated into a standard product or a control reagent, the hemoglobin content per packaging unit is extremely small, and the hemoglobin content varies greatly due to denaturation, and the standard product or control reagent containing denatured hemoglobin is used. However, there is a problem in that the error of the measured value is increased when the method is used, and the measurement accuracy is lacking.

As a method for stabilizing hemoglobin to solve such a problem, Japanese Patent Application Laid-Open No. 9-61431 and the like disclose amino acids and prevent oxidation of heme iron, thereby reducing hemoglobin formation based on heme iron oxidation. It suppresses metamorphosis.

Further, Japanese Patent Application Laid-Open Nos. Hei 5-281227 and Hei 8
No. 262020 and the like contain iron proteins other than iron protoporphyrin or human hemoglobin, and similarly prevent denaturation of hemoglobin caused by oxidation of heme iron.

Further, Japanese Patent Application Laid-Open No. 8-245421 shows that the same effect can be obtained by including albumin and an amino acid.

[0009]

The above methods are all effects brought about by the addition of other components such as drugs, and are not due to the stabilization of hemoglobin itself.

Although the above description has been directed to the detection of human hemoglobin in an occult blood test, the same applies to the detection of human hemoglobin in a diabetes test.

That is, in recent years, as a diabetes test method,
It is known that the amount of human glycated hemoglobin (HbA1c, HbA1), which is a non-enzymatic conjugate of human hemoglobin and glucose, is detected to estimate the amount of urinary glucose.

Specifically, the ratio of human glycated hemoglobin (HbA1c, HbA1) to human hemoglobin (HbA, HbA1, HbA2, HbF) in the blood is measured.
This is because bA1c and HbA1) are also denatured with time and the antigen activity decreases.

[0013] The present invention provides a method for detecting human hemoglobin excellent in measurement accuracy without adding other components such as drugs.
A first object is to provide a substance for detecting human hemoglobin and a method for producing the same.

As a method for detecting human hemoglobin,
Although it is conceivable to use a sensor for detecting the amount of human hemoglobin in the solution, the sensor sensitivity sometimes fluctuates due to environmental factors such as temperature.

A second object of the present invention is to reduce the influence of sensor sensitivity fluctuation in a human hemoglobin detection method and detection device using a sensor for detecting the amount of human hemoglobin in a solution.

It is a third object of the present invention to provide a detection device capable of easily detecting components contained in urine such as human hemoglobin even in a room other than a laboratory.

[0017]

According to the present invention, there is provided a first step of preparing a substance in which subunits of human hemoglobin are bound, a second step of detecting the amount of the substance, And a method for detecting human hemoglobin, comprising:

That is, it is known that one-time denaturation of human hemoglobin is caused by the dissociation of a subunit composed of an α-chain and a β-chain. Due to the binding, the change over time in the detected value of human hemoglobin can be reduced as much as possible.

In the case of using a substance to which subunits are bound as in the present invention, there is a concern that the properties of human hemoglobin itself may be lost.
Since the so-called crosslinked hemoglobin in which 142-β142 is covalently bound with a crosslinking reagent such as dibromosalicyl fumarate is used, the characteristics of human hemoglobin itself are not lost.

Incidentally, the use of crosslinked hemoglobin as a blood substitute is because human hemoglobin dissociated from a tetramer to a dimer causes a problem for the kidney. A stabilizing effect has been observed.

According to an embodiment of the present invention, it is desirable that the subunits be connected to each other by β chains. This is because the β-β chain is more easily dissociated than the α-β chain or the α-α chain.

The bonding between the subunits can be easily realized by bonding by cross-linking. The positions of the cross-linking are α1lys-99-α2lys-99, β1val
-1-β2lys-82, β1lys-82-β2ly
s-82, β1 val-146-β2 val-146, etc., and the reaction is carried out by introducing a specific reagent at a site adjacent to the higher order structure between subunits, or introducing a cysteine residue by genetic engineering. A covalent bond is formed.

Specifically, as the crosslinking reagent, 2-nor
-2-formylpyridoxal-5'-pho
sphere (NFPLF), bis (3,5-dib)
romosalicyl) fumarate, fuma
rylbis (dibromoaspirin), 4,
4'-diisothiocyanostilbene
−2, 2′-disulfonate, bis (pyr
idoxalphosphate), bis (pyri
doxaltraphosphate) and the like.

For the above method for detecting human hemoglobin, specifically, it is preferable to use an immunological technique using an anti-human hemoglobin antibody, but in addition, a protein quantification method based on ultraviolet absorption of a protein is preferable. , Detection by a change in absorbance based on the adsorption of a dye to a protein, detection by a quantitative method based on a wavelength specific to human hemoglobin, or detection by a quantitative method based on the peroxidase-like activity of human hemoglobin.

The present invention also includes a first step of preparing human hemoglobin and a second step of changing the molecular structure of human hemoglobin to produce a substance different from human hemoglobin. Provided is a method for producing a substance for detecting human hemoglobin, which has a property that the substance is less likely to be denatured with time than human hemoglobin. If the substance for detecting human hemoglobin is detected, a change over time in the detected value of human hemoglobin can be reduced as much as possible.

As the substance for detecting human hemoglobin, a substance in which subunits of human hemoglobin are bound, particularly a substance in which β chains of human hemoglobin are bound, is suitable.

As a specific form of the human hemoglobin described above, human glycated hemoglobin (Hb
Compounds such as A1) may be used. In this case, in the method for producing a substance for detecting human hemoglobin, in the first step, a step of preparing human hemoglobin (HbA) and a saccharide, and binding the human hemoglobin (HbA) to a saccharide to form a human glycated hemoglobin ( And a step of producing HbA1).

According to a second aspect of the present invention, a first step of preparing a solution containing human hemoglobin and a second step of detecting the amount of human hemoglobin in the solution based on the value detected by the sensor for detecting human hemoglobin And a method for detecting human hemoglobin, comprising: a third step of preparing a solution containing a standard substance for calibrating the detection value of the sensor, comprising: It is characterized in that the detection value in the second step is calibrated based on the standard detection value based on the standard detection value.

That is, even if the sensor sensitivity fluctuates due to environmental factors such as temperature, the effect of the sensor sensitivity fluctuation can be reduced because the sensor detection value is calibrated in the third step. Here, human hemoglobin Hb is HbA, Hb
bA2, HbF and HbA formed by binding of HbA to sugar
However, the target to be detected in the second step may be all of these Hb or a part of Hb such as HbA1.

When the solution containing human hemoglobin in the second step contains stool or urine, it is effective for detecting occult blood, and when the solution contains blood, for example, the ratio of HbA1 to the total Hb is detected. By doing so, it can be used for determining diabetes and the like. In this case, for example, by preparing an antigen-antibody reaction sensor provided with both an antibody against all of Hb and an antibody against only HbA1, the whole Hb and HbA1 can be simultaneously recognized only by preparing the solution once. .

As the sensor, a sensor having an anti-human hemoglobin antibody, in particular, a sensor utilizing surface plasmon resonance can be used.

As the standard substance, human hemoglobin itself may be used. However, if a known amount of a human hemoglobin substitute substance, which is less likely to denature with time than human hemoglobin, is used, the standard substance can be stored for a long time. The above-mentioned substance in which the molecular structure of human hemoglobin is changed, a substance in which subunits of human hemoglobin are bound, and a substance in which β chains of human hemoglobin are bound are suitable.

When the standard substance is stored, various additives can be contained as needed. Such additives include buffering agents such as HEPS and phosphate, inorganic salts such as sodium chloride, SDS, Trion X-1.
A surfactant such as 00 can be exemplified. The standard substance is also applied to a freeze-dried preparation, and can be used after being dissolved in purified water at the time of use.

Furthermore, if the standard detection value is repeatedly used for a plurality of solutions containing human hemoglobin, the third step does not have to be performed every time the amount of human hemoglobin is detected.

If the solution containing the standard substance is repeatedly used in the third step a plurality of times, it is not necessary to use a new standard substance every time the third step is performed. In this case, since it is necessary to store the standard substance for a long period of time, it is desirable to use a known amount of a human hemoglobin substitute substance that is less likely to be denatured over time than human hemoglobin.

The present invention also relates to a sensor for detecting human hemoglobin, a storage means for storing a sensor-containing value for a solution containing a standard substance for calibrating the detection value of the sensor, and a storage means for storing a solution containing human hemoglobin. A notifying means for notifying the detection result of the sensor in consideration of the contents stored in the storage means, and providing a human hemoglobin detection device.

That is, the provision of the storage means for storing the detection value of the sensor makes it possible to repeatedly use the standard detection value for a solution containing a plurality of types of human hemoglobin.

As the sensor, a sensor having an anti-human hemoglobin antibody, in particular, a sensor utilizing surface plasmon resonance can be used.

As the standard substance, human hemoglobin itself may be used. However, if a known amount of a human hemoglobin substitute substance, which is less likely to be denatured with time than human hemoglobin, is used, the standard substance can be stored for a long time. The above-mentioned substance in which the molecular structure of human hemoglobin is changed, a substance in which subunits of human hemoglobin are bound, and a substance in which β chains of human hemoglobin are bound are suitable.

Further, if the reference material is repeatedly used for updating the stored contents a plurality of times, it is not necessary to use a new reference material every time the detection value of the sensor is stored. In this case, since it is necessary to store the standard substance for a long period of time, it is desirable to use a known amount of a human hemoglobin substitute substance that is less likely to be denatured over time than human hemoglobin.

As an embodiment of a solution containing human hemoglobin, in the case of a solution containing stool and urine, if a human hemoglobin detecting device is provided in a toilet, occult blood in stool and urine can be detected regardless of the examination room or the like. it can. Further, as a specific form of the human hemoglobin described above, a compound such as human glycated hemoglobin (HbA1) may be used.

According to a third aspect of the present invention, there is provided a storage unit for storing urine, a sensor for detecting human hemoglobin, and a detecting means for detecting the amount of human hemoglobin in the stored urine using the sensor. To provide a toilet device comprising: According to the present invention, urinary occult blood can be detected without using an examination room or the like.

As a preferred embodiment, if the notifying means for notifying the detection result of the detecting means is provided in the toilet device, the detection result can be easily known.

If a sensor utilizing surface plasmon resonance is used as a sensor, even a small amount of human hemoglobin can be accurately detected.

The present invention also provides a storage unit for storing urine,
A sensor comprising an antibody against an antigen contained in urine,
A detecting device for detecting the amount of antigen in urine stored using the sensor;

According to the present invention, various components contained in urine can be detected by using an antigen-antibody reaction without using a test room or the like.

If a sensor utilizing surface plasmon resonance is used as a sensor, even a small amount of antigen can be accurately detected. When the antigen is linked to the sugar contained in the urine, the amount of the sugar in the urine can be reported based on the detected amount of the antigen in the urine.

The present invention further provides a storage unit for storing urine, a sensor utilizing surface plasmon resonance, and a detecting means for detecting an amount of a substance to be detected in the stored urine using the sensor. A toilet device comprising:

According to the present invention, even a very small amount of an analyte such as sugar in urine can be accurately detected.

[0050]

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 from the urine reservoir by the urine collecting mechanism 3, and red blood cells in the urine were mixed with a surfactant to cause hemolysis, 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 to dissolve 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 household inspection device 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 processing 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 the timing for updating the initial value comes (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 changes 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 was 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 unless the measurement is 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.

The saccharification of hemoglobin proceeds through a two-step reaction as shown in FIG. 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 manner, 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 is obtained by binding glucose to the end of β chain, and measurement using a monoclonal antibody having the binding site as an epitope is also possible. 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.

Incidentally, 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 solution, human whole blood was diluted 15-fold with a hemolysis reagent containing 0.33% (V / V) of octylphenoxy-polyethoxy-ethanol and 0.6 M borate.
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 draining completely, 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] A crosslinked hemoglobin can be prepared in the same manner as described above.

[Stability of Crosslinked Glycated Hemoglobin A1c—Measurement by 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 Glycosylated 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] Crosslinked glycated human hemoglobin and crosslinked glycated human hemoglobin A1c of known concentrations stored in the standard solution storage unit 11 are buffered via a valve 16 and a mixing chamber 17 into a buffer 14. 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 device 20, respectively. When a predetermined operation is performed, the processing device 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 unit 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] Urine stored in the sample storage section 12 is mixed with a buffer stored in a hemolysis buffer 13 via a valve 16 and a 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.

The processor 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 results

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 apparatus 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 saccharification 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.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Home test equipment, 2 ... Toilet bowl, 3 ... Urine collection mechanism, 4 ... Measuring device main body, 5 ... Display part, 11 ... Standard liquid storage part, 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 the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/53 G01N 33/53 K 33/72 33/72 A // G01N 33/50 33/50 N

Claims (42)

[Claims] 1. A method for detecting human hemoglobin, comprising: a first step of preparing a substance in which subunits of human hemoglobin are bound, and a second step of detecting the amount of the substance. 2. The method for detecting human hemoglobin according to claim 1, comprising using a substance in which β chains of human hemoglobin are bound. 3. The method for detecting human hemoglobin according to claim 1, wherein the subunits or the β chains are linked by crosslinking. 4. The method according to claim 1, wherein the detection is carried out by an immunological technique using an anti-human hemoglobin antibody.
4. The method for detecting human hemoglobin according to any one of 3. 5. The method for detecting human hemoglobin according to claim 1, wherein the detection is performed by a protein quantification method based on ultraviolet absorption of the protein. 6. The method for detecting human hemoglobin according to claim 1, wherein the detection is performed by a change in absorbance based on the adsorption of a dye to a protein. 7. The detection method according to claim 1, wherein the detection is performed by a quantitative method based on a wavelength specific to human hemoglobin.
The method for detecting human hemoglobin according to any one of the above. 8. The method for detecting human hemoglobin according to claim 1, wherein the detection is performed by a quantitative method based on a peroxidase-like activity of human hemoglobin. 9. The method for detecting human hemoglobin according to claim 1, wherein said human hemoglobin is human glycated hemoglobin (HbA1). 10. A first step of preparing human hemoglobin, and a second step of changing the molecular structure of human hemoglobin to produce a substance different from human hemoglobin, wherein the substance is a human hemoglobin. A method for producing a substance for detecting human hemoglobin, which has a property of being less likely to denature with time than hemoglobin. 11. The method for producing a substance for detecting human hemoglobin according to claim 10, wherein in the second step, a substance in which subunits of human hemoglobin are bound is generated. 12. The method for producing a substance for detecting human hemoglobin according to claim 11, wherein in the second step, a substance to which a β chain of human hemoglobin is bound is generated. 13. The method for detecting human hemoglobin according to claim 10, wherein the human hemoglobin is human glycated hemoglobin (HbA1). 14. The first step is a step of preparing human hemoglobin (HbA) and a saccharide, and a step of producing a human glycated hemoglobin (HbA1) by binding the human hemoglobin (HbA) to a saccharide; 14. The method for detecting human hemoglobin according to claim 13, comprising: A substance for detecting human hemoglobin produced by the production method according to any one of claims 10 to 14. 16. A method for preparing a solution containing human hemoglobin, comprising: a first step of preparing a solution containing human hemoglobin; and a second step of detecting the amount of human hemoglobin in the solution based on a detection value of a sensor for detecting human hemoglobin. A detection method, comprising a third step of preparing a solution containing a standard substance for calibrating the detection value of the sensor, wherein the detection value of the sensor for the solution containing the standard substance is used as a standard detection value, A method for detecting human hemoglobin, wherein the detection value in the second step is calibrated based on a standard detection value. 17. The method for detecting human hemoglobin according to claim 16, wherein the sensor has an anti-human hemoglobin antibody. 18. The method for detecting human hemoglobin according to claim 17, wherein the sensor utilizes surface plasmon resonance. 19. The method for detecting human hemoglobin according to any one of claims 16 to 18, wherein the standard substance is a known amount of a human hemoglobin replacement substance that is less likely to change over time than human hemoglobin. 20. The method for detecting human hemoglobin according to claim 19, wherein the standard substance is the substance for detecting human hemoglobin according to claim 15. 21. The method for detecting human hemoglobin according to claim 16, wherein the standard detection value is repeatedly used for a plurality of solutions containing the human hemoglobin. 22. The solution containing the standard substance, which is used repeatedly in the third step a plurality of times.
The method for detecting human hemoglobin according to the above. 23. The method for detecting human hemoglobin according to claim 16, wherein the solution containing human hemoglobin in the second step includes any of blood, stool, and urine. 24. The method for detecting human hemoglobin according to claim 16, wherein the human hemoglobin is human glycated hemoglobin (HbA1). 25. A sensor for detecting human hemoglobin, storage means for storing a detection value of the sensor for a solution containing a standard substance for calibrating a detection value of the sensor, and a sensor for detecting a solution containing human hemoglobin. A notification unit for notifying the detection result in consideration of the storage content of the storage unit when notifying the detection result. 26. The human hemoglobin detection device according to claim 25, wherein the sensor has an anti-human hemoglobin antibody. 27. The method according to claim 26, wherein the sensor utilizes surface plasmon resonance. 28. The human hemoglobin detection device according to claim 25, wherein the standard substance is a known amount of a human hemoglobin replacement substance that is less likely to be degenerated over time than human hemoglobin. 29. The apparatus for detecting human hemoglobin according to claim 28, wherein the standard substance is the substance for detecting human hemoglobin according to claim 15. 30. The human hemoglobin detection device according to claim 25, further comprising an updating unit that updates the stored content under a predetermined condition, wherein the reference material is repeatedly used for the updating a plurality of times. 31. The solution containing human hemoglobin,
The human hemoglobin detection device according to any one of claims 25 to 30, comprising blood. 32. The solution containing human hemoglobin,
The human hemoglobin detection device according to any one of claims 25 to 30, wherein the device includes any of stool and urine. 33. The human hemoglobin detection device according to claim 32, which is provided in a toilet. 34. The human hemoglobin detection device according to claim 25, wherein the human hemoglobin is human glycated hemoglobin (HbA1). 35. A toilet apparatus comprising: a storage section for storing urine; a sensor for detecting human hemoglobin; and a detecting means for detecting the amount of human hemoglobin in the stored urine using the sensor. 36. The toilet apparatus according to claim 35, further comprising a notifying means for notifying a detection result of said detecting means. 37. The toilet apparatus according to claim 35, wherein the sensor uses surface plasmon resonance. 38. A storage section for storing urine, a sensor having an antibody against an antigen contained in urine, and a detecting means for detecting the amount of antigen in the stored urine using the sensor. Toilet equipment. 39. The toilet apparatus according to claim 38, wherein the sensor uses surface plasmon resonance. 40. A notifying means for notifying the amount of sugar in urine based on the amount of antigen in urine detected by the detecting means, wherein the antigen is linked to sugar contained in urine. Claim 38.
Or a toilet device according to item 39. 41. A toilet comprising: a storage unit for storing urine; a sensor using surface plasmon resonance; and detection means for detecting an amount of a substance to be detected in the stored urine using the sensor. apparatus. 42. The substance to be detected is a sugar.
The toilet device according to 1.