JP5119202B2 - Hemoglobin A1c measuring device - Google Patents

Description

  The present invention relates to an apparatus for measuring hemoglobin A1c with high sensitivity.

  Measurement of hemoglobin A1c (hereinafter referred to as HbA1c) in blood is regarded as important as a test item for diabetes diagnosis. HbA1c is obtained by binding a sugar chain to hemoglobin (Hb), and clinically, the ratio of the HbA1c concentration to the total Hb concentration present in blood is used for diagnosis as a test value. Although the measurement of HbA1c has been conventionally performed using chromatography, there are spectroscopic measurement methods using an immunoturbidimetric method and an enzymatic method as simpler measurement methods. Further, Patent Document 1 discloses a method of detecting an electrochemical signal as a detection method using a so-called sensor. On the other hand, Hb is known to exhibit a high redox potential because it absorbs light of a wide wavelength and has an iron complex structure. Since HbA1c is a glycated peptide bound to Hb, in the measurement method as described above, Hb itself is a noise that decreases the absorbance or electrochemical signal S / N ratio derived from the target HbA1c. Is generated. Therefore, in the measurement method using the spectroscopic method, the amount of light absorbed by Hb is suppressed by diluting the whole blood sample containing Hb or shortening the optical path length, thereby enabling measurement of HbA1c.

JP 2007-155684 A

  On the other hand, in the measuring method for detecting an electrochemical signal derived from HbA1c, there is no effective means for reducing the electrical signal derived from Hb. Further, the spectroscopic measurement method has a problem in that the measurement sensitivity is lowered by diluting at a high magnification.

  In the present invention, Hb is precipitated by using a polymer precipitating agent, and Hb liberated the glycated peptide from HbA1c is removed by filtration or extraction of the supernatant, thereby improving measurement accuracy in HbA1c measurement.

  According to the measuring device disclosed in the present invention, by using a polymer precipitant, Hb is precipitated / removed under mild conditions without causing a change in temperature and pH, thereby improving measurement accuracy in HbA1c measurement. It becomes possible.

It is the figure which showed one Embodiment of the measuring apparatus to which this invention is applied. It is the figure which showed one Embodiment of the measurement mechanism in this invention. It is the figure which showed one Embodiment of the container in this invention. It is the figure which showed one Embodiment of the liquid feeding method in this invention. It is the figure which showed one Embodiment of the liquid feeding method in this invention. It is the figure which showed one Embodiment of the deposit separation mechanism in this invention. It is the figure which showed one Embodiment of the measurement mechanism in this invention. It is the figure which showed one Embodiment of the measurement mechanism in this invention. It is a figure which shows the result of Example 1. It is a figure which shows the result of Example 1. It is a figure which shows the result of Example 2. It is a figure which shows the result of Example 3.

  The desirable form for implementing this invention is illustrated below.

  In a desirable embodiment, a method and an apparatus for measuring HbA1c concentration with high sensitivity using whole blood as a measurement sample are provided.

  The measurement apparatus of the present invention has a pretreatment container in which a sample is introduced and glycated peptide is released from HbA1c by chemical reaction or physical means, and Hb cleaved from the glycated peptide in the pretreatment container or originally has no glycated peptide. It consists of a precipitation container for precipitating Hb, a precipitate separation mechanism for removing the precipitate from the measurement sample, a measurement container for quantifying the glycated peptide, and a measurement mechanism for quantifying the glycated peptide in the sample in the measurement container. . One container can also function as a plurality of containers. The container may be any structure that can hold the solution, and is preferably a cup, filter, membrane, tube, or the like.

  A whole blood sample is used as a measurement sample. The sample may be processed with an anticoagulant or a hemolyzed sample before the start of measurement. The amount of the sample solution introduced into the reaction vessel is desirably 0.1 μL or more, and in the most desirable embodiment, is 10 μL or more.

  After the measurement sample is added to the pretreatment container, a hemolytic agent and a degrading enzyme for cutting out the glycated peptide from HbA1c are added, and the glycated peptide is released from HbA1c in the sample hemolyzed by the hemolytic agent. At this time, the hemolytic agent and the degrading enzyme may be held in the container in advance. The container may contain other reagents such as an anticoagulant as long as it does not inhibit the reaction of a hemolytic agent or a degrading enzyme. As the hemolytic agent, an ionic or nonionic surfactant, an organic solvent, a salt, and an enzyme can be used. As the surfactant, polyoxyethylene octyl phenyl ether (trade name: Triton X100), sodium dodecyl sulfate (hereinafter referred to as SDS), saponin and the like are desirable. As the organic solvent, formaldehyde, hexane, acetone and the like are desirable. As the salt, ammonium chloride, aluminum chloride and the like are desirable. A more desirable example is SDS. In this case, a desirable concentration is 1 to 20 v / v%. In addition, hemolysis may be promoted by a change in salt concentration such as dilution with distilled water. As the degrading enzyme, an enzyme capable of releasing a glycated peptide unique to HbA1c from HbA1c can be used. For example, protease, proteinase, and endonuclease can be mentioned. Desirable examples include protease XIV (Sigma), bromelain F (Amano Enzyme), protease N (Amano Enzyme), Protamex (Novozyme), and most preferably Protease N (Amano Enzyme). is there.

  After the treatment in the pretreatment container is completed, the sample solution containing free glycated peptide and Hb in the pretreatment solution is moved to the precipitation container. In the precipitation container, a precipitant is added to the sample solution, and Hb is precipitated by reaction with the precipitant. The reaction solution in which the precipitation has occurred is removed through the precipitate separation mechanism, and the measurement sample containing the free glycated peptide is separated.

  As the precipitant, a surfactant inorganic salt, an organic salt, a polyelectrolyte, a protein denaturant and a mixture thereof can be used, and a combination of an anionic surfactant and a metal salt is particularly desirable. The surfactant is preferably a water-soluble surfactant having an anionic functional group and capable of binding to Hb to form a complex. SDS, sodium dextran sulfate and sodium heparin are desirable, and SDS is most desirable. The concentration of SDS is preferably 100 mmol / L or more, and most preferably about 640 mmol / L. The metal salt is preferably water-soluble and soluble in water to produce a polyvalent metal cation. Specifically, a salt of magnesium, manganese, calcium, zinc, aluminum, cesium or the like can be used, and most preferably magnesium chloride. The concentration is desirably 10 mmol / L or more, and most desirably 600 mmol / L or more. Furthermore, for the purpose of improving the precipitation performance, it is also effective to add polyethylene glycol (hereinafter referred to as PEG) to the mixture of SDS and metal salt. In this case, the average molecular weight of PEG is desirably about 1,000 to 10,000, and most desirably about 6,000. The concentration is preferably 100 to 1,000 g / L.

  The precipitate separation mechanism may be any structure that can separate the reaction solution component containing the glycated peptide and the precipitate containing Hb, and includes a filtration mechanism, a centrifugal separation mechanism, a supernatant extraction mechanism, and the like. The filtration mechanism is preferably a structure in which filters having a plurality of pore sizes are laminated for the purpose of removing clogging due to separated Hb. The filter may be provided in a cylindrical structure such as a syringe or a tube, and separation may be promoted by drawing pressure or pressurization. The centrifugal separation mechanism can be small and has a steady rotation speed of 3,000 rpm or more, and most preferably has a steady rotation speed of about 6,000 rpm. The supernatant extraction mechanism may be any mechanism that can partially suck the supernatant portion that does not contain the precipitate, and is preferably a pipetter or a tube. In order to facilitate the separation, a filter may be further provided in the suction part.

  The measurement mechanism may be any mechanism that can stably determine the HbA1c concentration with good reproducibility, and the measurement principle may be any of the spectroscopic method, current method, voltage method, AC impedance method, and conductivity method. . Desirably, it is a spectroscopic method that has been used in many devices and has established a measurement method, or a voltage measurement type sensor that can measure even a small amount of sample after Hb separation.

  The most preferred embodiment of the disclosed device is shown in FIG.

  In the most desirable embodiment, the pretreatment container 1, the sedimentation container 2, the sediment separation mechanism 3, and the measurement container 4 are formed of filters, and the liquid is transferred between the containers by capillary action by stacking them. The precipitation vessel contains a precipitation agent, and the sample solution permeates into the precipitation vessel and reacts with the precipitation agent to solidify. Since the solidified Hb significantly decreases the moving speed in the filter, which is a precipitate separation mechanism, the solution component containing the glycated peptide first reaches the measurement container. The glycated peptide in the solution component reacts with the reaction reagent contained in the measurement container to produce a reaction product. HbA1c can be quantified by detecting the reaction product with the measurement mechanism 5 in contact with the measurement container. In this case, the measurement mechanism is a sensor that quantifies HbA1c by detecting an electrical signal or absorbance.

  Precipitant composition: The concentration of each reagent is adjusted to the following concentration by adding the sample.

SDS: 640 mmol / L
Magnesium chloride: 600 mmol / L
PEG: 600 g / L

  As described above, the sensor is used as the measurement mechanism. However, the spectroscopic cell may be used as the measurement container, and the spectrophotometer may be used as the measurement mechanism to perform the measurement by spectroscopy (FIG. 2). Moreover, although the filter was used for the pretreatment container and the precipitation container, it may be moved to the next container by a dispensing mechanism using a cup-shaped container (FIG. 3). Further, as a driving force for the measurement sample to pass through the precipitate separation mechanism, liquid feeding may be promoted by pulling (FIG. 4) or pressurizing (FIG. 5) using a pump. As a precipitate separation mechanism, it is possible to provide a small centrifugal separator (FIG. 6). In addition, by connecting the Hb measurement unit and the HbA1c measurement unit in succession, it is possible to use the Hb measured sample as it is for the HbA1c measurement (FIG. 7). The introduced sample is divided into two or more. It is also possible to distribute the introduced sample to the Hb measurement unit and the HbA1c measurement unit and measure them in parallel (FIG. 8).

Example 1: Comparison of Measurement Value According to Most Desired Form and Apparatus Not Containing Removal Mechanism According to the most desirable embodiment, whole blood samples having different concentrations were measured using a potentiometric sensor, and a calibration curve was prepared. The slope of the calibration curve was defined as the measurement sensitivity, and the sensitivity of the most desirable embodiment (Example 1) and the sensitivity not including the Hb removal mechanism were compared. Compared with the theoretical value (−59.2 mV / dec) of the sensitivity calculated from the equation, the sensitivity was −4.3 mV / dec (FIG. 9). On the other hand, in the most desirable embodiment, a sensitivity close to the theoretical value of -56.3 mV / dec was obtained (FIG. 10), and a sensitivity improvement of about 13 times was confirmed as compared with the sensitivity in the form not including the removal mechanism. .

Example 2: Optimization of precipitant composition SDS and magnesium chloride were used as precipitants, and the precipitants of various compositions were prepared by mixing at different concentrations. Precipitation agent of various compositions was reacted with Hb-containing samples to cause precipitation, followed by centrifugation, and the concentration of Hb contained in the supernatant was measured to evaluate the precipitation ability. The Hb concentration was measured using an absorbance method. The results are shown in FIG. When the SDS concentration is in the range of 320 to 640 mmol / L and the magnesium chloride concentration is in the range of 300 to 600 mmol / L, there is a tendency that the remaining Hb concentration can be decreased (easily precipitated) as the concentration increases, and the SDS concentration is 640 mmol / L. , Hb could be precipitated most effectively at a magnesium chloride concentration of 600 mmol / L.

Example 3: Improving the precipitating ability of the precipitating agent To the precipitating agent having the composition optimized in Example 2, PEG (average molecular weight 6,000) generally used as a protein precipitating agent was further added. The precipitation ability was evaluated in the same manner as in No. 2. The results are shown in FIG. The higher the PEG concentration, the higher the Hb concentration that can be precipitated, and the Hb remaining at a PEG concentration of 600 g / L decreased below the detection limit value of the spectroscopic method.

1 HbA1c pretreatment container 2 Hb precipitation container 3 Precipitate separation mechanism 4 HbA1c measurement container 5 HbA1c measurement mechanism 6 Hb measurement mechanism 7 Hb measurement container 8 Sample distribution mechanism

Claims (3)

A filter containing a precipitant that precipitates hemoglobin, a filter that separates the precipitate precipitated by the precipitant and the liquid containing the glycated peptide, and a filter that reaches the liquid containing the glycated peptide are stacked,
The precipitating agent comprises a combination of a substance selected from the group consisting of dodecyl sulfate, dextran sulfate, heparin salt and a substance selected from the group consisting of magnesium salt, calcium salt, zinc salt,
The hemoglobin A1c measuring apparatus is characterized in that liquid is moved between the filters by capillary action. In the hemoglobin A1c measuring apparatus of Claim 1,
Furthermore, the hemoglobin A1c measuring apparatus is provided with a measuring mechanism for measuring hemoglobin A1c in contact with a filter that the liquid containing the glycated peptide reaches . The hemoglobin A1c measuring device according to claim 2,
The hemoglobin A1c measuring apparatus , wherein the measuring mechanism is a sensor that quantifies hemoglobin A1c by detecting an electrical signal or absorbance .

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