Chemistry: analytical and immunological testing – Hemoglobin – myoglobin – or occult blood
Reexamination Certificate
2003-03-20
2004-09-14
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Hemoglobin, myoglobin, or occult blood
C436S067000, C436S164000, C436S904000
Reexamination Certificate
active
06790665
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method of determining an amount of hemoglobin (Hb) in a sample.
BACKGROUND ART
Hb in the blood plays an important role in transporting oxygen from the lungs to organs and thus relates to diseases such as leukemia, anemia, and the like, for example. Therefore, determining an amount of Hb has been considered very important in the field of a clinical analysis. On the other hand, glycated Hb serves as an important index for the diagnosis, treatment, etc. of diabetes because it reflects previous blood glucose levels in vivo. Therefore, determining a ratio of glycated Hb also has been considered important. For determining the ratio of glycated Hb, it is necessary to determine the amount of Hb.
Examples of a method of determining Hb include measuring an absorbance of Hb. However, the Hb that is not yet denatured (hereinafter, referred to as “undenatured Hb”) exhibits an absorption maximum at different wavelengths depending on its state, e.g., the state where it is bound to oxygen, the state where it is not bound to oxygen, etc. Therefore, it is difficult to determine an amount of Hb accurately by merely measuring the absorbance of the Hb. On this account, conventionally, a method has been employed in which the absorbance of Hb is measured after the Hb has been denatured so as to be stabilized. Examples of such a method include a cyanmethemoglobin method (HiCN method), azide metohemoglobin method, sodium lauryl sulfate method (SLS method), alkaline hematin method, and the like. Among these, the HiCN method, which is an international standard method, is employed particularly widely. In this HiCN method, a reagent containing potassium ferricyanide and potassium cyanide is added to blood so that Hb is converted into stable cyanmethemoglobin, and the absorbance is measured at a predetermined wavelength (540 nm) to determine the amount of the Hb.
DISCLOSURE OF INVENTION
However, the HiCN method and the azide metohemoglobin method are not preferable from the viewpoint of environmental friendliness because the HiCN method produces a toxic liquid waste containing a cyanogen compound and the azide metohemoglobin method produces a liquid waste containing sodium azide. Further, the SLS method and the alkaline hematin method have the following problem. In these methods, a reagent such as SLS and a strong alkali is added to a sample as a protein-denaturing agent. Thus, when the sample containing such a reagent is used directly for the determination of a substance other than Hb, the reagent affects the system of determination utilizing an enzyme and the like, for example, which makes the determination difficult. Therefore, with respect to the sample that has been subjected to the treatment for denaturing Hb, it is difficult to carry out the determination of the denatured Hb and the determination of the substance other than the denatured Hb in a series of operations.
Therefore, it is an object of the present invention to provide a method of determining Hb, by which an amount of Hb can be determined easily and accurately without fear of damage to the environment.
In order to achieve the above object, a method of determining Hb according to the present invention includes: denaturing Hb in a sample with a tetrazolium compound to give denatured Hb; measuring an amount of an optical change in the sample at an absorption wavelength specific to the denatured Hb; and calculating an amount of the Hb in the sample from the amount of the optical change. The term “denatured Hb” as used in the present invention refers to Hb that has been denatured with a tetrazolium compound.
While the undenatured Hb exhibits various absorption wavelengths depending on its state, the denatured Hb obtained by the treatment with the tetrazolium compound is stable and exhibits the absorption maximum at a wavelength falling within a certain range. Therefore, according to the method of determining Hb of the present invention, an amount of Hb can be determined easily. In addition, the method of the present invention does not use a cyanogen compound, a strong alkali, or the like as used in the above-mentioned conventional methods. Therefore, the method of the present invention is a useful method without fear of damage to the environment.
In the method of determining Hb according to the present invention, the amount of the optical change may be an absorbance, reflectance, or the like, for example.
The tetrazolium compound is not specifically limited. For example, tetrazolium compounds described later can be used. Among these, 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium salt (e.g., available from Dojindo Laboratories under the trade name WST-3) is most preferable.
In the method of determining Hb according to the present invention, the wavelength for measuring the denatured Hb preferably is in the range from 440 to 700 nm, more preferably from 500 to 670 nm, and most preferably 540 to 670 nm.
In the method of determining Hb according to the present invention, the amount of the tetrazolium compound added to the sample is not specifically limited and can be decided as appropriate depending on the type of the sample and the like. More specifically, it is preferable that the tetrazolium compound is added to the sample so that a content of the tetrazolium compound per microliter of the sample is in the range from 0.001 to 100 &mgr;mol, more preferably from 0.01 to 10 &mgr;mol, and most preferably from 0.05 to 5 &mgr;mol, for example.
The sample is not specifically limited. However, as described later, a sample containing red blood cells, e.g., whole blood, preferably is used. In the case where the sample is whole blood, the tetrazolium compound preferably is added to the sample so that a content of the tetrazolium compound per microliter of the sample is in the range from 0.01 to 30 &mgr;mol, more preferably from 0.05 to 10 mol, and most preferably from 0.1 to 5 &mgr;mol, for example. In general, it is estimated that whole blood contains about 50 vol % of blood cells.
In the method of determining Hb according to the present invention, it is preferable that the Hb in the sample is treated with the tetrazolium compound in the presence of a surfactant. When the surfactant is present in addition to the tetrazolium compound, denaturation of the Hb can be accelerated still further so that the Hb can be determined quickly.
In the method of determining Hb according to the present invention, the amount of the surfactant added to the sample is not specifically limited, and can be decided as appropriate, for example, depending on the amount of the tetrazolium compound added to the sample and the like. More specifically, the surfactant is added to the sample so that a content of the surfactant per mole of the tetrazolium compound is in the range from 0.01 to 70 mol, preferably from 0.05 to 50 mol, and more preferably from 0.1 to 20 mol.
In the method of determining Hb according to the present invention, the sample is not specifically limited and can be, for example, blood samples such as whole blood, plasma, serum, blood cells, and the like. It is preferable to use a sample containing red blood cells, for example, a whole blood sample, a blood cell sample, etc.
Next, a method of determining a ratio of glycated Hb according to the present invention includes: determining an amount of Hb in a sample containing glycated Hb by the method of determining Hb according to the present invention; causing a redox reaction between a glycation site of the denatured Hb obtained and a fructosyl amino acid oxidase (hereinafter, referred to as “FAOD”); determining the redox reaction to determine an amount of the glycated Hb; and calculating a ratio of the glycated Hb from the amount of the Hb and the amount of the glycated Hb. It is to be noted here that the amount of Hb refers to the amount of both the glycated Hb and the Hb that is not glycated.
Similarly to the method of determining Hb described above, the method of determining a ratio of glycated Hb according to the present invention al
Yagi Yuji
Yonehara Satoshi
Arkray Inc.
Merchant & Gould P.C.
Wallenhorst Maureen M.
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