WO2006112339A1 - ヘモグロビン誘導体の測定方法、それに用いる試薬組成物、測定キット、分析デバイス、及び分析システム - Google Patents
ヘモグロビン誘導体の測定方法、それに用いる試薬組成物、測定キット、分析デバイス、及び分析システム Download PDFInfo
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- WO2006112339A1 WO2006112339A1 PCT/JP2006/307781 JP2006307781W WO2006112339A1 WO 2006112339 A1 WO2006112339 A1 WO 2006112339A1 JP 2006307781 W JP2006307781 W JP 2006307781W WO 2006112339 A1 WO2006112339 A1 WO 2006112339A1
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- hemoglobin
- derivative
- hemoglobin derivative
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
Definitions
- the present invention relates to a method for measuring a hemoglobin derivative in a blood sample, a reagent composition used therefor, a measurement kit, an analysis device, and an analysis system, and relates to a technique for quickly and reliably denaturing hemoglobin. . Background art
- Glycohemoglobin one of the hemoglobin derivatives, can be used for early detection of lifestyle-related diseases because it is possible to determine normal blood glucose levels without the effects of blood glucose fluctuations due to diet. It is an item that is often measured.
- Glucose hemoglobin is also called hemoglobin Ale, which is glucose bound to hemoglobin contained in erythrocytes, and is numerically expressed at a ratio of sugar hemoglobin to hemoglobin.
- a method for measuring glycated hemoglobin there is a method utilizing an immune reaction.
- the measurement method using this immune reaction first discriminates whether hemoglobin is non-glycohemoglobin, which is a non-glycohemoglobin, after hemoglobin is removed from the red blood cells by hemolyzing the blood sample. Therefore, by changing the three-dimensional structure of hemoglobin, the glycated part of the hemoglobin protein is exposed to the outside of the three-dimensional structure (degeneration of hemoglobin), and the glycated part is made specific. The amount of glycated hemoglobin is measured immunologically by reacting with an antibody to be recognized.
- this method is an analytical method for measuring the relative amount of a specific hemoglobin derivative in a blood sample, comprising: (a) a blood sample in (i) 0.5-6 A thiocyanate salt that can denature substantially all hemoglobin present in the blood sample at a concentration of OM and substantially all hemoglobin present in the GO blood sample are converted to the form of methemoglobin.
- Patent Document 3 there is a method of performing modification with an ionic surfactant. Specifically, this method is a method for measuring the content of a hemoglobin derivative in a blood sample, and (a) an ion having a pH of 5 to 9.5 at a temperature of 4 to 37 ° C. Treatment with a hemolysis reagent containing a detergent for up to 10 minutes, and (b) immunological measurement in a blood sample in which hemoglobin derivative is hemolyzed Patent Document 1: JP-A-3-51759 Gazette
- Patent Document 2 JP-A-1-155268
- Patent Document 3 Japanese Patent Application Laid-Open No. 6-11510
- the method using an ionic surfactant described in Patent Document 3 adversely affects the immune reaction system after hemoglobin denaturation treatment due to its powerful protein denaturation effect.
- a multi-step operation is required, for example, in which a hemoglobin-denatured solution is diluted with a buffer solution and the diluted hemoglobin solution is mixed with an immune reaction reagent.
- Such a complicated measurement method is inconvenient and may cause an error in the measurement value due to dilution variation.
- a dilution operation it will be difficult to construct a simple measurement system.
- the present invention solves these conventional problems.
- a hemoglobin derivative that can quickly and reliably denature hemoglobin while reducing the influence on the immune reaction by the denaturing reagent. It is an object of the present invention to provide a measurement method, a reagent composition used therefor, a measurement kit, an analysis device, and an analysis system. Means for solving the problem
- the method for measuring a hemoglobin derivative of the present invention comprises treating a sample containing a blood component with a nonionic surfactant and an oxidizing agent to remove hemoglobin in the sample. It includes a step of denaturing.
- the hemoglobin denaturation effect can be obtained quickly and reliably while minimizing the influence on the immune reaction.
- the method for measuring a hemoglobin derivative of the present invention is characterized in that the hemoglobin derivative modified by treatment with the nonionic surfactant and the oxidizing agent is specific to the modified site of the hemoglobin derivative.
- An immunoassay is performed using a typical antibody and detected.
- the hemoglobin derivative is glycated hemoglobin, and the glycated hemoglobin modified by treatment with the nonionic surfactant and an oxidizing agent is used.
- Antibodies specific for the denatured site of glycohemoglobin An immunoassay is performed and detected.
- sugar cane hemoglobin can be detected.
- the method for measuring a hemoglobin derivative of the present invention comprises treating the sample with the nonionic surfactant concentration that does not significantly interfere with the immunoassay. This eliminates the need for a dilution operation after the denaturation treatment, and in addition to preventing a decrease in measurement accuracy due to dilution, the user operability can be greatly improved.
- the method for measuring a hemoglobin derivative of the present invention further includes a step of measuring hemoglobin contained in the sample, and calculates the abundance ratio of the hemoglobin derivative to the hemoglobin.
- the abundance ratio of the hemoglobin derivative in the sample can be obtained.
- the hemoglobin derivative is sugar-hemoglobin.
- the reagent composition of the present invention is a reagent composition for measuring a hemoglobin derivative in a sample containing blood components, and contains at least a nonionic surfactant and an oxidizing agent.
- the reagent composition of the present invention further comprises an antibody specific for the modified site of the hemoglobin derivative.
- the hemoglobin derivative can be detected only by mixing the reagent composition and the sample.
- the reagent composition of the present invention is one in which the hemoglobin derivative is saccharide hemoglobin, and further comprises an antibody specific for the modified site of the saccharide hemoglobin.
- sugar hemoglobin can be detected only by mixing the reagent composition and the sample.
- the reagent composition of the present invention is such that the concentration of the nonionic surfactant does not significantly interfere with the immunity assay. This eliminates the need for a dilution operation after the denaturation treatment, and in addition to preventing a decrease in measurement accuracy due to dilution, the user operability can be greatly improved.
- the measurement kit of the present invention comprises an antibody specific to the denatured portion of the hemoglobin derivative.
- the measurement kit of the present invention comprises an antibody specific to the denatured portion of the hemoglobin derivative.
- the hemoglobin derivative can be easily measured even if the user has no specialized knowledge.
- the hemoglobin derivative is glycohemoglobin, and an antibody specific to the denatured portion of the glycohemoglobin is retained.
- the measurement kit of the present invention is such that the nonionic surfactant concentration is a concentration that does not significantly interfere with the immunoassay.
- the analysis device of the present invention is an analysis device for measuring a hemoglobin derivative in a sample containing a blood component, and is connected to at least a sample addition site to which the sample is added, and the sample addition site.
- a detection unit is an analysis device for measuring a hemoglobin derivative in a sample containing a blood component, and is connected to at least a sample addition site to which the sample is added, and the sample addition site.
- This can provide an analytical device that can denature hemoglobin more easily and quickly.
- the analysis device of the present invention provides a modified portion of the hemoglobin derivative. And a hemoglobin derivative in the sample is denatured with the reagent composition, and the denatured hemoglobin derivative is subjected to an immunoassay using the antibody. , To detect.
- the hemoglobin derivative in the sample can be detected more easily and rapidly.
- the analysis device of the present invention has an immunoassay part in which the hemoglobin derivative is saccharide hemoglobin, and carries an antibody specific to the denatured portion of the saccharide hemoglobin.
- sugar-hemoglobin in the sample is denatured with the reagent composition, and the denatured sugar-hemoglobin is detected by performing an immunoassay using the antibody.
- sugar-hemoglobin in the sample can be detected more easily and quickly.
- the analytical device of the present invention is such that the nonionic surface active concentration contained in the reagent composition is a concentration that does not significantly interfere with the immunoassay.
- the analysis device of the present invention further includes a detection unit that is connected to the sample addition site and detects hemoglobin contained in the sample, and calculates the abundance ratio of the hemoglobin derivative to the hemoglobin. is there.
- the abundance ratio of the hemoglobin derivative can be obtained more simply and rapidly by adding the sample to be detected.
- hemoglobin derivative is sugar-hemoglobin.
- the abundance ratio of glycated hemoglobin can be obtained more simply and quickly by adding the sample to be detected.
- the analysis system of the present invention includes an analysis device according to any one of claims 15 to 18, and a measurement unit that measures the amount of the hemoglobin derivative detected at a detection site of the analysis device. It is comprised from.
- the denaturation treatment of hemoglobin in a sample containing a blood component is performed using a nonionic surfactant and an acid detergent. Processing can be performed quickly and reliably.
- the nonionic surfactant has little inhibitory effect on the immunoassay when the hemoglobin derivative is measured by the immunoassay, so that it is not necessary to dilute the denaturing treatment solution, resulting in a variation in dilution. Therefore, it is possible to eliminate the problem that the measurement accuracy decreases. In addition, since a dilution operation is not required, a simpler form of immune assembly can be constructed.
- the abundance ratio of the hemoglobin derivative to the hemoglobin can be calculated.
- the hemoglobin denaturation treatment can be performed quickly and reliably.
- the reagent composition can be held in a more stable state for a long period of time if it is in a solid state, even if it is in a liquid state, a solid state, or a liquid in a dry state.
- a reagent composition containing at least a nonionic surfactant and an acidic agent is held in a part thereof, and the reagent composition and the reagent are mixed. Accordingly, since hemoglobin is denatured, the hemoglobin denaturation treatment can be performed quickly and reliably.
- the measurement kit is packed with reagents, blood collection devices, instructions for use, etc. necessary for the measurement of hemoglobin derivatives, simpler hemoglobin can be obtained without having specialized knowledge. Derivative measurement is possible.
- the analysis device of the present invention since at least a nonionic surfactant and an oxidizing agent are supported on a part of the analysis device, it is possible for the user to perform the hemoglobin denaturation process quickly and reliably. An apparatus that reduces complicated work can be provided.
- the modified part of the hemoglobin derivative in a part of the analytical device, the modified part of the hemoglobin derivative.
- an apparatus capable of detecting the hemoglobin derivative and reducing complicated operations for the user can be provided.
- the analysis system is configured by the analysis device carrying the reagent necessary for the measurement of the hemoglobin derivative and the measurement unit dedicated to the analysis device. Difficult and simple hemoglobin derivatives can be measured.
- FIG. 1 is a diagram showing a configuration of an analysis system according to Embodiment 4 of the present invention.
- FIG. 2 is a diagram showing a detailed configuration of the analysis device according to Embodiment 4 of the present invention
- FIG. (A) is an exploded perspective view thereof
- (b) is a reagent in the analysis device. It is a perspective view which shows the state which added.
- FIG. 3 is a diagram showing another configuration of the analysis system according to Embodiment 4 of the present invention.
- FIG. 4 is a diagram showing a detailed configuration of an analysis device in Embodiment 4 of the present invention
- FIG. 4 (a) is an exploded perspective view thereof
- FIG. It is a figure which shows the denaturation processing procedure in an analysis device.
- FIG. 5 is a view showing a control curve obtained by treating a standard solution with porcine pepsin in Example 1 of the present invention.
- FIG. 6 is a graph showing the relationship between the time taken to treat a blood sample with porcine pepsin and the hemoglobin denaturation rate in Example 1 of the present invention.
- FIG. 7 is a graph showing the relationship between each nonionic surfactant and the rate of denaturation of hemoglobin in Example 1 of the present invention.
- FIG. 8 is a graph showing the relationship between each nonionic surfactant and the modification rate of hemoglobin in Example 1 of the present invention.
- FIG. 9 is a graph showing the relationship between the concentration ratio of each nonionic surfactant to CMC and the hemoglobin denaturation rate in Example 1 of the present invention.
- FIG. 10 shows the concentration of each ferricyan potassium in Example 1 of the present invention.
- FIG. 10 (b) is a graph showing the relationship between the ratio of potassium ferricyanide to hemoglobin and the hemoglobin denaturation rate in Example 1 of the present invention. It is.
- FIG. 11 is a diagram for explaining the influence of each nonionic surfactant and ionic surfactant on the latex agglutination reaction in Example 1 of the present invention.
- Fig. 12 is a diagram comparing the measurement results obtained when the glycosylation hemoglobin was modified and the latex agglutination inhibition reaction was performed in Example 1 of the present invention with the measurement results obtained by the conventional method. is there.
- FIG. 13 is a diagram for explaining the results of hemoglobin measurement by hemoglobin B test kit in Example 2 of the present invention.
- FIG. 14 is a diagram showing the hemoglobin concentrations of blood specimens A and B, the sugar-hemoglobin concentration, and the abundance ratio of sugar-hemoglobin in Example 2 of the present invention.
- FIG. 15 is a diagram showing the measurement results of blood samples A and B by Tosohichi's automated sugar hemoglobin analyzer in Example 2 of the present invention.
- FIG. 16 is a diagram showing the results of measuring blood samples A and B by the analysis system in Example 3 of the present invention.
- Embodiment 1 of the present invention a method for measuring a hemoglobin derivative will be described, which comprises a step of treating a sample containing blood components with a nonionic surfactant and an oxidizing agent to denature hemoglobin in the sample. .
- the hemoglobin (hereinafter also referred to as "Hb") is a tetramer formed by the association of a chain and non- ⁇ chain (j8, ⁇ , ⁇ chain) globin with heme. Based on structure, About 90% of HbA (a ⁇ ), about 3% HbA2 (a ⁇ ), and about 1%
- HbF (a ⁇ ).
- the HbA has no sugar bonded to the amino acid terminal of the j8 chain.
- HbAO and sugar-bound HbAl includes HbAla, Hb Alb, and HbAlc (hereinafter also referred to as “sugar hemoglobin”). HbAl is called a hemoglobin derivative.
- a point for determining a hemoglobin derivative is whether or not there is a region in which amino acid residues or peptide ends are modified.
- the HbAla is modified with a phosphate sugar at the 8-chain N-terminus
- the HbAlb is aldehyde-modified at the j8-chain N-terminus
- the HbAlc is sugar-modified at the j8-chain N-terminus.
- the hemoglobin derivative according to the first embodiment is different from the structure of the partial region of hemoglobin as described above.
- hemoglobin derivatives there are various types of hemoglobin derivatives.
- acetaldehyde hemoglobin adduct due to alcohol abuse urea-hemoglobin adduct present in the blood of uremia patients
- aspirin-hemoglobin complex and carboxymethyl hemoglobin.
- hemoglobin derivative particularly, a sugar-hemoglobin produced by a non-enzymatic reaction between a reactive amin group of hemoglobin protein and dalcose is cited as a useful measurement item. It is not limited to this.
- the hemoglobin derivative of the first embodiment which differs only in the structure of a partial region of hemoglobin, a slightly different region of the hemoglobin derivative is distinguished and recognized. Thus, it is necessary to identify and quantify each hemoglobin derivative.
- to deviate (expose) a different part of the hemoglobin derivative, that is, a specific part of the hemoglobin derivative from the structure of the protein to the outside of the structure is called “denaturation”.
- the site exposed from the protein structure is called "denatured site”.
- the modification treatment is performed using a nonionic surfactant and an oxidizing agent.
- the subunits constituting the quaternary structure The degree to which the structure is dissociated, the degree to which the hydrophobic bonds, hydrogen bonds, van der Waalska, and ionic bonds that make up the tertiary structure are dissociated, the degree to which the structure of the oc-helix and ⁇ sheet that make up the secondary structure is changed, or Any degree of hemoglobin having a linear structure may be used.
- a protein exists as a functional substance in a living body because the protein maintains a precise three-dimensional structure formed from these structures. Therefore, changing the structure can be said to change the function of the protein and change its properties. This includes a decline in function or an improvement in function.
- the nonionic surfactant in the first embodiment is a compound composed of a hydrophobic group having a charge and a hydrophilic group having no charge, and is generally a membrane protein.
- the protein is used for the purpose of solubilizing the protein. It is required that the interference action is not exhibited.
- BIGCHAPJ N-Bis (3-D-gluconamidopropyl) cholamide
- aoxy— BIG CHAPJ N-Bis (3—D—gluconamidopropyl) deoxycholamide
- n— Decyl— ⁇ — D— maltopyranoside hereinafter referred to as “n— Decyl— ⁇ — D— maltosidej”.
- n Dodecyl— ⁇ —D— maltopyranoside (hereinafter “n—Dodecyl— ⁇ — D— malto side” and Ik c)
- n—Heptyl— ⁇ —D—thioglucopyranoside hereinafter referred to as “n—Heptyl— ⁇ —D—thio glucosidej”)
- MEGA-8 J n-Octanoyl— N— methylglucamide
- n-Decanoyl— N— methylglucamide hereinafter referred to as “MEG A— 10”
- MEG A— 10 n— Nonyl— ⁇ — D— thiomaltopyranoside
- n— Nonyl— ⁇ — D— thiom altoside '' and Iks myself
- n— Octyl— j8 D— glucopyranoside hereinafter referred to as “n— Octyl— ⁇ D— glucosidej”.
- n Octyl— ⁇ —D—thioglucopyranoside (hereinafter “n— Octyl— ⁇ — D— thioglu cosidej and 3 ⁇ 4 Self)
- nonionic surfactants that dissolve membrane proteins are particularly characterized in that the proteins are not denatured and inactivated, and all have little denaturation effect on proteins.
- hemoglobin can be actively denatured by combining a nonionic surfactant having the above-described characteristics with an oxidizing agent and selecting an appropriate concentration for each of the combined reagents. It was found that this can be done (see Example 1 (d) below). Therefore, in the first embodiment, the blood sample can be denatured quickly, reliably and more effectively.
- Conditions under which the denaturing reagent in the first embodiment can effectively denature hemoglobin vary depending on the type of the nonionic surfactant. This is due to the critical micelle concentration of the nonionic surfactant (hereinafter referred to as “CMC”). In the presence of an oxidizing agent, hemoglobin is at least CMC or more, more preferably about 2 times or more of CMC. It effectively denatures at a concentration of (see Example 1 (e) below).
- the concentration of hemoglobin should be 0.25% or higher.
- the concentration of the oxidant needs to be sufficient to oxidize all hemoglobin. For example, when denaturing hemoglobin in blood diluted 500 times, 0.1% The above potassium ferricyanide is necessary (see Example 1 (f) described later).
- the denaturation effect is improved by treatment with an oxidant.
- the hemoglobin force is converted to S by an oxidant, which makes it easier to receive a denaturation effect by a nonionic surfactant. This is probably because
- the oxidizing agent can be selected from substances having a charge sufficient to convert hemoglobin into the form of methemoglobin.
- K Fe ( CN) potassium ferricyanide
- treating with a nonionic surfactant and an oxidizing agent means that a non-containing oxidizing agent that satisfies a condition for obtaining a desired modification effect is included.
- An ionic surfactant solution is prepared and added to a specimen containing a hemoglobin derivative, or a nonionic surfactant solution containing an oxidant that satisfies the conditions for obtaining the desired denaturation effect is added to blood. It means putting a sample.
- the nonionic surfactant breaks down the erythrocyte membrane only by the denaturation effect and elutes hemoglobin (“hemolysis”! ) It must also be effective.
- a solid reagent composed of an oxidizing agent and a nonionic surfactant is used so as to satisfy a condition for obtaining a desired denaturing effect.
- a method of directly adding to the specimen containing the hemoglobin derivative or the blood specimen is also included.
- the “solid” may be a dried product, and examples thereof include air drying, heat drying, vacuum drying, and vacuum freeze drying.
- the hemoglobin derivative in the sample is measured using the modified hemoglobin derivative.
- the method for measuring a hemoglobin derivative is by using an antibody that recognizes a region outside the hemoglobin structure due to denaturation. For example, there is an immunoassay or a method using a borate salt having affinity for cis-diol of glucose.
- the great merit of the method for measuring a hemoglobin derivative in Embodiment 1 is that denaturation of a hemoglobin derivative treated with a nonionic surfactant and an oxidizing agent while minimizing the influence on the immune reaction. It is to be able to perform immunoassay using a specific antibody against the site. That is, in the first embodiment, the use of a nonionic surfactant in the hemoglobin denaturation step minimizes the influence on the immune reaction and minimizes the specific region (denaturation). (Site) can be taken out of the structure of hemoglobin, so that more specific measurement can be performed, and steric hindrance is reduced in the formation of the complex accompanying the antigen-antibody reaction. The reaction efficiency can be improved.
- the immunoassay in Embodiment 1 is generally known as long as it is a measurement principle based on an antigen-antibody reaction.
- the hemoglobin derivative described above is well measured for glycated hemoglobin, and its measurement method is a modified site of glycated hemoglobin, that is, outside the structure by modification.
- An immunoassay using an antibody specific to the sugar moiety of the exposed sugar glycohemoglobin is used.
- Glycohemoglobin is HbAlc, which is an index for managing diabetic patients, one of the three major adult diseases in recent years. It is a guideline. Specifically, HbAlc is denatured by acting with a nonionic surfactant and an oxidizing agent, and then the ⁇ -chain ⁇ terminal amino acid peculiar to the HbAlc is glycosylated. Perform immunoassay with specific antibodies.
- the method for measuring a hemoglobin derivative includes a step of measuring the amount of hemoglobin contained in a sample. And a hemoglobin derivative contained in the sample Measuring the amount, and calculating a ratio of the amount of hemoglobin derivative to the measured amount of hemoglobin.
- the step of measuring the amount of hemoglobin contained in this sample is based on the hemoglobin quantification method currently used in clinical tests. For example, in addition to the cyanmethemoglobin method or the SLS-hemoglobin method, 415 nm of hemoglobin is used. There is a method to measure the absorption. This may also be an immunoassay using an antibody against hemoglobin.
- the amount of hemoglobin measured in this step is the amount of partial hemoglobin obtained by hemolyzing some red blood cells, not the total amount of hemoglobin contained in the sample, obtained by completely hemolyzing all red blood cells. It may be. This is because the hemoglobin derivative is calculated by the ratio to the amount of hemoglobin as described above.
- the hemoglobin derivative is HbAlc
- a calculation method used in the IFCC (International Clinical Chemistry Union) reference method may be used. That is, it is a method that is calculated by the ratio of HbAlc to the sum of HbAO and HbAlc.
- the IFCC reference method is described in detail below. First, after washing a blood sample with physiological saline, blood cells are obtained by centrifugation, and incubated at 37 ° C for 4 hours to remove unstable HbAlc. Next, adjust the hemoglobin concentration to 6 gZdL and treat with endoprotease and ammonium acetate buffer (pH 4.0) at 37 ° C for 18 hours. This solution is injected into HPLC to obtain a glycated 6-chain peptide and a 6-chain peptide, and the peak area is obtained by the electrospray ion method.
- hemoglobin As described above, if a specimen containing a hemoglobin derivative or a blood specimen is treated with a nonionic surfactant and an oxidizing agent to denature hemoglobin, hemoglobin is ensured while ensuring safety. Can be quickly and reliably modified.
- the antibody used in the immunoassay can be prevented from being significantly denatured.
- the hemoglobin derivative, the antibody and the nonionic surfactant can coexist in one measurement system. It becomes. Therefore, in the first embodiment, one-step measurement that does not require a dilution operation can be realized, and operability can be greatly improved.
- a sample containing blood components is treated with a nonionic surfactant and an oxidizing agent, and the sample in the sample is washed. Since the step of denaturing moglobin is included, the denaturation can be performed quickly and reliably.
- the nonionic surfactant has a small inhibitory effect on the immunoassay, when the amount of hemoglobin derivative is measured by the immunoassay reaction after the denaturation treatment, a dilution operation for the solution after the denaturation treatment is unnecessary. , Prevents degradation of measurement accuracy due to dilution In addition, the operability for the user can be greatly improved.
- the concentration of the nonionic surfactant used is determined as the critical micelle. Since the concentration is more than the concentration, more preferably about twice the concentration of CMC, the denaturation effect of hemoglobin can be further improved.
- a reagent composition for measuring a hemoglobin derivative containing at least a nonionic surfactant and an oxidizing agent will be described.
- the reagent composition may be liquid or a solid mixture, and the liquid reagent composition may be dried. It is possible to denature hemoglobin simply by mixing this reagent composition with a sample solution containing hemoglobin. Therefore, a reagent composition containing a nonionic surfactant and an oxidizing agent can be the most basic element for easily measuring a hemoglobin derivative.
- the stability of the reagent can be further improved by refrigeration or light shielding.
- the dried reagent composition can be stored for a long period of time, which generally has better storage stability than that in a solution state.
- the reagent composition In the case of drying the reagent composition, it is possible to adopt a method such as air drying, heat drying, vacuum drying, vacuum freeze drying, etc. Especially when freeze-drying, the reagent composition can be frozen. Depending on the shape of the vessel, it is possible to prepare reagent compositions of various designs. Further, the solubility can be further improved by vacuum freeze-drying.
- a protein when contained in the reagent composition, it is effective to add a sugar to the reagent composition in order to enhance the stability, and in order to promote an immune reaction, It is also possible to contain an immune reaction promoter such as polyethylene glycol in the reagent composition.
- ferricyanium potassium is used as the oxidizing agent, it is possible to produce a reagent composition that is more stable for a longer period of time by shielding it from light and storing it in a dry state.
- the nonionic surfactant since the influence on the immunoassay is considerably small! /, since the nonionic surfactant is used, the antibody used for the immunoassay and the nonionic surfactant are used. May coexist in a single system, that is, the reagent composition may contain the antibody.
- a method for preparing a reagent composition in the case where an antibody is included in the reagent composition sufficient stability can be ensured by air drying or heat drying, but a method using vacuum freeze drying is most desirable.
- the reagent composition can be kept stable. In particular, since an antibody is a protein, adding a sugar leads to an improvement in the stability of the reagent composition.
- the reagent composition for measuring the hemoglobin derivative of the second embodiment at least the nonionic surfactant and the oxidizing agent are included.
- the denaturation treatment can be performed quickly and reliably.
- the reagent composition may be in a liquid state, a solid state, or a dry state of the liquid, and may be displaced. In particular, if it is in a solid state, it can be maintained in a more stable state for a long time. Become
- the reagent composition further contains an antibody specific for the denatured portion of the hemoglobin derivative
- the reagent composition and the sample can be mixed together. Since the hemoglobin derivative can be detected, user operability can be improved.
- Embodiment 3 a measurement kit for holding a reagent composition containing at least a nonionic surfactant and an oxidizing agent and measuring a hemoglobin derivative using the reagent composition will be described.
- the measurement kit refers to a kit in which reagents and members necessary for measuring a hemoglobin derivative are packed. Specifically, reagents necessary for the measurement of hemoglobin derivatives, blood collection tools such as instruction manuals, lancets or syringes, disinfecting equipment necessary before and after blood collection, weighing instruments such as dispenser syringes used to add reagents, etc. Samples to be examined are collected and diluted quantitatively using these reagents and components, and after subjecting the samples to denaturation, etc., a clinical automatic measuring machine, or Use a spectrophotometer, etc. Thus, the hemoglobin derivative can be easily measured.
- the reagent necessary for the measurement of the above-mentioned hemoglobin derivative is a reagent containing at least a nonionic surfactant and an oxidizing agent, which can quickly and reliably denature the hemoglobin derivative. it can.
- the measurement kit may carry an antibody specific for a hemoglobin derivative.
- it possesses a reagent composition containing a nonionic surfactant and an oxidizing agent that hemolyzes and denatures hemoglobin, and an antibody specific to the denatured site of the hemoglobin derivative for detecting the hemoglobin derivative.
- a measurement kit composed of a reagent (for example, a latex labeled antibody and an agglutinating reagent that is an aggregating multivalent antigen) when a latex agglutination inhibition reaction is used.
- each of these reagents is sealed in a container, and the hemoglobin derivative can be measured more easily by performing procedures for hemolysis and denaturation of the hemoglobin derivative and further immunoassay operation.
- the force exemplified by the latex agglutination inhibition reaction is used to denature hemoglobin, and the amount of the denatured hemoglobin derivative is measured using an antibody specific for the denatured site of the hemoglobin derivative. If it is an immunoassay reaction to be measured, it is not limited to the above.
- the reagent composition and the antibody to be retained in the measurement kit may be retained separately, or the antibody may be included in the reagent composition and retained.
- a reagent capable of measuring hemoglobin in a sample may be sealed in a container and added to the measurement kit. By doing so, it is possible to calculate the abundance ratio of the hemoglobin derivative. The calculation of the abundance ratio is particularly useful when measuring glycated hemoglobin.
- the ability to measure the peak wavelength near 415 nm using the absorption of hemoglobin itself is the methemoglobin method and the SLS-hemoglobin method that measure the wavelength near 540 nm.
- Other examples include the latex agglomeration method.
- a part or all of the reagents necessary for the measurement of the hemoglobin derivative in the sample are each enclosed in a container or the like. Therefore, it is possible to measure the amount of denatured hemoglobin by using a reagent that specifically recognizes the denatured hemoglobin derivative after performing hemolysis and degeneration of hemoglobin according to a predetermined procedure.
- the hemoglobin derivative can be measured more easily. More preferably, if a reagent capable of measuring the hemoglobin concentration in the sample is also attached to the measurement kit, the abundance ratio of the hemoglobin derivative to hemoglobin can be calculated, and in particular, sugar hemoglobin is measured. Useful in cases.
- At least a sample addition part for adding a sample, a modification part for modifying a hemoglobin derivative in the sample with a nonionic surfactant and an oxidizing agent, and the amount of the modified hemoglobin derivative An analysis device for measuring a hemoglobin derivative, comprising a detection unit for detecting selenium, will be described.
- the analysis device can take the form of an analysis system in combination with a measuring apparatus for evaluating the analysis device. By taking this form, the hemoglobin derivative can be measured more easily and rapidly.
- the analytical device of the fourth embodiment has a configuration in which a nonionic surfactant and an oxidizing agent are supported, and a reagent and an agglutinating reagent are supported on a denatured site of a hemoglobin derivative. Furthermore, it is possible to take a form in which they are carried at different locations.
- the measurement step first, after a denaturation step in which a blood sample is reacted with a nonionic surfactant and an oxidizing agent, the denatured hemoglobin derivative and the denatured site of the hemoglobin derivative are applied. And a reaction with a latex-labeled antibody and an agglutinating reagent in which latex is labeled with a specific antibody against a hemoglobin derivative.
- the latex labeled antibody and the agglutinating reagent may be reacted simultaneously with the sample solution treated with the nonionic surfactant and the oxidizing agent, but after reacting with the latex labeled antibody. It can be reacted with an agglutination reagent.
- the amount of hemoglobin derivative is calculated by measuring the change in absorbance of the reaction solution.
- the abundance ratio of the hemoglobin derivative to hemoglobin can be calculated by calculating the hemoglobin concentration.
- This hemoglobin concentration measurement is based on the absorption of hemoglobin itself, the ability to measure the peak wavelength near 415 nm, or the cyan methemoglobin method and SLS-hemoglobin method that measure the wavelength near 540 nm, as well as the latex agglutination method. For example.
- an example of the analysis device for example, a device using centrifugal force and capillary force is conceivable.
- An example of an apparatus for evaluating an analysis device having such a configuration includes a device equipped with a rotation mechanism capable of rotating the analysis device and an optical measurement function capable of measuring absorbance.
- FIG. 1 is a diagram showing the configuration of the analysis system.
- the analysis system 100 includes an analysis device 101, a measurement unit 110 that irradiates light from the light source 102 to the analysis device 101, and detects transmitted light by the detector 103, and a part of the analysis device 101.
- the rotating board 104 is fixed to the extracted portion, and the motor 105 is configured to rotate the rotating board 104.
- the driving mechanism of the motor 105, the power and the circuit configuration connected to the light source 102 and the detector 103 are omitted.
- FIG. 2 is a diagram showing a detailed configuration of the analysis device
- FIG. 2 (a) is an exploded perspective view thereof
- FIG. 2 (b) is a diagram showing a state where a reagent is added.
- the analysis device 101 has a lower substrate 201, an upper substrate 213, and an adhesive effect on both front and back surfaces.
- the adhesive layer 202 is formed by bonding them together.
- the lower substrate 201 a transparent resin substrate is used, and spaces of various shapes are accurately formed by injection molding or the like.
- the lower substrate 201 includes a dilution stirring unit 203 that is a denaturation site for denaturing the hemoglobin derivative, a diluent holding unit 204, a detection unit A205 that detects the amount of added hemoglobin, A recess for forming a detection part B206, which is a detection site for detecting the amount of the denatured hemoglobin derivative, is formed on the upper surface by injection molding.
- plastic resin such as polycarbonate and polystyrene can be cited as an example as long as it is a material that transmits light.
- the adhesive layer 202 has a pattern shape of the channel 207 that connects them. It has been pulled out. Further, the flow path 207 in front of the detection unit A205 and the detection unit B206 is cut out so as to expand a part thereof, thereby quantifying the amount of liquid transferred to the detection unit A205 and the detection unit B206.
- the quantification unit A208 and the quantification unit B209 are to be formed.
- a hot melt sheet that can be bonded by heating can be used, and the upper substrate 213 is made of a transparent resin substrate.
- the analytical device 101 before the upper substrate 213 is bonded, as shown in FIG.
- the denatured reagent 210 made of a nonionic surfactant and an oxidizing agent is added to the diluting stirring part 203 of the above, and the site where the hemoglobin derivative is denatured is added to the quantitative chamber B209 formed by the lower substrate 201 and the adhesive layer 202.
- each of the substrates is supported and then dried by vacuum freeze-drying, and then the upper substrate 213 is bonded to the upper surface of the adhesive layer 202. Further, the two openings of the channel 20 07 cut out in the adhesive layer 202, which are formed by bonding the upper substrate 213, the adhesive layer 202, and the lower substrate 201, respectively, 215 and diluent inlet 216. Next, the operation of the analysis system 100 will be described.
- 1 L of blood is injected from the sample injection port 215 of the analysis device 101, and 500 L of the dilution solution is injected from the dilution solution injection port 216.
- the blood is held in the flow path inside the specimen injection port 215, and the diluent is held in the diluent holding part 204.
- the analysis device 101 into which blood and diluent have been injected is set in the portion of the rotating substrate 104 that has been removed, and is rotated by the motor 105 at a predetermined rotation speed for a predetermined time.
- the diluted solution and blood are transferred to the dilution stirring unit 203 and mixed to form a diluted sample solution, and hemolysis and hemoglobin derivative denaturation are caused by the action of the nonionic surfactant and the oxidizing agent.
- the sample liquid is transferred to the quantification unit A208 and the quantification unit B209 through the channel 207 by capillary action.
- the sample solution transferred to the quantification unit B209 is held in advance in the quantification unit B209 and mixed with the latex reagent 211, so that the latex reagent 211 and the hemoglobin derivative in the sample solution are mixed. Join.
- the sample liquid transferred to the quantification unit A208 is sent to the detection unit A205, while the quantification unit B209 is used as a latex reagent.
- the sample liquid mixed with 211 is transferred to the detection unit B206.
- the agglutination reagent 212 carried on the detection unit B206 binds to the V and latex reagents that are not bound to the hemoglobin derivative, and a latex agglutination inhibition reaction occurs according to the concentration of the hemoglobin derivative. After a certain time, the latex agglutination inhibition reaction is detected by measuring the transmitted light of the quantitative unit B206.
- the absorption of hemoglobin can be measured, and the hemoglobin concentration can be calculated.
- the latex agglutination inhibition reaction in the detection unit B206 can be measured at a wavelength near 550 nm, and the hemoglobin measurement in the detection unit A205 can be performed by a method of measuring the maximum absorption near 415 nm. A method of measuring absorption at 540 nm may be used.
- the concentrations of hemoglobin and hemoglobin derivative can be calculated using the calibration curve. It is possible to calculate the abundance ratio of the hemoglobin derivative by correlating the concentration of the hemoglobin with the concentration of the hemoglobin derivative.
- the analysis device 101 is in the form of a chip, and an analysis system that controls the order of the measurement system, the amount of reagents, the reaction time, etc. by liquid transfer using centrifugal force and capillary force is taken as an example.
- liquid transfer is not limited to this configuration and method as long as the order of the measurement system, reagent amount, and reaction time can be controlled.
- a method of transferring liquid by pressure using a pump is also possible.
- the analytical device may be in the form of a chromatograph, for example, or more simply in the shape of a rectangular parallelepiped plastic cell, and can be sufficiently used by devising a method for supporting the reagent.
- FIG. 3 An analysis device having a simpler configuration and an analysis system using the analysis device will be described with reference to FIGS. 3 and 4.
- FIG. 3 An analysis device having a simpler configuration and an analysis system using the analysis device will be described with reference to FIGS. 3 and 4.
- FIG. 3 is a diagram showing another configuration of the analysis system according to the fourth embodiment.
- the analysis system 300 includes a measurement unit 310 that irradiates the analysis device 301 with light from the light source 308 and detects transmitted light by the light receiving unit 309.
- a circuit configuration for connecting the light source 308 and the light receiving unit 309 or a configuration for setting the analysis device 301 in the analysis system is omitted.
- FIG. 4 is a diagram showing a detailed configuration of the analysis device 301.
- FIG. 4 (a) is an exploded perspective view
- FIGS. 4 (b) to (d) show the reagent denaturation processing procedure in the analysis device 301.
- FIG. FIG. 4 (b) is an exploded perspective view
- FIGS. 4 (b) to (d) show the reagent denaturation processing procedure in the analysis device 301.
- FIG. FIG. 4 is a diagram showing a detailed configuration of the analysis device 301.
- FIG. 4 (a) is an exploded perspective view
- FIGS. 4 (b) to (d) show the reagent denaturation processing procedure in the analysis device 301.
- the analysis device 301 includes a lower case 302b having an inlet 306 into which a blood sample is injected, a solution reagent seal 305 for sealing the opened bottom surface of the lower case 302b, and an upper case 302a. And a case seal 307 for sealing the inlet 306, and is formed by bonding the open bottom surfaces of the upper case 302a and the lower case 302b with an adhesive.
- the lower case 302b is a plastic rectangular parallelepiped case having an open bottom.
- a reagent 304 in which an antibody specific to the denatured portion of the hemoglobin derivative is added to a reagent composed of a nonionic surfactant and an oxidizing agent, Sealed and held by a reagent seal 305.
- the upper case 302a is a plastic rectangular parallelepiped case having the same shape as the lower case 302b and having an open bottom surface. As shown in Fig. 4 (b), the upper case 302a has hemoglobin. Latex reagent 303 capable of reacting specifically with the derivative is vacuum-freeze-dried and supported.
- a dispenser or the like is used to inject a reagent 304 containing a nonionic surfactant, an oxidizing agent, and an agglutinating reagent into the lower case 302b, and the lower portion of the solution reagent seal 305 is used to After sealing the case 302b, as shown in FIG. 4 (b), the lower case 302b and the upper case 302a carrying the latex reagent are bonded together with an adhesive.
- a blood sample is injected through the injection port 306 using, for example, a dispenser or the like, and the case seal 307 is used to inject the blood sample as shown in FIG. 4 (c). Seal the inlet 306.
- the blood sample and the reagent 304 are gently mixed with the latex reagent 303 carried on the upper end of the upper case 302a so that the reagent 304 is not applied, and left for a predetermined time.
- the analysis device 301 is set in the analysis system 300 as shown in FIG. 3 and the absorbance at 540 nm is measured by the measurement unit 310 at this time.
- the lower case 302b of the analysis device 301 is squeezed upward, and the latex reagent 303 is mixed into the reagent 304 to which a blood sample has been added. , Leave for a predetermined time.
- the analysis device 301 is set in the analysis system 300 as shown in FIG. 3, and the absorbance at 550 nm is measured by the measurement unit 310 to thereby determine the concentration of the hemoglobin derivative. calculate.
- an analysis device carrying a reagent necessary for measuring a hemoglobin derivative is designed, and an analysis system combined with a measurement unit dedicated to the analysis device is provided. As it was constructed, it was simple and difficult to be affected by the procedure. Rapid measurement of hemoglobin derivatives can be performed.
- HbAlc a representative test item of hemoglobin derivative strength sugar hemoglobin, and the oxidizing power of potassium ferricyanide added together with a nonionic surfactant. Examine the denaturation effect of the active agent.
- Cobas reagent HbAlc commercially available from Roche Diagnostics Co., Ltd. was used.
- FIG. 5 is a graph in which the horizontal axis represents the sugar-hemoglobin concentration and the vertical axis represents the amount of change in absorbance.
- the calculation was performed with a hemoglobin molecular weight of 64,500.
- FIG. 6 is a plot in which the horizontal axis represents the denaturation treatment time and the vertical axis represents the hemoglobin denaturation rate. The rate of hemoglobin denaturation at each treatment time was calculated with the glycated hemoglobin concentration calculated when the blood sample was treated with pig-derived pepsin for 25 minutes as 100%.
- the denaturation method is to prepare 100 ⁇ L of an aqueous solution containing the above-mentioned 10 nonionic surfactants at a concentration of 0.1 to 9% and 0.25% ferricyanium potassium (oxidant). After adding 2 blood samples (diluted 51 times), they were left at 25 ° C for 3 minutes. Next, 14 ⁇ L of the reaction solution 102 IX L was reacted for 4 minutes with 560 ⁇ of a latex reagent solution labeled with glycated hemoglobin antibody. Then react with 112 L of agglutination reagent and after 3 minutes at 550 nm The amount of change in absorbance was measured.
- the sugar-hemoglobin concentration in the blood sample was calculated from the absorbance value in the latex agglutination inhibition reaction of the same blood sample treated with porcine pepsin (control value), and then From the absorbance value of the latex agglutination inhibition reaction when the same blood sample was treated with each of the 10 types of nonionic surfactants described above, the concentration of moglobin in sugar was determined. Further, to the control described above Pepsin treatment, blood samples said treated with the nonionic surfactant, calculated what 0/0 Ti ⁇ hemoglobin is detected, this denaturation proportion of hemoglobin It was.
- FIG. 7 is a graph showing a plot of the nonionic surfactant concentration on the horizontal axis and the denaturation ratio on the vertical axis.
- the denaturation effect is high depending on the type of nonionic surfactant, and the nonionic surfactant concentration is different. Both have the same hemoglobin denaturation effect as the control pepsin treatment. did it.
- the denaturation method is an aqueous solution containing the above-mentioned five kinds of nonionic surfactants in a concentration of 0.05% to 0.5% and 0.25% ferricyanium potassium (oxidant) lmL After 2 ⁇ L of blood sample was added (diluted 501 times), it was left at 25 ° C for 3 minutes. Next, 140 L of 1002 L of the reaction solution was reacted for 4 minutes with 560 L of a latex solution labeled with a glycohemoglobin antibody. Then, it was made to react with the 112 / zL agglutinating reagent, and the change in absorbance at 550 nm was measured after 3 minutes.
- the denaturation was confirmed based on the absorbance value at the time of latex aggregation inhibition of the same blood sample treated with porcine pepsin. From the absorbance value of the latex agglutination inhibition reaction when the same blood sample was treated with each of the 10 nonionic surfactants described above (control value). The concentration of glycated hemoglobin in the blood was determined, and the rate of denaturation of each nonionic surfactant was determined using the one modified with pepsin as a control.
- Fig. 8 is a graph in which the horizontal axis represents the nonionic surfactant concentration and the vertical axis represents the denaturation ratio of glycated hemoglobin.
- the horizontal axis represents each nonionic surfactant activity.
- FIG. 3 is a graph in which the ratio between the CMC (critical micelle concentration) of the agent and the concentration of the nonionic surfactant is plotted on the vertical axis, and the rate of change of sugar hemoglobin is plotted on the vertical axis.
- the concentration of the nonionic surfactant having a high hemoglobin denaturation effect varies depending on the type, but as shown in FIG.
- the concentration of the non-ionic surfactant to the CMC of the activator it was confirmed that the denaturation effect was increased at concentrations more than twice that of the CMC.
- the denaturation was confirmed by first calculating the moglobin concentration to glycation in the blood sample from the absorbance value (control value) in the rat agglutination inhibition reaction of the blood sample treated with porcine pepsin. Next, the concentration of glycated hemoglobin in the blood was determined from the absorbance value of the latex agglutination inhibition reaction when the same blood sample was treated with a nonionic surfactant. And the non-ionic field relative to the pepsin-treated control. In the blood sample treated with the surfactant, it was determined how many 0 / o sugar hemoglobin was detected, and this was used as the denatured ratio of hemoglobin.
- Fig. 10 (a) is a plot of the ferricyan potassium concentration on the horizontal axis and the rate of hemoglobin denaturation on the vertical axis.
- Fig. 10 (b) shows the potassium ferricyanide and hemoglobin levels on the horizontal axis. Is a diagram in which the ratio of the above is plotted with the rate of denaturation of hemoglobin on the vertical axis.
- the non-ionic surfactant alone has a weak hemoglobin-modifying effect, but it is simultaneously treated with at least 0.1% ferricyanium potassium at the same time as the non-ionic surfactant. As a result, it was found that even a blood sample with a hematocrit value of 70% was sufficiently denatured.
- each nonionic surfactant solution was added to the latex reagent solution described above, an agglutinating reagent was added, and the change in absorbance at 550 nm was measured 3 minutes later.
- SLS sodium lauryl sulfate
- Fig. 11 is a graph in which the horizontal axis represents the surfactant concentration, and the vertical axis represents the absorbance change ratio of each nonionic surfactant when the absorbance change of the control is 100%. is there.
- SLS sodium lauryl sulfate
- n-Decyl- ⁇ -D-maltosid n-No nyl-j8-D-thiomaltoside, MEGA10, sucrose monocaprate, Sucrose monolaurate, etc. are sufficient hemoglobin. It was confirmed that the concentration of the denaturation effect has little influence on the latex agglutination reaction. This suggests that the hemoglobin-modified solution can be used sufficiently as it is as a reagent composition for the reaction in the latex agglutination reaction or the latex agglutination inhibition reaction.
- FIG. 12 is a diagram in which the horizontal axis represents the sugar-hemoglobin concentration and the vertical axis represents the amount of change in absorbance.
- the denaturation reaction of the hemoglobin derivative was performed with 0.5% Sucrose monocaprate and 0.25% potassium ferricyanide solution.
- the latex agglutination-inhibiting reaction can be carried out in a state where the denaturing reagent composition is included.
- Hemoglobin B Test Sakai Koichi sold by Wako Pure Chemical Industries, Ltd. was used. This is a method of detecting hemoglobin by the SLS hemoglobin method.
- FIG. 13 is a plot of hemoglobin concentration on the horizontal axis and absorbance on the vertical axis, with the hemoglobin concentration of the reaction solution added with 40 g / z L of 15 gZdL hemoglobin standard solution being 30 gZdL. Control.
- each of blood samples A and B to be measured are added to 3.5 mM sodium lauryl sulfate.
- the volume was adjusted to 5 mL of the lithium solution.
- the absorbance at 540 nm of this reaction solution was measured, and the hemoglobin concentrations of the blood samples A and B were determined from the absorbance data with respect to the known hemoglobin concentration in FIG.
- 2 L of blood samples A and B were added to 100% Sucrose monocaprate / 0.25% potassium ferricyanide solution of 0.5% concentration, respectively, and left for 3 minutes.
- 14 L was reacted for 4 minutes with 560 L of a latex solution labeled with a glycohemoglobin antibody. Thereafter, it was reacted with 112 L of agglutinating reagent, and the change in absorbance at 550 nm was measured after 3 minutes.
- FIG. 14 is a diagram showing the hemoglobin concentration, glycated hemoglobin concentration of blood samples A and B, and the ratio of glycated hemoglobin in the total hemoglobin.
- Fig. 15 shows that blood samples A and B described above were saccharified with an automatic glycated hemoglobin analyzer (HLC-723GHbV) manufactured by Tosoh Corporation, which is the standard method for measuring glycated hemoglobin. It is the result of measuring the proportion of hemoglobin.
- HSC-723GHbV automatic glycated hemoglobin analyzer
- a latex reagent 303 having a solution power containing a latex reagent capable of specifically binding to saccharified hemoglobin and 5% sucrose is supported by vacuum freeze-drying.
- the solution reagent seal 305 is peeled off, and 0.5 ⁇ L of a blood sample is injected from the inlet 306 of the analysis device 301. As shown in FIG.
- the analysis device 301 is hermetically sealed.
- the latex reagent 303 carried on the upper case 302a is gently mixed so that the reagent 304 is not applied, and left in this state for 3 minutes. Then, as shown in FIG. It is set in the analysis system 300 and the absorbance at 540 nm is measured by the measurement unit 310.
- the analytical device 301 is inverted, the latex reagent 303 and the reagent 304 in the upper case 302a are mixed and dissolved, and left for 3 minutes.
- the analysis device 301 is set in the analysis system 300, and after 3 minutes from the dissolution of the latex reagent 303, the absorbance at 550 nm is measured by the measurement unit 310.
- the measurement unit 310 Although detailed description of the measurement unit 310 is omitted, light is emitted from the light source 308 to the analysis device 301 and transmitted light is detected by the light receiving unit 309. As an example of the measurement unit 310, the spectrophotometer function can be used satisfactorily, and a detailed description thereof will be omitted.
- cyan methemoglobin was measured by measuring 540 nm
- latex aggregation was measured by measuring 550 nm. This series of measurement operations is performed on the blood samples A and B.
- FIG. 16 is a diagram showing the glycated hemoglobin concentration and the hemoglobin concentration and the abundance ratio of glycated hemoglobin in each of blood samples A and B.
- the result of measuring the proportion of glycated hemoglobin in this analysis system 300 is the result of an automatic glycated hemoglobin analyzer (HLC- 723GHbV ) Confirmed that it is possible to accurately measure glycated hemoglobin concentration using this analysis system 300, which is very close to the result of measuring the proportion of glycated hemoglobin (see Fig. 15).
- HSC- 723GHbV automatic glycated hemoglobin analyzer
- hemoglobin in a sample solution can be denatured quickly and reliably, it is possible to accurately measure a hemoglobin derivative.
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Abstract
Description
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EP06731717.2A EP1873522B1 (en) | 2005-04-14 | 2006-04-12 | Method for determination of hemoglobin derivative, and reagent composition, assay kit, analysis device and analysis system for use in the method |
CA2604474A CA2604474C (en) | 2005-04-14 | 2006-04-12 | Hemoglobin derivative measurement method, and reagent composition, measurement kit, analysis device and analysis system for use in the method |
JP2007521210A JP4957547B2 (ja) | 2005-04-14 | 2006-04-12 | ヘモグロビン誘導体の測定方法、それに用いる試薬組成物、測定キット、分析デバイス、及び分析システム |
CN2006800121194A CN101160527B (zh) | 2005-04-14 | 2006-04-12 | 血红蛋白衍生物的测定方法、该法中使用的试剂组合物、测定试剂盒、分析设备以及分析*** |
US11/918,394 US8921052B2 (en) | 2005-04-14 | 2006-04-12 | Hemoglobin derivative measurement method, and reagent composition, measurement kit, analysis device and analysis system for use in the method |
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- 2006-04-12 US US11/918,394 patent/US8921052B2/en active Active
- 2006-04-12 WO PCT/JP2006/307781 patent/WO2006112339A1/ja active Application Filing
- 2006-04-12 CN CN2006800121194A patent/CN101160527B/zh active Active
- 2006-04-12 KR KR1020077023505A patent/KR20070116103A/ko not_active Application Discontinuation
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US8415140B2 (en) | 2007-10-04 | 2013-04-09 | Panasonic Corporation | Analysis device, and analysis apparatus and method using the same |
EP3447494A1 (en) | 2007-10-04 | 2019-02-27 | PHC Holdings Corporation | Analysis method using analysis device |
EP3141901A1 (en) | 2007-10-04 | 2017-03-15 | Panasonic Healthcare Holdings Co., Ltd. | Analysis device, and analysis apparatus and method using the same |
US8956879B2 (en) | 2007-10-04 | 2015-02-17 | Panasonic Healthcare Co., Ltd. | Analysis device and method using the same |
WO2009044552A1 (ja) | 2007-10-04 | 2009-04-09 | Panasonic Corporation | 分析用デバイスとこれを使用する分析装置および分析方法 |
EP2211175A4 (en) * | 2007-10-30 | 2010-11-10 | Panasonic Corp | METHOD OF MEASURING HEMOGLOBIN AND HEMOGLOBIN DERIVATIVE AND MEASUREMENT KIT |
CN103353534A (zh) * | 2007-10-30 | 2013-10-16 | 松下电器产业株式会社 | 血红蛋白及血红蛋白衍生物的测定方法、测定试剂盒 |
US8574913B2 (en) | 2007-10-30 | 2013-11-05 | Panasonic Corporation | Method for measurement of hemoglobin and hemoglobin derivative, and measurement kit |
JP5465008B2 (ja) * | 2007-10-30 | 2014-04-09 | パナソニック株式会社 | ヘモグロビン及びヘモグロビン誘導体の測定方法、測定キット |
EP2211175A1 (en) * | 2007-10-30 | 2010-07-28 | Panasonic Corporation | Method for measurement of hemoglobin and hemoglobin derivative, and measurement kit |
US9151765B2 (en) | 2007-10-30 | 2015-10-06 | Panasonic Healthcare Holdings Co., Ltd. | Method for producing agglutinating reagent, agglutinating reagent or product produced thereby, and method for measuring analysis object using the same, and test kit and analysis device |
WO2009057293A1 (ja) | 2007-10-30 | 2009-05-07 | Panasonic Corporation | ヘモグロビン及びヘモグロビン誘導体の測定方法、測定キット |
JP2009180688A (ja) * | 2008-02-01 | 2009-08-13 | Panasonic Corp | 分析用デバイスを用いた分析方法 |
JP2013036959A (ja) * | 2011-08-11 | 2013-02-21 | Toyobo Co Ltd | 全血検体の成分測定方法 |
JP2020046435A (ja) * | 2018-09-12 | 2020-03-26 | 積水メディカル株式会社 | ヘモグロビン類測定用試薬及びヘモグロビン類の測定方法 |
JP7340439B2 (ja) | 2018-09-12 | 2023-09-07 | 積水メディカル株式会社 | ヘモグロビン類測定用試薬及びヘモグロビン類の測定方法 |
Also Published As
Publication number | Publication date |
---|---|
EP1873522A4 (en) | 2009-08-19 |
CA2604474C (en) | 2012-07-17 |
US20080293074A1 (en) | 2008-11-27 |
US8921052B2 (en) | 2014-12-30 |
EP1873522A1 (en) | 2008-01-02 |
CN101160527B (zh) | 2013-04-03 |
KR20070116103A (ko) | 2007-12-06 |
JPWO2006112339A1 (ja) | 2008-12-11 |
CN101160527A (zh) | 2008-04-09 |
JP4957547B2 (ja) | 2012-06-20 |
CA2604474A1 (en) | 2006-10-26 |
EP1873522B1 (en) | 2014-08-13 |
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