Sample analysis method, capillary electrophoresis solution, and sample analysis kit

Abstract

To provide a sample analysis method, capillary electrophoresis solution, and a sample analysis kit, which enable more accurate isolation of protein compared with conventional methods.SOLUTION: A sample analysis method disclosed herein comprises an isolation step of isolating protein in a sample by capillary electrophoresis in alkaline solution, where the isolation of the protein is carried out under the presence of a cationic low-molecular compound having two or more primary amino groups, and the alkaline solution contains a cationic polymer.SELECTED DRAWING: Figure 1

Description

[[Technical Field]]

[0001] The present disclosure relates to a sample analysis method, a solution for capillary electrophoresis, and a kit for sample analysis. [[Background Art]]

[0002] In the field of clinical testing, analysis of hemoglobin contained in erythrocytes, and proteins such as albumin, globulin, transferrin contained in serum, etc., is performed on a daily basis. As one of protein analysis methods, capillary electrophoresis has been used. A sample analysis method using capillary electrophoresis has advantages in that only a small amount of sample is required, and the analysis apparatus can be miniaturized, etc. In recent years, improvements in analysis accuracy and shortening of analysis time have been desired for the above analysis method.

[0003] Patent Document 1 discloses a free solution capillary electrophoresis method at alkaline pH for analyzing a sample containing a protein component, characterized by comprising at least one step of introducing the sample into a capillary tube containing a buffer system, the buffer system further being capable of hydrophobically interacting with one or more protein components, imparting one or more negative charges to said protein components, and adjusting the electrophoretic mobility, the electrophoresis method described above, which comprises at least one additive, is described.

[0004] Patent Document 2 discloses, in a free solution capillary electrophoresis method at alkaline pH for analyzing a sample containing a protein component including one or more lipoprotein components, the method comprising at least one step of introducing the sample into a capillary tube containing an analysis buffer, said analysis buffer further containing at least one anionic surfactant additive capable of changing the electrophoretic mobility relative to the mobility of other protein components by hydrophobic interaction with lipoproteins, wherein the concentration of the additive in the buffer ranges from 0.001 mM to 0.2 mM is described.

[0005] Patent Document 3 describes a sample analysis method that includes separating various hemoglobins in an alkaline solution containing a cationic polymer by capillary electrophoresis. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Special Publication No. 2004-517339 [Patent Document 2] Special Publication No. 2005-326407 [Patent Document 3] Japanese Patent Publication No. 2016-136135 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] One embodiment of this disclosure aims to solve the problem of providing a sample analysis method, a capillary electrophoresis solution, and a sample analysis kit that enable the separation of proteins with higher precision than conventional methods. [Means for solving the problem]

[0008] A sample analysis method according to the embodiments of this disclosure includes a separation step of separating proteins in a sample by capillary electrophoresis in an alkaline solution, wherein the separation of proteins is carried out in the presence of a cationic low molecular weight compound having two or more primary amino groups, and the alkaline solution contains a cationic polymer. [Effects of the Invention]

[0009] According to one embodiment of the present disclosure, a sample analysis method, a capillary electrophoresis solution, and a sample analysis kit can be provided that enable the separation of proteins with higher precision than conventional methods. [Brief explanation of the drawing]

[0010] [Figure 1]Figure 1A is a top view showing one embodiment of a capillary electrophoresis chip. Figure 1B is a cross-sectional view of the electrophoresis chip shown in Figure 1A. [Figure 2] Figure 2 shows the electroferogram obtained in the sample analysis method of Example 1. [Figure 3] Figure 3 shows the electroferogram obtained in the sample analysis method of Example 2. [Figure 4] Figure 4 shows the electroferogram obtained in the sample analysis method of Example 3. [Figure 5] Figure 5 shows the electroferogram obtained in the sample analysis method of Example 4. [Figure 6] Figure 6 shows the electroferrogram obtained in the sample analysis method of Comparative Example 1. [Figure 7] Figure 7 shows the electroferogram obtained in the sample analysis method of Comparative Example 2. [Figure 8] Figure 8 shows the electroferogram obtained in the sample analysis method of Comparative Example 3. [Figure 9] Figure 9 shows the electroferogram obtained in the sample analysis method of Comparative Example 4. [Figure 10] Figure 10 shows the electroferogram obtained in the sample analysis method of Comparative Example 5. [Figure 11] Figure 11 shows the electroferogram obtained in the sample analysis method of Comparative Example 6. [Figure 12] Figure 12 shows the electroferogram obtained in the sample analysis method of Comparative Example 7. [Figure 13] Figure 13 shows the electroferogram obtained in the sample analysis method of Comparative Example 8. [Figure 14] Figure 14 shows the electroferogram obtained in the sample analysis method of Comparative Example 9. [Figure 15] Figure 15 shows the electroferogram obtained in the sample analysis method of Example 5. [Figure 16]Fig. 16 shows an electroferrogram obtained in the sample analysis method of Comparative Example 10. [Figure 17] Fig. 17 shows an electroferrogram obtained in the sample analysis method of Example 6. [Figure 18] Fig. 18 shows an electroferrogram obtained in the sample analysis method of Comparative Example 11. [Figure 19] Fig. 19 shows an electroferrogram obtained in the sample analysis method of Example 7. [Figure 20] Fig. 20 shows an electroferrogram obtained in the sample analysis method of Example 8. [Figure 21] Fig. 21 shows an electroferrogram obtained in the sample analysis method of Comparative Example 12. MODE FOR CARRYING OUT THE INVENTION

[0011] Hereinafter, embodiments which are examples of the present invention will be described. These descriptions and examples are for illustrating the embodiments, and do not limit the scope of the invention. In this specification, the symbol "~" used to indicate a numerical range means that it includes the numerical values described before and after it as the lower limit and upper limit. In the numerical ranges described stepwise in the present disclosure, the upper limit or lower limit described for one numerical range may be replaced with the upper limit or lower limit of another stepwise described numerical range. Further, in the numerical ranges described in the present disclosure, the upper limit or lower limit of the numerical range may be replaced with the values shown in the examples.

[0012] Each component may contain a plurality of types of corresponding substances. When referring to the amount of each component in the composition, if a plurality of substances corresponding to each component are present in the composition when present, it means the total amount of the plurality of substances present in the composition, unless otherwise specified. In this disclosure, the term "process" includes not only independent processes but also processes that cannot be clearly distinguished from other processes, as long as their intended purpose is achieved. In this disclosure, a combination of two or more preferred embodiments is a more preferred embodiment. When embodiments are described in this disclosure with reference to the drawings, the configuration of such embodiments is not limited to the configuration shown in the drawings. Furthermore, the sizes of the components in each figure are conceptual, and the relative relationships between the sizes of the components are not limited thereto.

[0013] [Sample Analysis Method] A sample analysis method according to the embodiments of this disclosure (hereinafter also referred to as the "specific sample analysis method") includes a separation step of separating proteins in a sample by capillary electrophoresis in an alkaline solution, wherein the separation of proteins is carried out in the presence of a cationic low molecular weight compound having two or more primary amino groups (hereinafter also referred to as the "specific low molecular weight compound"), and the alkaline solution (hereinafter also referred to as the "specific alkaline solution") contains a cationic polymer.

[0014] This specific sample analysis method allows for more accurate separation than conventional protein separation methods. While the reason for this effect is unclear, it is speculated to be as follows. Proteins such as hemoglobin, albumin, gamma globulin, and transferrin, which are the target substances for analysis, become negatively charged in alkaline solutions. When a voltage is applied with a negative electrode in contact with the sample introduction side, these analytes move towards the positive electrode side due to their charge and electroosmotic flow. On the other hand, cationic polymers, possessing cationic groups, move from the positive electrode side to the negative electrode side. During electrophoresis, the analyte and cationic polymer repeatedly bond and dissociate within the capillary, causing the analyte to become positively charged, thus generating a force that pulls the analyte back to the negative electrode side. Because cationic polymers have a large charge, the charge difference between the analyte interacting with the cationic polymer becomes larger than the charge difference inherent in the analyte. Therefore, it is presumed that the difference in electrophoretic speed between the analytes becomes larger, making it possible to efficiently separate the analytes in a short time. The inventors have discovered a new problem: the number of cationic polymers that interact with the protein, which is the substance to be analyzed, varies, which may lead to a decrease in separation accuracy. Furthermore, by separating the target protein in the presence of specific low-molecular-weight compounds, these compounds will compete with cationic polymers and interact with the target protein. This is expected to reduce the variability in the number of cationic polymers interacting with the target protein, thereby improving separation accuracy. Furthermore, in order to generate electroosmotic flow from the negative electrode side to the positive electrode side within the capillary channel, the inner wall of the capillary channel may be coated with a cationic substance. In such cases, negatively charged analyte may be adsorbed onto the inner wall. It is presumed that separating the analyte in the presence of a specific low-molecular-weight compound can suppress this adsorption and improve separation accuracy.

[0015] <Separation process> The specific sample analysis method includes a separation step of separating proteins in the sample by capillary electrophoresis in a specific alkaline solution. As the protein, at least one selected from the group consisting of hemoglobin, albumin, γ-globulin, and transferrin is preferred.

[0016] The separation of proteins in a sample by capillary electrophoresis in a specific alkaline solution can be performed by introducing the sample into a capillary channel filled with the specific alkaline solution, and then applying a voltage to the entire or a part of the capillary channel. By applying the above voltage, the proteins in the sample can be electrophoresed and separated. Voltage can be applied to the capillary channel by contacting the negative electrode with the sample introduction side of the capillary channel and the positive electrode with the supply side of the specific alkaline solution.

[0017] Furthermore, in the specific sample analysis method, protein separation is performed in the presence of a specific low-molecular-weight compound. From the viewpoint of improving separation accuracy, it is preferable to include a specific low-molecular-weight compound in the specific alkaline solution. The specific low-molecular-weight compound will be described later.

[0018] Capillary electrophoresis can be performed using an apparatus equipped with a capillary channel. The cross-sectional shape of the capillary channel is not particularly limited and may be circular, rectangular, or any other shape. In the case of a rectangular shape, the channel height and channel width of the capillary channel are preferably 1 to 1000 μm, more preferably 10 to 200 μm, and even more preferably 25 μm to 100 μm, respectively. In the case of a circular shape, the inner diameter of the capillary channel is preferably 10 μm or more or 25 μm or more, and preferably 100 μm or less or 75 μm or less. The channel length of the capillary channel is preferably 10 mm to 150 mm, and more preferably 20 mm to 60 mm.

[0019] Materials for the capillary channel include glass, fused silica, and plastic. Examples of plastics include polymethyl methacrylate (PMMA), polycarbonate, polystyrene, polytetrafluoroethylene (PTFE), and polyether ether ketone (PEEK).

[0020] In the separation process, a capillary electrophoresis chip with the capillary channel described above integrated into a microchip may be used. A capillary electrophoresis chip may have a sample holding tank, an electrophoresis solution holding tank, and a capillary channel, with the sample holding tank and the electrophoresis solution holding tank being connected by the capillary channel. The size of the capillary electrophoresis tip is not particularly limited and is preferably adjusted as appropriate. For example, the size of the capillary electrophoresis tip can be 10 mm to 200 mm in length, 1 mm to 60 mm in width, and 0.3 mm to 5 mm in thickness.

[0021] The volumes of the sample holding tank and the electrophoresis solution holding tank are determined appropriately according to the inner diameter and length of the capillary channel, but each is 1 mm 3 ~1000mm 3 Preferably, 5 mm 3 ~100mm 3 It is preferable that it be so. The amount of sample to be filled into the sample holding tank is not particularly limited and can be between 1 μL and 70 μL. The amount of specific alkaline solution to be filled into the electrophoresis solution holding tank is not particularly limited and can be between 1 μL and 70 μL.

[0022] The voltage applied across both ends of the capillary channel is preferably 500V to 10000V, and more preferably 500V to 5000V.

[0023] The electrophoresis time for capillary electrophoresis in the separation step is preferably 50 seconds or more and less than 250 seconds, and more preferably 60 seconds or more and 200 seconds or less.

[0024] A liquid flow may be generated within the capillary channel from the negative electrode side to the positive electrode side. Examples of such liquid flows include electroosmotic flow.

[0025] It is preferable that the inner wall of the capillary channel is coated with a cationic or anionic substance. By coating the inner wall of the capillary channel with a cationic substance, the inner wall of the capillary channel can be positively charged. As a result, electroosmotic flow from the negative electrode side to the positive electrode side can be easily generated within the capillary channel. When the inner wall of a capillary channel is coated with an anionic substance, the inner wall of the capillary channel becomes negatively charged. However, a cationic polymer contained in a specific alkaline solution binds to the negatively charged inner wall of the capillary channel. As a result, the inner wall of the capillary channel becomes positively charged, and, as described above, electroosmotic flow from the negative electrode side to the positive electrode side can be easily generated within the capillary channel.

[0026] The cationic substance is not particularly limited, and silane coupling agents having cationic functional groups can be used. From the viewpoint of improving separation accuracy, the cationic substance is preferably a polymer having a quaternary ammonium base.

[0027] The anionic substance is not particularly limited, and polysaccharides having anionic groups, silane coupling agents having anionic functional groups, etc., can be used. Examples of polysaccharides having anionic groups include sulfated polysaccharides, carboxylic acidized polysaccharides, sulfonated polysaccharides, and phosphorylated polysaccharides. Examples of sulfated polysaccharides include chondroitin sulfate, heparin, heparan, fucoidan, and their salts. Examples of carboxylated polysaccharides include alginic acid, hyaluronic acid, and their salts.

[0028] Figures 1A and 1B show one embodiment of a capillary electrophoresis chip. Figure 1A is a top view showing one embodiment of a capillary electrophoresis chip, and Figure 1B is a cross-sectional view of the electrophoresis chip shown in Figure 1A. The capillary electrophoresis chip shown in Figures 1A and 1B comprises a capillary channel 1, a sample holding tank 2, and an electrophoresis solution holding tank 3, with the sample holding tank 2 and the electrophoresis solution holding tank 3 being connected by the capillary channel 1. A detection unit 4 is formed in the capillary channel 1. The sample holding tank 2 and the electrophoresis solution holding tank 3 may each be equipped with electrodes for applying voltage to both ends of the capillary channel 1 (not shown). Specifically, the sample holding tank 2 (sample introduction side) may be equipped with a negative electrode, and the electrophoresis solution holding tank 3 (specific alkaline solution supply side) may be equipped with a positive electrode.

[0029] The position of the detection unit 4, that is, the length required for separation (distance from the sample holding tank 2 to the detection unit 4, x in Figure 1A), can be appropriately determined by the length of the capillary channel 1, etc. When the length of the capillary channel 1 (x+y in Figure 1A) is 10 mm to 150 mm, the distance (x) from the sample holding tank 2 to the detection unit 4 is preferably 5 mm to 140 mm, more preferably 10 mm to 100 mm, and even more preferably 15 mm to 50 mm.

[0030] -Specific alkaline solution- In this disclosure, "alkaline" means a pH greater than 7.0. The pH of a specific alkaline solution is preferably higher than the isoelectric point of proteins such as hemoglobin, albumin, γ-globulin, and transferrin, preferably between 8.5 and 12.0, more preferably between 9.0 and 11.0, and even more preferably between 9.0 and 10.0. In this disclosure, the pH of the specific alkaline solution is the pH of the specific alkaline solution at 25°C, and is measured using a pH meter 30 minutes after immersion of the electrode. A Horiba F-72 or a similar device can be used as the pH meter.

[0031] --Cationic polymer-- The specific alkaline solution contains a cationic polymer. The specific alkaline solution may contain two or more cationic polymers. In this disclosure, "cationic polymer" means a polymer having cationic groups and a weight-average molecular weight greater than 1000. In this disclosure, "cationic group" includes cationic groups and groups that are ionized to become cationic groups. Cationic groups include primary amino groups, secondary amino groups, tertiary amino groups, quaternary ammonium bases, and imino groups. Among these, primary or secondary amino groups are preferred, and secondary amino groups are more preferred, from the viewpoint of shortening the protein separation time.

[0032] The cationic polymer is preferably water-soluble. In this disclosure, "water-soluble" means that the substance dissolves in water at 25°C in an amount of 1% by mass or more.

[0033] Cationic polymers having primary to tertiary amino groups or cationic groups that can be ionized to primary to tertiary amino groups include polyallylamine, polyvinylamine, polylysine, polyarginine, polyhistidine, polyornithine, polydiallylamine, polymethyldiallylamine, polyethyleneimine, diallylamine-acrylamide polymer, dimethylamine-ammonia-epichlorohydrin polymer, allylamine-diallylamine polymer, and the like. Cationic polymers having an imino group or a cationic group that can be ionized into an imino group include polyethyleneimine. Examples of cationic polymers having a quaternary ammonium base or a cationic group that can be ionized by a quaternary ammonium base include polyquaternium, dimethylamine-ammonia-epichlorohydrin polymer, dimethylamine-epichlorohydrin polymer, and diallyldimethylammonium chloride polymer. In this disclosure, "polyquaternium" refers to a cationic polymer containing constituent units derived from monomers having quaternary ammonium groups. Polyquaternium can be identified in the INCI (International Nomenclature for Cosmetic Ingredients) directory. Examples of polyquaternium in one or more embodiments include polydiallyldimethylammonium salts such as polyquaternium-6 (poly(diallyldimethylammoniumchloride)), polyquaternium-7 (copolymer of acrylamide and diallyldimethylammoniumchloride), polyquaternium-4 (diallyldimethylammoniumchloride-hydroxyethylcellulose copolymer), and polyquaternium-22 (copolymer of acrylic acid and diallyldimethylammoniumchloride), as well as polyquaternium-2 (poly[bis(2-chloroethyl)ether-alt-1,3-bis[3-(dimethylamino)propyl]urea]). In addition to the ammonium salts mentioned above, cationic polymers of onium salts such as phosphonium salts, oxonium salts, sulfonium salts, fluoronium salts, and chloronium salts can also be used in one or more embodiments. Cationic polymers having a hydrazide group or a cationic group that can be ionized to a hydrazide group include aminopolyacrylamide and the like.

[0034] From the viewpoint of shortening the protein separation time, the cationic polymer preferably contains one or more selected from the group consisting of dimethylamine-ammonia-epichlorohydrin polymer, dimethylamine-epichlorohydrin polymer, diallylamine-acrylamide polymer, polyethyleneimine, polyallylamine, diallylamine polymer, allylamine-diallylamine polymer, methyldiallylamine polymer, and diallyldimethylammonium chloride polymer; more preferably contains one or more selected from the group consisting of dimethylamine-ammonia-epichlorohydrin polymer, dimethylamine-epichlorohydrin polymer, diallylamine-acrylamide polymer, polyethyleneimine, and polyallylamine; and even more preferably contains one or more selected from the group consisting of dimethylamine-ammonia-epichlorohydrin polymer and diallylamine-acrylamide polymer. The cationic polymers mentioned above may also be in the form of salts, such as hydrochloride salts.

[0035] From the viewpoint of shortening the protein separation time, the weight-average molecular weight of the cationic polymer is preferably 10,000 to 500,000, more preferably 12,000 to 300,000, even more preferably 15,000 to 150,000, particularly preferably 20,000 to 130,000, and most preferably 20,000 to 100,000. The reason why setting the weight-average molecular weight of the cationic polymer within the above numerical range shortens the protein separation time is not clear, but it is speculated as follows. For example, the isoelectric points of albumin and gamma globulin (IgG as a representative component) are around pH 5 and around pH 5-9, respectively. Since albumin has a lower isoelectric point than gamma globulin, its negative charge becomes greater than that of gamma globulin in alkaline solutions, resulting in stronger interactions with cationic polymers. Therefore, by setting the weight-average molecular weight of the cationic polymer to 500,000 or less, it is possible to suppress the excessively large difference between the force pulling albumin back to the negative electrode side and the force pulling γ-globulin back, resulting from the interaction with the cationic polymer, and thereby further shorten the separation time of albumin and γ-globulin. Furthermore, by setting the weight-average molecular weight of the cationic polymer to 10,000 or more, it is possible to suppress the excessive reduction in the difference between the force pulling albumin back to the negative electrode side and the force pulling γ-globulin back, which is caused by the interaction with the cationic polymer, thereby improving separation accuracy. In this disclosure, the weight-average molecular weight of cationic polymers is based on catalog values. If a catalog value for the weight-average molecular weight is unavailable, the weight-average molecular weight is determined as the weight-average molecular weight in terms of polyethylene glycol, measured by gel permeation chromatography (GPC).

[0036] From the viewpoint of shortening the protein separation time, the content of the cationic polymer relative to the total mass of the specific alkaline solution is preferably 0.01% to 10% by mass, more preferably 0.05% to 8.0% by mass, even more preferably 0.1% to 5.0% by mass, and even more preferably 1.0% to 3.0% by mass.

[0037] The cationic polymer may be synthesized by conventionally known methods or may be a commercially available product.

[0038] --Specified low molecular weight compounds-- From the viewpoint of improving the accuracy of protein separation, it is preferable that the specific alkaline solution contains a specific low molecular weight compound. In this disclosure, "specific low molecular weight compound" means a compound with a molecular weight of 1000 or less. If there is a distribution in molecular weight, the molecular weight refers to the mass-average molecular weight (Mw). From the viewpoint of improving the accuracy of protein separation, the molecular weight of the specific low molecular weight compound is preferably 30 to 1000, more preferably 40 to 800, even more preferably 50 to 400, and even more preferably 100 to 200.

[0039] From the viewpoint of improving the accuracy of protein separation, the number of primary amino groups in a specific low molecular weight compound is preferably 2 to 5, more preferably 2 to 4, and even more preferably 2 or 3.

[0040] From the viewpoint of improving the accuracy of protein separation, the specific low molecular weight compound is preferably one or more selected from the group consisting of 3,3'-diamino-N-methyldipropylamine, N,N'-bis(3-aminopropyl)ethylenediamine, 3,3'-diaminodipropylamine, tris(2-aminoethyl)amine, 1,2-diaminopropane, ethylenediamine, 1,3-diaminopentane, amidol, and 2,2'-oxybis(ethylamine). It is more preferably one or more selected from the group consisting of 3,3'-diamino-N-methyldipropylamine, N,N'-bis(3-aminopropyl)ethylenediamine, 3,3'-diaminodipropylamine, and tris(2-aminoethyl)amine, and even more preferably at least one of 3,3'-diamino-N-methyldipropylamine and 3,3'-diaminodipropylamine.

[0041] From the viewpoint of improving the accuracy of protein separation, the content of the specific low molecular weight compound relative to the total mass of the specific alkaline solution is preferably 0.01% to 10% by mass, more preferably 0.05% to 5% by mass, even more preferably 0.1% to 3% by mass, and even more preferably 0.3% to 1.0% by mass. In a specific alkaline solution, the ratio of the content of a specific low molecular weight compound to the content of a cationic polymer (content of specific low molecular weight compound / content of cationic polymer) is preferably 0.001 to 1000, more preferably 0.01 to 100, even more preferably 0.05 to 10, and still more preferably 0.1 to 5.

[0042] The specific low-molecular-weight compound may be one synthesized by conventionally known methods or one that is commercially available.

[0043] --water-- The specific alkaline solution may contain water. Examples of water include distilled water, deionized water, pure water, and ultrapure water. The water content relative to the total mass of the specific alkaline solution is not particularly limited and can range from 10% by mass to 99.9% by mass.

[0044] --Additives-- The specific alkaline solution may contain additives such as non-surfactant type amphoteric substances (such as non-surfactant type betaine), pH buffering substances, and preservatives to inhibit the growth of microorganisms. Examples of preservatives include sodium azide, ethylparaben, and procline. In this disclosure, “non-surfactant type zwionic substance” means a zwionic substance that does not form micelles. Furthermore, in this disclosure, “does not form micelles” means that it does not form micelles or substantially does not form micelles in an aqueous medium. Moreover, in this disclosure, “substantially does not form micelles” means that the critical micelle concentration is preferably 200 mmol / L or more, more preferably 300 mmol / L or more, and even more preferably the zwionic substance does not have a critical micelle concentration. In this disclosure, "amphoionic substance" means a compound having a positively charged group and a negatively charged group in non-adjacent positions within the same molecule, with no dissociable hydrogen atoms bonded to the positively charged atom, and the molecule as a whole having no charge.

[0045] -sample- The sample contains protein. The protein is preferably one or more selected from the group consisting of hemoglobin, albumin, γ-globulin, and transferrin. Examples of hemoglobin include HbA0, HbA1c, HbA2, HbE, HbG, HbA, HbS, HbF, HbC, and HbD. Examples of gamma globulins include IgG, IgM, IgA, IgD, and IgE. The protein content relative to the total mass of the sample is not particularly limited and can range from 0.001% by mass to 100% by mass. The form of the sample is not particularly limited; it may be a prepared sample material, or it may be the sample material itself. Examples of sample materials include protein-containing materials and biological samples. Examples of biological samples include blood, serum, plasma, or preparations made from these, as well as blood-derived products containing red blood cell components, etc. Blood, serum, and plasma refer to blood, serum, and plasma collected from living organisms, including blood, serum, and plasma from mammals other than humans, and human blood, serum, and plasma. Blood-derived products containing red blood cell components include those separated or prepared from blood and containing red blood cell components. Examples include blood cell fractions from which plasma has been removed, blood cell concentrates, freeze-dried blood or blood cells, hemolyzed samples obtained by hemolyzing whole blood, centrifuged blood, spontaneously settled blood, and washed blood cells.

[0046] The sample may contain specific low-molecular-weight compounds, but it is preferable that it does not contain them from the viewpoint of separation accuracy, etc.

[0047] From the viewpoint of improving the accuracy of protein separation, it is preferable that the sample contains an alkaline solution containing a cationic polymer. A sample containing the above-mentioned alkaline solution can be obtained by diluting the sample raw material with the above-mentioned alkaline solution. The dilution ratio is preferably 1.2 to 100 times by volume, more preferably 2 to 60 times, and even more preferably 3 to 50 times. The materials used for dilution are not particularly limited and include pH adjusters (e.g., hydrochloric acid), surfactants (e.g., Emulgen LS-110 (manufactured by Kao Corporation)), preservatives (e.g., sodium azide), ionic strength adjusters (e.g., sodium chloride), and refractive index adjusters (e.g., sugars such as sucrose). Furthermore, the above-mentioned alkaline solution may be the same as, or different from, the specific alkaline solution filled into the capillary channel.

[0048] The sample may contain a non-surfactant type sulfobetaine. In this disclosure, “non-surfactant sulfobetaine” means a sulfobetaine that does not form micelles. Furthermore, in this disclosure, “not forming micelles” means not forming micelles or substantially not forming micelles in an aqueous medium. Moreover, in this disclosure, “substantially not forming micelles” means that the critical micelle concentration is preferably 200 mmol / L or higher, more preferably 300 mmol / L or higher, and even more preferably the amphoteric substance does not have a critical micelle concentration. In this disclosure, "betaine" means a compound having a positively charged group and a negatively charged group in non-adjacent positions within the same molecule, with no dissociable hydrogen atoms bonded to the positively charged atom, and the molecule as a whole having no charge.

[0049] Positively charged groups found in non-surfactant sulfobetaines include quaternary ammonium cation groups, sulfonium groups, and phosphonium groups. Among these, quaternary ammonium cation groups are preferred from the viewpoint of shortening the separation time of albumin and γ-globulin and improving separation accuracy.

[0050] The number of carbon atoms between the positively charged group and the negatively charged group is preferably 1 to 10, more preferably 2 to 8, and even more preferably 2 to 5.

[0051] From the viewpoint of shortening the separation time of albumin and γ-globulin and improving separation accuracy, the molecular weight of the non-surfactant type sulfobetaine is preferably 100 to 500, more preferably 120 to 400, and even more preferably 170 to 300.

[0052] Examples of non-surfactant sulfobetaines include 3-(1-Pyridinio)propanesulfonate (left, first from the top in Chemical Formula 1), Dimethylethylammonium Propane Sulfonate (left, second from the top in Chemical Formula 1), 3-[(2-Hydroxyethyl)dimethylammonio]propane-1-sulfonate (left, third from the top in Chemical Formula 1), 3-(4-tert-Butyl-1-pyridinio)propanesulfonate (left, fourth from the top in Chemical Formula 1), N-Methyl-N-(3-sulfopropyl)morpholinium (left, fifth from the top in Chemical Formula 1), Dimethylbenzylammonium Propane Sulfonate (right, first from the top in Chemical Formula 1), and 3-(1-Methylpiperidinio)-1-propanesulfonate (right, second from the top in Chemical Formula 1). Furthermore, non-surfactant sulfobetaines are not limited to these compounds.

[0053] [ka]

[0054] Examples of commercially available compounds as exemplified above include NDSB-201 (3-(1-Pyridinio)propanesulfonate), NDSB-195 (Dimethylethylammonium Propane Sulfonate), NDSB-211 (3-[(2-Hydroxyethyl)dimethylammonio]propane-1-sulfonate), NDSB-256 (Dimethylbenzylammonium Propane Sulfonate), NDSB-221 (3-(1-Methylpiperidinio)-1-propanesulfonate), NDSB-256-4T (3-(4-tert-Butyl-1-pyridinio)propanesulfonate), and NDSB-223 (N-Methyl-N-(3-sulfopropyl)morpholinium), all manufactured by Merck.

[0055] Among the compounds exemplified above, the following compounds are preferred from the viewpoint of shortening the separation time of albumin and γ-globulin and improving separation accuracy.

[0056] [ka]

[0057] Non-surfactant sulfobetaine may be synthesized by conventionally known methods or may be commercially available.

[0058] From the viewpoint of shortening the separation time of albumin and γ-globulin and improving separation accuracy, the content of non-surfactant type sulfobetaine relative to the total mass of the sample is preferably 0.1% to 30% by mass, more preferably 0.5% to 20% by mass, even more preferably 1% to 15% by mass, and even more preferably 1% to 9% by mass.

[0059] <Detection Process> The specific sample analysis method may include a detection step to detect proteins separated in the separation step. Proteins can be detected by optical methods. Examples of optical detection methods include measuring absorbance. More specifically, the isolated protein can be detected by irradiating it with light at a wavelength of 280 nm and obtaining an absorbance spectrum with absorbance on the vertical axis and time on the horizontal axis. When using a capillary electrophoresis chip for protein separation, it is preferable to irradiate the detection unit with light at a wavelength of 280 nm.

[0060] Protein detection may also be performed using an electropherogram (differential waveform) obtained by differentiating the absorbance spectrum waveform described above with respect to time.

[0061] The superior separation accuracy of a specific sample analysis method compared to conventional methods can be confirmed by examining the value of the maximum absorbance change at the protein peak, whether the peak in the electropherogram is broadened, and other factors.

[0062] Referring to Figures 1A and 1B, one embodiment of the specific sample analysis method will be described. Note that the specific sample analysis method is not limited to the one described below.

[0063] First, a specific alkaline solution containing a cationic polymer is filled into the electrophoresis fluid holding tank 3 of the capillary electrophoresis tip as the electrophoresis fluid, and the specific alkaline solution is filled into the capillary channel 1 by capillary action.

[0064] Next, the sample is added to the sample holding tank 2 of the capillary electrophoresis tip, which is filled with a specific alkaline solution.

[0065] The sample to be added to the sample holding tank 2 can be prepared by diluting the biological sample, which is the raw material for the sample, with the above-mentioned alkaline solution.

[0066] A negative electrode is brought into contact with the sample holding tank 2 and a positive electrode is brought into contact with the electrophoresis solution holding tank 3 (not shown). A voltage is applied between both ends of the capillary channel 1, i.e., between the sample holding tank 2 and the electrophoresis solution holding tank 3. As a result, the sample is introduced from the sample holding tank 2 into the capillary channel 1, the protein-containing sample moves from the sample holding tank 2 towards the electrophoresis solution holding tank 3, and the proteins are separated.

[0067] Then, in the detection unit 4, light with a wavelength of 280 nm is irradiated, and the absorbance is measured by an absorbance measuring device to detect the protein.

[0068] The sample analysis method disclosed herein can be used for the prevention, diagnosis, and treatment of multiple myeloma, nephrotic syndrome, cirrhosis, nutritional deficiencies, abnormal hemoglobin, β-thalassemia, and other related applications.

[0069] [Capillary electrophoresis solution] The capillary electrophoresis solution according to the embodiments of this disclosure contains a cationic polymer and a specific low molecular weight compound, and is used for the separation of proteins by capillary electrophoresis. The preferred embodiment of the capillary electrophoresis solution is the same as that of the specific alkaline solution used in the specific sample analysis method, and therefore will not be described here.

[0070] [Sample Analysis Kit] A sample analysis kit according to the embodiment of the present disclosure includes a container containing the capillary electrophoresis solution, and an electrophoresis tip having a sample holding tank, an electrophoresis solution holding tank, and a capillary channel, wherein the sample holding tank and the electrophoresis solution holding tank are connected by a capillary channel. The preferred embodiment of the electrophoresis chip is the same as that used in the specific sample analysis method, so it will not be described here.

[0071] Examples of materials for containers containing capillary electrophoresis solutions include glass, fused silica, and plastic. Since plastic has been discussed above, it will not be mentioned here.

[0072] This disclosure may relate to the following embodiments. <1> The process includes a separation step in which proteins in a sample are separated by capillary electrophoresis in an alkaline solution, and The above protein separation is performed in the presence of a cationic low-molecular-weight compound having two or more primary amino groups. A method for analyzing a sample, wherein the above-mentioned alkaline solution contains a cationic polymer. <2> The above alkaline solution contains the above cationic low molecular weight compound. <1> The sample analysis method described below. <3> The content of the cationic low molecular weight compound relative to the total mass of the above alkaline solution is 0.01% by mass to 10% by mass. <2> The sample analysis method described below. <4> In the above alkaline solution, the ratio of the content of the cationic low molecular weight compound to the content of the cationic polymer is 0.001 to 1000. <2> or <3> The sample analysis method described below. <5> The above cationic low molecular weight compound is one or more selected from the group consisting of 3,3'-diamino-N-methyldipropylamine, N,N'-bis(3-aminopropyl)ethylenediamine, 3,3'-diaminodipropylamine, tris(2-aminoethyl)amine, 1,2-diaminopropane, ethylenediamine, 1,3-diaminopentane, amidol, and 2,2'-oxybis(ethylamine). <1> ~ <4> A sample analysis method described in any one of the following. <6> The above protein is one or more selected from the group consisting of hemoglobin, albumin, γ-globulin, and transferrin. <1> ~ <5> A sample analysis method described in any one of the following. <7> The weight-average molecular weight of the above cationic polymer is 15,000 to 150,000. <1> ~ <6> A sample analysis method described in any one of the following. <8> The content of the cationic polymer relative to the total mass of the above alkaline solution is 0.01% by mass to 10% by mass. <1> ~ <7> A sample analysis method described in any one of the following. <9> The pH of the above alkaline solution is 8.5 to 12.0. <1> ~ <8> A sample analysis method described in any one of the following. <10> A capillary electrophoresis solution containing a cationic polymer and a cationic low molecular weight compound having two or more primary amino groups, and used for separating proteins by capillary electrophoresis. <11> the above <10> A container containing the capillary electrophoresis solution described above, An electrophoresis chip having a sample holding tank, an electrophoresis solution holding tank, and a capillary channel, wherein the sample holding tank and the electrophoresis solution holding tank are connected by the capillary channel, A kit for sample analysis, including the following: [Examples]

[0073] Examples are described below, but this disclosure is not limited to these examples. In the following description, unless otherwise specified, "parts" and "%" all refer to mass. Furthermore, in the following examples and comparative examples, electrophoresis was performed using the continuous sample introduction method.

[0074] <Separation devices and measuring instruments> As the separation device, a resin tip with a capillary channel 1 having the structure shown in Figure 1 (channel width: 40 μm, channel height: 40 μm, channel length: 30 mm, distance (x) from sample holding tank 2 to detection unit 4: 20 mm) was used. The capacity of the sample holding tank 2 and the electrophoresis solution holding tank 3 was 60 μL. The inner wall of the capillary channel was coated with polydiallyldimethylammonium chloride. The measurement equipment used was an electrophoresis system manufactured in-house.

[0075] <<Example 1>> The following substances were mixed, and 3-hydroxypropanesulfonic acid and water were added until the pH reached 9.8 to prepare specific alkaline solution (capillary electrophoresis solution) 1. The content of cationic polymer in specific alkaline solution 1 was 1.5% by mass, and the content of specific low molecular weight compounds was 0.58% by mass. (Composition of specific alkaline solution 1) • Dimethylamine-ammonia-epichlorohydrin polymer 1 (cationic polymer, manufactured by Senka, KHE102L, weight-average molecular weight 20,000-100,000) 1.5% by mass • Specific low molecular weight compound 1 (3,3'-diamino-N-methyldipropylamine (hereinafter also referred to as "DAMDPA," and represented by the following chemical formula), molecular weight 145.25) 0.58% by mass • Sodium azide 0.02% by mass • Emulgen LS-110 (manufactured by Kao Corporation) 0.1% by mass • 3-hydroxypropanesulfonic acid ·water

[0076] [ka]

[0077] A human serum sample containing albumin and γ-globulin was prepared and diluted with diluent 1 (pH 8.8) of the following composition (dilution ratio 7 times (by volume)) to obtain sample A. In dilution 1, hydrochloric acid and water were added until the pH of the solution reached 8.8. (Dilution 1) • Polyethyleneimine (cationic polymer, manufactured by Fujifilm Wako Pure Chemical Industries, weight-average molecular weight 70,000) 0.75% by mass Sucrose 0.787% by mass • Sodium chloride 0.26% by mass • Sodium azide 0.018% by mass • Emulgen LS-110 (manufactured by Kao Corporation) 0.4% by mass ·hydrochloric acid ·water

[0078] 60 μL of specific alkaline solution 1 was added to the electrophoresis solution holding tank 3, and the specific alkaline solution 1 was filled into the capillary channel 1 by capillary action.

[0079] 60 μL of sample A was added to sample holding tank 2. Next, the negative electrode was brought into contact with the sample holding tank 2 and the positive electrode with the electrophoresis solution holding tank 3, and electrophoresis was started by applying a voltage with constant current control of 75 μA.

[0080] During electrophoresis, the detection unit 4 was irradiated with 280 nm light, and its absorbance was measured to obtain an absorbance spectrum. An electropherogram was obtained by differentiating the waveform of the absorbance spectrum with respect to time. The electrophoresis was performed for 200 seconds. The obtained electropherogram is shown in Figure 2. Furthermore, a prototype machine manufactured in-house was used for light irradiation, absorbance measurement, and electropherogram acquisition. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0081] <<Example 2>> An electroferrogram was obtained in the same manner as in Example 1, except that specific low molecular weight compound 1 was replaced with specific low molecular weight compound 2 (N,N'-bis(3-aminopropyl)ethylenediamine (hereinafter also referred to as "BAPEDA"), molecular weight 174.29), represented by the following chemical formula, and the content was changed to 0.70% by mass. The obtained electroferrogram is shown in Figure 3. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0082] [ka]

[0083] <<Example 3>> An electroferrogram was obtained in the same manner as in Example 1, except that specific low molecular weight compound 1 was replaced with specific low molecular weight compound 3 (3,3'-diaminodipropylamine (hereinafter also referred to as "DADPA"), molecular weight 131.22), represented by the following chemical formula, and the content was changed to 0.52% by mass. The obtained electroferrogram is shown in Figure 4. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0084] [ka]

[0085] <<Example 4>> An electroferrogram was obtained in the same manner as in Example 1, except that specific low molecular weight compound 1 was replaced with specific low molecular weight compound 4 (tris(2-aminoethyl)amine (hereinafter also referred to as "TAA"), molecular weight 146.24), represented by the following chemical formula, and the content was changed to 0.58% by mass. The obtained electroferrogram is shown in Figure 5. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0086] [ka]

[0087] <<Comparative Example 1>> An electroferrogram was obtained in the same manner as in Example 1, except that specific alkaline solution 1 was replaced with alkaline solution 10 having the following composition. The obtained electroferrogram is shown in Figure 6. In alkaline solution 10, sodium hydroxide and water were added until the pH of the solution reached 9.8. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively. (Composition of alkaline solution 10) • Dimethylamine-ammonia-epichlorohydrin polymer 1 (cationic polymer, manufactured by Senka, KHE102L, weight-average molecular weight 20,000-100,000) 1.5% by mass • Sodium azide 0.02% by mass • Emulgen LS-110 (manufactured by Kao Corporation) 0.1% by mass Sodium hydroxide ·water

[0088] <<Comparative Example 2>> An electroferrogram was obtained in the same manner as in Example 1, except that specific alkaline solution 1 was replaced with alkaline solution 11 having the following composition. The obtained electroferrogram is shown in Figure 7. In alkaline solution 11, sodium hydroxide and water were added until the pH of the solution reached 9.8. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively. Furthermore, the content of cationic polymers relative to the total mass of alkaline solution 11 was set to 1.5% by mass, and the content of low molecular weight compounds was set to 0.69% by mass. (Composition of alkaline solution 11) • Dimethylamine-ammonia-epichlorohydrin polymer 1 (cationic polymer, manufactured by Senka, KHE102L, weight-average molecular weight 20,000-100,000) 1.5% by mass • Low molecular weight compound 11 (N,N,N',N'',N''-pentamethyldiethylenetriamine (hereinafter also referred to as "PMDETA" and represented by the following chemical formula), molecular weight 173.30) 0.69% by mass • Sodium azide 0.02% by mass • Emulgen LS-110 (manufactured by Kao Corporation) 0.1% by mass Sodium hydroxide ·water

[0089] [ka]

[0090] <<Comparative Example 3>> An electroferrogram was obtained in the same manner as in Example 1, except that specific low molecular weight compound 1 was replaced with low molecular weight compound 12 represented by the following chemical formula (diethylamine (hereinafter also referred to as "DEA"), molecular weight 73.14), and the content was set to 0.29% by mass. The obtained electroferrogram is shown in Figure 8. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0091] [ka]

[0092] <<Comparative Example 4>> An electroferrogram was obtained in the same manner as in Example 1, except that specific low molecular weight compound 1 was replaced with low molecular weight compound 13 represented by the following chemical formula (tetraethylammonium hydroxide (hereinafter also referred to as "TEA"), molecular weight 147.26), and the content was set to 0.59% by mass. The obtained electroferrogram is shown in Figure 9. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0093] [ka]

[0094] <<Comparative Example 5>> An electroferrogram was obtained in the same manner as in Example 1, except that low molecular weight compound 11 was replaced with low molecular weight compound 14 (diethanolamine (hereinafter also referred to as "DEtOHA"), molecular weight 105.14), represented by the following chemical formula, and the content was set to 0.42% by mass. The obtained electroferrogram is shown in Figure 10. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0095] [ka]

[0096] <<Comparative Example 6>> An electroferrogram was obtained in the same manner as in Comparative Example 2, except that low molecular weight compound 12 was replaced with low molecular weight compound 15 (N-methyldiethanolamine (hereinafter also referred to as "MDEA"), molecular weight 119.16), represented by the following chemical formula, and the content was set to 0.48% by mass. The obtained electroferrogram is shown in Figure 11. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0097] [ka]

[0098] <<Comparative Example 7>> An electroferrogram was obtained in the same manner as in Comparative Example 2, except that low molecular weight compound 12 was replaced with low molecular weight compound 16 (2-amino-2-hydroxymethyl-1,3-propanediol (hereinafter also referred to as "TRIS"), molecular weight 121.14), represented by the following chemical formula, and the content was set to 0.48% by mass. The obtained electroferrogram is shown in Figure 12. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0099] [ka]

[0100] <<Comparative Example 8>> An electroferrogram was obtained in the same manner as in Comparative Example 2, except that low molecular weight compound 12 was replaced with low molecular weight compound 17 (N-(2-hydroxypropyl)ethylenediamine (hereinafter also referred to as "HPEDA"), molecular weight 118.18), represented by the following chemical formula, and the content was set to 0.47% by mass. The obtained electroferrogram is shown in Figure 13. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0101] [ka]

[0102] <<Comparative Example 9>> An electroferrogram was obtained in the same manner as in Example 1, except that specific low molecular weight compound 1 was replaced with low molecular weight compound 18 (N,N'-dimethylethylenediamine (hereinafter also referred to as "DMEDA"), molecular weight 88.15), represented by the following chemical formula, and the content was set to 0.35% by mass. The obtained electroferrogram is shown in Figure 14. Table 1 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0103] [ka]

[0104] [Table 1]

[0105] From Figures 2 to 14 and Table 1, it can be seen that the sample analysis method in the example, in which albumin and γ-globulin were separated using an alkaline solution containing a cationic polymer and a cationic low-molecular-weight compound having two or more primary amino groups, exhibits superior separation accuracy compared to the sample analysis method in the comparative example, in which albumin and γ-globulin were separated using an alkaline solution that did not contain a cationic low-molecular-weight compound having two or more primary amino groups. The maximum absorbance changes for albumin and γ-globulin are larger, and the peaks in the figures are not broadened.

[0106] <<Example 5>> A sample containing hemoglobin was prepared and diluted with the above-mentioned diluent 1 (pH 8.8) (dilution ratio 7 times (by volume)) to obtain sample C. An electropherogram was obtained in the same manner as in Example 1, except that sample A was replaced with sample C. The obtained electropherogram is shown in Figure 15. Table 2 summarizes the change in maximum absorbance for hemoglobin.

[0107] <<Comparative Example 10>> An electroferrogram was obtained in the same manner as in Example 1, except that sample A was replaced with sample C. The obtained electroferrogram is shown in Figure 16. Table 2 summarizes the change in maximum absorbance for hemoglobin.

[0108] [Table 2]

[0109] From Figures 15-16 and Table 2, it can be seen that the sample analysis method in the example, in which hemoglobin was separated using an alkaline solution containing a cationic polymer and a cationic low-molecular-weight compound having two or more primary amino groups, exhibits superior separation accuracy compared to the sample analysis method in the comparative example, in which hemoglobin was separated using an alkaline solution that does not contain a cationic low-molecular-weight compound having two or more primary amino groups. The change in the maximum absorbance of hemoglobin is larger, and the peak in the figure is not broadened.

[0110] <<Example 6>> A sample containing transferrin was prepared and diluted with the above-mentioned diluent 1 (pH 8.8) (dilution ratio 7 times (by volume)) to obtain sample D. An electroferrogram was obtained in the same manner as in Example 1, except that sample A was replaced with sample D. The obtained electroferrogram is shown in Figure 17. Table 3 summarizes the change in maximum absorbance for transferrin.

[0111] <<Comparative Example 11>> An electroferrogram was obtained in the same manner as in Comparative Example 1, except that sample A was replaced with sample D. The obtained electroferrogram is shown in Figure 18. Table 3 summarizes the change in maximum absorbance for transferrin.

[0112] [Table 3]

[0113] From Figures 17-18 and Table 3, it can be seen that the sample analysis method in the example in which transferrin was separated using an alkaline solution containing a cationic polymer and a cationic low molecular weight compound having two or more primary amino groups showed superior separation accuracy compared to the sample analysis method in the comparative example in which transferrin was separated using an alkaline solution that did not contain a cationic low molecular weight compound having two or more primary amino groups. The maximum absorbance change of transferrin was larger, and the peak in the figure was not broadened.

[0114] <<Example 7>> An electropherogram was obtained in the same manner as in Example 1, except that human biological serum samples obtained from different donors were used. The obtained electropherogram is shown in Figure 19. Table 4 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0115] <<Example 8>> An electroferrogram was obtained in the same manner as in Example 7, except that Diluent 1 was replaced with Diluent 2 described below. The obtained electroferrogram is shown in Figure 20. Table 4 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively. In dilution 2, hydrochloric acid and water were added until the pH of the solution reached 8.8. (Dilution 2) • Polyethyleneimine (cationic polymer, manufactured by Wako, weight-average molecular weight 70,000) 0.75% by mass • Non-surfactant type sulfobetaine (manufactured by Tokyo Chemical Industry Co., Ltd., NDSB-201, 3-(1-pyridino)propanesulfonic acid) 8.06% by mass Sucrose 0.787% by mass • Sodium chloride 0.26% by mass • Sodium azide 0.018% by mass • Emulgen LS-110 (manufactured by Kao Corporation) 0.4% by mass ·hydrochloric acid ·water

[0116] <<Comparative Example 12>> An electroferrogram was obtained in the same manner as in Example 7, except that specific alkaline solution 1 was replaced with alkaline solution 10. The obtained electroferrogram is shown in Figure 21. Table 4 summarizes the maximum absorbance changes for albumin and γ-globulin, respectively.

[0117] [Table 4]

[0118] From Figures 19 to 21 and Table 4, it can be seen that the sample analysis method in the example, in which albumin and γ-globulin were separated using an alkaline solution containing a cationic polymer and a cationic low molecular weight compound having two or more primary amino groups, exhibits superior separation accuracy compared to the sample analysis method in the comparative example, in which albumin and γ-globulin were separated using an alkaline solution that did not contain a cationic low molecular weight compound having two or more primary amino groups. The maximum absorbance change for albumin and γ-globulin is larger, and the peaks in the figures are not broadened. Furthermore, from Figures 19-20 and Table 4, it can be seen that the sample analysis method of Example 8, in which albumin and γ-globulin were separated using a sample containing non-surfactant sulfobetaine, exhibits superior separation accuracy compared to the sample analysis method of Example 7, in which albumin and γ-globulin were separated using a sample not containing non-surfactant sulfobetaine. This is because the maximum absorbance change for albumin and γ-globulin is larger, and the peaks in the figures are not broadened. Additionally, the separation time is shorter. [Explanation of Symbols]

[0119] 1: Capillary channel, 2: Sample holding tank, 3: Electrophoresis solution holding tank, 4: Detection unit

Claims

1. The process includes a separation step in which proteins in a sample are separated by capillary electrophoresis in an alkaline solution, and The separation of the aforementioned protein is carried out in the presence of a cationic low-molecular-weight compound having two or more primary amino groups. The alkaline solution contains a cationic polymer, The cationic low molecular weight compound is one or more selected from the group consisting of 3,3'-diamino-N-methyldipropylamine, N,N'-bis(3-aminopropyl)ethylenediamine, 3,3'-diaminodipropylamine, tris(2-aminoethyl)amine, 1,2-diaminopropane, ethylenediamine, 1,3-diaminopentane, amidol, and 2,2'-oxybis(ethylamine). Sample analysis method.

2. The sample analysis method according to claim 1, wherein the alkaline solution contains the cationic low molecular weight compound.

3. The sample analysis method according to claim 2, wherein the content of the cationic low molecular weight compound relative to the total mass of the alkaline solution is 0.01% by mass to 10% by mass.

4. The sample analysis method according to claim 2 or 3, wherein in the alkaline solution, the ratio of the content of the cationic low molecular weight compound to the content of the cationic polymer is 0.001 to 1000.

5. The sample analysis method according to claim 1 or claim 2, wherein the protein is one or more selected from the group consisting of hemoglobin, albumin, γ-globulin, and transferrin.

6. The sample analysis method according to claim 1 or claim 2, wherein the weight-average molecular weight of the cationic polymer is 15,000 to 150,000.

7. The sample analysis method according to claim 1 or claim 2, wherein the content of the cationic polymer relative to the total mass of the alkaline solution is 0.01% by mass to 10% by mass.

8. The sample analysis method according to claim 1 or claim 2, wherein the pH of the alkaline solution is 8.5 to 12.

0.

9. A capillary electrophoresis solution containing a cationic polymer and a cationic low molecular weight compound having two or more primary amino groups, wherein the cationic low molecular weight compound is one or more selected from the group consisting of 3,3'-diamino-N-methyldipropylamine, N,N'-bis(3-aminopropyl)ethylenediamine, 3,3'-diaminodipropylamine, tris(2-aminoethyl)amine, 1,2-diaminopropane, ethylenediamine, 1,3-diaminopentane, amidol, and 2,2'-oxybis(ethylamine), and used for the separation of proteins by capillary electrophoresis.

10. A container containing the capillary electrophoresis solution described in Claim 9, An electrophoresis chip having a sample holding tank, an electrophoresis solution holding tank, and a capillary channel, wherein the sample holding tank and the electrophoresis solution holding tank are connected by the capillary channel, A kit for sample analysis, including the following:

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