Patent Application
20240159769
This application is based on and claims priority under 35 USC 119 from Japanese Patent Application No. 2022-173449, filed on Oct. 28, 2022, the disclosure of which is incorporated by reference herein.
The present disclosure relates to a sample analysis method, a sample production method, a diluent for diluting an albumin- and γ-globulin-containing sample, and a sample analysis kit.
In the field of clinical examination, proteins such as hemoglobin contained in erythrocytes, albumin, globulin or the like contained in serum, and the like are routinely analyzed.
As one of protein analysis methods, a capillary electrophoresis method is used. The sample analysis method using the capillary electrophoresis method is advantageous, for example, in that a small amount of sample is required, and an analyzer can be downsized. In recent years, improvement of analysis accuracy and shortening of analysis time are desired for the above analysis method.
Japanese Patent Application Laid-Open (JP-A) No. 2014-160058 discloses a sample analysis method for separating a substance to be analyzed such as hemoglobin, in an acidic solution (pH 5.3) by capillary electrophoresis in the presence of a non-surfactant-type zwitterionic substance.
JP-A No. 2016-136135 discloses a sample analysis method for separating various kinds of hemoglobin in an alkaline solution containing a cationic polymer by capillary electrophoresis.
Japanese National-Phase Publication (JP-A) No. 2004-517339 discloses an alkaline pH, free solution capillary electrophoresis method for analyzing a sample containing a protein constituent, the method including at least one step of introducing the sample into a capillary tube including a buffer system, the buffer system further containing at least one additive capable of hydrophobically interacting with one or more protein constituents and capable of providing the protein constituent or constituents with one or more negative charge and of modifying the electrophoretic mobility.
JP-A No. 2005-326407 discloses a free solution capillary electrophoresis method at alkaline pH for analyzing a sample containing protein constituents including one or more lipoprotein constituents, the method including at least one step of introducing a sample into a capillary tube containing an analysis buffer, the analysis buffer further containing at least one anionic surfactant-based additive that hydrophobically interacts with the one or more lipoprotein constituents and modifies the electrophoretic mobility with respect to the mobility of the other protein constituents, a concentration of the additive in the buffer in a range of from 0.001 mM to 0.2 mM.
An object of an embodiment of the disclosure is to provide a sample analysis method capable of separating albumin and γ-globulin with higher accuracy in a shorter time than in a conventional method, a sample production method, a diluent for diluting an albumin- and γ-globulin-containing sample, and a sample analysis kit.
A sample analysis method according to an embodiment of the disclosure includes: a separation step of separating albumin and γ-globulin from a sample containing albumin and γ-globulin, in an alkaline solution by capillary electrophoresis,
According to an embodiment of the disclosure, it is possible to provide a sample analysis method capable of separating albumin and γ-globulin from a sample containing albumin and γ-globulin with higher accuracy in a shorter time than in a conventional method, a sample production method, a diluent for diluting an albumin- and γ-globulin-containing sample, and a sample analysis kit.
Exemplary embodiments of the present disclosure will be described in detail based on the following figures, wherein:
An exemplary embodiment of the present invention will be described. These descriptions and examples illustrate embodiments and do not limit the scope of the invention.
In the present specification, “to” indicating a numerical range is used to mean that numerical values which are described before and after “to” are included as a lower limit value and an upper limit value, respectively.
In a numerical range described in a stepwise manner in the disclosure, an upper limit value or a lower limit value described in one numerical range may be replaced with an upper limit value or a lower limit value described in another numerical range described in a stepwise manner. In a numerical range described in the disclosure, an upper limit value or a lower limit value of the numerical range may be replaced with a value shown in Examples.
Each component may contain a plurality of corresponding substances.
When the amount of each component in a composition is referred to, In a case in which a plurality of substances corresponding to each component are present in the composition, the amount of each component in the composition means the total amount of the plurality of substances present in the composition unless otherwise specified.
In the disclosure, the term “step” includes not only an independent step but also a step by which an intended action of the step is achieved, though the step cannot be clearly distinguished from other steps.
In the disclosure, a combination of two or more preferred aspects is a more preferred aspect.
When an embodiment is described with reference to the drawings in the disclosure, the configuration of the embodiment is not limited to the configuration illustrated in the drawings. The sizes of members in each drawing are conceptual, and the relative relationship between the sizes of the members is not limited thereto.
A sample analysis method according to an embodiment of the disclosure (hereinafter, also referred to as “specific sample analysis method”) includes: a separation step of separating albumin and γ-globulin from a sample containing albumin and γ-globulin, in an alkaline solution by capillary electrophoresis, in which the sample contains a non-surfactant-type sulfobetaine, and the alkaline solution (hereinafter, also referred to as “specific alkaline solution”) contains a cationic polymer.
According to the specific sample analysis method, it is possible to separate albumin and γ-globulin from the sample containing albumin and γ-globulin with higher accuracy in a shorter time than in a conventional separation method. The reason why the above effect is exhibited is not clear, but it is presumed as follows.
Albumin and γ-globulin, which are substances to be analyzed, are negatively charged in an alkaline solution. When a negative electrode is brought into contact with the sample introduction side and a voltage is applied to the substance to be analyzed, these substances to be analyzed move to the positive electrode side due to the charge of the substance to be analyzed and the electroosmotic flow.
On the other hand, since the cationic polymer has a cationic group, the cationic polymer moves from the positive electrode side to the negative electrode side. During electrophoresis, the substance to be analyzed and the cationic polymer repeatedly interact with each other through bonding and dissociation in the capillary, whereby the substance to be analyzed is positively charged, so that a force for pulling back the substance to be analyzed to the negative electrode side is generated. Since the cationic polymer has a large charge, a charge difference between the substances to be analyzed, which have interacted with the cationic polymer, is larger than a charge difference inherent in the substance to be analyzed. Therefore, it is assumed that albumin and γ-globulin can be efficiently separated from the sample in a short time since a difference in electrophoretic velocity between the substances to be analyzed increases.
The present inventors have found a new problem in that a plurality of substances may interact with albumin and γ-globulin to be analyzed, and in this case, the degree of interaction between albumin and γ-globulin and the cationic polymer varies, and the separation accuracy tends to decrease.
When the sample contains a non-surfactant-type sulfobetaine in addition to albumin and γ-globulin, the positively charged group of the non-surfactant-type sulfobetaine is bound to albumin and γ-globulin negatively charged, and a sulfo group (—SO3−) exists on the surfaces of albumin and γ-globulin. As described above, it is assumed that the negative charge sites of albumin and γ-globulin are substituted with the sulfo group of the non-surfactant-type sulfobetaine, thereby leveling the degree of interaction with the cationic polymer and improving the separation accuracy.
The non-surfactant-type sulfobetaine is known to have an action of reducing hydrophobic interaction by binding to a hydrophobic moiety. The substance to be analyzed contained in the sample may form a complex with the substance to be analyzed or other contaminants via a hydrophobic moiety, or may nonspecifically interact with the hydrophobic moiety of the cationic polymer without depending on the charge of the substance to be analyzed.
As described above, it is assumed that the separation accuracy is improved by mixing albumin and γ-globulin with the non-surfactant-type sulfobetaine to suppress these unintended interactions.
In the sample analysis method disclosed in JP-A No. 2014-160058, since the substance to be analyzed is separated in an acidic solution, it is presumed that the above-described effect is not exhibited or is not sufficient.
Specifically, albumin and γ-globulin are positively charged in an acidic solution. The sulfo group of the non-surfactant-type sulfobetaine is bonded to these albumin and γ-globulin, and the positively charged group of the non-surfactant-type sulfobetaine exists on the surfaces of albumin and γ-globulin.
Since albumin and γ-globulin to which the non-surfactant-type sulfobetaine is bonded and the cationic polymer both move to the negative electrode side, and separation due to interaction with the cationic polymer does not occur, it is presumed that the above-described effect is not exhibited or is not sufficient.
The specific sample analysis method includes a separation step of separating albumin and γ-globulin from a sample containing albumin and γ-globulin, in a specific alkaline solution by capillary electrophoresis.
Separation of albumin and γ-globulin from the sample in the specific alkaline solution by capillary electrophoresis can be performed by introducing the sample into a capillary flow path in which the specific alkaline solution is filled, and applying a voltage to the whole or a part of the capillary flow path after introduction of the sample. By applying the voltage, albumin and γ-globulin in the sample can be electrophoresed and separated from the sample.
The voltage can be applied to the capillary flow path by bringing a negative electrode into contact with the sample introduction side of the capillary flow path and bringing a positive electrode into contact with the specific alkaline solution supply side.
In the specific sample analysis method, the sample contains a non-surfactant-type sulfobetaine. The non-surfactant-type sulfobetaine will be described below.
The cross-sectional shape of the capillary flow path is not particularly limited, and may be a circular shape, a rectangular shape, or other shapes.
In the case of a rectangular shape, each of the flow path height and the flow path width of the capillary flow path is preferably from 1 μm to 1000 μm, more preferably from 10 μm to 200 μm, and further preferably from 25 μm to 100 μm. In the case of a circular shape, the inner diameter of the capillary flow path is preferably 10 μm or more or 25 μm or more and is preferably 100 μm or less or 75 μm or less.
The flow path length of the capillary flow path is preferably from 10 mm to 150 mm and more preferably from 20 mm to 60 mm.
Examples of the material for the capillary flow path include glass, fused silica, and plastic. Examples of the plastic include polymethyl methacrylate (PMMA), polycarbonate, polystyrene, polytetrafluoroethylene (PTFE), and polyetheretherketone (PEEK).
In the separation step, a capillary electrophoresis chip in which the above-described capillary flow path is made into a microchip may be used.
The capillary electrophoresis chip can include a sample holding tank, an electrophoretic liquid holding tank, and a capillary flow path, in which the sample holding tank and the electrophoretic liquid holding tank are communicated with each other via the capillary flow path.
The size of the capillary electrophoresis chip is not particularly limited, and is preferably appropriately adjusted. The size of the capillary electrophoresis chip can be, for example, from 10 mm to 200 mm in length, from 1 mm to 60 mm in width, and from 0.3 mm to 5 mm in thickness.
The volume of each of the sample holding tank and the electrophoretic liquid holding tank is appropriately determined according to the inner diameter and the length of the capillary flow path, and is preferably from 1 mm3 to 1000 mm3 and more preferably from 5 mm3 to 100 mm3.
The amount of the sample to be filled in the sample holding tank is not particularly limited, and can be from 1 μL to 70 μL.
The amount of the specific alkaline solution to be filled in the electrophoretic liquid holding tank is not particularly limited, and can be from 1 μL to 70 μL.
The voltage applied to both ends of the capillary flow path is preferably from 500 V to 10000 V, and more preferably from 500 V to 5000 V.
The electrophoresis time of capillary electrophoresis in the separation step is preferably from 50 seconds to less than 250 seconds, and more preferably from 60 seconds to 200 seconds.
In the capillary flow path, a liquid flow from the negative electrode side toward the positive electrode side may be generated. Examples of the liquid flow include an electroosmotic flow.
The inner wall of the capillary flow path is preferably coated with a cationic substance or an anionic substance.
By coating the inner wall of the capillary flow path with a cationic substance, the inner wall of the capillary flow path can be positively charged. As a result, an electroosmotic flow from the negative electrode side toward the positive electrode side can be easily generated in the capillary flow path.
When the inner wall of the capillary flow path is coated with the anionic substance, the inner wall of the capillary flow path is negatively charged, but the cationic polymer contained in the specific alkaline solution is bonded to the inner wall of the negatively charged capillary flow path. As a result, the inner wall of the capillary flow path is positively charged, and the electroosmotic flow from the negative electrode side toward the positive electrode side can be easily generated in the capillary flow path as described above.
The cationic substance is not particularly limited, and a silane coupling agent having a cationic functional group and the like can be used. From the viewpoint of improving the separation accuracy, the cationic substance is preferably a polymer having a quaternary ammonium base.
The anionic substance is not particularly limited, and polysaccharides having an anionic group, a silane coupling agent having an anionic functional group, and the like can be used.
Examples of the polysaccharides having an anionic group include sulfated polysaccharides, carboxylated polysaccharides, sulfonated polysaccharides, and phosphorylated polysaccharides.
Examples of the sulfated polysaccharides include chondroitin sulfate, heparin, heparan, fucoidan, and salts thereof. Examples of the carboxylated polysaccharides include alginic acid, hyaluronic acid, and salts thereof.
The capillary electrophoresis chip illustrated in
Each of the sample holding tank 2 and the electrophoretic liquid holding tank 3 may include an electrode for applying a voltage to both ends of the capillary flow path 1 (not illustrated). Specifically, the sample holding tank 2 (sample introduction side) can include a negative electrode, and the electrophoretic liquid holding tank 3 (specific alkaline solution supply side) can include a positive electrode.
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
In the disclosure, “alkaline” means that the pH is more than 7.0. The pH of the specific alkaline solution is preferably higher than the isoelectric points of albumin and γ-globulin, and is preferably from 8.5 to 12.0, more preferably from 9.0 to 11.0, and further preferably from 9.0 to 10.0.
In the 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 the electrode is immersed. As the pH meter, F-72 manufactured by HORIBA, Ltd. or a device similar thereto can be used.
The specific alkaline solution contains a cationic polymer. The specific alkaline solution may contain two or more cationic polymers.
In the disclosure, the “cationic polymer” means a polymer having a cationic group.
In the disclosure, the “cationic group” includes a cationic group and a group that is ionized to become a cationic group.
Examples of the cationic group include a primary amino group, a secondary amino group, a tertiary amino group, a quaternary ammonium base, and an imino group. Among them, from the viewpoint of shortening the separation time of albumin and γ-globulin, a primary amino group or a secondary amino group is preferable, and a secondary amino group is more preferable.
The cationic polymer is preferably water-soluble. In the disclosure, “water-soluble” means that a target substance is dissolved in an amount of 1 mass % or more in water at 25° C.
Examples of the cationic polymer having primary to tertiary amino groups or a cationic group that can be ionized into primary to tertiary amino groups include polyallylamine, polyvinylamine, polylysine, polyarginine, polyhistidine, polyornithine, polydiallylamine, polymethyldiallylamine, polyethyleneimine, diallylamine-acrylamide polymer, dimethylamine-ammonia-epichlorohydrin polymer, and allylamine-diallylamine polymer.
Examples of the cationic polymer having an imino group or a cationic group that can be ionized into an imino group include polyethyleneimine.
Examples of the cationic polymer having a quaternary ammonium base or a cationic group that can be ionized into a quaternary ammonium base include polyquaternium, dimethylamine-ammonia-epichlorohydrin polymer, dimethylamine-epichlorohydrin polymer, and diallyldimethylammonium chloride polymer.
In the disclosure, “polyquaternium” refers to a cationic polymer including a constituent unit derived from a monomer having a quaternary ammonium group. Polyquaternium can be confirmed by INCI (International Nomenclature for Cosmetic Ingredients) directory. In one or more embodiments, examples of the polyquaternium include polydiallyl dimethyl ammonium salts such as polyquaternium-6 (poly(diallyldimethylammoniumchloride), polyquaternium-7 (copolymer of acrylamide and diallyl dimethyl ammonium chloride), polyquaternium-4 (diallyl dimethyl ammonium chloride-hydroxyethyl cellulose copolymer), and polyquaternium-22 (copolymer of acrylic acid and diallyl dimethyl ammonium chloride), and polyquaternium-2 (poly [bis(2-chloroethyl)ether-alt-1,3-bis[3-(dimethylamino)propyl]urea]).
As the cationic polymer, in addition to the ammonium salt, in one or more embodiments, a cationic polymer of an onium salt such as a phosphonium salt, an oxonium salt, a sulfonium salt, a fluoronium salt, or a chloronium salt can also be used.
Examples of the cationic polymer having a hydrazide group or a cationic group that can be ionized into a hydrazide group include aminopolyacrylamide.
From the viewpoint of shortening the separation time of albumin and γ-globulin, the cationic polymer preferably includes 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 includes one or more selected from the group consisting of dimethylamine-ammonia-epichlorohydrin polymer, dimethylamine-epichlorohydrin polymer, diallylamine-acrylamide polymer, polyethyleneimine, and polyallylamine, and further preferably includes one or more selected from the group consisting of dimethylamine-ammonia-epichlorohydrin polymer and diallylamine-acrylamide polymer.
The cationic polymer described above may be in the form of a salt, and examples thereof include hydrochloride.
From the viewpoint of shortening the separation time of albumin and γ-globulin, the weight average molecular weight of the cationic polymer is preferably more than 1000, more preferably from 10,000 to 500,000, further preferably from 12,000 to 300,000, particularly preferably from 15,000 to 150,000, and most preferably from 20,000 to 130,000, and may be from 20,000 to 100,000.
The reason why the separation time of albumin and γ-globulin is shortened by setting the weight average molecular weight of the cationic polymer within the above numerical range is not clear, but it is presumed as follows.
The isoelectric points of albumin and γ-globulin (IgG as a representative component) are around pH 5 and around pH 5 to 9, respectively.
Since albumin has a lower isoelectric point than γ-globulin, the negative charge of albumin is larger than that of γ-globulin in the specific alkaline solution, so that the interaction with the cationic polymer is strong.
Therefore, it is presumed that by setting the weight average molecular weight of the cationic polymer to 500,000 or less, it is possible to suppress an excessive increase in difference between the force for pulling back albumin to the negative electrode side and the force for pulling back γ-globulin to the negative electrode side, which are generated by the interaction with the cationic polymer, and it is possible to further shorten the separation time of albumin and γ-globulin.
It is presumed that by setting the weight average molecular weight of the cationic polymer to 10,000 or more, it is possible to suppress an excessive decrease in difference between the force for pulling back albumin to the negative electrode side and the force for pulling back γ-globulin to the negative electrode side, which are generated by the interaction with the cationic polymer, and the separation accuracy can be improved.
In the disclosure, the weight average molecular weight of the cationic polymer refers to the catalogue value. When there is no catalog value of the weight average molecular weight, the weight average molecular weight is a weight average molecular weight in terms of polyethylene glycol measured by a gel permeation chromatography (GPC) method.
From the viewpoint of shortening the separation time of albumin and γ-globulin, the content ratio of the cationic polymer with respect to the total mass of the specific alkaline solution is preferably from 0.01 mass % to 10 mass %, more preferably from 0.05 mass % to 8.0 mass %, further preferably from 0.1 mass % to 5.0 mass %, and still more preferably from 1.0 mass % to 3.0 mass %.
As the cationic polymer, one synthesized by a conventionally known method may be used, or a commercially available one may be used.
From the viewpoint of improving the separation accuracy of proteins, the specific alkaline solution preferably contains
a cationic low-molecular compound having two or more primary amino groups (hereinafter, also referred to as “specific low-molecular compound”).
In the disclosure, the “specific low-molecular compound” means a compound having a molecular weight of 1000 or less. When there is a distribution in molecular weight, the molecular weight represents a mass average molecular weight (Mw).
From the viewpoint of improving the separation accuracy of proteins, the molecular weight of the specific low-molecular compound is preferably from 30 to 1000, more preferably from 40 to 800, further preferably from 50 to 400, and still more preferably from 100 to 200.
From the viewpoint of improving the separation accuracy of proteins, the number of primary amino groups of the specific low-molecular compound is preferably from 2 to 5, more preferably from 2 to 4, and further preferably 2 or 3.
From the viewpoint of improving the separation accuracy of proteins, the specific low-molecular 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), 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 further preferably at least one of 3,3′-diamino-N-methyldipropylamine and 3,3′-diaminodipropylene.
From the viewpoint of improving the separation accuracy of proteins, the content ratio of the specific low-molecular compound with respect to the total mass of the specific alkaline solution is preferably from 0.01 mass % to 10 mass %, more preferably from 0.05 mass % to 5 mass %, further preferably from 0.1 mass % to 3 mass %, and still more preferably from 0.3 mass % to 1.0 mass %.
In the specific alkaline solution, the ratio of the content ratio of the specific low-molecular compound to the content ratio of the cationic polymer (the content ratio of the specific low-molecular compound/the content ratio of the cationic polymer) is preferably from 0.001 to 1000, more preferably from 0.01 to 100, further preferably from 0.05 to 10, and still more preferably from 0.1 to 5.
As the specific low-molecular compound, one synthesized by a conventionally known method may be used, or a commercially available one may be used.
The specific alkaline solution may contain water. Examples of water include distilled water, ion-exchanged water, pure water, and ultrapure water.
The content ratio of water with respect to the total mass of the specific alkaline solution is not particularly limited, and can be from 10 mass % to 99.9 mass %.
The specific alkaline solution may contain additives such as a pH buffering substance and a preservative for suppressing proliferation of microorganisms, and the like. Examples of the preservative include sodium azide, ethylparaben, and Proclin.
The sample contains albumin, γ-globulin, and a non-surfactant-type sulfobetaine.
Examples of γ-globulin include IgG, IgM, IgA, IgD, and IgE. The sample may contain at least one selected from the group consisting of IgG, IgM, IgA, IgD, and IgE, and may contain at least two selected from the group consisting of IgG, IgM, IgA, IgD, and IgE.
The total content of albumin and γ-globulin with respect to the total mass of the sample is not particularly limited, and is preferably from 0.001 mass % to 99 mass %, and more preferably from 0.001 mass % to 95 mass %.
The form of the sample is not particularly limited, and may be those prepared from a sample raw material or may be a sample raw material itself.
Examples of the sample raw material include a raw material containing albumin and γ-globulin, and a biological sample.
Examples of the biological sample include blood, serum, plasma, and a biological sample prepared from blood, serum, or plasma. The biological sample is preferably serum or plasma, and is more preferably serum.
Examples of blood, serum, and plasma include blood, serum, and plasma that are collected from a living body, and more specifically include blood, serum, and plasma of mammals other than human, and blood, serum, and plasma of human.
The sample contains a non-surfactant-type sulfobetaine.
In the disclosure, the “non-surfactant-type sulfobetaine” means sulfobetaine that does not form micelles. In the disclosure, “not forming micelles” means not forming micelles in an aqueous medium or not substantially forming micelles. In the disclosure, “not substantially forming micelles” means that the critical micelle concentration is preferably 200 mmol/L or more and more preferably 300 mmol/L or more, and further preferably, a zwitterionic substance has not critical micelle concentration.
In the disclosure, “betaine” means a compound having a positively charged group and a negatively charged group at positions not adjacent to each other in the same molecule, having no hydrogen atom capable of being dissociated bonded to an atom having a positive charge, and having no charge as a whole molecule.
Examples of the positively charged group of the non-surfactant-type sulfobetaine include a quaternary ammonium cation group, a sulfonium group, and a phosphonium group. Among them, from the viewpoint of shortening the separation time of albumin and γ-globulin and improving the separation accuracy, a quaternary ammonium cation group is preferable.
The number of carbon atoms present between the positively charged group and the negatively charged group is preferably from 1 to 10, more preferably from 2 to 8, and further preferably from 2 to 5.
From the viewpoint of shortening the separation time of albumin and γ-globulin and improving the separation accuracy, the molecular weight of the non-surfactant-type sulfobetaine is preferably from 100 to 500, more preferably from 120 to 400, and further preferably from 170 to 300.
Examples of the non-surfactant-type sulfobetaine include 3-(1-pyridinio)propanesulfonate (the first one from top in left in the chemical formulae as set forth below), dimethylethylammonium propane sulfonate (the second one from top in left in the chemical formulae as set forth below), 3-[(2-hydroxyethyl)dimethylammonio]propane-1-sulfonate (the third one from top in left in the chemical formulae as set forth below), 3-(4-tert-butyl-1-pyridinio)propanesulfonate (the fourth one from top in left in the chemical formulae as set forth below), N-methyl-N-(3-sulfopropyl)morpholinium (the fifth one from top in left in the chemical formulae as set forth below), dimethylbenzylammonium propane sulfonate (the first one from top in right in the chemical formulae as set forth below), and 3-(1-methylpiperidinio)-1-propanesulfonate (the second one from top in right in the chemical formulae as set forth below). The non-surfactant-type sulfobetaine is not limited to these compounds.
Examples of commercially available products of the compounds 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) manufactured by Merck Corporation.
Among the compounds exemplified above, from the viewpoint of shortening the separation time of albumin and γ-globulin and improving the separation accuracy, the following compounds are preferable.
As the non-surfactant-type sulfobetaine, one synthesized by a conventionally known method may be used, or a commercially available one may be used.
From the viewpoint of shortening the separation time of albumin and γ-globulin and improving the separation accuracy, the content ratio of the non-surfactant-type sulfobetaine with respect to the total mass of the sample is preferably from 0.1 mass % to 30 mass %, more preferably from 0.5 mass % to 20 mass %, further preferably from 1 mass % to 15 mass %, and still more preferably from 1 mass % to 9 mass %.
From the viewpoint of improving the separation accuracy, the sample preferably contains an alkaline solution containing a cationic polymer.
The sample containing the alkaline solution can be obtained by diluting the sample raw material using the alkaline solution. The dilution rate is preferably from 1.2 times to 100 times, more preferably from 2 times to 60 times, and further preferably from 3 times to 50 times on a volume basis. The material used for dilution is not particularly limited, and examples thereof include a pH adjusting agent (for example, hydrochloric acid or the like), a surfactant (for example, EMULGEN LS-110 (manufactured by Kao Corporation) or the like), an antiseptic (for example, sodium azide or the like), an ionic strength adjusting agent (for example, sodium chloride or the like), and a refractive index adjusting agent (for example, saccharides such as sucrose).
The alkaline solution may be the same as or different from the specific alkaline solution filled in the capillary flow path.
<Detection step>
The specific sample analysis method can include a detection step of detecting albumin and γ-globulin separated in the separation step.
Albumin and γ-globulin can be detected by an optical method. Examples of the detection by an optical method include measurement of absorbance.
More specifically, albumin and γ-globulin can be detected by irradiating the separated albumin and γ-globulin with light having a wavelength of 280 nm to obtain an absorbance spectrum in which the vertical axis represents absorbance and the horizontal axis represents time.
When a capillary electrophoresis chip is used for separating albumin and γ-globulin, it is preferable to irradiate the detection unit with light having a wavelength of 280 nm.
Albumin and γ-globulin may be detected using an electropherogram (differential waveform) obtained by differentiating the waveform of the absorbance spectrum with respect to time.
The fact that the specific sample analysis method is excellent in separation accuracy as compared with the conventional method can be confirmed by the value of the maximum absorbance variation at the peak of albumin, whether the peak in the electropherogram is broad, and the like.
The fact that the separation time is shortened in the specific sample analysis method as compared with the conventional method can be confirmed by the peak detection time of albumin.
An embodiment of the specific sample analysis method will be described with reference to
First, the specific alkaline solution containing a cationic polymer is filled as an electrophoretic liquid in the electrophoretic liquid holding tank 3 of the capillary electrophoresis chip, and the specific alkaline solution is filled in the capillary flow path 1 by capillary action.
Next, the sample is added to the sample holding tank 2 of the capillary electrophoresis chip in which the specific alkaline solution is filled.
The sample to be added to the sample holding tank 2 can be prepared by diluting serum, which is a sample raw material, with the alkaline solution.
A negative electrode is brought into contact with the sample holding tank 2 and a positive electrode is brought into contact with the electrophoretic liquid holding tank 3 (not illustrated), and a voltage is applied to both ends of the capillary flow path 1, that is, between the sample holding tank 2 and the electrophoretic liquid holding tank 3. As a result, the sample is introduced from the sample holding tank 2 into the capillary flow path 1, the sample containing albumin and γ-globulin moves from the sample holding tank 2 toward the electrophoretic liquid holding tank 3, and albumin and γ-globulin are separated.
In the detection unit 4, albumin and γ-globulin are detected by irradiation with light having a wavelength of 280 nm and measurement of absorbance by an absorbance measuring device.
The sample analysis method of the disclosure can be used for diagnosis, treatment, and the like of multiple myeloma, nephrotic syndrome, liver cirrhosis, nutritional disorder, and the like.
A sample production method according to an embodiment of the disclosure (hereinafter, also referred to as “specific sample production method”) includes a diluting step of diluting a mixture containing albumin and γ-globulin with a solution containing a non-surfactant-type sulfobetaine (hereinafter, also referred to as “specific solution”).
The non-surfactant-type sulfobetaine has been described above, and thus the description thereof is omitted here.
From the viewpoint of shortening the separation time of albumin and γ-globulin and improving the separation accuracy, the content ratio of the non-surfactant-type sulfobetaine with respect to the total mass of the specific solution is preferably from 0.1 mass % to 35 mass %, more preferably from 0.5 mass % to 25 mass %, further preferably from 1.0 mass % to 20 mass %, and still more preferably from 1.0 mass % to 10 mass %.
The specific solution may contain a cationic polymer. The cationic polymer has been described above, and thus the description thereof is omitted here.
From the viewpoint of shortening the separation time of albumin and γ-globulin, the content ratio of the cationic polymer with respect to the total mass of the specific solution is preferably from 0.01 mass % to 10 mass %, more preferably from 0.05 mass % to 8.0 mass %, further preferably from 0.1 mass % to 5.0 mass %, and still more preferably from 1.0 mass % to 3.0 mass %.
The specific solution may contain additives such as a pH buffering substance, a surfactant, a pH adjusting agent, an ionic strength adjusting agent, an antiseptic, a preservative for suppressing proliferation of microorganisms, and the like. Examples of the preservative include sodium azide, ethylparaben, and Proclin.
The pH of the specific solution is preferably higher than the isoelectric points of albumin and γ-globulin, and is preferably from 8.0 to 12.0, more preferably from 8.2 to 11.0, and further preferably from 8.5 to 9.5.
The dilution factor of the sample containing albumin and γ-globulin is preferably from 1.2 times to 100 times, more preferably from 2 times to 60 times, and further preferably from 3 times to 50 times on a volume basis.
A diluent according to an embodiment of the disclosure (hereinafter, also referred to as “specific diluent”) contains a non-surfactant-type sulfobetaine, is used for separation of albumin and γ-globulin by capillary electrophoresis, and is used for diluting a sample containing albumin and γ-globulin.
A preferred embodiment of the specific diluent is the same as the sample used in the specific sample analysis method, and thus the description thereof is omitted here.
A sample analysis kit according to an embodiment of the disclosure includes a container containing the above-described diluent and an electrophoresis chip including a sample holding tank, an electrophoretic liquid holding tank, and a capillary flow path, in which the sample holding tank and the electrophoretic liquid holding tank are communicated with each other via the capillary flow path.
The sample analysis kit according to the embodiment of the disclosure can further include a container containing a capillary electrophoresis solution containing a cationic polymer.
A preferred embodiment of the electrophoresis chip is the same as that of the electrophoresis chip used in the specific sample analysis method, and thus the description thereof is omitted here.
A preferred embodiment of the diluent is the same as that of the diluent according to the embodiment of the disclosure, and thus the description thereof is omitted here.
Examples of the material for the container containing the capillary electrophoresis solution include glass, fused silica, and plastic. The plastic has been described above, and thus the description thereof is omitted here.
The disclosure may relate to the following embodiments.
Examples will be described below, but the disclosure is not limited to these Examples at all. In the following description, unless otherwise specified, “part(s)” and “%” are all on a mass basis.
In the following Examples and Comparative Examples, electrophoresis was performed by a continuous sample introduction method.
As a separation device, a resin chip having the capillary flow path 1 with the structure illustrated in
As a measuring device, an electrophoresis device self-manufactured was used.
The following substances were mixed, and sodium hydroxide and water were added until the pH reached 9.8 to prepare a specific alkaline solution (capillary electrophoresis solution) 1. The content ratio of the cationic polymer with respect to the total mass of the specific alkaline solution 1 was set to 1.5 mass %.
Dimethylamine-ammonia-epichlorohydrin polymer (cationic polymer, KHE102L manufactured by SENKA corporation, weight average molecular weight: from 20,000 to 100,000) 1.5 mass %
A human biological serum sample containing albumin and γ-globulin was prepared, and diluted (dilution rate 7 times (volume basis)) with a specific diluent 1 (pH 8.8) having the following composition to obtain Sample A.
In the specific diluent 1, hydrochloric acid and water were added until the pH of the solution reached 8.8.
The content ratio of the non-surfactant-type sulfobetaine with respect to the total mass of the specific diluent 1 was set to 4.02 mass %.
To the electrophoretic liquid holding tank 3, 60 μL of the specific alkaline solution 1 was added, and the specific alkaline solution 1 was filled in the capillary flow path 1 by capillary action.
To the sample holding tank 2, 60 μL of Sample A was added.
Next, a negative electrode was brought into contact with the sample holding tank 2, a positive electrode was brought into contact with the electrophoretic liquid holding tank 3, and electrophoresis was started by applying a voltage under constant current control of 75 μA.
During electrophoresis, the detection unit 4 was irradiated with light of 280 nm, and the absorbance was measured to obtain an absorbance spectrum. An electropherogram was obtained by differentiating the waveform of the absorbance spectrum with respect to time. Electrophoresis was performed for 200 seconds. The obtained electropherogram is shown in
A prototype self-manufactured was used for light irradiation, absorbance measurement, and electropherogram acquisition.
In Table 1, the maximum absorbance variation and the peak detection time for each of albumin and γ-globulin were summarized.
A human biological serum sample containing albumin and γ-globulin was prepared, and diluted (dilution rate 7 times (volume basis)) with a specific diluent 2 (pH 8.8) having the following composition to obtain Sample B.
In the specific diluent 2, hydrochloric acid and water were added until the pH of the solution reached 8.8.
The content ratio of the non-surfactant-type sulfobetaine with respect to the total mass of the specific diluent 2 was set to 8.05 mass %.
An electropherogram using Sample B was obtained in the same manner as in Example 1, except that Sample A was changed to Sample B. The obtained electropherogram is shown in
In Table 1, the maximum absorbance variation and the peak detection time for each of albumin and γ-globulin were summarized.
A human biological serum sample containing albumin and γ-globulin was prepared, and diluted (dilution rate 7 times (volume basis)) with a diluent a (pH 8.8) having the following composition to obtain Sample a.
In the diluent a, hydrochloric acid and water were added until the pH of the solution reached 8.8.
An electropherogram using Sample a was obtained in the same manner as in Example 1, except that Sample A was changed to Sample a. The obtained electropherogram is shown in
In Table 1, the maximum absorbance variation and the peak detection time for each of albumin and γ-globulin were summarized.
From
A human biological serum sample containing albumin and γ-globulin was prepared, and diluted (dilution rate 7 times (volume basis)) with a specific diluent 3 (pH 8.8) having the following composition to obtain Sample C.
In the specific diluent 3, hydrochloric acid and water were added until the pH of the solution reached 8.8.
The content ratio of the non-surfactant-type sulfobetaine with respect to the total mass of the specific diluent 3 was set to 3.91 mass %.
An electropherogram using Sample C was obtained in the same manner as in Example 1, except that Sample A was changed to Sample C.
The obtained electropherogram is shown in
In Table 2, the maximum absorbance variation and the peak detection time for each of albumin and γ-globulin were summarized.
A human biological serum sample containing albumin and γ-globulin was prepared, and diluted (dilution rate 7 times (mass basis)) with a specific diluent 4 (pH 8.8) having the following composition to obtain Sample D.
In the specific diluent 4, hydrochloric acid and water were added until the pH of the solution reached 8.8.
The content ratio of the non-surfactant-type sulfobetaine with respect to the total mass of the specific diluent 4 was set to 7.81 mass %.
An electropherogram using Sample D was obtained in the same manner as in Example 1, except that Sample A was changed to Sample D. The obtained electropherogram is shown in
In Table 2, the maximum absorbance variation and the peak detection time for each of albumin and γ-globulin were summarized.
A human biological serum sample containing albumin and γ-globulin was prepared, and diluted (dilution rate 7 times (volume basis)) with a diluent b (pH 8.8) having the following composition to obtain Sample b.
In the diluent b, hydrochloric acid and water were added until the pH of the solution reached 8.8.
An electropherogram using Sample b was obtained in the same manner as in Example 1, except that Sample A was changed to Sample b. The obtained electropherogram is shown in
In Table 2, the maximum absorbance variation and the peak detection time for each of albumin and γ-globulin were summarized. Since the donor of the human serum was different, the result was slightly different from that of Comparative Example 1.
From
A human biological serum sample containing albumin and γ-globulin was prepared, and diluted (dilution rate 7 times (mass basis)) with a diluent c (pH 8.8) having the following composition to obtain Sample c.
In the diluent c, hydrochloric acid and water were added until the pH of the solution reached 8.8.
An electropherogram using Sample c was obtained in the same manner as in Example 1, except that Sample A was changed to Sample c. The obtained electropherogram is shown in
In Table 3, the maximum absorbance variation and the peak detection time for each of albumin and γ-globulin were summarized.
A human biological serum sample containing albumin and γ-globulin was prepared, and diluted (dilution rate 7 times (volume basis)) with a diluent d (pH 8.8) having the following composition to obtain Sample d.
In the diluent d, hydrochloric acid and water were added until the pH of the solution reached 8.8.
An electropherogram using Sample d was obtained in the same manner as in Example 1, except that Sample A was changed to Sample d. The obtained electropherogram is shown in
In Table 3, the maximum absorbance variation and the peak detection time for each of albumin and γ-globulin were summarized.
A human biological serum sample containing albumin and γ-globulin was prepared, and diluted (dilution rate 7 times (volume basis)) with a diluent e (pH 8.8) having the following composition to obtain Sample e.
In the diluent e, hydrochloric acid and water were added until the pH of the solution reached 8.8.
An electropherogram using Sample e was obtained in the same manner as in Example 1, except that Sample A was changed to Sample e. The obtained electropherogram is shown in
In Table 3, the maximum absorbance variation and the peak detection time for each of albumin and γ-globulin were summarized.
A human biological serum sample containing albumin and γ-globulin was prepared, and diluted (dilution rate 7 times (volume basis)) with a diluent f (pH 8.8) having the following composition to obtain Sample f.
In the diluent f, hydrochloric acid and water were added until the pH of the solution reached 8.8.
An electropherogram using Sample f was obtained in the same manner as in Example 1, except that Sample A was changed to Sample f. The obtained electropherogram is shown in
In Table 3, the maximum absorbance variation and the peak detection time for each of albumin and γ-globulin were summarized.
From
As shown in
From
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-173449 | Oct 2022 | JP | national |
