JP2016031250A - Target protein measurement reagent, and measurement method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a reagent that measures a target protein without the centrifugation of whole blood by a latex agglutination method.SOLUTION: The present invention provides protein measurement reagents and others, comprising insoluble carrier particles with an average particle diameter of 0.02 μm-2.0 μm, in which 1.0 μg-110 μg of hydrophilic polymers cover the particles per 1 mg.SELECTED DRAWING: None

Description

  The present invention relates to a measurement reagent for a target protein in a whole blood sample and a method for measuring the target protein in a whole blood sample using the reagent.

  The value of glycated hemoglobin (glycohemoglobin) in which sugar is bound to hemoglobin in erythrocytes, particularly hemoglobin A1c in which the N-terminal valine residue of the hemoglobin β chain is glycated (hereinafter sometimes referred to as “HbA1c”) is 1 in the past. Since it reflects the average blood glucose level of ˜2 months, it is widely used as an index suitable for diabetes diagnosis and diabetes follow-up.

  Known methods for measuring HbA1c include HPLC, capillary electrophoresis, enzymatic method, and immunological measurement. In immunoassay, HbA1c is measured using an antibody specific for the glycated portion of HbA1c (hemoglobin β-chain N-terminus), but the glycated portion is not present on the surface of the hemoglobin protein. It has been found that it is buried in the hemoglobin protein and exists in a place where the antibody is difficult to bind under physiological conditions. Due to such properties, in order to perform measurement by efficiently reacting an antibody that recognizes the glycation site as an epitope, a technique for exposing the epitope to the surface of a hemoglobin protein has been developed. As a typical means for exposing the epitope to the surface of hemoglobin protein, a method of treating hemoglobin by adsorbing hemoglobin to insoluble carrier particles such as latex particles has been reported (Patent Document 1). Since the particles adsorbed with hemoglobin protein aggregate in the reaction solution by using an antibody against the exposed epitope, the hemoglobin protein is quantified by optically detecting the turbidity of the aggregated particles as scattered light or transmitted light. (Latex aggregation method).

  However, when hemoglobin protein is quantified by latex agglutination, non-specific aggregation occurs in the particles due to the presence of plasma protein contained in whole blood when whole blood is hemolyzed and hemoglobin is extracted from erythrocytes and used as a specimen sample. There is a problem that optical detection based on turbidity cannot be performed. As a result, an extra step of centrifuging whole blood and removing plasma components prior to hemolysis was necessary. In this regard, bovine serum albumin (BSA) has been known to have a property that proteins and cells do not adhere to the surface, so the surface of the particles is inhibited for the purpose of inhibiting nonspecific aggregation of particles by proteins other than hemoglobin protein. Attempts to coat with BSA are being considered. However, coating the particle surface with BSA also affects the exposure of the epitope of the hemoglobin protein itself, so that the problem that whole blood cannot be used as a sample in the latex agglutination method remains unsolved.

Japanese Patent No. 2,677,753

  An object of the present invention is to provide a reagent for detecting a target protein using whole blood as a sample in the latex agglutination method, and a method for measuring the target protein using the reagent.

  The present inventors have found that when latex particles are coated with a specific range of BSA, the latex particles adsorb HbA1c, but the adsorbed HbA1c is not affected by BSA for epitope exposure. Furthermore, it was found that even when a whole blood sample was used as a sample when HbA1c was adsorbed to latex particles, nonspecific aggregation was not caused when an agglutination reaction was caused in the latex particles. As a result of further investigation based on the above findings, the present inventors have coated insoluble carrier particles with a specific range of amount of hydrophilic polymer, and used the whole blood sample as it is. It came to complete the method of measuring the target protein contained, and the reagent which can be used for this method.

That is, the present invention
[1] A reagent for measuring a target protein, comprising insoluble carrier particles having an average particle size of 0.02 μm to 2.0 μm and coated with 1.0 μg to 110 μg of hydrophilic polymer per 1 mg of the particles;
[2] The reagent according to [1], comprising insoluble carrier particles having an average particle diameter of 0.05 μm to 0.3 μm, wherein the insoluble carrier particles are coated with 2 μg to 70 μg of hydrophilic polymer per 1 mg of the particles. ;
[3] Polymer or copolymer particles of a molecule in which the insoluble carrier particles are at least one selected from the group consisting of styrene, vinyl chloride, acrylic esters, methacrylic esters and vinyl acetate, or styrene units and butadiene units The reagent according to [1] or [2], which is a copolymer particle having:
[4] The reagent according to [3], wherein the insoluble carrier particles are polystyrene particles;
[5] The reagent according to any one of [1] to [4], wherein the hydrophilic polymer is selected from the group consisting of dextran, albumin, casein, cellulose, and albumin;
[6] The reagent according to [5], wherein the hydrophilic polymer is bovine serum albumin;
[7] The reagent according to any one of [1] to [6], which contains an immune complex specific for the target protein;
[8] The reagent according to any one of [1] to [7], wherein the target protein is glycated hemoglobin;
[9] A method for immunologically measuring a target protein comprising the following steps:
(1) A step of coating insoluble carrier particles having an average particle diameter of 0.02 μm to 2.0 μm with 1.0 μg to 110 μg of hydrophilic polymer per mg,
(2) contacting the whole blood sample with the insoluble carrier particles;
(3) contacting the insoluble carrier particles with an immune complex specific for a target protein;
(4) a step of measuring the turbidity of the produced aggregates;
[10] The method according to [9], wherein the step (1) is a step of coating insoluble carrier particles having an average particle diameter of 0.05 μm to 0.3 μm with 2 μg to 70 μg of highly hydrophilic molecules per mg;
[11] Polymer or copolymer particles of molecules in which the insoluble carrier particles are at least one selected from the group consisting of styrene, vinyl chloride, acrylic esters, methacrylic esters and vinyl acetate, or styrene units and butadiene units The method according to [9] or [10], which is a copolymer particle having:
[12] The method according to [11], wherein the insoluble carrier particles are polystyrene particles;
[13] The method according to any one of [9] to [12], wherein the hydrophilic polymer is selected from the group consisting of dextran, albumin, casein, and cellulose;
[14] The method according to [13], wherein the hydrophilic polymer is bovine serum albumin;
[15] The method according to any one of [9] to [14], wherein the target protein is glycated hemoglobin;
Etc.

  According to the present invention, HbA1c can be quantified directly using whole blood as a sample without going through the steps of previously centrifuging whole blood and removing plasma components. Furthermore, according to the present invention, it is not necessary to remove the plasma component, and at the same time, the protein in the plasma component can be selected as the target protein.

It is a figure which shows the relationship between hemoglobin concentration and a light absorbency (660 nm).

  The present invention relates to a target protein measurement reagent comprising insoluble carrier particles having an average particle diameter of 0.02 μm to 2.0 μm and coated with 1.0 μg to 110 μg of hydrophilic polymer per 1 mg of the particles. (Hereinafter, the reagent of the present invention) is provided.

In the present specification, the insoluble carrier particles are not particularly limited as long as they are generally used as an immunological measurement reagent, and may be appropriately colored with a dye.
The insoluble carrier can be obtained by polymerizing or copolymerizing various monomers. Examples of monomers to be polymerized or copolymerized here include unsaturated aromatics such as styrene, chlorostyrene, α-methylstyrene, divinylbenzene, and vinyltoluene, such as acrylic acid, methacrylic acid, itaconic acid, maleic acid, and fumaric acid. Unsaturated carboxylic acids such as methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, acrylic acid-n-butyl, methacrylic acid-n-butyl and the like, acrylic acid esters or methacrylic acid esters, acrylonitrile , Acrylonitriles or methacrylonitriles such as methacrylonitrile, acrylic aldehydes or methacrylates such as acrolein and methacrolein, acrylamide, methacrylamide, N-methylol-acrylamide, N Acrylic amides or methacrylamides such as methylol-methacrylamide, methylenebisacrylamide, methylenebismethacrylamide, conjugated dienes such as butadiene and isoprene, vinyl acetate, vinylpyridine, N-vinylpyrrolidone, vinyl chloride, vinylidene chloride And vinyl monomers such as vinyl bromide, preferably styrene, vinyl chloride, vinyl acetate, acrylic acid esters or methacrylic acid esters, and more preferably polystyrene. The monomer to be copolymerized includes a combination of two or more selected from styrene, butadiene, isobutylene, isoprene and the like, and preferably a combination of styrene and butadiene. These monomers are appropriately selected depending on surface characteristics, specific gravity and the like, and can be used alone or in combination of two or more.

  The shape of the insoluble carrier is in the form of particles, and the average particle size is desirably large enough to allow the aggregates resulting from the aggregation reaction between the protein on the particle surface and the substance to be measured to be detected visually or optically. Such an average particle diameter is usually 0.02 μm to 2.0 μm, preferably 0.05 μm to 0.3 μm.

  In the present specification, the insoluble carrier particles may be latex. Here, latex refers to a suspension in which insoluble carrier particles are suspended in a buffer solution. The buffer solution in which the insoluble carrier particles are suspended is preferably a phosphate buffer solution, a glycine buffer solution, a borate buffer solution, an imidazole buffer solution or a Tris buffer solution from the viewpoint of stability. The concentration of the insoluble carrier particles in the latex is usually 0.01 to 1.5 weight percent, preferably 0.05 to 0.5 weight percent.

As already mentioned, when using the unsensitized latex agglutination method for whole blood samples, because non-specific aggregation occurs in the particles due to the presence of plasma proteins contained in the whole blood, the whole blood is centrifuged in advance, An extra step of removing plasma components was necessary. BSA has been reported not to adsorb proteins and cells, and can suppress nonspecific aggregation of particles by plasma proteins. However, if the surface of the particles is covered with an excessive amount of BSA, the epitope of the target protein is exposed. In addition, the target protein cannot be measured because the adsorption of the target protein to the particle surface is inhibited. Therefore, the idea of coating the particles with BSA could not be considered by those skilled in the art.
However, in order to solve the above problems, the present inventors can measure HbA1c using a whole blood sample without centrifugation when the latex particles are coated with a specific range of amounts of BSA. I found. According to the present invention, HbA1c can be quantified without going through the steps of previously centrifuging whole blood and removing plasma components. Furthermore, since it is not necessary to remove the plasma component, at the same time, the protein in the plasma component can be selected as the target protein.
In order to solve the above problems, insoluble carrier particles having an average particle diameter of 0.02 μm to 2.0 μm of the present invention are coated with 1.0 μg to 110 μg of hydrophilic polymer per 1 mg of the particles.

In the present specification, the hydrophilic polymer is a natural polymer or a non-natural polymer, and may be any polymer as long as it has hydrophilicity. Examples of such hydrophilic polymers include dextran, albumin (egg albumin, serum albumin, lactalbumin), casein, cellulose and the like, preferably serum albumin, particularly preferably bovine serum albumin.
The insoluble carrier particles are coated with 1.0 to 110 μg of the hydrophilic polymer per 1 mg of the particles having an average particle diameter of 0.02 to 2.0 μm. Preferably, when the average particle size of the insoluble carrier particles of the present invention is 0.05 μm to 0.3 μm, 2 mg to 70 μg of the hydrophilic polymer is coated per 1 mg of the particles.
As a method for coating the insoluble carrier particles with the hydrophilic polymer, for example, it can be carried out according to the method described in the following Examples. Specifically, the insoluble carrier particles having the above average particle diameter and the hydrophilic polymer are mixed in the buffer solution, and stirred at 25 to 30 ° C. for 30 minutes to 4 hours. Centrifuge for 15 minutes to 1 hour at 4 ° C, 15,000-20,000 rpm, remove the supernatant, add buffer again, re-disperse insoluble carrier particles, and coat with hydrophilic polymer Insoluble carrier particles can be prepared.

  The reagent of the present invention may further contain an immune complex specific for the target protein. In this specification, the target protein may be any protein as long as it is a protein contained in whole blood (blood cell component, plasma component). For example, glycated albumin, glycated hemoglobin, fructosamine and other hemoglobin proteins, Examples thereof include sugar chain binding proteins such as tumor markers.

  Examples of the glycated hemoglobin include hemoglobin A1c. Hemoglobin A1c is obtained by non-enzymatically binding glucose to the α-amino group of valine, which is the amino acid residue at the N-terminal of the β chain of hemoglobin, to form glycosylated hemoglobin. The blood level of hemoglobin A1c reflects the relatively long-term glycemic control status of diabetes. Therefore, measuring this hemoglobin A1c is very significant clinically for knowing the glycemic control state.

  As a tumor marker, a tumor-specific marker in which a sugar chain moiety is present inside a protein is assumed. The blood level of this tumor marker reflects the presence and progression of the tumor. Therefore, measuring this tumor marker is of great clinical significance in determining early tumor detection and treatment strategy.

  An immune complex that specifically recognizes a target protein comprises a primary antibody that specifically recognizes the target protein and a secondary antibody that recognizes the primary antibody. Other than the immune complex, a conjugate of a biotinylated primary antibody and avidin can also be used.

A primary antibody that specifically recognizes a target protein can be produced by an existing general production method using the target protein or a partial peptide having antigenicity as an immunogen. In the present specification, the primary antibody includes a polyclonal antibody, a natural antibody such as a monoclonal antibody (mAb), a chimeric antibody that can be produced using a gene recombination technique, a humanized antibody, a single chain antibody, and a binding thereof. Sex fragments are included, but not limited to. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody or a binding fragment thereof. The binding fragment means a partial region of the aforementioned antibody having specific binding activity, and specifically includes, for example, F (ab ′) ₂ , Fab ′, Fab, Fv, sFv, dsFv, sdAb and the like. (Exp. Opin. Ther. Patents, Vol.6, No.5, p.441-456, 1996). The class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of ease of purification. In the present invention, it is also preferable to use a commercially available antibody as an antibody that specifically recognizes the target protein.

  The secondary antibody that recognizes the primary antibody that specifically recognizes the target protein may be an antibody that specifically recognizes the primary antibody, and a partial peptide (for example, Fc region) having the antigenicity of the primary antibody may be used. It can be used as an immunogen and can be produced by existing general production methods. In the present specification, the secondary antibody may be a polyclonal antibody, a monoclonal antibody (mAb), a binding fragment, or the like, like the primary antibody, and the class of the antibody is not particularly limited. In the present invention, it is also preferable to use a commercially available antibody as the secondary antibody.

  The immune complex that specifically recognizes the target protein contained in the reagent of the present invention may be stored in a state where the primary antibody and the secondary antibody are complexed in advance, or may be stored in a state where they exist individually. Or just before use as an immune complex.

  The reagent of the present invention further contains, in addition to the immune complex, other substances necessary for the reaction for measuring the target protein, which do not adversely affect the reaction when stored in the coexisting state. be able to. Alternatively, the reagent may be provided with a separate reagent containing other substances necessary in the reaction for measuring the target protein. Examples of the other substance include a reaction buffer solution.

The present invention also provides a method for immunological measurement of a target protein (hereinafter, the method of the present invention) using the reagent. The method of the present invention comprises the following steps:
(1) A step of coating insoluble carrier particles having an average particle diameter of 0.02 μm to 2.0 μm with 1.0 μg to 110 μg of hydrophilic polymer per mg,
(2) contacting the whole blood sample with the insoluble carrier particles;
(3) contacting the insoluble carrier particles with an immune complex specific for a target protein;
(4) A step of measuring the turbidity of the generated aggregate.

  In the step (1) of the method of the present invention, the method for coating the insoluble carrier particles, the hydrophilic polymer and the insoluble carrier particles with the hydrophilic polymer may be the same as described in the reagent of the present invention.

  In the step (2) of the method of the present invention, insoluble carrier particles having an average particle size of 0.02 μm to 2.0 μm obtained in the step (1), and having a high hydrophilicity of 1.0 μg to 110 μg per 1 mg of the particles Insoluble carrier particles coated with molecules (preferably insoluble carrier particles having an average particle size of 0.05 μm to 0.3 μm, and coated with 2 μg to 70 μg of hydrophilic polymer per 1 mg of the particles) The whole blood sample can be contacted, for example, according to the method described in the examples below. Specifically, when targeting a protein present in plasma in a whole blood sample, the insoluble carrier particles obtained in the step (1) are directly mixed with the whole blood sample and agitated, thereby stirring the plasma. Target protein can be adsorbed on the surface of the insoluble carrier particles. Further, when targeting a protein present in blood cells in a whole blood sample, the whole blood sample is previously hemolyzed with purified water or the like, and then the treated whole blood sample is obtained in the step (1). The target protein in the blood cells can be adsorbed on the surface of the insoluble carrier particles by mixing and stirring with the insoluble carrier particles.

  In the step (3) of the method of the present invention, the contact between the insoluble carrier particles adsorbed with the target protein obtained in the step (2) and the immune complex specific for the target protein is described in, for example, the Examples described later. It can be carried out according to the methods described. Specifically, the insoluble carrier particles adsorbed with the target protein obtained in the step (2) are mixed and stirred with a primary antibody specific for the target protein and a secondary antibody specific for the primary antibody. Thus, the insoluble carrier particles having the target protein adsorbed via the immune complex can be aggregated. The primary antibody specific for the target protein and the secondary antibody specific for the primary antibody may be mixed sequentially with the insoluble carrier particles or simultaneously. Alternatively, an immune complex composed of a primary antibody specific for the target protein and a secondary antibody specific for the primary antibody may be formed in advance and then mixed with the insoluble carrier particles.

  In the step (4) of the method of the present invention, the turbidity of the produced aggregate obtained in the step (3) can be measured according to a known method. For example, the method described in the examples described later is used. Can be implemented according to Specifically, the absorbance can be measured by irradiating the generated aggregate with a measurement wavelength of 660 nm. The absorbance can be determined based on a standard curve relating to the concentration and absorbance of the target protein prepared in advance.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

Latex Reagent Preparation Method A 1% latex suspension was prepared by adding 9 volumes of 16.7 mM phosphate buffer (PH7.2) to 1 volume of a 10% polystyrene latex suspension (manufactured by Fujikura Kasei) having a particle size of 0.15 μm. To this was added BSA-containing 16.7 mM phosphate buffer (PH7.2) so that 0.008 mg of BSA was added per 1 mg of latex particles, and the mixture was stirred at 25 ° C. for 2 hours. Then, it centrifuged at 4 degreeC and 18000 rpm for 45 minutes. Tris buffer (PH8.2) was added to the obtained latex precipitate to redisperse the latex particles to prepare a 0.1% BSA-coated latex reagent.
Measurement method Normal whole blood sample was centrifuged and divided into blood cell layer and plasma layer. The blood cell layer was regarded as a whole blood sample with excessive red blood cells, and double dilutions (x1, x2, x4, x8, x16) were made with the previously separated plasma. These were regarded as whole blood samples having different hemoglobin concentrations, and 200 μL, 400 μL, 800 μL, and 1 mL of purified water were added to 10 μL, respectively, for hemolysis. Each sample thus hemolyzed was used as a test sample.
Using a biochemical analyzer, mix 3.2 μL of test sample and 120 μL of BSA-coated latex reagent, stir for 5 minutes, add 40 μL of immune complex of anti-HbA1c monoclonal antibody and anti-mouse IgG goat antiserum, and measure at 660 nm Aggregation of the BSA-coated latex reagent was confirmed by measuring the absorbance at
Measurement result Regardless of the dilution ratio of the specimen, even if the hemoglobin concentration changed, the absorbance was almost constant, and it was confirmed that the difference in hemoglobin concentration did not affect the measured value.
Further, it was found that the plasma concentration was changed with the change of the hemoglobin concentration, and it was hardly affected by the plasma (FIG. 1).

  By using the protein measurement reagent of the present invention, it is possible to quantify HbA1c directly using whole blood as a sample without going through the steps of previously centrifuging whole blood and removing plasma components. Furthermore, according to the present invention, it is not necessary to remove the plasma component, and at the same time, the protein in the plasma component can be selected as the target protein.

Claims (15)

  A reagent for measuring a target protein, comprising insoluble carrier particles having an average particle diameter of 0.02 μm to 2.0 μm and coated with 1.0 μg to 110 μg of hydrophilic polymer per 1 mg of the particles.   The reagent according to claim 1, comprising insoluble carrier particles having an average particle diameter of 0.05 μm to 0.3 μm and coated with 2 μg to 70 μg of hydrophilic polymer per 1 mg of the particles.   The insoluble carrier particles have a polymer or copolymer particle of a molecule selected from the group consisting of styrene, vinyl chloride, acrylic acid esters, methacrylic acid esters and vinyl acetate, or styrene units and butadiene units. The reagent according to claim 1, which is a copolymer particle.   The reagent according to claim 3, wherein the insoluble carrier particles are polystyrene particles.   The reagent according to any one of claims 1 to 4, wherein the hydrophilic polymer is selected from the group consisting of dextran, albumin, casein, cellulose, and albumin.   The reagent according to claim 5, wherein the hydrophilic polymer is bovine serum albumin.   The reagent according to any one of claims 1 to 6, comprising an immune complex specific for the target protein.   The reagent according to any one of claims 1 to 7, wherein the target protein is glycated hemoglobin. A method for immunoassay of a target protein comprising the following steps:
(1) A step of coating insoluble carrier particles having an average particle diameter of 0.02 μm to 2.0 μm with 1.0 μg to 110 μg of hydrophilic polymer per mg,
(2) contacting the whole blood sample with the insoluble carrier particles;
(3) contacting the insoluble carrier particles with an immune complex specific for a target protein;
(4) A step of measuring the turbidity of the generated aggregate.   The method according to claim 9, wherein the step (1) is a step of coating insoluble carrier particles having an average particle diameter of 0.05 μm to 0.3 μm with 2 μg to 70 μg of highly hydrophilic molecules per mg.   The insoluble carrier particles have a polymer or copolymer particle of a molecule selected from the group consisting of styrene, vinyl chloride, acrylic acid esters, methacrylic acid esters and vinyl acetate, or styrene units and butadiene units. The method according to claim 9 or 10, which is a copolymer particle.   The method according to claim 11, wherein the insoluble carrier particles are polystyrene particles.   The method according to any one of claims 9 to 12, wherein the hydrophilic polymer is selected from the group consisting of dextran, albumin, casein and cellulose.   The method according to claim 13, wherein the hydrophilic polymer is bovine serum albumin.   The method according to any one of claims 9 to 14, wherein the target protein is glycated hemoglobin.