JP5584993B2 - Method and kit for measuring protein in sample - Google Patents

Description

The present invention relates to a measurement method and a measurement kit capable of measuring a protein contained in a trace amount in a sample with high sensitivity and ease.
The present invention is useful in the fields of analytical chemistry, life science and medicine, and particularly useful in the field of clinical examination.

The protein concentration of serum or plasma becomes high in dehydration, reticuloendothelial disease or chronic infection.
Moreover, it becomes low in leakage of plasma protein, malnutrition or liver dysfunction.
Thus, since the protein concentration of serum or plasma changes with various diseases or various pathological conditions, the measurement is emphasized in the diagnosis and treatment of diseases.

Moreover, protein in urine appears in kidney disease, heart disease, blood disease, jaundice, high fever, and the like.
Measuring the protein in the urine is also important for the diagnosis and treatment of the disease.
In particular, measuring a small amount of albumin in urine is very important for early detection of diabetic nephropathy and the like.

As a conventional method for measuring protein (measurement kit) in a sample, there is a method of using a test piece in which a carrier is impregnated with a dye that changes its color tone when bound to protein.
However, this method using a test piece has a measurement limit of 10 to 20 mg / dL and cannot measure a protein contained in a trace amount in a sample.
In addition, this measurement method (measurement kit) may have a measurement error due to the influence of other substances in the sample and unbound dyes.

  In addition, the Biuret method (burette method) using a reaction in which four peptide bonds in a protein chelate bond with a divalent copper ion in an alkaline solution and develop a purple color has a measurement limit of 10 mg / dL. The measurement operation is complicated and takes time.

  The Lowry method using a divalent copper ion and a phenol reagent (Lowry method) has a measurement limit of 2.5 mg / dL and high sensitivity, but the measurement operation is still complicated and requires time for measurement. there were.

  In addition, the pyrogallol red / molybdenum complex coloring method, which uses a reaction that turns blue-purple when bound to protein under acidic conditions, has a high sensitivity of 2 mg / dL, but the measurement is still high. This operation is complicated and takes time to measure.

Kinoshita et al. Reported a method of concentrating proteins in a sample on a membrane filter, and then fluorescently staining the filter with fluorescein isothiocyanate (FITC), and measuring the fluorescence intensity of the spot after washing.
However, in this method, the reaction between the protein and FITC takes 30 minutes, and it is complicated that washing is required after staining in order to remove interfering substances (see Non-Patent Document 1). ).

Fujita et al. Reported a method of measuring the absorbance of a solution obtained by physically collecting a precipitate aggregate of a protein and a dye-metal complex with a filter and then re-dissolving with a sodium hydroxide aqueous solution. .
However, in this method, it cannot be said that the quantitativeness is never good due to clogging of the precipitate, and the color on the filter is not observed, but the redissolved solution is measured. It was not suitable for analysis (see Non-Patent Document 2).

In addition, as a method (measuring reagent) for measuring trace albumin in urine, there is a method using an immunoturbidimetric method or a latex turbidimetric method as a measurement principle.
Although this method is highly sensitive, it requires an expensive analyzer for the measurement, and the cost of the measuring reagent is high.

  Recently, a test piece in which a carrier that is impregnated with a dye that changes its color tone upon binding to a protein has come out with a measurement limit of 2-3 mg / dL. However, the test piece can be directly used without diluting the sample. Since the measurement is performed by bringing the sample into contact with the test piece, it is easily affected by various interfering substances contained in the sample. Especially when the sample is urine, the pH is adjusted by a substance having a buffering capacity contained in urine. The change in pH affected the change in the color tone of the dye, which sometimes caused an error in the measurement of the protein in the sample.

  The above-described conventional protein measurement methods and measurement kits have high measurement limits, that is, low measurement sensitivity, complicated measurement operations, time-consuming measurement, and expensive analysis. An apparatus is required, the cost of the measurement reagent is high, or the measurement result is easily affected by the interference substance in the sample.

  In addition, the present inventors firstly mixed a sample that may contain a protein and a dye that forms an ion aggregate with the protein, and the ion aggregate solution without precipitating the ion aggregate. B) passing the ion aggregate solution through a hydrophobic filter to adsorb the aggregate dissolved in the ion aggregate solution to the filter; and c) the above the filter on the filter A method for analyzing proteins in a sample and an analysis kit have been developed that sequentially perform a step of analyzing the aggregates qualitatively and quantitatively (see Patent Document 1).

  In addition, the inventors of the present invention firstly contact a sample, a dye that forms an aggregate with a protein, and a surfactant; after the contact, add a certain amount thereof on the porous membrane; and the porous A method for measuring protein in a sample and a measurement kit including a step of measuring a colored region on a sex membrane were developed (see Patent Document 2).

JP-A-11-304803 JP 2005-55417 A

"Analytical Chemistry", Volume 23, pages 1543 to 1544, 1974 “Analytical Chemistry”, 44, 733-739, 1995

  The problem of the present invention is that no expensive apparatus, instrument or measurement reagent is required for the measurement, the measurement operation is simple, the time required for the measurement is extremely short, and the measurement results are affected by the interference substance in the sample. An object of the present invention is to provide a highly sensitive method for measuring a protein in a sample and a measurement kit that are difficult to measure and can measure even a small amount of protein in the sample.

The inventors of the present invention have made various studies based on the fact that the applicant has received a development contract from the Japan Science and Technology Agency, and completed the present invention.
The present invention includes the following inventions.
(1) 1) Aggregates that contact a sample and a dye that associates with a protein to form an aggregate, thereby associating the protein contained in the sample with the dye and forming these aggregates Forming step;
2) After the formation of the aggregate, a flow step of passing a certain amount of the solution containing the aggregate through a porous carrier made of an organic compound and having a pore size of 3 μm or more (however, the fixed amount is the solution in the porous carrier) And 3) a color measurement step for measuring the color of the surface of the porous carrier, and a method for measuring a protein in a sample.
(2) The method for measuring protein in the sample according to (1), wherein the certain amount is 2 mL or more.
(3) The method for measuring protein in the sample according to (1) or (2), wherein the certain amount is 5 mL or more.
( 4 ) The method for measuring a protein in a sample according to any one of (1) to (3) , wherein a surfactant is allowed to coexist in the aggregate formation step.
(5) The method for measuring protein in the sample according to (4) above, wherein the surfactant is a surfactant.
(6) The protein in the sample according to (5), wherein the surfactant is a nonionic surfactant, an amphoteric surfactant, an anionic surfactant, or a cationic surfactant. Measuring method.
(7) The method for measuring a protein in a sample according to (5) or (6) above, wherein the surfactant is a nonionic surfactant.
(8) In the sample according to any one of (4) to (7), the surfactant does not contain any surfactant other than the surfactant, polysaccharide or derivative thereof. Protein measurement method.
(9) The method for measuring protein in the sample according to (8), wherein the other surfactant is polyalkylene glycol.

  The method and kit for measuring protein in a sample of the present invention do not require expensive equipment, instruments or measurement reagents for measurement, are easy to operate, have a very short time for measurement, and are contained in the sample. It is a highly sensitive method for measuring a protein in a sample and a measurement kit capable of measuring a protein contained in a trace amount.

  In addition, the method and kit for measuring a protein in a sample of the present invention are a method in which a sample and a dye that associates with the protein to form an aggregate are brought into contact with each other, whereby the sample is diluted, that is, an interfering substance in the sample. Since the concentration of the interfering substance becomes low, the method for measuring a protein in the sample and the measurement kit of the present invention are not easily affected by the measurement result of the interfering substance in the sample.

It is the figure which showed the effect of using the porous support | carrier which consists of an organic compound and has a pore diameter of 3 micrometers or more in the measuring method and measuring kit of the protein in a sample.

I. Method for measuring protein in sample 1. Sample The sample in the present invention is not particularly limited as long as it may contain a protein.

  Examples of the sample include a sample derived from a human or an animal, a sample derived from a plant, a sample derived from a microorganism, a food, a beverage, drinking water, a drug, a reagent, or an environmental sample.

  Samples derived from humans or animals are not particularly limited, for example, human or animal body fluids such as blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites, amniotic fluid, stool, etc. Excreta; organs such as brain, heart, kidney or liver; tissues such as hair, skin, nails, muscles or nerves; or cells.

  The food is not particularly limited, and examples thereof include meat, vegetables, grains, fruits, eggs, marine products, processed foods, and the like.

  A drink is not specifically limited, For example, juice, milk, tea, coffee, liquor etc. can be mentioned.

  The drug is not particularly limited, and examples thereof include an infusion solution, an injection solution, a powder, or a tablet.

In addition, since the sample used for measurement in the present invention needs to be a liquid, if the sample is not a liquid, pretreatment such as extraction or solubilization may be performed according to a known method to obtain a liquid sample. Good.
Further, the sample may be diluted or concentrated as necessary.

2. Dye that associates with protein to form an aggregate In the present invention, the dye that associates with a protein to form an aggregate can be associated with the protein by electrostatic attraction, hydrophobic interaction, or the like. The associated proteins can form aggregates.
If it is such a pigment | dye, it can be used without being specifically limited.

  Examples of the dye that associates with this protein to form an aggregate include xanthene dyes such as erythrosin B, eosin B, eosin Y, phloxine B, or rose bengal; coomassie brilliant blue (CBB), bromochlorophenol blue, tetra And triphenylmethane dyes such as bromophenol blue, bromophenol blue, or bromocresol purple; or azo dyes such as buffalo black, acid orange 12, methyl orange, orange G, or azosulfathiazole.

  The dye that associates with the protein to form an aggregate is preferably a highly hydrophobic dye.

  Moreover, as a pigment | dye which associates with this protein and forms an aggregate, that whose value of the acid dissociation constant (pKa) is lower than the value of the isoelectric point (pI) of the protein to measure is preferable.

  Thus, when the value of the acid dissociation constant (pKa) of a dye that associates with a protein to form an aggregate is lower than the isoelectric point value of the protein to be measured, When the pH at which the aggregate is formed is less than the isoelectric point of the protein to be measured, the charge of the protein to be measured becomes positive, and the negative charge of the dye can be maintained. This is preferable because the protein contained in the sample and the dye are likely to associate with each other, thereby increasing the sensitivity of the protein measurement.

  The dye that associates with the protein to form an aggregate is preferably a xanthene dye, more preferably erythrosin B, eosin B, eosin Y, phloxine B, or rose bengal, and more preferably erythrosine B. Particularly preferred.

  The dye that associates with the protein to form an aggregate is more preferably a reagent containing a dye that associates with the protein to form an aggregate, more preferably a solution containing a dye that associates with the protein to form an aggregate. The reagent is preferably mixed with the sample and brought into contact.

  In addition, since the density | concentration of the pigment | dye which associates with this protein and forms an aggregate is too low, since the sensitivity of a measurement is not acquired and the protein in a sample cannot be measured, when contacting with the protein in a sample normally The concentration is preferably 1 μM or more, more preferably 10 μM or more, and particularly preferably 20 μM or more.

  In addition, if the concentration of the dye that associates with the protein to form an aggregate is too high, color development occurs on the surface of the porous carrier even if the protein is not present in the sample, and the protein is present in the sample. In general, it is preferably 500 μM or less, more preferably 100 μM or less, and particularly preferably 50 μM or less, as the concentration when contacting with the protein in the sample.

3. Porous carrier made of an organic compound and having a pore size of 3 μm or more The porous carrier made of an organic compound and having a pore size of 3 μm or more is made of an organic compound, has a number of fine pores, and has a pore size of 3 μm or more. Any carrier can be used without particular limitation as long as the solution can be passed through when the carrier is added to the carrier.

Although it is a porous carrier made of this organic compound and having a pore size of 3 μm or more, any porous carrier made of an organic compound and having a pore size of 3 μm or more can be used without particular limitation.
The organic compound is not particularly limited. For example, cellulose acetate, cellulose mixed ester, cellulose mixed ester-mixed ester, cellulose fiber, regenerated cellulose, hydrophilic reinforced cellulose, nitrocellulose, reinforced cellulose, polypropylene, polyethersulfone. , Polysulfone, polyvinylidene fluoride, hydrophilic polyvinylidene fluoride, hydrophilic polytetrafluoroethylene, polycarbonate, nylon, polyamide, polyester, polyester-polyethylene terephthalate, or cellulose acetate-coated polyester nonwoven fabric.

The pore size (or particle retention capacity, retention particle size, etc.) of the porous carrier needs to be 3 μm or more.
When measurement is performed using a porous carrier made of an organic compound and having a pore size of less than 3 μm, the color of the surface of the porous carrier when measuring a sample that does not contain protein becomes dark. Will be erroneously determined.
That is, when a porous carrier made of an organic compound and having a pore diameter of less than 3 μm is used as the porous carrier, it cannot be correctly determined that no protein is present in the sample.
Therefore, when a protein in a sample is measured using a porous carrier made of an organic compound, the pore size needs to be 3 μm or more.

In particular, the pore diameter is preferably 5 μm or more, more preferably 8 μm or more, and particularly preferably 10 μm or more.
If this is also mentioned, it is preferable that the pore diameter is 20 μm or less.

  The porous carrier made of an organic compound and having a pore size of 3 μm or more in the present invention may be made of a filter paper having a pore size of 3 μm or more, a filter made of an organic compound, or the like, and a plurality of these filter papers or filters may be used. It may be combined, or these filter papers or filters may be set in a filter device, or these filter papers or filters etc. are housed in a housing. Further, the filter paper or filter may be combined with a prefilter made of an organic compound or other material.

  Among the porous carriers made of this organic compound and having a pore diameter of 3 μm or more, those made of filter paper having a pore diameter of 3 μm or more, a filter made of an organic compound, etc. Etch / Cyclopore Membrane, Catalog Number: 7061-1313, Pore Diameter: 5.0 μm, Membrane: Polyester ”,“ Product Name: Track Etch / Cyclopore Membrane, Catalog Number: 7062-4712, Pore Diameter: 3.0 μm, Membrane: "Polycarbonate", "Trade name: Track Etch / Cyclopore Membrane, catalog number: 7062-2513, Pore diameter: 5.0 µm, Membrane: Polycarbonate, Thickness: 15 µm", "Product name: Track Etch / Cyclopore membrane, Catalog No. : 7060-2 14, pore size: 8.0 μm, membrane: polycarbonate, thickness: 12 μm ”,“ trade name: track etch cyclopore membrane, catalog number: 7060-2515, pore size: 10.0 μm, membrane: polycarbonate ”,“ trade name : Track Etch Cyclopore Membrane, Catalog Number: 7060-2516, Pore Diameter: 12.0 μm, Membrane: Polycarbonate, Thickness: 8 μm ”,“ Product Name: Track Etch Nuclepore Membrane, Catalog Number: 110612, Pore Diameter: 3 0.0 μm, Membrane: Polycarbonate ”,“ Product Name: Track Etch New Clipore Membrane, Catalog Number: 110613, Pore Diameter: 5.0 μm, Membrane: Polycarbonate ”,“ Product Name: Track Etch New Clipore Membrane, Catalog No. 110614, pore size: 8.0 μm, membrane: polycarbonate ”,“ Product Name: Track Etch New Clipore Membrane, Catalog No. 110615, pore size: 10.0 μm, Membrane: Polycarbonate ”,“ Product Name: Track Etch ・Nuclepore membrane, catalog number: 110616, pore size: 12.0 μm, membrane: polycarbonate ”,“ Product name: Track Etch New Clipore membrane, catalog number: 150446, pore size: 8.0 μm, membrane: polycarbonate / PVP free ”, “Product name: Nitrocellulose membrane WCN, catalog number: 7193-002, pore size: 3.0 μm, membrane: Nitrocellulose, thickness: 125 μm”, “Product name: Nitrocellulose membrane WC , Catalog number: 7195-002, pore size: 5.0 μm, membrane: nitrocellulose, thickness: 125 μm ”,“ trade name: nitrocellulose membrane AE98, catalog number: 10400206, pore size: 5.0 μm, membrane: nitrocellulose, “Thickness: 125 μm”, “trade name: nitrocellulose membrane AE99, catalog number: 10400106, pore size: 8.0 μm, membrane: nitrocellulose, thickness: 125 μm”, “trade name: nitrocellulose membrane AE100, catalog number: 10400012” , Pore size: 12.0 μm, membrane: nitrocellulose, thickness: 125 μm ”, or“ trade name: cellulose mixed ester membrane ME29, catalog number: 10400772, pore size: 3.0 μm, membrane: cellulose Mixed esters, thickness: 150 [mu] m ", and the like. As products of Advantech Toyo Co., Ltd., “trade name: polycarbonate type membrane filter / K800A013A, product code: 16180001, pore size: 8.00 μm, membrane: polycarbonate, thickness: 7 μm”, “trade name: cellulose mixed ester type” Membrane filter A500A025A, product code: 10150002, pore size: 5.00 μm, membrane: cellulose mixed ester, thickness: 160 μm, “product name: cellulose mixed ester type membrane filter A300A025A, product code: 10130002, pore size: 3. 00 μm, membrane: cellulose mixed ester, thickness: 155 μm ”, or“ trade name: cellulose acetate type membrane filter C300A025A, product Over de: 11130002, pore size: 3.00, membrane: cellulose acetate, thickness: 135 .mu.m "and the like.

  In addition, among porous carriers made of an organic compound and having a pore diameter of 3 μm or more, filter paper having a pore diameter of 3 μm or more, or a filter made of an organic compound, etc. are housed in a housing. GD / X Syringe Filter, Catalog Number: 6870-2550, Pore Diameter: 5.0 μm, Membrane: Nylon, “Product Name: Pladisc FP30 CN-S, Sterilized Individual Packaging, Catalog Number: 10462000, Pore Diameter: 5.0 μm , Membrane: Nitrocellulose ”,“ Trade name: Pladisc FP30 CN, non-sterile, catalog number: 10462520, pore size: 5.0 μm, Membrane: Nitrocellulose ”, or“ Product name: Polydisc HD, catalog number: 6728- 5050, pore size: 5.0 μm, membrane: polypropylene Down "and the like can be mentioned.

4). Surfactant In the present invention, the coexistence of a surfactant in the aggregate formation step means that the color development degree (darkness) of the surface of the porous carrier in the sample containing protein in the color measurement step. As a result, the measurement of the sample not containing protein (negative control; or reagent blank) has no color development on the surface of the porous carrier or only a very thin color development. Since the effect of facilitating the discrimination between the protein-containing sample and the protein-free sample in the color measurement described above is produced, it is preferable that a surfactant is allowed to coexist.

As the surfactant, any surfactant can be used without particular limitation.
This surfactant may be a natural surfactant or may be a surfactant produced by synthesis or the like.

  Examples of this surfactant include surfactants, polysaccharides or derivatives thereof, and other surfactants.

  Examples of the surfactant include nonionic surfactants, amphoteric surfactants, anionic surfactants, and cationic surfactants.

  More specific examples of this surfactant include, for example, sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxy Nonionic surfactants such as ethylene alkyl ether, polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin Amphoteric surfactants such as betaine acetate; polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ether Anionic surfactants such as Le acetate; alkyltrimethylammonium salts, and cationic surfactants such as such as dialkyl dimethyl ammonium salts or benzalkonium chloride.

  Specific examples of the nonionic surfactant include Triton X-100, Triton X-405, and Tween 20.

  Examples of the polysaccharide include saccharides produced by dehydration condensation of several monosaccharides, and examples thereof include cyclodextrin (CD), dextran, and heparin.

As the polysaccharide derivative, a functional group such as hydrogen or hydroxyl group of the polysaccharide is an aliphatic hydrocarbon group such as hydroxypropyl group, hydroxybutyl group, glucosyl group, maltosyl group, diethylaminoethyl group, methyl group or ethylidene group. , Alicyclic hydrocarbon groups such as cyclopropyl group, aromatic hydrocarbon groups such as phenyl group, benzyl group or benzylidene group, ether groups such as methoxy group or phenoxy group, ester groups such as acetoxy group or benzoyloxy group, acetyl Substituted with an acyl group such as a group, propionyl group or benzoyl group, sulfhydryl group, sulfo group, sulfonyl group, carboxyl group, amino group, imino group, nitrile group, carbonyl group, oxo group, hydroxyl group, or nitro group Etc.
Furthermore, as a derivative | guide_body of a polysaccharide, the crosslinked material of the said polysaccharide or the said substituted body etc. can be mentioned.

Examples of the cyclodextrin include α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), and γ-cyclodextrin (γ-CD).
In addition, as the cyclodextrin derivative, for example, a hydroxyl group such as α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin has a hydroxypropyl group, a hydroxybutyl group, a glucosyl group, a maltosyl group, a benzyl group, or a sulfonyl group. Or a cross-linked product of these cyclodextrins or derivatives thereof.

  As the cyclodextrin, β-cyclodextrin or γ-cyclodextrin is preferable, and as the cyclodextrin derivative, β-cyclodextrin or γ-cyclodextrin in which a hydroxyl group or the like is substituted, or β-cyclodextrin or γ-cyclodextrin is substituted. A cross-linked product of dextrin is preferred.

  The dextran sulfate or derivative thereof preferably has a molecular weight in the range of 1,000 to 5,000,000, and particularly preferably has a molecular weight in the range of 5,000 to 1,000,000.

Examples of other surfactants include polyalkylene glycols such as polyethylene glycol and polypropylene glycol.
As the polyalkylene glycol such as polyethylene glycol or polypropylene glycol, those having a molecular weight of 200 to 4,000,000 can be used, for example. Those having a molecular weight of 1,000 to 400,000 are more preferred, and those having a molecular weight of 2,000 to 40,000 are particularly preferred.

  The surfactant is preferably a nonionic surfactant such as Triton X-100, Triton X-405 or Tween 20, or a polyalkylene glycol such as polyethylene glycol or polypropylene glycol, and more preferably Triton X-100.

  In addition, since the coexistence effect cannot be obtained if the concentration of the surfactant is too low, the concentration is usually 0.0001% (W / V) or more when coexisting in the aggregate forming step. Preferably, it is 0.001% (W / V) or more, more preferably 0.005% (W / V) or more.

  In addition, if the concentration of the surfactant is too high, the viscosity of the solution containing the surfactant may increase. In such a case, addition to a porous carrier made of an organic compound in the flow-through process and having a pore size of 3 μm or more, etc. Since an error may occur in the amount, normally, the concentration when coexisting in the aggregate forming step is preferably 5% (W / V) or less, preferably 1% (W / V) or less. More preferably, it is 0.1% (W / V) or less.

  In the agglomerate forming step, a plurality of types of surfactant substances may be combined and coexist.

5. pH
In the present invention, in the aggregate formation step, the protein contained in the sample is associated with the dye that forms an aggregate by associating with the protein, and the pH at which these aggregates are formed should be measured. The pH is preferably less than the isoelectric point (pI) of the protein.
Thus, when the pH is less than the isoelectric point of the protein to be measured,
The charge of the protein to be measured is on the positive side, and it is easy to associate with the dye, which is preferable because the sensitivity of the protein measurement can be increased.

Examples of isoelectric point values of this protein are: human albumin pH 4.8, α1-globulin pH 2.0, γ1-globulin pH 5.8, γ2-globulin pH 7.4, haptoglobulin. PH 4.1, ovalbumin may include pH 4.6.
Therefore, for example, when measuring human albumin, it is preferable that the pH in the aggregate formation step is less than pH 4.8.

Further, in the present invention, in the aggregate formation step, the protein contained in the sample is associated with the dye that associates with the protein to form an aggregate, and the pH at which these aggregates are formed is It is preferably near the pH of the acid dissociation constant (pKa) of the dye that associates with this protein to form an aggregate.
More preferably, the pH is within a range of ± 2 of the value of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate in the aggregate formation step, and is particularly preferably a protein. The pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate, and more preferably the acid of the dye that associates with the protein to form an aggregate. The pH is within a range of ± 1 of the value of the dissociation constant (pKa).

  Thus, in the aggregate formation step, the negative charge of the dye is adjusted by setting the pH to be close to the pH of the acid dissociation constant (pKa) of the dye that associates with the protein and forms an aggregate as described above. This makes it easy to associate with the protein contained in the sample, thereby increasing the sensitivity of the protein measurement, which is preferable.

  An example of the acid dissociation constant (pKa) of the dye that associates with this protein to form an aggregate is as follows: erythrosin B is pKa3.2, eosin B is pKa2.2, eosin Y is pKa2.8, and rose bengal Is pKa3.51 or the like.

  Thus, for example, when erythrosine B is used as a dye that associates with a protein to form an aggregate, the pH in the aggregate formation step is set to a pH in the vicinity of the acid dissociation constant (pKa) 3.2 of erythrosine B. It is preferable that the pH is in the range of pH 1.2 to pH 5.2, more preferably pH is in the range of pH 1.7 to pH 4.7, and pH 2.2 to pH 4.2. More preferably, the pH is within the range.

  In addition, for example, when eosin B is used as a dye that associates with a protein to form an aggregate, the pH in the aggregate formation step is set to a pH near the acid dissociation constant (pKa) 2.2 of eosin B. It is preferable to set the pH within the range of pH 1.2 to pH 4.2, more preferably set to the pH within the range of pH 1.4 to pH 3.7, and pH 1.7 to pH 3.2. More preferably, the pH is within the range.

  For example, when eosin Y is used as a dye that associates with a protein to form an aggregate, the pH in the aggregate formation step is set to a pH near the acid dissociation constant (pKa) 2.8 of eosin Y. The pH is preferably within the range of pH 1.2 to pH 4.8, more preferably within the range of pH 1.5 to pH 4.3, and pH 1.8 to pH 3.8. More preferably, the pH is within the range.

  For example, when rose bengal is used as a pigment that associates with a protein to form an aggregate, the pH in the aggregate forming step is set to a pH in the vicinity of the acid dissociation constant (pKa) 3.51 of rose bengal. It is preferable to set the pH within the range of pH 1.5 to pH 5.5, more preferably set to the pH within the range of pH 2.0 to pH 5.0, and pH 2.5 to pH 4.5. More preferably, the pH is within the range.

  From the above, in the aggregate formation step, the pH is set to a pH lower than the isoelectric point of the protein to be measured, and the acid dissociation constant (pKa of the dye that associates with the protein to form an aggregate) It is particularly preferred that the pH be in the vicinity of the pH value of.

  For example, when erythrosine B is used as a dye that associates with a protein to form an aggregate and human albumin in a sample is to be measured, in the above-described aggregate formation step, pH 4 which is the isoelectric point of human albumin. It is preferable that the pH is less than 0.8, and the pH is in the vicinity of the acid dissociation constant (pKa) 3.2 of erythrosin B, and the pH is in the range of 1.2 or more and less than 4.8. More preferably, the pH is particularly preferably in the range of pH 1.7 to pH 4.5, and more preferably in the range of pH 2.2 to pH 4.2.

  For example, when eosin B is used as a dye that associates with a protein to form an aggregate and human albumin in a sample is to be measured, the isoelectric point of human albumin is used in the above-described aggregate formation step. It is preferable that the pH is less than a certain pH of 4.8 and is in the vicinity of the acid dissociation constant (pKa) of 2.2 of eosin B, and the pH is within the range of pH 1.2 to pH 4.2. More preferably, the pH is in the range of pH 1.4 to pH 3.7, particularly preferably, and the pH is more preferably in the range of pH 1.7 to pH 3.2.

  For example, when eosin Y is used as a dye that associates with a protein to form an aggregate, and human albumin in a sample is to be measured, the isoelectric point of human albumin is used in the aggregate formation step. It is preferable that the pH is less than a certain pH of 4.8 and is in the vicinity of the acid dissociation constant (pKa) of 2.8 of eosin Y, and the pH is in the range of 1.2 or more and less than 4.8. More preferably, the pH is particularly preferably in the range of pH 1.5 to pH 4.3, and more preferably in the range of pH 1.8 to pH 3.8.

  For example, when rose bengal is used as a dye that associates with a protein to form an aggregate, and human albumin in a sample is to be measured, the isoelectric point of human albumin is determined in the aggregate formation step. It is preferable that the pH is less than a certain pH of 4.8 and is in the vicinity of Rose Bengal's acid dissociation constant (pKa) of 3.51, and the pH is in the range of 1.5 or more and less than 4.8. More preferably, the pH is particularly preferably in the range of pH 2.0 to pH 4.65, and more preferably in the range of pH 2.5 to pH 4.5.

  In the agglomerate forming step, if the pH is too low, for example, less than pH 1, the protein is undesirably denatured or insolubilized.

Then, in the aggregate formation step, the protein contained in the sample is associated with the dye that associates with the protein to form an aggregate, and the pH at which these aggregates are formed is the pH as described above. In order to achieve this, it is preferable to use a buffering agent having such a pH (buffering agent having a buffering ability in these pH ranges).
As this buffer, a known buffer can be used.

  For example, glycine (pKa1: 2.35), phosphoric acid (pKa1: 2.15), phthalic acid (pKa1: 2.95, pKa2: 5.41), citric acid (pKa1: 3.13, pKa2: 4.76), barbituric acid (pKa1: 4.04), succinic acid (pKa1: 4.21, pKa2: 5.64), acetic acid (pKa2: 4.76), various good buffers, etc. Can be mentioned.

6). Measurement Operation The measurement operation of the method for measuring a protein in a sample according to the present invention will be described below.
(1) Aggregate formation step The sample and a dye that associates with the protein to form an aggregate are brought into contact, thereby associating the protein contained in the sample with the dye and forming these aggregates. In the aggregate forming step, any method may be used as long as the sample and the dye that forms an aggregate by associating with the protein can be brought into contact with each other.
By this contact, the protein contained in the sample is associated with the dye that associates with the protein to form an aggregate, and an aggregate of these associated “dyes that associate with protein-protein to form an aggregate” is formed. Is done.
The concentration at this time is as described above.

  As described above, it is preferable to coexist a surface active substance in this aggregate formation step.

  In this aggregate formation step, in order to form the above-mentioned aggregate, it is a time for contacting the sample and a dye that associates with the protein and forms an aggregate. However, this is not particularly limited, and can be appropriately set. good.

  Normally, in this aggregate formation step, the sample can be brought into contact with a dye that associates with a protein to form an aggregate, and the next flow-through step can be performed immediately. The contact time of the dye that associates with the protein to form an aggregate is preferably 2 seconds or longer, more preferably 5 seconds or longer, particularly preferably 10 seconds or longer, and 20 seconds or longer. More preferably it is.

In this agglomerate forming step, the temperature is the temperature at which the sample and the dye that associates with the protein and forms an agglomerate are contacted to form the agglomerate, but there is no particular limitation.
However, since the protein may be denatured when the temperature is too high, it is preferably 60 ° C. or lower, more preferably 50 ° C. or lower, particularly preferably 40 ° C. or lower, and further preferably 30 ° C. or lower.

  Also, in this aggregate formation step, if the temperature when contacting the sample and the dye that forms an aggregate with the protein is too low, the protein is denatured, or the association between the protein and the dye and the formation of the aggregate Since it may take time for this reaction, 5 ° C. or higher is preferable, 10 ° C. or higher is more preferable, 15 ° C. or higher is particularly preferable, and 20 ° C. or higher is even more preferable.

  The sample and the dye that associates with the protein to form an aggregate are brought into contact with each other, whereby the protein and the dye contained in the sample are associated with each other to form these aggregates. It may be performed in one stage, but may be performed in a plurality of stages including two or more stages.

  When performing this aggregate formation process by 1 step, it can implement by the following aspect, for example.

(A) A reagent containing a “dye that associates with a protein to form an aggregate” and a sample are mixed and brought into contact with each other, whereby the protein contained in the sample and the dye are associated with each other. Aggregates are formed.

(B) Contains a “dye that associates with a protein to form an aggregate”, and has a pH lower than the isoelectric point of the protein to be measured, and associates with the protein to form an aggregate. The reagent having a pH near the pH of the acid dissociation constant (pKa) of the dye to be mixed is brought into contact with the sample, so that the pH is less than the isoelectric point of the protein to be measured. In addition, the protein contained in the sample and the dye are associated with each other at a pH near the acid dissociation constant (pKa) value of the dye that associates with the protein to form an aggregate, thereby forming an aggregate. Let

(C) A reagent containing “a dye that associates with a protein to form an aggregate” and “surfactant” and a sample are mixed and brought into contact with each other, whereby the protein contained in the sample and the dye are mixed with each other. Associate to form these aggregates. Or

(D) containing a “dye that associates with a protein to form an aggregate” and a “surfactant”, and has a pH lower than the isoelectric point of the protein to be measured, The reagent having the acid dissociation constant (pKa) of the dye that associates and forms an aggregate is mixed with and brought into contact with the reagent, and thereby the isoelectric point of the protein to be measured is measured. The protein contained in the sample and the dye are associated with each other at a pH lower than the value and near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate, These aggregates are formed.

  Moreover, when performing this aggregate formation process in two or more steps | paragraphs, it can implement by the following aspect, for example.

(A) First step: A reagent containing a “dye that associates with a protein to form an aggregate” and a sample are mixed and brought into contact with each other.
Second stage: A reagent containing a “buffering agent” in the mixed liquid of the first stage [the pH of which is lower than the isoelectric point value of the protein to be measured and is associated with the protein and aggregates. The acid dissociation constant (pKa) of the dye to be formed is mixed, and the pH of the mixed solution is set to a pH lower than the isoelectric point of the protein to be measured, And a pH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate ”. At this pH, the protein contained in the sample and the dye are associated with each other, A collection is formed.

(B) First stage: A reagent containing a “dye that associates with a protein to form an aggregate” and a “surfactant” is mixed and brought into contact with the sample.
Second stage: A reagent containing a “buffering agent” in the mixed liquid of the first stage [the pH of which is lower than the isoelectric point value of the protein to be measured and is associated with the protein and aggregates. The acid dissociation constant (pKa) of the dye to be formed is mixed, and the pH of the mixed solution is set to a pH lower than the isoelectric point of the protein to be measured, And a pH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate ”. At this pH, the protein contained in the sample and the dye are associated with each other, A collection is formed.

(C) First stage: A reagent containing a “dye that associates with a protein to form an aggregate” and a sample are mixed and brought into contact with each other.
Second stage: A reagent containing “surfactant” and “buffer” in the mixed liquid of the first stage [the pH of which is lower than the isoelectric point of the protein to be measured, and the protein The pH of the acid dissociation constant (pKa) of the dye that associates with the dye to form an aggregate is mixed, and the pH of this mixture is less than the value of the isoelectric point of the protein to be measured. PH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate, and at this pH, the protein contained in the sample and the dye Associate to form these aggregates.

(D) First stage: a reagent containing a “buffering agent” [the acid of the dye whose pH is less than the isoelectric point of the protein to be measured and which associates with the protein to form an aggregate A sample having a pH close to the pH of the dissociation constant (pKa) is mixed with the sample.
Second stage: A reagent containing “a dye that associates with a protein to form an aggregate” is mixed with the mixed liquid in the first stage. In this mixed liquid, “less than the isoelectric point of the protein to be measured” At the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate, and the sample and the “dye that associates with the protein to form an aggregate” Thereby, the protein contained in the sample and the dye are associated to form an aggregate of these.

(E) First stage: a reagent containing “buffer” and “surfactant” [pH of the pH is less than the isoelectric point of the protein to be measured, and aggregated with the protein And a sample having a pH in the vicinity of the pH of the acid dissociation constant (pKa) of the dye forming the compound.
Second stage: A reagent containing “a dye that associates with a protein to form an aggregate” is mixed with the mixed liquid in the first stage. In this mixed liquid, “less than the isoelectric point of the protein to be measured” At the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate, and the sample and the “dye that associates with the protein to form an aggregate” Thereby, the protein contained in the sample and the dye are associated to form an aggregate of these.

(F) First stage: a reagent containing a “buffering agent” [a pH of a dye whose pH is less than the isoelectric point of the protein to be measured, and which is associated with the protein to form an aggregate. A sample having a pH close to the pH of the dissociation constant (pKa) is mixed with the sample.
Second stage: A reagent containing “dye that forms an aggregate by associating with protein” and “surfactant” is mixed with the mixed liquid in the first stage, and in this mixed liquid, “the protein to be measured” is mixed. The pH is less than the value of the isoelectric point and the pH is close to the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate. The “dye to be formed” is brought into contact, thereby associating the protein contained in the sample with the dye and forming an aggregate thereof.

(G) First stage: A reagent containing a “dye that associates with a protein to form an aggregate” and a sample are mixed and brought into contact with each other.
Second stage: A reagent containing “surfactant” is mixed in the mixed liquid in the first stage.
Third stage: A reagent containing a “buffering agent” in the mixed liquid in the second stage [the pH of which is lower than the isoelectric point of the protein to be measured, and is associated with the protein and aggregates. The acid dissociation constant (pKa) of the dye to be formed is mixed, and the pH of the mixed solution is set to a pH lower than the isoelectric point of the protein to be measured, And a pH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate ”. At this pH, the protein contained in the sample and the dye are associated with each other, A collection is formed.

(H) First stage: A reagent containing a “dye that associates with a protein to form an aggregate” and a sample are mixed and brought into contact with each other.
Second stage: A reagent containing a “buffering agent” in the mixed liquid of the first stage [the pH of which is lower than the isoelectric point value of the protein to be measured and is associated with the protein and aggregates. In the vicinity of the pH of the acid dissociation constant (pKa) of the dye to be formed].
Third stage: A reagent containing “surfactant” is mixed in the mixed liquid in the second stage, and in this mixed liquid, “the pH is less than the value of the isoelectric point of the protein to be measured, and The protein contained in the sample is associated with the dye at a pH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate, and these aggregates are formed.

(I) First stage: A reagent containing a “surfactant” is mixed with a sample.
Second stage: A reagent containing a “dye that associates with a protein to form an aggregate” is mixed with the mixed liquid in the first stage and brought into contact therewith.
Third stage: A reagent containing a “buffering agent” in the mixed liquid in the second stage [the pH of which is lower than the isoelectric point of the protein to be measured, and is associated with the protein and aggregates. The acid dissociation constant (pKa) of the dye to be formed is mixed, and the pH of the mixed solution is set to a pH lower than the isoelectric point of the protein to be measured, And a pH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate ”. At this pH, the protein contained in the sample and the dye are associated with each other, A collection is formed.

(J) First stage: A reagent containing “surfactant” is mixed with a sample.
Second stage: A reagent containing a “buffering agent” in the mixed liquid of the first stage [the pH of which is lower than the isoelectric point value of the protein to be measured and is associated with the protein and aggregates. In the vicinity of the pH of the acid dissociation constant (pKa) of the dye to be formed].
Third stage: A reagent containing a “dye that associates with a protein to form an aggregate” is mixed with the mixed liquid in the second stage and brought into contact therewith. In this mixed liquid, “the isoelectric point of the protein to be measured” At a pH lower than the pH value, and a pH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate ”, and associates the dye contained in the sample with the dye. To form these aggregates.

(K) First step: a reagent containing a “buffering agent” [the pH of the dye that is less than the isoelectric point of the protein to be measured, and an acid of a dye that associates with the protein to form an aggregate A sample having a pH close to the pH of the dissociation constant (pKa) is mixed with the sample.
Second stage: A reagent containing a “dye that associates with a protein to form an aggregate” is mixed with the mixed liquid in the first stage and brought into contact therewith.
Third stage: A reagent containing “surfactant” is mixed in the mixed liquid in the second stage, and in this mixed liquid, “the pH is less than the value of the isoelectric point of the protein to be measured, and The protein contained in the sample is associated with the dye at a pH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate, and these aggregates are formed.

(L) First step: a reagent containing a “buffering agent” [the acid of the dye whose pH is less than the isoelectric point of the protein to be measured and which forms an aggregate by associating with the protein A sample having a pH close to the pH of the dissociation constant (pKa) is mixed with the sample.
Second stage: A reagent containing “surfactant” is mixed in the mixed liquid in the first stage.
Third stage: A reagent containing a “dye that associates with a protein to form an aggregate” is mixed with the mixed liquid in the second stage and brought into contact therewith. In this mixed liquid, “the isoelectric point of the protein to be measured” At a pH lower than the pH value, and a pH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate ”, and associates the dye contained in the sample with the dye. To form these aggregates.

(M) First stage: A reagent containing a “dye that associates with a protein to form an aggregate” and a reagent containing a “surfactant” are mixed.
Second stage: The sample is mixed and brought into contact with the liquid mixture of the first stage.
Third stage: A reagent containing a “buffering agent” in the mixed liquid in the second stage [the pH of which is lower than the isoelectric point of the protein to be measured, and is associated with the protein and aggregates. The acid dissociation constant (pKa) of the dye to be formed is mixed, and the pH of the mixed solution is set to a pH lower than the isoelectric point of the protein to be measured, And a pH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate ”. At this pH, the protein contained in the sample and the dye are associated with each other, A collection is formed.

(N) First stage: a reagent containing a “dye that associates with a protein to form an aggregate” and a reagent containing a “buffer” [the pH of which is less than the isoelectric point of the protein to be measured pH and a pH in the vicinity of the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate] is mixed.
Second stage: The sample is mixed and brought into contact with the liquid mixture of the first stage.
Third stage: A reagent containing “surfactant” is mixed in the mixed liquid in the second stage, and in this mixed liquid, “the pH is less than the value of the isoelectric point of the protein to be measured, and The protein contained in the sample is associated with the dye at a pH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate, and these aggregates are formed.

(O) First stage: a reagent containing “surfactant” and a reagent containing “buffer” [pH of which is lower than the isoelectric point of the protein to be measured, and protein In the vicinity of the pH of the acid dissociation constant (pKa) of the dye that aggregates and forms an aggregate].
Second stage: The sample is mixed with the mixed liquid of the first stage.
Third stage: A reagent containing a “dye that associates with a protein to form an aggregate” is mixed with the mixed liquid in the second stage and brought into contact therewith. In this mixed liquid, “the isoelectric point of the protein to be measured” At a pH lower than the pH value, and a pH near the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate ”, and associates the dye contained in the sample with the dye. To form these aggregates.

(P) First stage: A reagent containing a “dye that associates with a protein to form an aggregate” and a reagent containing a “surfactant” are mixed.
Second stage: A reagent containing a “buffering agent” in the mixed liquid of the first stage [the pH of which is lower than the isoelectric point value of the protein to be measured and is associated with the protein and aggregates. In the vicinity of the pH of the acid dissociation constant (pKa) of the dye to be formed].
Third stage: The sample is mixed with the liquid mixture of the second stage and brought into contact therewith. In this liquid mixture, “the pH is lower than the value of the isoelectric point of the protein to be measured and the protein is associated with The protein contained in the sample is associated with the dye at a pH near the pH of the acid dissociation constant (pKa) of the dye forming the aggregate, and these aggregates are formed.

(Q) First stage: a reagent containing a “dye that associates with a protein to form an aggregate” and a reagent containing a “buffer” [the pH of which is less than the isoelectric point of the protein to be measured pH and a pH in the vicinity of the pH of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate] is mixed.
Second stage: A reagent containing “surfactant” is mixed in the mixed liquid in the first stage.
Third stage: The sample is mixed with the liquid mixture of the second stage and brought into contact therewith. In this liquid mixture, “the pH is lower than the value of the isoelectric point of the protein to be measured and the protein is associated with The protein contained in the sample is associated with the dye at a pH near the pH of the acid dissociation constant (pKa) of the dye forming the aggregate, and these aggregates are formed.

(R) First stage: a reagent containing “surfactant” and a reagent containing “buffer” [the pH of which is lower than the isoelectric point of the protein to be measured, and the protein In the vicinity of the pH of the acid dissociation constant (pKa) of the dye that aggregates and forms an aggregate].
Second stage: A reagent containing “a dye that associates with a protein to form an aggregate” is mixed with the mixed liquid in the first stage.
Third stage: The sample is mixed with the liquid mixture of the second stage and brought into contact therewith. In this liquid mixture, “the pH is lower than the value of the isoelectric point of the protein to be measured and the protein is associated with The protein contained in the sample is associated with the dye at a pH near the pH of the acid dissociation constant (pKa) of the dye forming the aggregate, and these aggregates are formed.

  In the case where the aggregate formation process is performed in two or more stages, the pH of the mixed solution is “below the value of the isoelectric point of the protein to be measured and The pH is preferably near the pH of the value of the acid dissociation constant (pKa) of the dye that associates with the dye to form an aggregate.

(2) Flowing step In this flow step, after the formation of the aggregate in the aggregate formation step, a certain amount of the solution containing the aggregate is passed through a porous carrier made of an organic compound and having a pore diameter of 3 μm or more. As long as a certain amount of the solution containing the aggregate can be passed through a porous carrier made of the organic compound and having a pore diameter of 3 μm or more, there is no particular limitation, and any method may be used.
By passing the solution containing the aggregate, the aggregate is made of an organic compound and captured by a porous carrier having a pore size of 3 μm or more.

  When a certain amount of the solution containing the aggregate is passed through a porous carrier made of an organic compound and having a pore size of 3 μm or more, if the fixed amount is very small, the organic compound is made of an organic compound and has a pore size of 3 μm or more. Although it is held inside the porous carrier, when the maximum amount of the carrier is exceeded, it flows through the inside of the porous carrier made of this organic compound and having a pore diameter of 3 μm or more, and flows out from the side opposite to the added side.

  By the way, although it is a fixed amount of the solution containing the agglomerates, which is made of the organic compound and passed through a porous carrier having a pore size of 3 μm or more, the added amount of the organic compound becomes the pore size. However, the amount of the aggregate to be captured by a porous carrier having a particle size of 3 μm or more is increased, and as a result, even a lower concentration of protein can be measured, and the measurement becomes highly sensitive.

  The fixed amount is usually preferably 0.1 mL or more, more preferably 0.5 mL or more, particularly preferably 2 mL or more, and further preferably 5 mL or more.

  The upper limit of the certain amount is not particularly limited, but to be strong, it is the amount until reaching the maximum trapping amount (upper limit) of the substance made of the organic compound to be trapped by the porous carrier having a pore diameter of 3 μm or more. .

  In this flow-through step, a certain amount of the solution containing the aggregate is passed through a porous carrier made of an organic compound and having a pore size of 3 μm or more, but there is no particular limitation.

  For example, a solution containing the aggregate is added to a porous carrier made of an organic compound and having a pore size of 3 μm or more, and the solution containing the aggregate is passed through the porous carrier by gravity without applying pressure or suction force. May be.

  Further, the solution containing the aggregate is added to, injected or sucked into a porous carrier made of an organic compound and having a pore size of 3 μm or more, and is pressurized or sucked to obtain the solution containing the aggregate. You may pass through.

  Alternatively, in the case of a syringe filter in which a porous carrier made of an organic compound and having a pore diameter of 3 μm or more is housed in a housing that can be connected to a syringe (for example, in the case of “GD / X syringe filter” of Whatman Japan, etc.) The solution containing the aggregate is put into a syringe, the syringe and the syringe filter are connected, and the piston portion of the syringe is pushed, whereby the solution containing the aggregate is pushed out from the organic compound of the syringe filter. The solution containing the aggregate can be passed through a porous carrier having a pore diameter of 3 μm or more.

  In the case of a syringe filter housed in a housing in which a porous carrier made of an organic compound and having a pore size of 3 μm or more can be connected to a syringe (for example, in the case of “GD / X syringe filter” of Whatman Japan), a syringe and Connect the syringe filter, bring the other mouth part of the syringe filter opposite to the side where the syringe is connected into contact with the solution containing the aggregate, and pull the piston part of the syringe Thus, the solution containing the aggregate can be sucked into the syringe, whereby the solution containing the aggregate can be passed through a porous carrier made of an organic compound of the syringe filter and having a pore diameter of 3 μm or more.

(3) Color measurement step The color of the surface of the porous carrier made of an organic compound through which a certain amount of the solution containing the aggregate is passed in the flow-through step and having a pore diameter of 3 μm or more, that is, the color of the surface of the porous carrier There is no particular limitation on measuring the color in the color measurement step as long as the color of the surface of the porous carrier can be measured, and any method may be used.

  This is a measurement of the color of the surface of the porous carrier. The color generated on the surface of the porous carrier made of an organic compound through which a certain amount of the solution containing the aggregate is passed in the flow-through step has a pore diameter of 3 μm or more. When no protein is present in the sample, there is no coloration or light coloration.

On the other hand, when protein is present in the sample, the surface of the porous carrier made of an organic compound and having a pore diameter of 3 μm or more becomes deeply colored. Therefore, the color of the surface of the porous carrier is measured. Thus, it can be determined whether or not the protein is present in the sample.
That is, in this color measurement step, the color of the surface of the porous carrier is measured, and when there is no coloration or the coloration is light, there is no protein in the sample or less than a certain concentration (cutoff value) It is determined that (That is, it is determined as “negative”.)
Further, the color of the surface of the porous carrier is measured, and when the color is deep, it is determined that the protein is present in the sample or is at a certain concentration (cutoff value) or more. (That is, “positive” is determined.)

  Then, the measurement of the color of the surface of the porous carrier may be performed by visual observation, or may be performed by using a device such as a densitometer, a spot meter, or a CCD device. .

  Further, the color density of the surface of the porous carrier obtained by measuring a sample containing no protein (negative control sample or reagent blank sample) and / or a sample containing protein (positive control sample), and the sample, The presence of protein in the sample may be determined by comparing the color density of the surface of the porous carrier obtained by measurement.

  In addition, when quantitatively measuring the concentration of protein contained in a sample, a sample containing no protein (negative control sample or reagent blank sample) and / or one concentration of a sample having a known protein concentration (standard sample) or The color density of the surface of the porous carrier obtained by measuring multiple concentrations and the color density of the surface of the porous carrier obtained by measuring a sample with an unknown protein concentration. A quantitative value can be obtained by comparison (or calculation by proportional calculation, etc.).

  After passing a certain amount of the solution containing the aggregate in the flow step through a porous carrier made of an organic compound and having a pore size of 3 μm or more, until the color of the surface of the porous carrier is measured in this color measurement step The time depends on conditions such as the type, area and thickness of a porous carrier made of an organic compound to be used and having a pore size of 3 μm or more, a certain amount or viscosity of the solution containing the agglomerates, and the presence or absence of pressure or suction. Because it is different, I cannot say it in general.

  However, usually, a certain amount of the solution containing the aggregate is passed through a porous carrier made of an organic compound and having a pore size of 3 μm or more, and the color of the surface of the porous carrier can be measured immediately without taking any time. it can.

  Although the coloring of the surface of the porous carrier is stable, a certain amount of the solution containing the aggregate is passed through the porous carrier made of an organic compound and having a pore diameter of 3 μm or more, and the porous carrier is within 6 hours. It is preferable to measure the color of the surface.

II. A kit for measuring a protein in a sample The kit for measuring a protein in a sample according to the present invention comprises at least a “reagent containing a dye that associates with a protein to form an aggregate” and “a porous material composed of an organic compound and having a pore size of 3 μm or more. Sex carrier ".

1. Reagent The kit for measuring a protein in a sample of the present invention contains a reagent containing a dye that associates with a protein to form an aggregate.

(1) A dye that associates with a protein to form an aggregate In the reagent for measuring a protein in a sample of the present invention, a reagent that contains a dye that associates with a protein to form an aggregate forms an aggregate that associates with the protein. The dye is as described in detail in “2. Dye that associates with protein and forms aggregate” in “I. Method for measuring protein in sample”.

  If the concentration of the dye containing the protein to form aggregates in the reagent is too low, the sensitivity of the measurement cannot be obtained and the protein in the sample cannot be measured. It is preferable to contain in the reagent so that the concentration when it is made to be 1 μM or more, and it is more preferable to make it contained in the reagent so that the concentration when contacting with the protein in the sample is 10 μM or more. It is particularly preferable that the reagent is contained so that the concentration when contacting with a protein is 20 μM or more.

  In addition, if the concentration at which the reagent contains a dye that associates with the protein to form an aggregate is too high, coloration occurs on the surface of the porous carrier even if no protein is present in the sample. Since it is difficult to discriminate from the presence of protein in the medium, it is usually preferable that the reagent be contained in the reagent so that the concentration when contacting with the protein in the sample is 500 μM or less, and contact with the protein in the sample. It is more preferable that the reagent be contained in the reagent so that the concentration thereof is 100 μM or less, and it is particularly preferable that the reagent be contained so that the concentration when contacting with the protein in the sample is 50 μM or less.

(2) Surfactant A surfactant may be contained in a reagent containing a dye that forms an aggregate by associating with a protein, or other reagent, contained in the protein measurement kit of the sample of the present invention.
Reagents containing dyes that associate with proteins to form aggregates by containing surfactants in reagents or other reagents that contain dyes that associate with proteins to form aggregates (and other reagents) The color development degree (darkness) of the surface of the porous carrier after passing the mixed solution of the sample and the sample through a porous carrier made of an organic compound and having a pore size of 3 μm or more is the degree of color development in the sample containing protein. (Density) becomes high, and measurement can be performed with high sensitivity, and in the measurement of a sample not containing protein (negative control; or reagent blank), there is no color development on the surface of the porous carrier or very little The effect of facilitating the distinction between the sample containing protein and the sample not containing protein in the color measurement is limited. The "protein and association to a reagent containing a dye to form aggregates," or "other reagent" contained in the click quality measurement kit preferably contains a surfactant.
The surfactant is as described in detail in “4. Surfactant” in “I. Method for measuring protein in sample”.

  If the concentration of the surface active substance is too low, the effect of containing the surface active substance cannot be obtained. Usually, the surface active substance, the dye that associates with the protein to form an aggregate, and the protein in the sample are brought into contact with each other. The reagent is preferably contained in the reagent so that the concentration at the time is 0.0001% (W / V) or higher, and the reagent is added so that the concentration at the time of contact is 0.001% (W / V) or higher. It is more preferable to make it contain, and it is especially preferable to make it contain in the said reagent so that the density | concentration at the time of the said contact may be 0.005% (W / V) or more.

  If the concentration of the surfactant is too high, the viscosity of the solution containing the surfactant may increase. In such a case, the aggregate is formed by associating with the protein to a porous carrier made of an organic compound and having a pore size of 3 μm or more. Since an error may occur in the amount of the liquid mixture of the reagent containing the dye to be formed and the sample, the dye that forms an aggregate by associating with the surface active substance or protein is usually used. It is preferable to include a surfactant in the reagent so that the concentration when the protein is brought into contact is 5% (W / V) or less, and the concentration during the contact is 1% (W / V) or less. Thus, it is more preferable that the reagent is contained in the reagent, and it is particularly preferable that the reagent is contained so that the concentration at the time of contact is 0.1% (W / V) or less.

(3) pH
In the present invention, the protein contained in the sample is associated with a dye that associates with the protein to form an aggregate, and the pH at which these aggregates are formed is determined by the isoelectric point of the protein to be measured. The pH is preferably less than the value.
Thus, when the pH is less than the isoelectric point of the protein to be measured,
The charge of the protein to be measured is on the positive side, and it is easy to associate with the dye, which is preferable because the sensitivity of the protein measurement can be increased.

  Therefore, the pH at which the protein contained in the sample is associated with the dye that associates with the protein to form an aggregate and forms these aggregates is less than the isoelectric point of the protein to be measured. It is preferable to set the pH of the reagent contained in the protein measurement kit in the sample of the present invention so that the pH of the reagent of the present invention is adjusted.

Further, in the present invention, the protein contained in the sample is associated with the dye that forms an aggregate by associating with the protein, and the pH at which these aggregates are formed is determined by associating the aggregate with the protein. It is preferably near the pH of the acid dissociation constant (pKa) of the dye to be formed.
This is more preferably set to a pH within the range of ± 2 of the value of the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate during the formation of the aggregate. The pH is within the range of ± 1.5 of the value of acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate, and more preferably the dye that associates with this protein to form an aggregate. The pH is within a range of ± 1 of the value of the acid dissociation constant (pKa).

  Thus, at the time of forming the aggregate, the negative charge of the dye is reduced by setting the pH to be close to the pH of the acid dissociation constant (pKa) of the dye that associates with the protein and forms an aggregate as described above. This makes it easy to associate with the protein contained in the sample, thereby increasing the sensitivity of the protein measurement, which is preferable.

  Therefore, at the time of the formation of the aggregate, the protein in the sample of the present invention is adjusted so that the pH is close to the pH of the acid dissociation constant (pKa) of the dye that associates with the protein and forms an aggregate as described above. It is preferable to set the pH of the reagent contained in the measurement kit.

  From the above, at the time of the formation of the aggregate, the pH is set to a pH lower than the isoelectric point of the protein to be measured, and the acid dissociation constant (pKa) of the dye that associates with the protein to form the aggregate. It is particularly preferred that the pH be in the vicinity of the pH value of.

  Therefore, at the time of the formation of the aggregate, the pH is set to a pH lower than the isoelectric point of the protein to be measured, and the acid dissociation constant (pKa) of the dye that associates with the protein to form an aggregate is determined. It is particularly preferable to set the pH of the reagent contained in the protein measurement kit in the sample of the present invention so that the pH is close to pH.

  In addition, when the aggregate is formed, if the pH is too low, for example, less than pH 1, the protein is undesirably denatured or insolubilized.

  Therefore, it is preferable to set the pH of the reagent contained in the protein measurement kit in the sample of the present invention so that the pH does not become too low, for example, less than pH 1, during the formation of the aggregate. .

  When the aggregate is formed, the protein contained in the sample is associated with the dye that forms an aggregate by associating with the protein, and the pH at which these aggregates are formed is the pH as described above. In order to achieve this, it is preferable to use a buffering agent having such a pH (buffering agent having a buffering ability in these pH ranges).

As this buffer, a known buffer can be used.
For example, glycine (pKa1: 2.35), phosphoric acid (pKa1: 2.15), phthalic acid (pKa1: 2.95, pKa2: 5.41), citric acid (pKa1: 3.13, pKa2: 4.76), barbituric acid (pKa1: 4.04), succinic acid (pKa1: 4.21, pKa2: 5.64), acetic acid (pKa2: 4.76), various good buffers, etc. Can be mentioned.

  Therefore, at the time of forming the aggregate, the pH is set as described above, and thus a buffering agent (buffering agent having a buffering ability in these pH ranges) that is the pH is added to the protein in the sample of the present invention. It is preferable to make it contain in the reagent contained in a measurement kit.

  The other matters of this pH are as described in detail in “5. pH” of “I. Method for measuring protein in sample”.

(4) Configuration of Reagent The protein measurement kit in the sample of the present invention may contain one reagent, or may contain two or more plural reagents.
In the case of containing only one reagent, the reagent is a reagent containing a dye that associates with a protein to form an aggregate.
In the case of containing a plurality of reagents of two or more, the plurality of reagents contain a dye that forms one or more aggregates by associating one or two or more reagents with the protein.

Furthermore, the protein measurement kit in the sample of the present invention may contain a reagent containing a component other than a dye that associates with a protein to form an aggregate.
The kit for measuring a protein in a sample of the present invention includes, for example, a reagent containing a dye that associates with a protein to form an aggregate, a reagent that associates with a protein to form an aggregate, and a surfactant, a protein, Reagents containing dyes and buffers that associate to form aggregates, dyes that associate with proteins to form aggregates, reagents containing surfactants and buffers, reagents containing surfactants, contain buffers And a reagent containing a surfactant and a buffer, or a reagent comprising a sample diluent.
The embodiment of the reagent included in the kit for measuring the protein in the sample of the present invention is described in “(1) Aggregate formation step” in “6. Measurement operation” in “I. Method for measuring protein in sample”. As described in the above.

(5) Other reagent components In the present invention, the reagents contained in the protein measurement kit of the present invention include, for example, salts; antiseptics such as sodium azide, antibiotics or synthetic antibacterial agents as necessary. Agents; Stabilizers such as saccharides; Reaction activators; Substances related to elimination or influence suppression of measurement interfering substances contained in a sample; or other reagent components can be appropriately contained.

  In addition, as said salt, the compound etc. which consist of a cation and an anion can be mentioned, for example.

As the cation, any ion having a positive charge can be used without particular limitation.
The cation may be a monovalent cation or a divalent or higher valent cation.
And as this cation, a metal ion, an ammonium ion, or another cation etc. can be mentioned, for example.

  Examples of the metal ions include alkali metal ions, alkaline earth metal ions, transition metal ions, and other metal ions.

  Examples of alkali metal ions include lithium ions, sodium ions, or potassium ions.

  Examples of the alkaline earth metal ion include beryllium ion, magnesium ion, calcium ion, barium ion, and the like.

  Examples of transition metal ions include manganese ions, iron ions, cobalt ions, nickel ions, or copper ions.

  Examples of other metal ions include zinc ions and aluminum ions.

  Examples of ammonium ions include primary ammonium ions, secondary ammonium ions, tertiary ammonium ions, and quaternary ammonium ions.

  As other cations, for example, carbon atoms, silicon atoms, boron atoms, nitrogen atoms (in cases other than ammonium ions), phosphorus atoms, sulfur atoms, etc. are positively charged atoms or atomic groups Can be mentioned.

  Specific examples thereof include a choline ion in which a carbon atom has a positive charge.

  In addition, as this cation, a monovalent cation is preferable.

  And as this monovalent cation, an alkali metal ion is preferable. In particular, sodium ion is preferable.

    In addition, as this cation, only one type may be used, or a plurality of types may be used simultaneously.

  Moreover, as an anion, if it is an ion which has a negative charge, it will not be specifically limited, It can use.

  The anion may be a monovalent anion or a divalent or higher polyvalent anion.

  Examples of the anion include a halogen ion, an acid group made of an organic compound, or an acid group made of another inorganic compound.

  As a halogen ion, a fluorine ion, a chlorine ion, a bromine ion, an iodine ion etc. can be mentioned, for example.

  Examples of the acid group made of an organic compound include acetate ion, citrate ion, gluconate ion, or oxalate ion.

  Examples of acid groups composed of other inorganic compounds include sulfate ion, sulfite ion, disulfite ion, dithionite ion, thiosulfite ion, nitrate ion, nitrite ion, hyponitrite ion, peroxonitrite ion, Examples include phosphate ion, phosphite ion, diphosphite ion, hypophosphite ion, diphosphate ion, borate ion, carbonate ion, cyanate ion, isocyanate ion, or silicate ion. it can.

  In addition, as this anion, a monovalent anion is preferable.

  And as this monovalent anion, a halogen ion is preferable. In particular, a chlorine ion is preferable.

  Moreover, as this anion, only one type may be used, or a plurality of types may be used simultaneously.

2. Porous carrier The “porous carrier made of an organic compound and having a pore size of 3 μm or more” in the protein measurement kit of the present invention is made of an organic compound, has a large number of fine pores, and has a pore size of 3 μm or more. Any carrier can be used without particular limitation as long as it can pass the solution when added to the carrier.
As for the “porous support made of an organic compound and having a pore size of 3 μm or more”, “3. Porous support made of an organic compound and having a pore size of 3 μm or more” in “I. As described in detail in.

3. Others The kit for measuring the protein in the sample of the present invention contains, as necessary, “a reagent containing a dye that associates with a protein to form an aggregate” and “a porous material composed of an organic compound and having a pore size of 3 μm or more. Substances other than "sexual carrier" can be included.

  For example, “negative control sample (reagent blank sample)”, “positive control sample”, “standard substance (standard sample)”, “control table of measurement results (color of the porous carrier surface)”, etc. Can be mentioned.

4). Measurement of protein in sample The measurement kit for protein in a sample of the present invention is suitable for the above-mentioned "method for measuring protein in sample" of the present invention.
The measurement of protein in a sample according to the “method for measuring protein in sample” of the present invention using the protein measurement kit in the sample of the present invention is described in “I. Measurement of protein in sample” above. As described in "Method".

  EXAMPLES Hereinafter, although an Example demonstrates this invention more specifically in detail, this invention is not limited by description of this Example.

[Example 1] (Confirmation of the effect of using a porous carrier made of an organic compound and having a pore size of 3 μm or more)
The effect of using a porous carrier made of an organic compound and having a pore diameter of 3 μm or more in the method and kit for measuring protein in the sample of the present invention was confirmed.

1. Reagent (1) Reagent A
Preparation of an aqueous solution containing erythrosine B as a dye that associates with a protein to form an aggregate at a concentration of 40 μM and Triton X-100 as a surfactant at a concentration of 0.01% (W / V) Reagent A.

(2) Reagent B
A 0.2 M citrate-potassium hydroxide buffer (pH 2.3 (20 ° C.)) was prepared and used as reagent B.

2. Porous carrier As the porous carrier, the following porous carriers were used.
(1) A porous carrier made of an organic compound and having a pore diameter of less than 3 μm (cellulose mixed ester)
It is a porous carrier made of an organic compound and having a pore size of less than 3 μm, “trade name: cellulose mixed ester type membrane filter A010A025A, product code: 10010002, pore size: 0.10 μm, membrane: cellulose mixed ester, thickness: 110 μm, “Advantech Toyo Co., Ltd.” was designated as “a porous carrier made of an organic compound and having a pore diameter of less than 3 μm (cellulose mixed ester)”.

(2) A porous carrier made of an organic compound and having a pore size of 3 μm or more (cellulose mixed ester)
A porous carrier made of an organic compound and having a pore size of 3 μm or more, “trade name: cellulose mixed ester type membrane filter A500A025A, product code: 10150002, pore size: 5.00 μm, membrane: cellulose mixed ester, thickness: 160 μm, “Advantech Toyo Co., Ltd.” was defined as “a porous carrier made of an organic compound and having a pore size of 3 μm or more (cellulose mixed ester)”.

(3) A porous carrier made of an organic compound and having a pore diameter of less than 3 μm (cellulose acetate)
It is a porous carrier made of an organic compound and having a pore size of less than 3 μm. Was made of “a porous carrier made of an organic compound and having a pore diameter of less than 3 μm (cellulose acetate)”.

(4) A porous carrier made of an organic compound and having a pore size of 3 μm or more (cellulose acetate)
It is a porous carrier made of an organic compound and having a pore diameter of 3 μm or more. Was made of “a porous carrier made of an organic compound and having a pore size of 3 μm or more (cellulose acetate)”.

3. Sample (1) Protein-containing sample As a protein-containing sample, an aqueous solution containing 30 mg / L, that is, 3 mg / dL of human serum albumin (HSA) was prepared.

(2) Protein-free sample Pure water was prepared as a protein-free sample, that is, a protein-free sample.

4). Measurement (1) 500 μL of reagent (1) (2) B was added to 5,000 μL of reagent (1) (1) and mixed.

(2) Next, 150 μL of the protein-containing sample of (1) above was added to the mixed solution of (1) above, mixed, and contacted.

(3) Then, it was left at room temperature for 20 seconds.

(4) Next, the “porous support (cellulose mixed ester) made of an organic compound and having a pore size of less than 3 μm” of (1) above is set in a filtration device, and the upper surface of the porous support The total amount (5,650 μL) of the mixed solution after leaving (3) was added to and passed through.

(5) Immediately after the addition of the mixed solution in (4), the color of the surface of the porous carrier was measured visually.

  If the surface of the porous carrier is highly colored, it is determined that protein is present in the sample. If the surface of the porous carrier is not colored or lightly colored, the sample It was determined that no protein was present.

(6) Further, the measurement was performed by performing the operations as in (1) to (5) except that the sample was changed from the protein-containing sample of (3) to the protein-free sample of (2). .

(7) Further, the porous carrier is changed from “a porous carrier made of an organic compound and having a pore diameter of less than 3 μm (cellulose mixed ester)” in (2) above to “a porous carrier made of an organic compound and having a pore diameter of 3 μm or more”. The measurement was performed by performing the operations as described in (1) to (6) except that the porous support (cellulose mixed ester) was changed.

(8) Further, the porous carrier is changed from “a porous carrier made of an organic compound (cellulose mixed ester) having a pore diameter of less than 3 μm” (2) to a “pore made of an organic compound and having a pore diameter of less than 3 μm”. The measurement was carried out by performing the operations as described in (1) to (6) except that the porous support (cellulose acetate) was changed.

(9) Furthermore, the porous carrier is changed from “a porous carrier made of an organic compound (cellulose mixed ester) having a pore diameter of less than 3 μm” (2) to a “pore made of an organic compound and having a pore diameter of 3 μm or more”. The measurement was carried out by performing the operations as described in (1) to (6) except that the porous support (cellulose acetate) was changed.

5. Measurement Results (1) The measurement results are shown in FIG.
This figure shows the following.

(2) First, when a “porous carrier made of an organic compound and having a pore size of less than 3 μm (cellulose mixed ester)” is used as the porous carrier, protein (30 mg / L, ie, 3 mg / dL human serum albumin) The color of the surface of the porous carrier when the sample containing) is measured is dark, and it can be determined that protein is present in the sample.

  However, when this “porous carrier made of an organic compound and having a pore size of less than 3 μm (cellulose mixed ester)” is used, the color of the surface of the porous carrier when measuring a sample not containing protein (pure water) is: This was also dark, and it was determined that protein was present in the sample.

  That is, when “a porous carrier made of an organic compound and having a pore diameter of less than 3 μm (cellulose mixed ester)” is used as the porous carrier, it cannot be correctly determined that there is no protein in the sample. I understand.

  Therefore, it is confirmed that the protein in the sample cannot be measured in the method and the kit for measuring the protein in the sample using the “porous support made of an organic compound and having a pore size of less than 3 μm” as the porous carrier. It was.

(3) Next, when “a porous carrier made of an organic compound and having a pore size of 3 μm or more (cellulose mixed ester)” is used as the porous carrier, protein (30 mg / L, ie, 3 mg / dL human serum) When the sample containing albumin) is measured, the color of the surface of the porous carrier is dark, and it can be determined that the protein is present in the sample.

  In addition, when this “porous support made of an organic compound and having a pore size of 3 μm or more (cellulose mixed ester)” is used, the surface of the porous support is lightly colored when a sample containing no protein (pure water) is measured. It can be determined that no protein is present in the sample.

  That is, when “a porous carrier made of an organic compound and having a pore size of 3 μm or more (cellulose mixed ester)” is used as the porous carrier, the presence of protein in the sample and the absence of protein in the sample It can be seen that each can be correctly determined.

  Therefore, it was confirmed that the protein in the sample can be accurately measured in the protein measurement method and measurement kit of the present invention using a porous carrier made of an organic compound and having a pore size of 3 μm or more.

(4) Next, when “a porous carrier made of an organic compound and having a pore size of less than 3 μm (cellulose acetate)” is used as the porous carrier, protein (30 mg / L, ie 3 mg / dL human serum albumin) The color of the surface of the porous carrier when the sample containing) is measured is dark, and it can be determined that protein is present in the sample.

  However, when this “porous support made of an organic compound and having a pore size of less than 3 μm (cellulose acetate)” is used, the color of the surface of the porous support when a sample not containing protein (pure water) is measured. Was also dark, and it was determined that protein was present in the sample.

  That is, when “a porous carrier made of an organic compound and having a pore diameter of less than 3 μm (cellulose acetate)” is used as the porous carrier, it may not be possible to correctly determine that there is no protein in the sample. I understand.

  Therefore, the protein measurement method and measurement kit using a “porous support made of an organic compound and having a pore diameter of less than 3 μm” as the porous support cannot measure the protein in the sample. It was confirmed in some cases.

(5) Next, when “a porous carrier made of an organic compound and having a pore size of 3 μm or more (cellulose acetate)” is used as the porous carrier, protein (30 mg / L, ie, 3 mg / dL human serum albumin) The color of the surface of the porous carrier when the sample containing) is measured is dark, and it can be determined that protein is present in the sample.

  In addition, when this “porous support made of an organic compound (cellulose acetate) having a pore size of 3 μm or more” is used, the surface of the porous support when the sample not containing protein (pure water) is measured is thin, It can be determined that no protein is present in the sample.

  That is, when a porous carrier made of an organic compound and having a pore size of 3 μm or more (cellulose acetate) is used as a porous carrier, the presence of protein in the sample and the absence of protein in the sample It can be seen that each can be correctly determined.

  Therefore, the protein measuring method and the measuring kit of the present invention using a porous carrier made of an organic compound and having a pore diameter of 3 μm or more can confirm the protein in the sample accurately even in this case. It was.

  As described above, the protein measurement method and measurement kit of the present invention using a porous carrier made of an organic compound and having a pore size of 3 μm or more can accurately measure the protein in the sample. Thus, it was confirmed that when a “porous support made of an organic compound and having a pore size of less than 3 μm” was used, the protein in the sample could not be accurately measured.

Claims (6)

1) Aggregate formation step in which a sample and a dye that associates with a protein to form an aggregate are brought into contact, thereby associating the protein contained in the sample with the dye and forming these aggregates;
2) After the formation of the aggregate, a flow step of passing a certain amount of the solution containing the aggregate through a porous carrier made of an organic compound and having a pore size of 3 μm or more (however, the fixed amount is an amount of 2 mL or more ) And 3) a method for measuring a protein in a sample, comprising a color measurement step of measuring the color of the surface of the porous carrier. The predetermined amount is an amount of more than 5 mL, the measurement methods of protein in a sample according to claim 1 Symbol placement. In aggregate formation step, the coexistence of surfactant, method of measuring protein in a sample according to claim 1 or claim 2 Symbol placement. The method for measuring a protein in a sample according to claim 3 , wherein the surfactant is a surfactant. The method for measuring a protein in a sample according to claim 4 , wherein the surfactant is a nonionic surfactant, an amphoteric surfactant, an anionic surfactant, or a cationic surfactant. The method for measuring a protein in a sample according to claim 4 or 5 , wherein the surfactant is a nonionic surfactant.