WO2014057880A1 - Measurement reagent and measurement method for measuring c-reactive protein - Google Patents

Abstract

A measurement reagent for use in the measurement of C-reactive protein by a latex coagulation nephelometry method comprises particles each having, on the surface thereof, a group capable of reacting specifically with C-reactive protein. In a particle size distribution curve wherein the horizontal axis represents the particle diameter (x (nm)) of each of the particles and the vertical axis represents the frequency (y (%)) of the particles in terms of mass, at least one peak appears in a region in which x is less than 200 and at least one peak appears in a region in which x is equal to or more than 200, and the area bounded by a line connecting a coordinate point P1 (x1, y1) to a coordinate point V1 (x3, ymin), a line connecting the coordinate point V1 to a coordinate point V2 (x4, ymin), a line connecting the coordinate point V2 to a coordinate point P2 (x2, y2) and a line connecting the coordinate point P2 to the coordinate point P1 in the particle size distribution curve is 300 or more.

Description

Measuring reagent and measuring method for C-reactive protein measurement

The present invention relates to a measuring reagent and a measuring method for measuring C-reactive protein.
This application claims priority based on Japanese Patent Application No. 2012-227105 for which it applied to Japan on October 12, 2012, and uses the content here.

In the field of clinical tests, various diseases are diagnosed using biological samples (blood, urine, etc.). One criterion for this diagnosis is C-reactive protein (hereinafter also referred to as “CRP”). It is known that CRP is hardly contained in the blood in a normal state, but rapidly increases when inflammation or tissue destruction occurs.
As a CRP measurement method, an immunoassay method utilizing an antigen-antibody reaction is generally used, and in particular, a latex agglutination turbidimetry is widely used. In the latex agglutination turbidimetry, latex in which latex particles carrying an antibody that specifically reacts with CRP or a fragment thereof are dispersed is used as a measurement reagent. A test sample is added to this measurement reagent, and the degree of aggregation of latex particles due to the reaction between CRP and an antibody or fragment thereof is optically measured to detect and quantify CRP as a measurement target substance.
As an antibody used when measuring CRP in a test sample by latex agglutination turbidimetry, a polyclonal antibody or a monoclonal antibody is usually used. Latex particles are inexpensive, available in large quantities, and have consistent quality (for example, polystyrene or co-polymer of styrene and styrene sulfonic acid, acrylic acid, acrylic ester, methacrylic ester, acrylonitrile, etc.). Particles made of polymers and the like are used.

Since the CRP concentration in the test sample varies depending on the disease state, the CRP measurement is required to be able to measure a wide range of measurement concentrations.
However, in the latex agglutination turbidimetry, it is difficult to cover a wide measurement range when the particle size of the contained latex particles is single. Thus, it has been proposed to use latex particles having different particle diameters together or to contain free monoclonal antibodies or fragments thereof in the latex (for example, Patent Document 1).
However, it is not always possible to stably measure CRP in a test sample from a low concentration range to a high concentration range simply by using large and small particles together. For example, there is a problem that the small particle size particles are first aggregated in the low concentration region of CRP, and the large particle size necessary for promoting the growth of the aggregated particles is not aggregated in the low concentration region of CRP.

On the other hand, a method of using phosphorylcholine labeled with a fluorescent substance or the like has been proposed as a rapid and highly sensitive method for measuring CRP in serum in the normal range (Patent Document 2). In this method, measurement by a sandwich type measurement method is performed using the specific binding property between phosphorylcholine and CRP. However, since this method uses labeled phosphorylcholine, the reagent is more expensive than the latex aggregation turbidimetric method.

Japanese Unexamined Patent Publication No. 2004-191332 Japanese Unexamined Patent Publication No. 2000-249703

The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide a measuring reagent and a measuring method that can stably measure CRP in a test sample from a low concentration range to a high concentration range by latex agglutination turbidimetry. And

In order to achieve the above object, the present invention employs the following configuration.
The measuring reagent according to the first aspect of the present invention is a measuring reagent for measuring C-reactive protein by latex agglutination turbidimetry, and has particles having groups on the surface that specifically react with C-reactive protein. A particle size distribution curve containing the particle diameter x (nm) of the particles as a horizontal axis and the mass conversion frequency y (%) as a vertical axis, at least one peak in a region of x <200, and a region of 200 ≦ x In the particle size distribution curve, coordinates P1 (x1, y1), V1 (x3, _ymin ), V2 (x4, _ymin ), P2 (x2, y2), and P1 The area of the region surrounded by the straight line connecting sequentially is 300 or more.
However, x1 and y1 respectively indicate the particle size and frequency of the peak having the largest particle size among the peaks in the region of x <200.
Each of x2 and y2 represents the particle size and frequency of the peak having the smallest particle size among the peaks in the region of 200 ≦ x.
y _min represents the minimum value of y in the region of x1 <x <x2.
x3 indicates the minimum value of x where y = y _min in the region of x1 <x <x2.
x4 indicates the maximum value of x where y = y _min in the region of x1 <x <x2.

In the measurement reagent according to the first aspect of the present invention, the group that specifically reacts with the C-reactive protein may be a phosphorylcholine group.
The measuring reagent according to the first aspect of the present invention has a surfactant having a steroid skeleton hydrocarbon group to which a substituent may be bonded and a group represented by the following general formula (2) in the molecular structure. An agent may further be included.

[Wherein, X represents a linear alkylene group having 3 to 5 carbon atoms to which a hydroxyl group may be bonded. ]

The measurement reagent according to the first aspect of the present invention may be a kit including a first liquid that is a particle dispersion in which the particles are dispersed, and a second liquid that includes the surfactant.
In the measurement method according to the second aspect of the present invention, the CRP concentration in the test sample is measured by the latex agglutination nephelometry using the measurement reagent according to the first aspect.
The measurement method according to the second aspect of the present invention includes a step of mixing a test sample and the measurement reagent according to the first aspect to prepare a reaction solution, and measuring the absorbance of the reaction solution, The concentration of the surfactant in it may be 0.15 to 0.40 mass%.

According to the above aspect of the present invention, it is possible to provide a measuring reagent and a measuring method capable of stably measuring CRP in a test sample from a low concentration region to a high concentration region by latex agglutination turbidimetry.

Coordinates P1, V1, and V2 in a particle size distribution curve with the horizontal axis representing the particle size x (nm) of the particles contained in the measurement reagent according to the first embodiment of the present invention and the vertical axis representing the frequency y (%) in terms of mass. , P2, and the area of a region surrounded by a straight line connecting those coordinates sequentially. 2 is a particle size distribution curve of RA1 to RA15 produced in Examples. FIG. 5 is a graph (CRP concentration is 0 to 30 mg / dL) with the measurement result of the absorbance change amount Abs in Examples 1 to 27 in the examples as the vertical axis and the CRP concentration (mg / dL) of the test sample as the horizontal axis. 5 is a graph (CRP concentration is 0 to 0.94 mg / dL) with the measurement result of the absorbance change amount Abs of Examples 1 to 27 in the example as the vertical axis and the CRP concentration (mg / dL) of the test sample as the horizontal axis.

<Measurement reagent>
The measurement reagent according to the first embodiment of the present invention is a measurement reagent for CRP measurement by latex agglutination turbidimetry, and a group that specifically reacts with CRP (hereinafter also referred to as CRP-reactive group). Includes particles (hereinafter also referred to as latex particles) on the surface.
As the latex particles, known latex particles, for example, particles in which an anti-CRP antibody or a fragment thereof is supported on the surface of the carrier particles can be used.
However, anti-CRP antibodies are derived from living organisms, and particles carrying anti-CRP antibodies and fragments thereof on the surface are expensive.
Therefore, the present inventors have come up with the use of particles having phosphorylcholine groups on the surface as CRP-reactive groups. As will be described later, these particles can be produced at low cost using a monomer having a phosphorylcholine group in the molecule. Therefore, an inexpensive measurement reagent can be realized by using particles having phosphorylcholine groups on the surface as latex particles.
The phosphorylcholine group is a group represented by the following chemical formula (1) and specifically reacts with CRP.

The method for producing the particles having phosphorylcholine groups on the surface is not particularly limited. For example, a particle having a phosphorylcholine group on its surface is obtained by suspension polymerization or emulsion polymerization of a monomer having a phosphorylcholine group in the molecule alone or together with another monomer copolymerizable with this monomer. Obtainable.
Examples of the monomer having a phosphorylcholine group in the molecule include compounds having a phosphorylcholine group and a polymerizable double bond in the molecule (for example, 2- (meth) acryloyloxyphosphorylcholine).
As the other monomer, for example, any monomer having a polymerizable double bond in the molecule and not having a phosphorylcholine group can be used, and the type thereof is not particularly limited. Specific examples include vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylic acid ester, and methacrylic acid ester. Among these, at least one selected from styrene, vinyltoluene, and methyl methacrylate is preferable because it is difficult to inhibit the reactivity of the phosphorylcholine group present on the surface.
When the monomer having a phosphorylcholine group is copolymerized with another monomer, the ratio of the monomer having a phosphorylcholine group is 1.5 to 7.5 parts by mass with respect to 100 parts by mass of the other monomer. It is preferable to be in the range of parts. If it is 1.5 mass parts or more, sufficient reactivity with respect to CRP is securable. If it is within 7.5 parts by mass, the reactivity is not inhibited by steric hindrance between phosphorylcholine groups.

In the measurement reagent according to the first embodiment of the present invention, the particle size distribution with the horizontal axis representing the particle size x (nm) of the latex particles contained in the measurement reagent and the vertical axis representing the mass conversion frequency y (%). The curve has at least one peak in the region of x <200 and at least one peak in the region of 200 ≦ x.
The particle size distribution curve can be obtained by analyzing a particle dispersion obtained by dispersing the latex particles in a dispersion medium such as water by a dynamic light scattering (DLS) method. Usually, submicron particles have Brownian motion in a dispersion medium, and the intensity of scattered light changes (fluctuates) with time when irradiated with laser light. For this fluctuation of the scattered light intensity, for example, an autocorrelation function is obtained by using a photon correlation method (JIS Z8826), a diffusion coefficient indicating a Brownian motion velocity is calculated by a cumulant analysis, and Einstein-Stokes's The average particle size can be determined using the formula. Further, by analyzing the fluctuation of the scattered light intensity using, for example, a polydispersity index (PI) by the cumulant method, the particle size distribution can be obtained.

When the particle size distribution curve has at least one peak in the region of x <200, CRP can be stably detected in the high concentration region.
In the particle size distribution curve, the number of peaks present in the region of x <200 is preferably 1 or 2, and more preferably 1.
The particle diameter of the peak existing in the region of x <200 is preferably in the range of 100 nm or more and less than 200 nm.
The peak particle size refers to the particle size corresponding to the peak top of the peak (the particle size with the highest frequency).

When the particle size distribution curve has at least one peak in the region of 200 ≦ x, CRP can be stably detected in the low concentration region.
In the particle size distribution curve, the number of peaks present in the region of 200 ≦ x is preferably 1 or 2, and more preferably 1.
The peak particle size existing in the region of 200 ≦ x is preferably in the range of 200 nm to 350 nm.

In the embodiment of the present invention, in the particle size distribution curve, coordinates P1 (x1, y1), V1 (x3, _ymin ), V2 (x4, _ymin ), P2 (x2, y2), and P1 are sequentially set. The area of the region surrounded by the connecting straight line is 300 or more, preferably 500 to 2000, and more preferably 1000 to 2000.
Each of x1 and y1 represents the particle size and frequency of the peak having the largest particle size (hereinafter also referred to as peak 1) among the peaks in the region of x <200.
Each of x2 and y2 represents the particle size and frequency of the peak having the smallest particle size (hereinafter also referred to as peak 2) among the peaks in the region of 200 ≦ x.
y _min represents the minimum value of y in the region of x1 <x <x2.
x3 indicates the minimum value of x where y = y _min in the region of x1 <x <x2.
x4 indicates the maximum value of x where y = y _min in the region of x1 <x <x2.
That is, the coordinate P1 indicates the position of the peak top of peak 1, and the coordinate P2 indicates the position of the peak top of peak 2. Y _min is the bottom of the valley between peak 1 and peak 2, coordinate V1 is the position closest to peak 1 at the bottom of the valley, and coordinate V2 is closest to peak 2 at the bottom of the valley. Indicates the position.

On the particle size distribution curve, V1 and V2 are different when there is not one value of x satisfying y = y _min between P1 and P2. When V1 and V2 are different (when x3 ≠ x4), the area surrounded by the straight line connecting the coordinates P1, V1, V2, P2, and P1 is a quadrangle.
On the particle size distribution curve, when there is one value of x where y = y _min between P1 and P2, V1 and V2 are the same. When V1 and V2 are the same (when x3 = x4), a region surrounded by a straight line connecting the coordinates P1, V1, V2, P2, and P1 is a triangle.

The larger the area, the clearer the two peaks 1 and 2 that are closest to each other with a particle size of around 200 nm. If this area is 300 or more, CRP in the test sample can be stably measured from a low concentration range to a high concentration range. The reason why the CRP in the test sample can be stably measured from the low concentration range to the high concentration range when this area is 300 or more is as follows. (1) Particles having a particle size near P1 and particles near P2 Due to the generation of composite agglomerates of particles having a diameter, large agglomerates are efficiently generated even in a low concentration range of CRP, and (2) the growth of a single agglomerate of particles having a particle size near P1 is Since it is slow, it is considered that agglomerates proportional to the concentration are synergistically acting even in the high concentration region.
On the other hand, when the area of the region is 2000 or less, in the low concentration region of CRP, from the size of the surface area, the antigen-antibody reaction is likely to cause aggregation and the particle size near P1 is likely to aggregate. Complex aggregation with particles having a particle size in the vicinity of P2 that tends to increase the absorbance when aggregated tends to occur. Therefore, large aggregates are efficiently generated in a low concentration range, and the change in absorbance is easy to observe.

The area of a region surrounded by a straight line that sequentially connects the coordinates P1, V1, V2, P2, and P1 can be obtained from the value of each coordinate by the following formula in either case of a square or a triangle.
Area of region = {(x2−x1) × (larger value of y1 and y2−y _min )} − {(x3−x1) × (y1−y _min ) / 2} − {(x2−x4) × (y2-y _min ) / 2}-{(x2-x1) × (| y1-y2 |) / 2}

With reference to FIG. 1, a specific example will be described to describe the areas of the coordinates P1, V1, V2, P2, and the area surrounded by a straight line connecting these coordinates in sequence. FIG. 1 is a particle size distribution curve (horizontal axis: particle diameter x (nm), vertical axis: frequency y (%) in terms of mass) of latex particles contained in RA1 and RA4 manufactured in Examples described later.
The particle size distribution curve of latex particles contained in RA1 has one peak in the region of x <200 and one peak in the region of 200 ≦ x. P1 is (100, 14.57) and P2 is (300, 5.77). The minimum value y _min of the frequency y between P1 and P2 is 0, V1 is (126.519, 0), and V2 is (193.604, 0). Therefore, the area of the quadrangular region surrounded by the straight line that sequentially connects P1, V1, V2, P2, and P1 is about 1420.
The particle size distribution curve of latex particles contained in RA4 has one peak in the region of x <200 and one peak in the region of 200 ≦ x. P1 is (180, 14.69), and P2 at the peak top of the peak in the region having a particle diameter of 200 nm or more is (300, 3.93).
The minimum value y _min of the frequency y between P1 and P2 is 0.44, and V1 and V2 coincide with each other (229.412, 0.44). Therefore, the area surrounded by the straight lines connecting P1, V1, V2, P2, and P1 in sequence is a triangle, and its area is about 489.

In the particle size distribution curve, the ratio of the particle size x1 of peak 1 to the particle size x2 of peak 2 is preferably x1: x2 = 1: 1.3 to 1: 3.5 (x2 with respect to x1 is 1. Is preferably in the range of 3 to 3.5), more preferably 1: 1.5 to 1: 3.5 (x2 with respect to x1 is more preferably in the range of 1.5 to 3.5). .
When x2 is 1.3 times or more of x1, the area of the region tends to be 300 or more. When x2 is 3.5 times or less of x1, a large aggregate is efficiently generated, and it becomes easy to observe a change in absorbance in a low concentration region of CRP.

In the particle size distribution curve, the ratio of the frequency y1 of peak 1 to the frequency y2 of peak 2 is preferably y1: y2 = 8: 2 to 5: 5 (y1 is 0.8 relative to the sum of y1 and y2). To 0.5 and y2 is preferably in the range of 0.2 to 0.5).
When y2 is equal to or smaller than y1, a change in absorbance at a high concentration range of CRP is easily observed. If y1 is 4 times or less of y2, a change in absorbance at a low concentration range of CRP is easily observed.

The latex particles are usually dispersed in water or the like and used as a particle dispersion.
The method for preparing the latex particles or the particle dispersion in which they are dispersed is not particularly limited. For example, a particle dispersion in which the latex particles are dispersed can be prepared by preparing a plurality of particle dispersions having different average particle diameters as a particle dispersion for preparing a measurement reagent and mixing them. Thereafter, drying, dilution, or the like may be performed as necessary.
At least one of the plurality of particle dispersions to be mixed at this time is a particle dispersion having an average particle diameter of less than 200 nm, and at least one is a particle dispersion having an average particle diameter of 200 nm or more. As described above, the average particle diameter is obtained by analyzing the particle dispersion by the dynamic light scattering method. In the particle dispersion for preparing the measurement reagent, the particle size distribution curve measured in the same manner as described above preferably has one peak.
The above-mentioned reaction liquid obtained by suspension polymerization or emulsion polymerization of a monomer having a phosphorylcholine group and a polymerizable double bond in the molecule can be used as a particle dispersion for preparing a measurement reagent. . By carrying out suspension polymerization or emulsion polymerization a plurality of times under different polymerization conditions, a plurality of particle dispersions having different average particle diameters can be obtained.
By adjusting the average particle diameter, particle size distribution, etc. of the particle dispersion for preparing the measurement reagent, the area of the region surrounded by the straight line connecting the coordinates P1, V1, V2, P2, and P1 can be adjusted. For example, when two particle dispersions are mixed, the area of the region increases as the difference between the average particle diameters increases. In addition, even if the difference in average particle diameter is the same, the narrower the particle size distribution of the respective particle dispersions, the larger the area of the region.

When the latex particle is a particle having a phosphorylcholine group on the surface, the measurement reagent according to the first embodiment of the present invention has a steroid skeleton hydrocarbon group to which a substituent may be bonded in the molecular structure. And a surfactant having a group represented by the following general formula (2) (hereinafter referred to as “surfactant (A)”).

[Wherein, X represents a linear alkylene group having 3 to 5 carbon atoms to which a hydroxyl group may be bonded. ]

As described above, the latex particles are preferably particles having phosphorylcholine groups on the surface. However, according to the study by the present inventors, when particles having phosphorylcholine groups on the surface are applied to CRP measurement by latex agglutination turbidimetry, particles having anti-CRP antibodies or fragments thereof supported on the surface are used. Compared to the above, the measurement efficiency of CRP is lowered. Therefore, as a result of further investigation, when particles having phosphorylcholine groups on the surface are used as latex particles, particles having an anti-CRP antibody or fragment thereof supported on the surface are used in combination with the surfactant (A). It has been found that measurement of CRP by the latex agglutination turbidimetry can be stably and efficiently performed from a low concentration range to a high concentration range even without this.
In addition, even if the surfactant (A) is combined with an antibody or fragment thereof generally used for CRP measurement by latex agglutination turbidimetry, the above effect cannot be obtained.

Surfactant (A) is a compound having a steroid skeleton hydrocarbon group to which a substituent may be bonded and a group represented by the general formula (2) in the molecular structure.

The hydrocarbon group of the steroid skeleton in the hydrocarbon group of the steroid skeleton possessed by the surfactant (A) is represented by the following formula (3).

Examples of the substituent which may be bonded to the hydrocarbon group of the steroid skeleton include a hydroxyl group and an alkyl group, and a hydroxyl group is preferable.

The surfactant (A) has a hydrocarbon group having a steroid skeleton to which the substituent may be bonded at one end of the molecular structure, and a group represented by the general formula (2) at the other end. A compound is preferable, and a compound represented by the following general formula (4) is particularly preferable.

[Wherein R ¹ represents a linear or branched alkylene group having 1 to 9 carbon atoms. R ² to R ³ each independently represents a group represented by the following general formula (I), a hydrogen atom, or an alkyl group having 1 to 9 carbon atoms, and one or both of R ² and R ³ are It is group represented by the said general formula (I). ]

[Wherein, X is the same as defined above, and R ⁴ represents a linear or branched alkylene group having 1 to 9 carbon atoms. ]

Typical examples of the compound represented by the formula (4) include compounds represented by the following formulas (4-1) to (4-5).

A commercially available compound can be used for the compound represented by Formula (4). For example, as a commercial product of the compound represented by the formula (4-1) (3-[(3-colamidopropyl) dimethylammonio] propanesulfonate), CHAPS (product name) manufactured by Dojindo Laboratories Co., Ltd. Can be mentioned. As a commercial product of the compound represented by the formula (4-2) (3-[(3-colamidopropyl) dimethylammonio] -2-hydroxypropanesulfonate), CHAPSO (product of Dojindo Laboratories) Name).

The measurement reagent according to the first embodiment of the present invention may further contain other components other than the latex particles and the surfactant (A) as necessary, as long as the effects of the present invention are not impaired. Good. As other components, known additives can be used.
For example, a surfactant, a preservative such as sodium azide, a stabilizer such as sucrose or bovine serum albumin, a buffering agent such as sodium acetate, etc. can be included in order to enhance reactivity, stability and the like.
When particles having a phosphorylcholine group on the surface are used as latex particles, it is preferable to further contain a calcium ion source such as calcium chloride because the phosphorylcholine group easily binds specifically to CRP in the presence of Ca ²⁺ .

When the measurement reagent according to the first embodiment of the present invention includes other components other than the latex particles, the measurement reagent according to the first embodiment of the present invention includes one component containing all components in one agent. It may be a measuring reagent of a type. In addition to the agent containing latex particles, it may be a multi-drug type measuring reagent further comprising one or more agents containing part or all of other components.
When a surfactant is included as another component, the surfactant may be adsorbed on the surface of the latex particle and the storage stability may be impaired, so the latex particle and the surfactant may be blended in a different agent. preferable.

The measurement reagent according to the first embodiment of the present invention is preferably a kit including a first liquid that is a particle dispersion in which the latex particles are dispersed, and a second liquid that includes a surfactant. Such a kit can be used for CRP measurement by latex agglutination turbidimetry as it is. For example, what added and mixed the 2nd liquid and the 1st liquid to the test sample sequentially or simultaneously can be used for the measurement of absorbance as it is.
In this kit, the first liquid preferably does not contain a surfactant. The second liquid preferably does not contain the latex particles.

The explanation of the latex particles in the first liquid is the same as described above.
The latex particles are preferably particles having phosphorylcholine groups on the surface.
The amount of latex particles in the first liquid is preferably such that the concentration when mixed with the second liquid is 1% by mass or less with respect to the total mass of the obtained liquid mixture. If it is 1% by mass or less, it can be applied to many existing measuring instruments. The minimum of content is not specifically limited, What is necessary is just more than 0 mass%.
The first liquid contains preservatives such as sodium azide, stabilizers such as sucrose, and buffering agents such as sodium acetate in order to enhance reactivity, stability, etc., as long as the effects of the present invention are not impaired. Also good.

As the surfactant in the second liquid, the same surfactant as described above can be used.
When the latex particles contained in the first liquid are particles having a phosphorylcholine group on the surface, the surfactant is preferably the surfactant (A).
The content of the surfactant (A) in the second liquid is preferably 10% by mass or less with respect to the total mass of the second liquid. If it exceeds 10% by mass, there is a possibility that it cannot be completely dissolved. The minimum of content is not specifically limited, What is necessary is just more than 0 mass%.
The second liquid contains a preservative such as sodium azide, a stabilizer such as bovine serum albumin, and a buffer such as sodium acetate in order to enhance reactivity, stability, etc. within the range not impairing the effects of the present invention. May be.
When the latex particles are particles having a phosphorylcholine group as a CRP-reactive group, the second liquid preferably contains a calcium source such as calcium chloride. Storage stability improves by making a 2nd liquid contain a calcium source instead of a 1st liquid.
The second liquid can be prepared by dissolving or dispersing each component in water or the like.

<Measurement method>
In the measurement method according to the second embodiment of the present invention, the CRP concentration in the test sample is measured by the latex agglutination nephelometry using the measurement reagent according to the first embodiment of the present invention.
As the test sample, any test sample that has been conventionally subjected to CRP measurement by latex agglutination turbidimetry can be used, and examples thereof include human serum samples.
Measurement of the CRP concentration in the test sample can be carried out in the same procedure as in the usual latex agglutination turbidimetry.
For example, there is a method of preparing a reaction solution by mixing a test sample and a measurement reagent and measuring the absorbance of the obtained reaction solution.
If the reaction solution is allowed to stand under conditions where the CRP reactive group on the latex particle surface and CRP react specifically, if CRP is contained in the test sample, latex particles aggregate in the reaction solution. The turbidity of the reaction solution increases and the absorbance increases. The higher the CRP concentration in the test sample, the greater the amount of change in absorbance. A calibration curve is prepared in advance using a standard sample with a known CRP concentration, and immediately after mixing the test sample and the measuring reagent, and after mixing, the CRP reactive group on the latex particle surface and CRP react specifically. The CRP concentration in the test sample can be quantified from the amount of change in the absorbance by measuring the absorbance of the reaction solution after being allowed to stand for an arbitrary time below.

The amount of the measurement reagent used is set in consideration of the desired latex particle concentration, surfactant (A) concentration, etc. in the reaction solution at the time of absorbance measurement.
The latex particle concentration in the reaction solution during the absorbance measurement is preferably 1.0% by mass or less with respect to the total mass of the reaction solution. If it is 1.0% by mass or less, it can be used for many measuring instruments, and a difference in absorbance due to aggregation can be sufficiently detected. The minimum of content is not specifically limited, What is necessary is just more than 0 mass%.
When the measurement reagent contains a surfactant (A), the concentration of the surfactant (A) in the reaction solution at the time of absorbance measurement is in the range of 0.15 to 0.40 mass% with respect to the total mass of the reaction solution. Is preferred. The effect by containing surfactant (A) is fully acquired as the surfactant (A) density | concentration at the time of a measurement is 0.15 mass% or more. When the content is 0.40% by mass or less, aggregation inhibition due to the dispersion stabilizing action of the surfactant does not occur.

When the measurement reagent according to the first embodiment of the present invention is the kit, the test sample and the measurement reagent according to the first embodiment of the present invention are mixed with the second liquid and the first liquid. Can be carried out by mixing them sequentially or simultaneously. Preferably, the test sample and the second liquid are mixed and allowed to stand for an arbitrary period of time, and then the first liquid is added thereto, or the test sample, the second liquid, and the first liquid are collectively This is done by mixing. In particular, it is preferable that the test sample and the second liquid are mixed and allowed to stand for an arbitrary time, and then the first liquid is added thereto. By preliminarily mixing the test sample with the second solution, the CRP in the test sample is solubilized and the reactivity is improved.
The standing after the test sample and the second liquid are mixed is preferably performed under conditions of 15 to 40 ° C. The standing time is not particularly limited, but is usually about 1 to 15 minutes.

The standing conditions after mixing the test sample and the measurement reagent according to the first embodiment of the present invention (conditions in which the CRP-reactive group on the latex particle surface and CRP react specifically) are the CRP on the latex particle surface. It can be set according to the reactive group. For example, when the CRP-reactive group is a phosphorylcholine group, the standing temperature is preferably 15 to 40 ° C., and the standing time is preferably 1 to 15 minutes.
The absorbance of the mixed solution can be measured by a conventional method, and can be measured, for example, under the conditions shown in Examples described later.
These series of operations can be carried out using a general-purpose automatic measuring apparatus used for measurement by latex agglutination turbidimetry. It may be performed manually.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.
In the following examples, the particle size distribution and the average particle size were measured by the following procedure.
(Particle size distribution and average particle size)
The particle size distribution and average particle size of the latex particles were determined by diluting the latex particle dispersion with an aqueous NaCl solution having a concentration of 10 mM so that the concentration of the latex particles was 1% by mass, and a special concentrated particle size analyzer FPAR-1000 (Otsuka It was calculated | required by analyzing by the electronic company make.

<Manufacture of latex (particle dispersion)>
69.4 g of pure water, 20 g of styrene, 0.6 g of 2-methacryloyloxyethyl phosphorylcholine, 10 g of ethanol and 0.2 g of sodium p-styrenesulfonate are put into a 200 mL separable flask, and nitrogen is poured into the liquid with stirring. However, after heating up to 72 ° C., 0.06 g of potassium persulfate was added, and a polymerization reaction was performed for 3.5 hours. Thereafter, the temperature was raised to 93 ° C., and a heat treatment was performed at this temperature for 3 hours to obtain Latex 1.
It was 100 nm when the average particle diameter of the latex particle contained in the obtained latex 1 was measured by the above procedure.

Latex 2 to 9 were obtained in the same manner as in Production Example 1 except that the amount (g) of each raw material used in Production Example 1 was changed according to the formulation shown in Table 1.
The average particle size of latex particles contained in each latex 2-9 was measured by the above procedure. The results are shown in Table 1.

<Manufacture of measuring reagent> [Preparation of first reagent for measuring reagent]
Latex 1-9 obtained above were mixed according to the blending ratio (mass ratio) shown in Table 2, and further sodium azide, sucrose, sodium acetate, and pure water were mixed, dissolved, and diluted, Measurement reagent first liquids RA1 to RA15 having the composition (unit: mass%) shown in Table 3 were obtained.
Among the latexes used for the preparation of RA1 to RA12 and RA15, the average particle size ratio of the small particle size latex having an average particle size of less than 200 nm and the large particle size latex having an average particle size of 200 nm or more (large particle size latex The average particle size / average particle size of the small particle size latex) is also shown in Table 2.

The particle size distribution of RA1 to RA15 was measured.
FIG. 2 shows the particle size distribution curves of RA1 to RA15. The particle size distribution curves of RA1 to RA12 and RA15 in which two types of latex were mixed had two peaks, and the peak top particle size of each peak corresponded to the average particle size of each of the two types of latex. The particle size distribution curve of RA13 and RA14 using one type of latex alone had one peak.
Among the two peaks in the particle size distribution curves of RA1 to RA15, the maximum frequency y1 (%) of the smaller peak (peak 1) of the particle size x, the corresponding particle size x1 (nm), and the particle size Maximum frequency y2 (%) of the larger peak (peak 2) and the corresponding particle size x2 (nm), minimum frequency y _mim between peak 1 and peak 2, and x1 < Table 4 shows the minimum value x3 of x satisfying y = y _min in the region of x <x2, and the maximum value x4 of x satisfying y = y _min in the region of x1 <x <x2. In addition, from these values, coordinates P1 (x1, y1), V1 (x3, _ymin ), V2 (x4, _ymin ), P2 (x2, y2), and a region surrounded by a straight line connecting P1 in turn The area was calculated. The results are shown in Table 4.

[Preparation of second reagent for measurement reagent]
As surfactants, CHAPS (product name, manufactured by Dojindo Laboratories, Inc., 3-[(3-colamidopropyl) dimethylammonio] propanesulfonate), CHAPSO (product name, manufactured by Dojindo Laboratories, Inc., 3 -[(3-Colamidopropyl) dimethylammonio] -2-hydroxypropanesulfonate), laurylsulfobetaine, and cholic acid were used.
These surfactants, calcium chloride, sodium acetate, bovine serum albumin, sodium azide, and pure water were mixed and dissolved, and the measurement reagents of the composition (unit: mass%) shown in Table 5 Two liquids 1-1 to 1-12 were obtained.

[Evaluation of CRP measurement performance]
As a CRP for a test sample, C. a recombinant protein of E. coli is used. Using REACTIVE PROTEIN (Roche Diagnostics Co., Ltd.), a test sample in which the concentration was halved stepwise by adding physiological saline from 30 mg / dL was prepared and used for measurement.
CRP measurement was performed in the following procedure using the measurement reagent first solution and the measurement reagent second solution prepared above in the combinations shown in Tables 6 to 8.

(Examples 1 to 4, 6 to 12, 14, 16 to 27)
2.1 μL of the test sample is mixed with 120 μL of the second reagent for measurement and then allowed to stand for 5 minutes. After that, 120 μL of the first reagent for measurement is added, and the absorbance immediately after the addition of the first reagent for measurement ( (Absorbance at 0.00 hour) and absorbance after 5 minutes from the addition of the first reagent for measurement.
The absorbance was measured using an automatic general-purpose measuring instrument Hitachi 7170E (manufactured by Hitachi, Ltd.) at a measurement wavelength of 570 nm and a measurement temperature of 37 ° C.
From the above measurement results, the difference between the absorbance after 5 minutes and the absorbance at 0.00 hours (absorbance after 5 minutes minus absorbance at 0.00 hours) was calculated, and the value was calculated as the absorbance change Abs. did. The results are shown in Tables 6-8.

(Examples 5, 13, 15)
2.1 μL of the test sample, 120 μL of the measurement reagent second liquid, and 120 μL of the measurement reagent first liquid are mixed at once, and the absorbance immediately after mixing (absorbance at 0.00) is 5 minutes after mixing. The absorbance after the lapse was measured as described above.
From the above measurement results, the difference between the absorbance after 5 minutes and the absorbance at 0.00 hours (absorbance after 5 minutes minus absorbance at 0.00 hours) was calculated, and the value was used as the absorbance change Abs. . The results are shown in Tables 6-8.

A graph was prepared with the measurement result of the absorbance change amount Abs of Examples 1 to 27 as the vertical axis and the CRP concentration (mg / dL) of the test sample as the horizontal axis. This graph is shown in FIGS. FIG. 3 is a graph of the entire measurement region (CRP concentration is 0 to 30 mg / dL), and FIG. 4 is a graph of a low concentration region (CRP concentration is 0 to 0.94 mg / dL).

(Evaluation)
From the measurement result of the absorbance change amount Abs, the low concentration region change rate and the high concentration region change rate were calculated by the following formulas.
Low concentration region change rate = [Absorbance change amount Abs when CRP concentration is 0.5 mg / dL] − [Absorbance change amount Abs when CRP concentration is 0.0 mg / dL]
High concentration range change rate = [Absorbance change Abs when CRP concentration is 30.0 mg / dL] − [Absorbance change Abs when CRP concentration is 0.0 mg / dL]

It shows that CRP measurement in a low concentration area | region can be implemented favorably, so that the value of a low concentration area change rate is large. That is, the larger the value of the low concentration region change rate, the better the graph rises in the low concentration region, and a slight change in the CRP concentration is reflected in the absorbance change amount Abs. The low concentration region change rate is preferably 30 or more, and more preferably 50 or more.
It shows that CRP measurement in a high concentration area | region can be implemented favorably, so that the value of a high concentration area | region change rate is large. The rate of change in the high concentration region is preferably 3000 or more, and more preferably 4000 or more.
In the present invention, it is particularly preferable that the following conditions 1 and 2 are satisfied.
Condition 1: Low concentration region change rate ≧ 30
Condition 2: High density region change rate ≧ 3000

As shown in the above results, in Example 22, in which only one RA13 having a particle size distribution curve having only one peak in the region of x <200 nm was used as the measurement reagent first solution, the rate of change in the low concentration region was low.
In Example 23 in which only one RA14 was used in the region where the particle size distribution curve had x ≧ 200 nm as the measurement reagent first solution, the rate of change in the high concentration region was low.
Even if the particle size distribution curve has one peak each in the region of x <200 nm and the region of x ≧ 200 nm as the measurement reagent first solution, the straight line that sequentially connects the coordinates P1, V1, V2, P2, and P1 Example 27 using RA15 in which the area of the region surrounded by <300> was less than 300 in both the low concentration region and the high concentration region.

The particle size distribution curve has one peak each in the region of x <200 nm and the region of x ≧ 200 nm, and the area of the region surrounded by straight lines sequentially connecting the coordinates P1, V1, V2, P2, and P1 is 300 or more. Example 1 wherein the measurement reagent first solution and the measurement reagent second solution containing the surfactant (A) were used in combination, and the concentration of the surfactant (A) during measurement was 0.15 to 0.40 mass%. In -18, both the low concentration region change rate and the high concentration region change rate were high, and the conditions 1 and 2 were satisfied.
The particle size distribution curve has one peak each in the region of x <200 nm and the region of x ≧ 200 nm, and the area of the region surrounded by straight lines sequentially connecting the coordinates P1, V1, V2, P2, and P1 is 300 or more. Example 19 was used in combination with the measurement reagent second liquid containing no surfactant (A), but the high concentration range change rate of Example 19 was improved over Examples 22 and 23, and the low concentration range The rate of change was also improved from Example 22, but the rate of change in the low concentration region did not satisfy Condition 1.
The particle size distribution curve has one peak each in the region of x <200 nm and the region of x ≧ 200 nm, and the area of the region surrounded by straight lines sequentially connecting the coordinates P1, V1, V2, P2, and P1 is 300 or more. The measurement reagent first liquid and the measurement reagent second liquid containing the surfactant (A) were used in combination, but the concentration of the surfactant (A) at the time of measurement was 0.1% by mass. The area change rate improved from Examples 22 and 23, and the low concentration area change rate improved from Example 22, but the low concentration area change rate did not satisfy Condition 1.
The particle size distribution curve has one peak each in the region of x <200 nm and the region of x ≧ 200 nm, and the area of the region surrounded by straight lines sequentially connecting the coordinates P1, V1, V2, P2, and P1 is 300 or more. The measurement reagent first solution and the measurement reagent second solution containing the surfactant (A) were used in combination, but the surfactant (A) concentration at the time of measurement was 0.45% by mass. The area change rate improved from Examples 22 and 23, and the low concentration area change rate improved from Example 22, but the low concentration area change rate did not satisfy Condition 1.

The particle size distribution curve has one peak each in the region of x <200 nm and the region of x ≧ 200 nm, and the area of the region surrounded by straight lines sequentially connecting the coordinates P1, V1, V2, P2, and P1 is 300 or more. In Example 24 using lauryl sulfobetaine and cholic acid as surfactants for the first reagent and the second reagent, the low concentration range change rate was improved over Example 22, but the high concentration range change rate was It became lower than Examples 22 and 23. In Examples 25 and 26 in which lauryl sulfobetaine and cholic acid were each used alone as the surfactant of the second reagent for the measurement reagent, both the low concentration range change rate and the high concentration range change rate were lower than those in Examples 22 and 23.
From these results, low concentration range change rate and high concentration of lauryl sulfobetaine with sulfobetaine structure but no steroid skeleton and cholic acid with steroid skeleton but no sulfobetaine structure It was confirmed that the improvement effect of the area change rate was not obtained.

Claims (6)

1. A measuring reagent for measuring C-reactive protein by latex aggregation turbidimetry,
   Containing particles on the surface with groups that react specifically with C-reactive protein,
   The particle size distribution curve with the particle diameter x (nm) of the particles as the horizontal axis and the mass conversion frequency y (%) as the vertical axis has at least one peak in the region of x <200 and at least 1 in the region of 200 ≦ x. Has two peaks,
   In the particle size distribution curve, a region surrounded by a straight line connecting coordinates P1 (x1, y1), V1 (x3, _ymin ), V2 (x4, _ymin ), P2 (x2, y2), and P1 in turn. A measuring reagent having an area of 300 or more.
   However, x1 and y1 respectively indicate the particle size and frequency of the peak having the largest particle size among the peaks in the region of x <200.
   Each of x2 and y2 represents the particle size and frequency of the peak having the smallest particle size among the peaks in the region of 200 ≦ x.
   y _min represents the minimum value of y in the region of x1 <x <x2.
   x3 indicates the minimum value of x where y = y _min in the region of x1 <x <x2.
   x4 indicates the maximum value of x where y = y _min in the region of x1 <x <x2.
2. The measurement reagent according to claim 1, wherein the group that specifically reacts with the C-reactive protein is a phosphorylcholine group.
3. The measurement according to claim 2, further comprising a surfactant having a steroid skeleton hydrocarbon group to which a substituent may be bonded and a group represented by the following general formula (2) in the molecular structure. reagent.
   

   

   [Wherein, X represents a linear alkylene group having 3 to 5 carbon atoms to which a hydroxyl group may be bonded. ]
4. The measurement reagent according to claim 3, which is a kit comprising a first liquid, which is a particle dispersion in which the particles are dispersed, and a second liquid containing the surfactant.
5. A measurement method for measuring the C-reactive protein concentration in a test sample by latex agglutination turbidimetry using the measurement reagent according to any one of claims 1 to 4.
6. Mixing a test sample and the measurement reagent according to claim 3 or 4 to prepare a reaction solution, and measuring the absorbance of the reaction solution,
   The measurement method according to claim 5, wherein the concentration of the surfactant in the reaction solution is 0.15 to 0.40 mass%.

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