JP2003344409A - Immuno-measurement reagent and immuno-measurement method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immuno-measurement reagent having greatly improved sensitivity than before. <P>SOLUTION: This immuno-measurement reagent has as constituents, an immuno-measurement reagent main component formed by carrying an antigen or an antibody against a measuring object on a polymer spherule having the surface charge density in the form of suspension of 0.01-0.3 μmol/m<SP>2</SP>in terms of the anionic dissociation concentration and the particle size of 0.1-0.7 μm, which is a polymer of a polymeric monomer having a phenyl group or an anionic polymeric monomer, and dextran having a weight-average molecular weight of 200,000-1,000,000. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunoassay reagent and an immunoassay method using an insoluble carrier composed of a polymer spherical body, and more specifically, an immunoassay reagent and an immunoassay reagent capable of improving the sensitivity of measurement. Concerning the law.

[0002]

2. Description of the Related Art In the field of clinical testing, an antigen or antibody is carried (adsorbed or bound) on a carrier composed of a polymer spherical body such as latex particles, and is specifically generated between the antibody or the antigen in a sample. A method is widely used in which latex particles are aggregated by an antibody reaction, and thereby an antibody or an antigen as a substance to be measured is measured.

Conventionally, the amount of hydrophilic functional groups per 1 m ² of the surface area of latex particles, that is, the surface charge density represented by the dissociation concentration of anions is 0.3 to 8 μmol / m ² , and the latex particles have an antigen or an antibody. By carrying the above, an immunoreagent in which the above-mentioned antigen or antibody specifically binds to the substance to be measured in the sample has been prepared. In this immunoassay reagent, latex particles aggregate depending on the amount of the substance to be measured, and the amount of the substance to be measured can be determined by measuring the amount of optical change due to aggregation (for example, JP-A-57-57). 168163 and JP-A-58-9765.
No. 6, etc.).

In the above-mentioned immunoassay method, a method for measuring a substance to be measured with higher sensitivity is desired.
Therefore, various methods for measuring the substance to be measured with higher sensitivity by increasing the amount of optical change have been tried.

For example, a method of increasing the particle size of latex has been considered. In this method, although the amount of optical change is increased, when the amount of the substance to be measured is large, the optical particles are agglomerated. The amount of dynamic change exceeds the measurable range. Therefore, when the concentration of the substance to be measured is high, there is a problem that the amount of optical change corresponding to the amount of the substance to be measured cannot be obtained.

On the other hand, a method of increasing the amount of optical change by increasing the amount of the sample at the time of measurement has been attempted. However, when the amount of the sample is increased, components other than the substance to be measured in blood are added. There is a problem that interference with the measured value is likely to occur due to the influence of.

On the other hand, as a method of reliably and simply increasing the amount of optical change, a method of using a reaction accelerator composed of various water-soluble polymers is generally used. As such a reaction accelerator, polyethylene glycol or the like is used. Also, Japanese Patent No. 1777481
In JP-A No. 2004-163, an aqueous solvent for agglutination test containing 1 to 30% by weight of dextran having an average molecular weight of 30,000 to 100,000 is proposed. However, dextran having such a molecular weight range cannot sufficiently enhance the sensitivity of the immunoassay reagent, and cannot achieve the currently required high measurement sensitivity.

[0008]

In view of the above-mentioned state of the art, an object of the present invention is to provide an immunoassay reagent containing a reagent component in which an antigen or antibody is supported on macromolecular spheres and a reaction promoter. In addition, it is to provide an immunoassay reagent and an immunoassay method that are more excellent in measurement sensitivity and that enable simple and easy measurement.

[0009]

[Means for Solving the Problems] The inventors of the present application have made further studies as to an immunoassay reagent having a reagent component in which an antigen or an antibody is supported on a polymer sphere, and as a result, a specific surface charge density and particle size have been obtained. The inventors have found that the combination of a reagent component in which an antigen or an antibody is supported on a macromolecular spherical body having a dextran and a dextran having a specific molecular weight can dramatically improve the measurement sensitivity, and have completed the present invention.

That is, the immunoassay reagent according to the present invention comprises:
1) A polymer of a phenyl group-containing polymerizable monomer and / or an anionic polymerizable monomer, which has a surface charge density of 0.01 as an anion dissociation concentration when made into a suspension. ~
0.3 μmol / m ² and a particle size of 0.1 to 0.
A main component of an insoluble reagent in which an antigen or an antibody corresponding to an object to be measured is carried on a polymeric sphere having a size of 7 μm, and 2) a weight average molecular weight of 200,000 to 1,000,000
And dextran as a constituent component.

In a particular aspect of the immunoassay reagent according to the present invention, the anionic polymerizable monomer is styrene sulfonate and / or (meth) acrylic acid. The immunoassay method according to the present invention is an assay method using the immunoassay reagent of the present invention, wherein the antigen or antibody carried on the macromolecular spheres and the antigen-antibody reaction of the substance to be measured in the sample are reacted as described above. It is characterized in that the substance to be measured is measured by detecting the degree of aggregation of the reagent main component.

The details of the present invention will be described below. The polymer spherical body used in the present invention comprises a polymer of a polymerizable monomer having a phenyl group and / or an anionic polymerizable monomer. The polymerizable monomer having a phenyl group is not particularly limited, and examples thereof include styrene, divinylbenzene, ethylstyrene, α-methylstyrene,
Examples thereof include p-methylstyrene, p-chlorostyrene, chloromethylstyrene and the like. These may be used alone or in combination of two or more.

The anionic polymerizable monomer is not particularly limited, and examples thereof include styrene sulfonate, (meth) acrylic acid, divinylbenzene sulfonate, ethylstyrene sulfonate, α-methyl sulfonic acid. Examples include salt. Further, the salt in this case is not particularly limited, and examples thereof include sodium salt, potassium salt, lithium salt, ammonium salt and the like. These may be used alone or in combination of two or more. Among them, styrenesulfonate and / or (meth) acrylic acid are preferable as the anionic polymerizable monomer used in the polymer spherical body of the present invention. By including styrene sulfonate and / or (meth) acrylic acid, the obtained polymer spherical body itself has good emulsifying power.

Since the above-mentioned anionic polymerizable monomer is contained, the obtained polymer spherical body has a surface charge. Therefore, the polymeric spheres are well dispersed in the solution without using an emulsifier. When the emulsifier is present in the suspension of the polymeric spheres, when an immunoassay reagent containing a reagent component in which an antigen or an antibody is supported on the polymeric spheres and dextran described below is constituted. , The emulsifier inhibits the aggregation of macromolecular spheres due to the specific antigen-antibody reaction,
In some cases, it may be involved in nonspecific reactions.
Therefore, it is preferable that no emulsifier is contained.

When the polymer spherical body is composed of a copolymer, the contents of the polymerizable monomer having a phenyl group and the anionic polymerizable monomer are not particularly limited, but have a phenyl group. With respect to 100 parts by weight of the copolymerizable component of the polymerizable monomer, the copolymerizable component of the anionic polymerizable monomer is 5 parts.
It is preferably 0 parts by weight or less. When the amount of the copolymerizable component of the anionic polymerizable monomer exceeds 50 parts by weight based on 100 parts by weight of the copolymerizable component of the phenyl group-containing polymerizable monomer, the obtained polymer spherical particles are dispersed. It can be difficult. More preferably, the copolymerization component of the anionic polymerizable monomer is 30 parts by weight or less based on 100 parts by weight of the copolymerization component of the polymerizable monomer having a phenyl group. Further, a polymer composed only of the above-mentioned polymerizable monomer having a phenyl group can also be used as the polymer spherical body of the present invention.

The method for polymerizing the above polymer is not particularly limited, and emulsion polymerization, suspension polymerization, and
Polymerization can be carried out by various polymerization methods such as seed polymerization and dispersion polymerization. The polymerization initiator is not particularly limited, and examples thereof include persulfates such as potassium persulfate.

The above-mentioned polymer spherical body has a surface charge, and this surface charge is used at the time of polymerization with that based on the anionic polymerizable monomer which is the above-mentioned copolymerization component. One is based on the anion of the section of the polymerization initiator. What is based on the anion of the section of the above-mentioned polymerization initiator means, for example, when a persulfate such as potassium persulfate is used, the sulfate group (-OSO _3- ) as the section exists on the surface of the copolymer particles. The sulfate radicals are gradually hydrolyzed and changed as shown in the following formula (1). −OSO _{3 −} + H ₂ O → −OH + HOSO _{3 −} (1)

In the above-mentioned polymer spheres according to the present invention, the surface charge density when made into a suspension is in the range of 0.01 to 0.3 μmol / m ² as the dissociation concentration of anions. In addition, as a medium of the suspension, an appropriate liquid medium having a spherical polymer as a carrier and used for an immunological measurement test is used, and examples thereof include water, physiological saline, and serum. If the charge density is less than 0.01 μmol / m ^{2 as} the dissociation concentration of anions, the repulsive force between particles becomes weak, and the emulsifying power of the spherical polymer itself is impaired. Therefore, stable dispersion does not occur in the suspension, self-aggregation occurs, and the practicality as a diagnostic reagent is impaired. The above charge density is 0.3μ at the dissociation concentration of anions
When it exceeds mol / m ² , the electric repulsive force between the polymer spheres becomes strong, and it is stable without self-aggregation, but it also prevents aggregation due to the antigen-antibody reaction. Therefore, the measurement cannot be performed with high sensitivity.

The surface charge density can be easily obtained by measuring the electric conductivity of the suspension, for example. The particle size of the polymer spherical body is 0.1 to
It is 0.7 μm. The particle size of the polymer spherical body is 0.1
When it is less than μm, the amount of optical change due to aggregation is small,
High sensitivity required for measurement cannot be obtained. If it exceeds 0.7 μm, the amount of optical change due to the aggregation of latex particles exceeds the measurable range, and the measurement range becomes small. It is preferably 0.2 to 0.5 μm.

In the immunoassay reagent of the present invention, the antigen or antibody carried on the macromolecular spheres is not particularly limited, and is generally a physiologically active substance that can utilize the antigen-antibody reaction and an antigen such as a pathogen such as a virus or a bacterium. , All of the antibodies can be used. Examples of the physiologically active substance include, for example,
Examples include various in-vivo receptors and enzymes existing in the living body. Examples of the pathogen antigen and antibody include
Syphilis-derived antigen, anti-syphilis antibody, HBs antigen, anti-H
Bs antibody, HCV antigen, HIV antigen, ATLA antigen, chlamydia antigen, herpes antigen, Helicobacter pylori antigen and the like can be mentioned. These are derived from biological components,
It may be a product obtained by culture, a product chemically synthesized, a product obtained by a technique such as gene recombination, or a processed product thereof. As the antibody,
In addition to the immunoglobulin molecule itself, for example, F (ab ') 2
It may be a fragment such as.

In obtaining the immunoassay reagent of the present invention, the method for supporting the antigen or antibody on the macromolecular spheres is not particularly limited, and the macromolecular spheres may be an antibody or an antigen against an antigen to be measured. May be supported physically or chemically by a known method.

In the immunoassay reagent of the present invention, a protein such as albumin, casein, gelatin, or a protein degradation product is used in order to suppress the nonspecific agglutination reaction of macromolecular spheres or to enhance the stability of the reagent. ,amino acid,
A surfactant or the like may be further supported on the above-mentioned polymer spherical body.

Further, the immunoassay reagent of the present invention comprises a reagent component in which an antigen or an antibody is carried on the above-mentioned polymer spherical body as a constituent component. For example, the above-mentioned polymer spherical body is suspended on the reagent component. A buffer solution for turbidity, a buffer solution used in the reaction, or the like may be added.

The dextran used in the immunoassay reagent of the present invention acts as a reaction accelerator, and the molecular weight of dextran is 200,000 to 1,000,000, preferably 300,000 to 700,000.
If the molecular weight is less than 200,000, the desired reaction-promoting effect cannot be obtained, and if it exceeds 1,000,000, the viscosity of the solution increases, which causes variations in measurement and decreases the measurement sensitivity.

The dextran is preferably used in a state of being dispersed or dissolved in an appropriate medium. In this case, the one-part immunoassay reagent may be constructed by dispersing and dissolving dextran in the same medium as the polymeric spheres supporting the above-mentioned antigen or antibody. Further, a two-part immunoassay reagent comprising a latex reagent and a dextran-containing reagent may be constituted by dispersing or dissolving dextran and a macromolecular spherical body carrying the above-mentioned antigen or antibody in separate media. Good.

The medium is not particularly limited, and for example,
Examples thereof include a phosphate buffer solution, a glycine buffer solution, a Tris salt buffer solution, and a Good's buffer solution. The pH of the medium is
It is preferably 5.5 to 8.5. More preferably 6.5-
It is 8.5.

Bovine serum albumin, sucrose, sodium chloride for adjusting the salt concentration, a surfactant and the like may be appropriately dissolved in the one-component immunoassay reagent. Also, when used as a two-component immunoassay reagent having the above-mentioned latex reagent and solution reagent, bovine serum albumin, sucrose, sodium chloride for adjusting salt concentration, surfactant, etc. are appropriately dissolved in each. You may let me.

Examples of the sample used in the immunoassay method of the present invention include serum and plasma. However, the sample is not limited to these and may be applied to various biological samples and non-biological samples. It is possible.

In the immunoassay reagent of the present invention, the degree of aggregation of the spherical polymer particles produced by the agglutination reaction is detected by optical measurement or visual observation. The method for optically observing the degree of aggregation of the polymer spherical body is not particularly limited, and for example, scattering intensity, absorbance, or increase or decrease in transmitted light intensity may be measured. Moreover, you may use these methods together.

In the case of visually observing the degree of aggregation of macromolecular spheres, a solution containing a sample and the above-mentioned macromolecular spheres carrying the protein is usually mixed on a judgment plate, and 1 to 5 is added.
After rocking for a minute, the presence or absence of aggregation is determined. The aggregation determination may be performed by photographing the aggregation state with a video camera and performing image processing in addition to the naked eye determination.

The temperature condition of the above antigen-body reaction is not particularly limited as long as it is a temperature at which the antigen-antibody reaction is carried out, but it is 0-5.
0 degreeC is preferable and 20-40 degreeC is more preferable. The reaction time is appropriately determined.

In order to enhance specificity during the reaction,
A quaternary ammonium salt such as choline chloride, EDTA, polyanion, chaotropic ion such as C _{1 −} , I ₋ , SCN ₋ , gelatin and the like may be added to the reaction system.

Since the immunoassay reagent of the present invention has the above-mentioned constitution, when measuring a low-concentration substance to be measured, the amount of optical change due to agglutination is larger than that of the conventional reagent. Further, when measuring a high-concentration substance to be measured, it is less likely to cause an optical change that exceeds the measurable range optically due to aggregation, as compared with a conventional reagent using a large latex. Further, an immunoassay reagent with good sensitivity and reproducibility can be obtained even if the amount of the antibody or antigen carried on the antigen or antibody as the substance to be measured is small. In addition, it is possible to reduce interference due to blood components.

[0034]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail by describing examples of the present invention. However, the present invention is not limited to the following examples. In the following examples and comparative examples, anti-treponemal antibody measuring reagents were prepared and evaluated.

Example 1 (1) Preparation of Sample Dilution Buffer A phosphate buffer containing 1% by weight of bovine serum albumin was added,
Dextran 500000 (manufactured by SIGMA, average molecular weight 500000) was added by 1.3% by weight and dissolved.

(2) Preparation of a liquid of antigen-sensitized latex derived from Treponema pallidum. An antigen derived from Treponema pallidum was dissolved in 0.01 M phosphate buffer (pH 7.4) at a concentration of 30 μg / ml to give 0.4 ml. , A polystyrene latex having an average particle size of 0.400 μm and a sulfone group amount of 0.11 μmol / m ² (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.) was added, and
The mixture was stirred at 0 ° C for 60 minutes.

Next, 0.5 ml of 0.1 M phosphate buffer (pH 7.4) containing 1% by weight of bovine serum albumin was added to this solution, and the mixture was stirred at 4 ° C. for 90 minutes, then 15
Centrifuged at 000 rpm for 15 minutes.

1 bovine serum albumin was added to the obtained precipitate.
4 ml of phosphate buffer containing 4 wt% was added and the latex was resuspended to prepare a Treponema pallidum-derived antigen-sensitized latex solution.

(3) Anti-treponemal antibody measuring reagent The anti-treponemal antibody measuring reagent of this Example is the same as the above (1).
Is a two-component system reagent comprising a first reagent consisting of the sample dilution buffer and a second reagent consisting of a latex liquid sensitized with the Treponema pallidum-derived antigen of the above (2).

(4) Anti-treponemal antibody standard solution 1 syphilis-positive standard serum (5 concentrations, Sekisui Chemical Co., Ltd.)
It was dissolved in purified water (ml) and used. Antibody titer is 0, 3
9, 121, 237 and 405T. U. There are 5 types.

Incidentally, T. U. Is a unit showing the antibody titer of the anti-treponemal antibody when the serum is measured with an anti-treponemal antibody measuring reagent, Mediace TPLA (manufactured by Sekisui Chemical Co., Ltd.). With this reagent, the antibody titer was 10 T.I.
U. In the above cases, it is determined to be positive.

(5) Sample for measurement 10T. U. Syphilis-positive standard serum was prepared.

(6) Measurement of anti-treponemal antibody standard solution and preparation of anti-treponemal antibody standard curve 20 μl of anti-treponemal antibody standard solution was taken, and 210 μl of the buffer solution (first reagent) described in the above item (1) was mixed with it. Three
After being kept at 7 ° C for an appropriate time, the anti-Treponemal varidami-derived antigen-sensitized latex liquid (second reagent) 30 of the above (2)
μl was added and stirred, and then the amount of change in absorbance at a wavelength of 700 nm for about 80 seconds to 300 seconds was measured and defined as the amount of change in absorbance (ΔAbs). Hitachi automatic analyzer 7 for measurement
Type 150 was used. Obtained amount of change in absorbance (ΔAb
s) and the standard anti-treponemal antibody titer (TU), a calibration curve for anti-treponemal antibody titer was prepared.

(7) Measurement of measurement sample Using the serum prepared in the above item (5), the same operation as in the above item (6) was carried out to determine the amount of change in absorbance. Using the calibration curve prepared in (6) above, the anti-treponemal antibody titer was determined from the change in absorbance of each sample. The measurement was repeated 10 times, the average value, the standard deviation (sd), and the variation (cv) (%) were obtained, and the simultaneous reproducibility test was performed.

(Comparative Example 1) In the preparation of (2) Treponema pallidum-derived antigen-sensitized latex liquid in Example 1, polystyrene latex was used as an average particle diameter of 0.40.
0 μm, sulfo group amount 0.31 μmol / m ² (solid content 10% by weight, manufactured by Sekisui Chemical Co., Ltd.), except that the operation was performed in the same manner as in Example 1 and the change in absorbance of the anti-treponemal antibody standard solution was performed. (ΔAbs) evaluation and simultaneous reproducibility test were performed.

Comparative Example 2 (1) In the preparation of the sample dilution buffer (1) in Example 1, except that 1.3 wt% dextran was changed to 0.8 wt% polyglucosylethylmethacrylate (GEMA). In the same manner as in Example 1, the amount of change in the absorbance of the anti-treponemal antibody standard solution (ΔA
bs) Evaluation and simultaneous reproducibility test were performed.

(Comparative Example 3) In the preparation of (1) Sample dilution buffer in Example 1, 1.6 wt% dextran 500000 (average molecular weight 500000) was added to 1.3 wt% dextran 35000-45000 (average molecular weight). (35000-45000) 1% by weight except that the procedure was the same as in Example 1 to evaluate the amount of change in absorbance (ΔAbs) of the anti-treponemal antibody standard solution. In addition,
For the simultaneous reproducibility test, 10T. U. It was not carried out because the amount of change in absorbance in the vicinity was insufficient.

The results of Example 1 and Comparative Examples 1 to 3 are shown in Table 1.
Shown in 2.

[0049]

[Table 1]

[0050]

[Table 2]

As is clear from Table 1, dextran having a weight average molecular weight of 500000 and surface charge density of 0.11
It can be seen that in Example 1 using μmol / m ² of latex, the amount of optical change is significantly increased as compared with Comparative Examples 1 to 3 using conventional additives and latex. Further, as is clear from Table 2, in Example 1, the variation in measured values was smaller than in Comparative Examples 1 and 2, and therefore the reproducibility of measurement was dramatically improved, and the object to be measured having a low concentration was further measured. It can be seen that can be reliably detected.

[0052]

INDUSTRIAL APPLICABILITY The immunoassay reagent according to the present invention is a polymer having a phenyl group and / or a polymer of an anionic polymerizable monomer, and has a surface charge density when made into a suspension. A reagent main component in which an antigen or an antibody against an object to be measured is carried on a polymer spherical body having an anion dissociation concentration in the above-mentioned specific range and a particle diameter in the above-mentioned specific range, and a weight average molecular weight of 200,000. Since dextran in the range of up to 1,000,000 is used as a component, the sensitivity in the immunoassay can be dramatically improved.

Therefore, according to the immunoassay method using the immunoassay reagent of the present invention, the substance to be measured in various biological samples can be immunologically highly sensitively measured.

Claims (3)

[Claims] 1. A polymer of a polymerizable monomer having a phenyl group and / or an anionic polymerizable monomer,
When the surface charge density of the suspension was 0.01 to 0.3 μmol / m ^{2 as} the dissociation concentration of anions and the particle size was 0.1 to 0.7 μm, the polymer spherical body was covered with An insoluble reagent main component carrying an antigen or antibody corresponding to the measurement target, and 2) a weight average molecular weight of 200,000 to 10
An immunoassay reagent comprising 00000 dextran as a constituent. 2. The immunoassay reagent according to claim 1, wherein the anionic polymerizable monomer is styrene sulfonate and / or (meth) acrylic acid. 3. An immunoassay method using the immunoassay reagent according to claim 1 or 2, wherein an antigen-antibody reaction between an antigen or antibody supported on a macromolecular sphere and a substance to be measured in a sample. An immunoassay method characterized in that the substance to be measured is measured by detecting the degree of aggregation of the reagent main component caused by.