JPH07133232A - Immunological diagnostic and carrier - Google Patents

Abstract

PURPOSE:To provide a carrier easy to immobilize antigen or antibody, and to provide an immunological diagnostic high in refractive index, thus enabling assays in high sensitivity by light scattering analysis or integrating-sphere turbidimetry. CONSTITUTION:The objective carrier for immunological diagnostics comprising polymer particles prepared by polymerization, using a radical polymerization initiator in an aqueous medium, of monomer component composed of (A) 5-99.9 (pref. 30-99.9) wt.% of a styrene derivative of the formula (R is H or methyl; X is F, Cl, Br, I or methyl; k is 1 or 2; m and n are each O or 1), e.g. m-[(p- chlorophenylthio)methyl]styrene and (B) 0.1-95 pref. 0.1-70) wt.% of a vinyl compound, styrene, sodium p-styrenesulfonate and/or divinylbenzene. The other objective immunological diagnostic consisting of an aqueous dispersion of immobilized antigen or immobilized antibody by immobilizing an antigen or antibody on the above carrier. The average particle diameter of the carrier is pref. 0.02-20mum (esp. 0.05-1mum).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a carrier for immunological diagnostic agents and an immunological diagnostic agent using the same, and particularly to an immunological diagnostic method which gives high sensitivity to measurement by a light scattering measurement method or an integrated sphere turbidity measurement method. The present invention relates to a carrier for a diagnostic agent and an immunological diagnostic agent using the carrier.

[0002]

2. Description of the Related Art An immunoassay utilizing an antigen-antibody reaction is
It has become the mainstream in the clinical laboratory field. There are already many immunological microassays, but the particle immunoassay is active. This is because a reagent (immunological diagnostic agent) sensitized with antibodies (or antigens) on the surface of carrier particles reacts with an antigen (or antibody) in a specimen to form a complex, that is, an aggregate, and this aggregate It is a method of optically measuring the turbidity of. Thereby, the antigen or antibody can be detected or quantified.

According to Japanese Industrial Standards (JIS), four methods of optical measurement of turbidity are 1) visual turbidity, 2) transmitted light turbidity, 3) scattered light turbidity, and 4) integrated sphere turbidity. It is classified into. Among them, in the integrating sphere turbidity method, parallel transmitted light is measured at the same time as forward scattered light, and the ratio thereof is calculated to make a compensation measure for interference factors other than the original amount of change in the antigen-antibody reaction.

In the carrier particles used in such a method, the surface characteristics on the carrier surface are controlled, and they are stable and do not cause non-specific reaction or non-specific aggregation. Properties such as excellent dispersibility and high diagnostic sensitivity are required.

Conventionally, as carrier particles used for immunological diagnostic agents, for example, polymer latex such as styrene polymer, styrene-acrylic acid copolymer, styrene-butadiene copolymer, or metal particles, liposomes. There are various particles such as styrene polymer latex having a uniform particle size distribution and uniform surface shape characteristics, but most of them are used for clinical application (JP-A-54-109494).
issue).

However, in the immunological diagnostic agent using the above-mentioned styrene polymer latex as a carrier,
There is a problem that reliability is poor in measurement for an analyte (analyte) in a low concentration (range of ng / ml) region. This is because the styrene polymer has a low refractive index.

The light-scattering properties of a particle suspension depend on the particle size, the wavelength of the light used for measurement, and the difference in refractive index between the dispersion medium and the particles, the higher the refractive index of the particles, the light The scattered signal is higher, and a highly sensitive measurement can be performed. Therefore, in light scattering measurement, a carrier having a higher refractive index than the conventional styrene polymer latex and having a uniform particle size distribution and surface characteristics is required.

[0008] On the other hand, Japanese Patent Publication No. 2-20067 discloses a particle reagent for light scattering immunoassay in which an antibody is bound to a carrier consisting of an outer shell having a functional group and an inner core having a high refractive index. However, this reagent has a problem that it is necessary to introduce a functional group corresponding to the antibody in order to chemically bond the antibody and a reaction for binding the antibody is required.

By the way, JP-A-3-273011 describes a compound represented by the following general formula [1] as a high refractive index organic glass. However, the above publication does not describe that this compound can be used as a material for carriers for immunological diagnostic agents.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above problems by providing an immunological assay having a high refractive index, capable of performing highly sensitive measurement, and easily immobilizing an antigen or an antibody. It is intended to provide a carrier for a diagnostic agent and an immunological diagnostic agent using the carrier.

[0011]

The present invention provides the following immunological diagnostic agents and immunological diagnostic agents using them. (1) (A) General formula [1]

[Chemical 2] (In the formula, R represents a hydrogen atom or a methyl group, X represents F,
A halogen atom or a methyl group selected from Cl, Br and I is shown. k represents 1 or 2, m and n are 0
Or 1 is shown. ) Styrene derivatives 5-9
9.9% by weight, and (B) vinyl compound 0.1-9
A carrier for an immunological diagnostic agent, comprising polymer particles obtained by polymerizing 5% by weight of a monomer component in an aqueous medium using a radical polymerization initiator. (2) An immunological diagnostic agent, which comprises an aqueous dispersion of an immobilized antigen or immobilized antibody in which the antigen or antibody is immobilized on the carrier for immunological diagnostic agent according to (1) above.

The styrene derivative (A) represented by the general formula [1] used in the present invention is, for example, p-
[(Phenylthio) methyl] styrene, m-[(phenylthio) methyl] styrene, p-[(p-chlorophenylthio) methyl] styrene, m-[(p-chlorophenylthio) methyl] styrene, p-[(m- Chlorophenylthio) methyl] styrene, m-[(m-chlorophenylthio) methyl] styrene, p-[(p-bromophenylthio) methyl] styrene, m-[(p-bromophenylthio) methyl] styrene, p- [(M-Bromophenylthio) methyl] styrene, m-[(m-bromophenylthio) methyl] styrene, p-[(p-iodophenylthio) methyl] styrene, m-[(p-iodophenylthio) Methyl] styrene, p-[(m-iodophenylthio) methyl] styrene, m-[(m-iodophenylthio) methyl] Tylene, p-[(p-methylphenylthio) methyl] styrene, m-[(p-methylphenylthio) methyl] styrene, p-[(m-methylphenylthio) methyl] styrene, m-[(m- Methylphenylthio) methyl] styrene, p-[(phenylthio) methyl] -α-methylstyrene, m-[(phenylthio)
Methyl] -α-methylstyrene, p-[(p-chlorophenylthio) methyl] -α-methylstyrene, m-
[(P-Chlorophenylthio) methyl] -α-methylstyrene, p-[(m-chlorophenylthio) methyl]-
α-methylstyrene, m-[(m-chlorophenylthio) methyl] -α-methylstyrene, p-[(p-bromophenylthio) methyl] -α-methylstyrene, m-
[(P-Bromophenylthio) methyl] -α-methylstyrene, p-[(m-bromophenylthio) methyl]-
α-methylstyrene, m-[(m-bromophenylthio) methyl] -α-methylstyrene, p-[(p-iodophenylthio) methyl] -α-methylstyrene, m-
[(P-iodophenylthio) methyl] -α-methylstyrene, p-[(m-iodophenylthio) methyl]-
α-methylstyrene, m-[(m-iodophenylthio) methyl] -α-methylstyrene, p-[(p-methylphenylthio) methyl] -α-methylstyrene, m-
[(P-Methylphenylthio) methyl] -α-methylstyrene, p-[(m-methylphenylthio) methyl]-
Examples include α-methylstyrene and m-[(m-methylphenylthio) methyl] -α-methylstyrene.

These styrene derivatives (A) can be used alone or in combination of two or more. By using such a styrene derivative (A) as a monomer component, a high refractive index copolymer can be obtained. The amount of the styrene derivative (A) represented by the general formula [1] is
5 to 99.9% by weight of all monomer components, preferably 30 to
It is 99.9% by weight. When the amount used is less than 5% by weight, the increase in the refractive index is insufficient, which is not preferable.

As the vinyl compound (B) used in the present invention, a vinyl compound which can be polymerized with the styrene derivative (A) represented by the above-mentioned general formula [1] can be used without limitation. Examples thereof include monofunctional vinyl compounds having a saturated double bond or polyfunctional vinyl compounds.

Examples of the monofunctional vinyl compound include aromatic vinyl compounds such as styrene, chloromethylstyrene and α-methylstyrene; (meth) acrylic acid, methyl (meth) acrylate, ethyl (meth) acrylate,
(Meth) acrylic acid-n-butyl, (meth) acrylic acid-n-hydroxyethyl, (meth) acrylic acid polyoxyethylene, (meth) acrylic acid glycidyl, (meth)
(Meth) acrylic compounds such as tribromophenyl acrylate, (meth) acrylonitrile, (meth) acrolein, (meth) acrylamide; sodium styrenesulfonate, potassium styrenesulfonate, lithium styrenesulfonate, ammonium styrenesulfonate, etc. Styrene sulfonate; vinyl acetate, vinyl pyridine, N-vinyl pyrrolidone, vinyl chloride, vinylidene chloride, vinyl bromide and the like.

Examples of polyfunctional vinyl compounds include divinyl compounds such as divinylbenzene and divinylsulfone; diallyl phthalate, diallyl acrylamide,
Allyl compounds such as triallyl (iso) cyanurate and triallyl trimellitate; (poly) oxyalkylene glycol di (meth) acrylates such as (poly) ethylene glycol di (meth) acrylate and (poly) propylene glycol di (meth) acrylate Pentaerythritol (meth) acrylates such as pentaerythritol di (meth) acrylate, pentaerythritol tri (meth) acrylate, pentaerythritol tetra (meth) acrylate, dipentaerythritol hexa (meth) acrylate, dipentaerythritol penta (meth) acrylate Derivatives: glycerol tri (meth) acrylate, glycerol di (meth) acrylate, 2,2-bis [(meth) acryloyloxyethoxyphenyl] p Such as bread and the like. When a polyfunctional vinyl compound is used, a crosslinkable polymer is obtained.

These vinyl compounds (B) can be used alone or in combination of two or more. The amount of the vinyl compound (B) used is 0.1 to 95% by weight, preferably 0.1 to 70% by weight, based on the total monomer components.

The carrier for immunological diagnostic agents of the present invention comprises the styrene derivative (A) and the vinyl compound (B) as monomer components, and these are used in the presence of a radical polymerization initiator.
It contains polymer particles obtained by polymerizing in an aqueous medium.

As a method for polymerizing in an aqueous medium, an emulsion polymerization method, a suspension polymerization method, a soap-free polymerization method, a seed polymerization method or the like can be adopted. These polymerization methods can be carried out according to known methods. For example, a method of charging all of the monomer components (A) and (B) into a reaction system at once and polymerizing them, After charging and reacting, the remaining monomer components may be continuously or dividedly added and polymerized, or the entire amount of the monomer components may be continuously charged and polymerized. In these polymerization methods, the method of adding the radical polymerization initiator can be arbitrarily selected, and a method of charging the whole amount at once, a method of continuously or dividingly adding, and the like can be adopted.

As the radical polymerization initiator used for the polymerization, any water-soluble or oil-soluble radical polymerization initiator can be used. Examples of the water-soluble radical polymerization initiator include persulfates such as potassium persulfate, ammonium persulfate and sodium persulfate, and hydrogen peroxide. When a water-soluble radical polymerization initiator is used, a reducing agent can be used together. Examples of such reducing agents include sodium pyrobisulfite, sodium hydrogen sulfite, sodium thiosulfate, L-ascorbic acid and salts thereof, sodium formaldehyde sulfoxylate, and the like.

Examples of the oil-soluble radical polymerization initiator include 2,2'-azobisisobutyronitrile,
2,2'-azobis (4-methoxy-2,4-dimethylvaleronitrile), 2,2'-azobis-2,4-dimethylvaleronitrile, 1,1'-azobis-cyclohexane-1-carbonitrile, per Examples thereof include benzoyl oxide, dibutyl peroxide, cumene hydroperoxide and the like. These oil-soluble radical polymerization initiators are used by being dissolved in a monomer or dispersed in an aqueous medium. The amount of the radical polymerization initiator used is 0.01 to 30 with respect to the monomer component.
It is desirable to set the content by weight, preferably 0.1 to 10% by weight.

As the aqueous medium serving as a reaction medium, water may be used alone or an organic solvent mixed water obtained by mixing an organic solvent with water may be used. Such organic solvents include ethanol, methanol, acetone,
A water-miscible organic solvent such as tetrahydrofuran or dimethylformamide can be used. The amount of the organic solvent added is preferably 0 to 30% by weight with respect to water.

The concentration of the monomer component in the aqueous medium is
The total amount of (A) and (B) is 0.2 to 30% by weight, preferably 1 to 10% by weight. The reaction temperature and the reaction time will differ depending on the synthesis conditions such as the type of radical polymerization initiator, but it is usually preferable to carry out the reaction at 5 to 50 hours and 30 to 90 ° C.

When the seed polymerization method is adopted as the polymerization method, the seed particles include styrene polymer and styrene-
Butadiene copolymer, styrene-acrylic acid copolymer,
A styrene-based copolymer such as a styrene-methacrylic acid copolymer, a (meth) acrylic acid-based polymer such as a methyl methacrylate polymer, or a copolymer can be used.
These seed particles may be crosslinked.
The particle size of the seed particles can be appropriately selected in the range of 0.05 to 2 μm depending on the conditions such as polymer particles to be obtained.

By polymerizing the styrene derivative (A) and the vinyl compound (B) in the aqueous medium under the above-mentioned conditions using a radical polymerization initiator, the polymer particles which are solid at room temperature are dispersed in the aqueous system. Obtained in the state of the body (latex). A condensate such as a polymerized product of a crosslinkable monomer generated at this time is separated by filtration or the like, and an unreacted monomer, a radical polymerization initiator, or the like is separated and purified by centrifugation. The separated polymer particles are used as they are or in the form of an aqueous dispersion by redispersing in an aqueous medium as a carrier for an immunological diagnostic agent, but in some cases, they can be used after being powdered by freeze-drying or the like. . When used as an aqueous dispersion, the concentration of polymer particles in the aqueous dispersion is 0.0
The amount is 1 to 30% by weight, preferably 0.1 to 10% by weight.

The average particle size of the carrier for immunological diagnostic agents thus obtained can be arbitrarily set depending on the purpose, but when used as a carrier for immunological diagnostic agents,
Usually 0.02 to 20 μm, preferably 0.05 to 1 μm
Is desirable. With such an average particle size, the carrier particles are dispersed in the aqueous medium and a stable aqueous dispersion is formed without adding an emulsifier.

The average particle diameter can be adjusted by selecting the amounts of the radical polymerization initiator and the water-soluble monomer component, their addition method, and other reaction conditions. The larger the amount of the radical polymerization initiator and the water-soluble monomer component, the smaller the average particle size tends to be, but a large fluctuation of the radical polymerization initiator greatly affects other reaction conditions and the physical properties of the polymer. Therefore, it is preferable to control the average particle diameter by adjusting the water-soluble monomer component.

The above carrier for immunological diagnostic agent can be arbitrarily set so that the refractive index measured by sodium D line is in the range of 1.59 to 1.70. The higher the composition ratio of the styrene derivative (A), the higher the refractive index, and the higher the composition ratio of the vinyl compound (B), the lower the refractive index. Therefore, determine the composition ratio according to the target refractive index. You can

The immunological diagnostic agent of the present invention comprises an aqueous dispersion (latex) in which an immobilized antigen or immobilized antibody in which an antigen or antibody is immobilized on the carrier for immunological diagnostic agent is dispersed in an aqueous medium. . The antigen immobilized on the carrier for immunological diagnostic agent is not particularly limited as long as it is a substance showing antigenicity.

The antibody immobilized on the carrier for immunological diagnostic agents is IgG mainly derived from all animal species such as bovine, goat, rabbit, mouse, chicken and human, but other than this, IgA and IgM are also available. , IgD, IgE, and any other class of immunoglobulins, and either polyclonal or monoclonal antibodies. It may also be various antibody-derived segments such as Fab, Fc, F (ab) ′.

Immobilization of the antigen or antibody on the carrier for immunological diagnosis can be carried out by mixing the two in an aqueous medium. In this case, since the antigen or antibody and the carrier are bound by the physicochemical bonding force between the hydrophobic groups,
Immobilization and desorption of the antigen or antibody is easy, and the carrier can be regenerated by the desorption.

The immobilization can be carried out by adding an antigen or an antibody to the reaction solution in which the carrier for immunological diagnostic agent is prepared, or the carrier for immunological diagnostic agent is isolated from the reaction solution and the carrier is washed with water or water. It is also possible to disperse in an organic solvent-mixed water prepared by mixing the above-mentioned water-miscible organic solvent or an aqueous medium such as a buffer and add an antigen or an antibody thereto.

By immobilizing the antigen or antibody on the carrier in this manner, the immunological diagnostic agent of the present invention can be obtained as an aqueous dispersion, which can be used for detection or quantification of the antigen or antibody. When a carrier having an average particle size of 0.02 to 20 μm is used as a carrier for an immunological diagnostic agent, the carrier on which an antigen or an antibody is immobilized can be easily dispersed in an aqueous medium without an emulsifier, resulting in a stable aqueous dispersion. An immunological diagnostic agent is obtained. The concentration of the antigen or antibody-immobilized carrier in the aqueous dispersion is 0.01 to 30% by weight, preferably 0.1 to 10%.
Weight%

For the detection or quantification of the antigen or antibody, it is preferable to measure the aggregate in which the immunological diagnostic agent of the present invention has undergone the antigen-antibody reaction by the light scattering measurement method or the integrated sphere turbidity measurement method. In this case, since the carrier for the immunological diagnostic agent has a high refractive index, highly sensitive measurement is possible, for example, ng /
Highly sensitive measurement is possible even for analytes with a concentration of ml.

[0035]

According to the present invention, since the styrene derivative represented by the general formula [1] is used as the monomer component, it has a high refractive index and facilitates immobilization of an antigen or antibody. A carrier for an immunological diagnostic agent is obtained.

Since the antigen or antibody is immobilized on the above carrier, an immunological diagnostic agent can be obtained which can perform measurement with high sensitivity even for low concentration of analyte.

[0037]

EXAMPLES The present invention will be described in more detail with reference to examples below, but these examples do not limit the scope of the present invention. Example 1 2000 parts by weight of ion-exchanged water, 0.15 parts by weight of potassium persulfate, and a styrene derivative of the component (A) and a vinyl-based simple substance of the component (B) having the composition shown in Table 1 were placed in a reaction vessel substituted with nitrogen gas. The monomer was added, the stirrer was driven at 200 rpm, and the copolymerization was carried out for 24 hours while controlling the reaction temperature at 70 ° C. After the copolymerization reaction, the reaction was terminated by cooling to 25 ° C. 8 after removing coagulum by filtration
After centrifuging at 000 rpm for 15 minutes and removing the supernatant, it was purified by redispersion with ion-exchanged water to obtain Samples 1 to 5 consisting of an aqueous dispersion of polymer particles as a carrier for immunological diagnostic agents. .

The particle size of the obtained sample was measured by a submicron particle size distribution analyzer MODEL N4SD manufactured by COULTER. Moreover, the refractive index of the sample was measured with the sodium D line using an Abbe refractometer. The sample for refractive index measurement at this time, after freeze-drying the aqueous dispersion,
It was diluted to 10 wt% using tert-butyl bromoacetate as a solvent, and the refractive index of the polymer was calculated by subtracting the value corresponding to the refractive index of the solvent from the measured value. The results are shown in Table 1. Further, using the device EL-1000 manufactured by Kyowa Medex Co., Ltd., the integrated sphere turbidity at each latex concentration was measured for Samples 2 and 3. The results are shown in Table 2.

Comparative Example 1 A monomer was added in the composition shown in Table 1 and polymerization and purification were carried out in the same manner as in Example 1 to obtain Comparative Samples 1 and 2 which were styrene particles containing no component (A). Obtained. The particle size, refractive index, and integrated sphere turbidity of the obtained comparative sample were measured in the same manner as in Example 1. The results are shown in Tables 1 and 2.

[0040]

[Table 1]

[0041]

[Table 2]

From the results shown in Table 1, it is understood that the refractive indexes of Samples 1 to 5 of Example 1 are higher than those of Comparative Samples 1 and 2 of Comparative Example 1. Further, from the results in Table 2, it can be seen that the integrated sphere turbidity of Samples 2 and 3 of Example 1 is higher than the integrated sphere turbidity of Comparative Sample 2 of Comparative Example 1.

Example 2, Comparative Example 2 Sample 2 or Comparative Sample 2 (1.0% by weight of aqueous dispersion 2
0.1 ml by weight of anti-CEA (carcino-em)
Bronic antigen) antibody solution (1 ml) was added to the sample to immobilize the antibody by adsorbing the antibody.
Antibody sensitization was performed to obtain a diagnostic agent. The antibody binding rate of Sample 2 was 92% of that of Comparative Sample 2.

Using a diagnostic agent sensitized with such an anti-CEA antibody, the change in integrated turbidity at each CEA concentration was measured using EL-1200 manufactured by Kyowa Medex Co., Ltd. For the measurement, 0.1 ml of buffer solution is added to 0.25 ml of diagnostic agent.
5 ml and 0.1 ml of each CEA concentration sample were added. The results are shown in Table 3.

[0045]

[Table 3]

From the results of Table 3, in Comparative Example 2 using Comparative Sample 2, the integrated sphere turbidity is generally low, and the integrated sphere turbidity does not significantly change with a change in CEA concentration.
In Example 2 using the sample 2 having a high refractive index, a high integrated sphere turbidity is shown, and the integrated sphere turbidity also greatly changes with a change in CEA concentration, which shows that the sensitivity is high.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location G01N 33/545 B (72) Inventor Yasumi Koinuma 32-16 Higashiarai, Tsukuba, Ibaraki (72) Inventor Oguri Alone 2-5-45 Yoki, Yokkaichi-shi, Mie Inventor Tsutsugu Imai 1-33-12 Okuradai, Yokkaichi-shi, Mie (72) Inventor Hiroshi Kishioka 4817 Nishihino-cho, Yokkaichi-shi, Mie Inventor Shohei Konishi 1-12-5 Nozawa, Setagaya-ku, Tokyo

Claims (2)

[Claims] 1. (A) General formula [1] (In the formula, R represents a hydrogen atom or a methyl group, X represents F,
A halogen atom or a methyl group selected from Cl, Br and I is shown. k represents 1 or 2, m and n are 0
Or 1 is shown. ) Styrene derivatives 5-9
9.9% by weight, and (B) vinyl compound 0.1-9
A carrier for an immunological diagnostic agent, comprising polymer particles obtained by polymerizing 5% by weight of a monomer component in an aqueous medium using a radical polymerization initiator. 2. An immunological diagnostic agent comprising an aqueous dispersion of an immobilized antigen or an immobilized antibody in which the antigen or antibody is immobilized on the carrier for immunological diagnostic agent according to claim 1.