JPH06160394A - Insoluble carrier and immunologically detecting method using the same carrier - Google Patents

Abstract

PURPOSE:To provide an insoluble carrier for immunological detection and a method for immunologically detecting by using the same carrier. CONSTITUTION:An insoluble carrier for an immunological detection is formed at least on its surface of polymer electrolyte complex. The carrier is formed at least on its surface of the polymer electrolyte complex by fixing immunologically active substance to be immunologically bonded to a substance to be detected to the surface in a state to be bonded to the substance to be detected. A method for immunologically detecting by using the same carrier effectively suppresses a conventional nonspecific reaction to improve a measuring accuracy and accurately detects and measures the substance to be inspected which is contained in an extremely small quantity in a living specimen.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an insoluble carrier for immunological detection, at least the surface of which is composed of a polyelectrolyte complex, and an immunological detection method using the insoluble carrier.

[0002]

2. Description of the Related Art In the field of clinical diagnosis, immunological measurement of a substance to be detected in a test sample is a routinely used means, and items to be measured are various antigens, antibodies and polypeptides. A wide variety of haptens such as drugs, steroids, and drugs. The amounts of these components contained in biological fluids are often extremely small, and accurate measurement of these components is important for accurate disease diagnosis and confirmation of therapeutic effects. Higher precision is progressing.

As a specific measuring method, an immunoturbidimetric method,
There are radioimmunoassay, latex agglutination, enzyme immunoassay (EIA), solid-phase enzyme immunoassay (ELISA), fluorescent immunoassay, luminescent immunoassay (CLIA) and the like. Particularly in recent years, so-called latex such as polystyrene, which is an organic synthetic polymer, or an insoluble carrier such as glass or silica of an inorganic substance, which carries an immunologically active substance capable of immunologically binding to a substance to be detected, A method is often used in which an immune reaction is performed with a substance to be detected in a test sample, and an agglutination reaction at that time and a signal due to a labeling substance are detected by visual or optical means.

However, most of the substances to be detected have a very small content, and the biological fluid as a test sample has physical properties such as properties and viscosity depending on its origin and type, composition components contained in the test sample, etc. There is a big difference in the variety. Therefore, a reaction unrelated to the originally intended immunological reaction,
There is a problem in that a so-called non-specific reaction occurs, which affects the measurement result.

[0005]

There are various possible causes of the non-specific reaction, and one of them is the involvement of the material of the insoluble carrier and the surface structure in the reaction system. For example, most of the latex particles currently in widespread use are commercially available polystyrene latex particles. Polystyrene is synthesized by a so-called emulsion polymerization method by a radical polymerization mechanism in the presence of an emulsion polymerization agent (for example, a surfactant) as a polymerization initiator and potassium persulfate, using a styrene-based aromatic vinyl compound as a monomer. ing. Further, in order to impart hydrophilicity to the produced polymer by further carrying out a polymerization reaction by combining a styrene monomer and an unsaturated carboxylic acid such as acrylic acid or methacrylic acid, or to adjust the specific gravity of the produced polymer. Intricate compounding and processing such as polymerization reaction including halogen atom-containing acrylates are performed. Therefore, although the produced polymer thus obtained is simply referred to as a styrene-based polymer, there are large differences in the composition, the type and amount of the surface functional group, the affinity and the charge. It is considered that substances other than the substance to be detected interfere with each other in response to various physical properties of such a carrier to cause a nonspecific reaction. In addition, granular materials such as latex particles sometimes cause self-aggregation during storage before use in the reaction, and this is also considered to be influenced by the physical properties of the carrier surface.

When performing an immunological assay using such an insoluble carrier having various properties, first, an immunologically active substance capable of immunologically binding to the substance to be detected is used as the insoluble carrier. To combine. The way to combine them is
A method of bringing an insoluble carrier and an immunologically active substance into contact with each other in a solution to bind to each other, a so-called physical adsorption method (hydrophobic bond due to interaction between surface hydrophobicity of insoluble carrier and hydrophobicity of immunologically active substance, or insoluble The binding between the negative charge on the surface of the carrier and the positively charged site of the immunologically active substance) and the introduction of a crosslinkable group (for example, amino group, carboxyl group or thiol group) on the surface of the insoluble carrier, There is a method of covalently bonding, a so-called chemical bonding method.

When immunological measurement is performed using the insoluble carrier to which the immunologically active substance is bound, substances other than the substance to be detected (for example, various proteins coexisting in the sample, other substances, or detection) In order to suppress the so-called non-specific reaction in which the labeled antibody used in the system) interferes with the insoluble carrier, measures have been taken to add inhibitors and modify the properties of the insoluble carrier itself. For example, as a non-specific reaction inhibitor, normal mammalian serum protein, albumin, skim milk, collagen, gelatin, etc. are added to treat the surface of the insoluble carrier to which the immunologically active substance is bound. It is being appreciated. Further, regarding the improvement of the method for producing the insoluble carrier itself, various methods are disclosed in, for example, JP-A-54-59988, JP-A-56-61410, and JP-A-61-218946. .

However, in particular, when quantifying a component contained in a biological sample in an extremely small amount, extremely sensitive measurement is required, whereas in the conventional method, a substance other than a substance to be detected is detected. It was difficult to sufficiently suppress substance interference, that is, nonspecific reaction. The present inventor has conducted various studies to solve the drawbacks of the prior art, and surprisingly, when an insoluble carrier formed using a polyelectrolyte complex or an insoluble carrier coated with a polyelectrolyte complex is used. , It was found that the above-mentioned non-specific reaction can be effectively suppressed, the measurement accuracy can be improved, and the substance to be inspected, which is contained in the biological sample in an extremely small amount, can be detected or measured accurately and highly accurately. It was The present invention is
It is based on these findings.

[0009]

Accordingly, the present invention relates to an insoluble carrier for immunological detection, which is characterized in that at least the surface is formed by a polyelectrolyte complex. The present invention also relates to an immunological detection method using the insoluble carrier. Hereinafter, the present invention will be described in detail.

The polyelectrolyte complex used in the present invention (p
The polyelectrolyte complex (hereinafter, also referred to as PEC) is a substance known per se as described in WO92 / 09198. PEC can be instantaneously formed by mixing a solution of a cationic polymer which is a polyelectrolyte having a positive charge and a solution of an anion polymer which is a polyelectrolyte having a negative charge. The PEC thus obtained is soluble in a special ternary solvent (for example, water / acetone / low-molecular-weight salt having a specific composition), but is insoluble in general solvents. PEC can provide various polyelectrolyte complexes having various properties depending on the types of starting polymers (polyelectrolytes), their mixing ratio, preparation conditions, and the like.

The cationic polymer used in the present invention is
A polymer electrolyte containing N ⁺ atoms in a repeating unit, and specifically,

(A1) General formula (I)

[Chemical 1]

[Chemical 2] A cationic polymer represented by: quaternary ammonium salt polymer,

(A2) General formula (II)

[Chemical 3]

[Chemical 4] A cationic polymer represented by: quaternary ammonium salt polymer,

(A3) General formula (III)

[Chemical 5] A cationic polymer represented by: a basic amino acid polymer, and

(A4) At least one compound selected from the group consisting of cationic polysaccharides.

The anionic polymer used in the present invention is
-COO in the repeating unit ^- based, -SO ₃ ^- group, -PO
A polymer electrolyte containing a ₃ H ⁻ group or a —PO ₃ ²⁻ group, and specifically,

(B1) General formula (IV)

[Chemical 6] Anionic polymer represented by, that is, an acidic amino acid polymer,

(B2) General formula (V)

[Chemical 7] Is at least one compound selected from the group consisting of an anionic polymer represented by the formula (i.e., an acrylic acid-based polymer) and (b3) an anionic polysaccharide.

The cationic polymer (a) of the general formula (I)
Specific examples of 1) include quaternary polyethyleneimine chloride and poly (N, N, N ', N'-tetramethyl-
Alkylene-p-xylylene diammonium chloride), poly (N, N, N ', N'-tetramethyl-alkylene-diammonium dichloride), poly (N,
N, -dimethyl-3-hydroxypropylammonium chloride), poly (2-hydroxy-3-methacryloyloxypropyltrimethylammonium chloride), poly (2-methacryloyloxyethyltrimethylammonium chloride), poly (glycidyltrimethylammonium chloride) , Poly [(dimethyliminio) ethylene (dimethyliminio) methylene-1,4-
Phenylene methylene dichloride] [generally referred to as 2X], poly [(dimethylinio) hexamethylene (dimethyliminio) methylene-1,4-phenylenemethylene dichloride] [generally referred to as 6X], poly [(dimethyl (Iminio) hexamethylene chloride]
[Generally referred to as 6,6], poly (N-ethyl-4)
-Vinylpyridinium bromide), poly (dimethyldiallylammonium chloride), and the like.

The cationic polymer (a) represented by the general formula (II)
Specific examples of 2) include poly (vinylbenzyltrimethylammonium chloride), polyvinylpyridinium chloride, and poly (N-benzyl-4-vinylpyridinium chloride). Specific examples of the cationic polymer (a3) of the general formula (III) include polylysine, polyarginine or copolymers of monomers constituting these polymers, and further, these monomers and glycine, alanine,
It is a copolymer with phenylalanine, tyrosine, valine, leucine, isoleucine, serine, threonine, methionine, cysteine, histidine, proline, and / or tryptophan. Cationic polysaccharide (a4)
Specific examples thereof include chitosan and derivatives thereof, and a diethylaminoethyl derivative of a neutral polysaccharide, as described in WO92 / 09198. Examples of neutral polysaccharides include dextran, cellulose, mannan, starch, agarose and the like. The degree of diethylaminoethyl substitution of these derivatives is 0.5 to 2.0 groups, preferably 0.7 to 1.5 groups, per sugar residue, and the degree of polymerization is 50 to 500, preferably 100 to 1000. Is. The nitrogen atom of the diethylaminoethyl group and the anionic group of the anionic polymer are bonded.

The anionic polymer (b) of the general formula (IV)
Specific examples of 1), polyglutamic acid, polyaspartic acid or a copolymer of monomers constituting these polymers, further, these monomers and glycine, alanine, phenylalanine, tyrosine, valine, leucine, isoleucine, serine, It is a copolymer with threonine, methionine, cysteine, histidine, proline, and / or tryptophan. The polyamino acids represented by the general formulas (III) and (IV) can be synthesized by a general acid anhydride monomer method, active esterification method, Maryfield method or the like.

The anionic polymer (b) of the general formula (V)
Specific examples of 2) include polyacrylic acid, polymethacrylic acid, polyitaconic acid monoester, polymaleic acid monoester, polyvinylsulfonic acid, polystyrenesulfonic acid, and any two or more kinds of monomers constituting these polymers. Further, it is a copolymer of these monomers and a carboxylic acid derivative having an alkyl group having 6 to 18 carbon atoms which is bonded to the carboxyl group of the monomer by an ester bond.

Specific examples of the anionic polysaccharide (a3) include hyaluronic acid and its derivatives, alginic acid and its derivatives, chondroitin sulfate A, chondroitin sulfate C and chondroitin sulfate, as described in WO92 / 09198. B (dermatan sulfate) and its derivatives, chondroitin sulfate D and its derivatives, chondroitin sulfate E and its derivatives, heparan sulfate and its derivatives, heparin and its derivatives, κ-carrageenan and its derivatives, λ-carrageenan and its derivatives, cellulose derivatives , Chitin derivatives, carboxymethyl starch and its derivatives, amylose derivatives, amylopectin derivatives, β-1,3′-glucan derivatives (eg curdlan), β-1,2′-glucan derivatives, β-1,3 ′
-; Β-1,6'-glucan derivative (for example, lentinan, schizophyllan, choriolan), dextran derivative, pullulan derivative, agarose derivative, β-1,4 '
Mention may be made of galactan derivatives, mannan derivatives and inulin derivatives.

PEC can be easily prepared by reacting these cationic polyelectrolytes and anionic polyelectrolytes in a conventional manner. That is, each aqueous solution of the above-mentioned cation polymer and anion polymer (preferably 10 ⁻⁵ mol / liter as an ionic seat)
10 ^-2 mol / liter), the concentration ratio of the cation seat of the cation polymer and the anion seat of the anion polymer (cation seat / anion seat) is in the range of 0.25 to 4.0, preferably 0.4 to 2 Within the range of 0.5, the reaction may be carried out by mixing in an aqueous solution. When the concentration ratio between the cation seat and the anion seat is out of the range of 0.25 to 4.0, it becomes difficult to form PEC, which is not preferable. As a solvent for dissolving each polymer, purified water, various buffer solutions (eg, phosphate buffer solution, etc.), or a mixed solution thereof with a water-miscible organic solvent (eg, methanol, ethanol, acetone, etc.) can be used. . Since this reaction has a relatively high activity, the pH, ionic strength, temperature, etc. of the solution can be in a relatively wide range, but generally, the pH is 3 to 9 and the ionic strength is 0.
It is carried out at ˜1.0 and 0 to 100 ° C. The polyelectrolyte complex thus obtained can be directly formed as an insoluble carrier or coated on a suitable substrate (for example, polystyrene, erythrocyte, silica, glass, gelatin, or a conventional insoluble carrier material) for immunological treatment. It can be used as an insoluble carrier for detection.

In the present invention, by changing the blending ratio of the cationic polymer and the anionic polymer, the charge balance of the PEC to be produced can be easily changed and adjusted. That is, PE having various charge balances
By using C, for example, the surface charge of the insoluble carrier coated with it can be adjusted to improve the immobilization efficiency of the immunologically active substance to be bound and to suppress the non-specific reaction during preparation of the insoluble carrier. It becomes possible to select appropriately. The charge balance of the PEC used in the present invention is preferably in the range of -6 to +6. Here, the charge balance expresses the charge state of PEC by the concentration ratio of each cation seat and anion seat of the cation polymer and the anion polymer which are the starting materials. For example, when the concentration ratios of the cation seat of the cationic polymer and the anion seat of the anionic polymer used are the same, the charge balance of the generated PEC is ± 0. If the concentration ratio is higher than this (that is, the concentration at the cation seat is higher), the charge balance is positive, and if the concentration ratio is lower (that is, when the anion seat is higher), the charge balance is negative. When the concentration ratio is 1.5, the charge balance is +2, and when the concentration ratio is 0.5, the charge balance is -3.3. The charge balance can be easily adjusted by changing the mixing amount of the cationic polymer solution and the anionic polymer solution having the same concentration.

By changing the pH of the solution, the salt concentration, and the content of the water-miscible organic solvent during the preparation of PEC, the physical properties (eg hardness and elasticity) of the produced PEC can be freely adjusted. Since it is possible to easily form particles, plates, films, etc., the entire insoluble carrier of the present invention can be formed by PEC itself.

Since PEC has the property of easily and easily coating various materials, it is possible to produce the insoluble carrier of the present invention by coating the entire surface of a suitable substrate with PEC. it can. As the base material, a synthetic polymer compound such as polystyrene or polyvinyl chloride, an organic material such as red blood cells, an inorganic material such as silica, or papers can be used. Further, it is used as an insoluble carrier in the conventional method. The carrier itself may be coated with PEC before use. For example, the latex particles used as an insoluble carrier in the latex agglutination method have a measurement sensitivity that varies depending on the particle size, so that the commercially available product has already been controlled in its particle size. Therefore, when the commercially available product is used as a substrate and coated with PEC, the insoluble carrier according to the present invention in which the particle size is controlled can be easily produced.

Further, according to the finding of the present inventor, PEC also has a function of protecting an immunologically active substance. For example, it is possible to coat the substrate after incorporating the immunologically active substance to be bound to the insoluble carrier into the PEC solution. Therefore, the following method can be mentioned as a typical method for preparing the insoluble carrier according to the present invention using PEC. (1) A method of binding an immunologically active substance to a substrate (in particular, an insoluble carrier in the conventional method), and subsequently coating PEC. (2) A method of coating a substrate with PEC and subsequently binding an immunologically active substance. (3) A method of mixing an anion polymer solution and a cation polymer solution, then adding an immunologically active substance, and bringing the mixed solution into contact with the substrate. (4) A method in which the anionic polymer solution, the cationic polymer solution, and the immunologically active substance-containing liquid are added at the same time and mixed, and the mixed liquid is brought into contact with the substrate. (5) A method in which a liquid containing an immunologically active substance is added to a cationic polymer solution, the mixed liquid is brought into contact with a substrate, and then an anionic polymer solution is added and mixed. (6) A method of adding a liquid containing an immunologically active substance to an anionic polymer solution, bringing the mixed liquid into contact with a substrate, and subsequently adding a cationic polymer solution and mixing.

To coat the substrate with PEC, for example,
It can be performed by a method such as coating, spraying or dipping. P
The EC solution may simply be contacted with the substrate. For example, a cationic polymer solution and an anionic polymer solution are mixed, the substrate is immersed in the mixed solution for about 0.5 to 48 hours, and the PEC-coated substrate thus treated is washed with physiological saline or purified water. Air dry at room temperature, or 50
Warm to about 100 ° C and dry. When styrene beads, latex, etc. are used as the substrate, they are coated with PEC, washed by a centrifugation method, etc., and stored in a washing solution or a solution such as an appropriate buffer solution without air-drying. You can leave it alone. When the base material is immersed in the PEC solution, the salt concentration and temperature can be appropriately adjusted as necessary depending on the material of the base material and the characteristics of the immunologically active substance bound to the base material. For example, when the polystyrene beads are immersed in the PEC solution, for example, a salt such as NaCl is added in an atmosphere range of 0.01 to 5M and a temperature of 0 to 100 ° C.
By generating the EC, contacting it with the substrate, and immersing it, the coating time of the PEC on the substrate is shortened (for example by increasing the temperature) or, conversely, the immunologically active substance is bound. When PEC is coated on the base material, the PEC can be coated under mild conditions that do not impair the activity of the immunologically active substance.

In the present specification, the substance to be detected refers to a physiologically active substance that can be detected by an immunological reaction, and particularly refers to a physiologically active substance contained in a biological component. Specifically, there are proteins, enzymes, polysaccharides, lipids, nucleic acids and the like, and examples thereof include various antigens, antibodies and receptors. More specifically, fibrinogen, albumin, C-reactive protein, anti-streptolysin O, rheumatoid factor, alpha-fetoprotein (AFP), Treponema pallidum antibody, HBs antibody, H
Bc antibody, HBe antibody, antibody against HTLV-1, H
There are antibodies against IV and the like. Furthermore, it refers to haptens, which are low molecular weight compounds, such as hormones, various drugs such as antiepileptic drugs, and antibodies to haptens. Thus, an immunologically active substance capable of immunologically binding to these refers to the other immunological partner of said substance.
In addition, the test sample in the present specification is not particularly limited as long as it is a sample that may contain the substance to be detected, but in particular blood, serum, plasma, urine, saliva, spinal fluid, etc. Biological fluids, cell and tissue extracts, and the like.

The immunologically active substance can be prepared and obtained by a conventionally known means. For example, in the case of measuring a certain antigenic substance in a test sample, an immunologically active substance such as an antiserum or a polyclonal antibody or a monoclonal antibody capable of specifically recognizing it or a product containing the same is conventionally used. Known operations (eg immunization of mammals)
Can be prepared by When the immunologically active substance is bound to a substrate not previously coated with PEC, or when bound to an insoluble carrier coated with PEC,
Both known physical adsorption methods or chemical bonding methods can be used.

The insoluble carrier of the present invention to which the immunologically active substance thus prepared is bound can be used in the same manner as the conventionally known insoluble carrier. For example, the insoluble carrier of the present invention to which an immunologically active substance is bound is directly contacted with a test sample or a treatment solution of the test sample, and an immune reaction accompanied therewith is detected by a conventionally known means to detect the sample. The substance to be detected in the sample can be accurately measured. For example, latex agglutination method, enzyme immunoassay,
It can be applied to general immunological detection methods using conventional insoluble carriers such as solid-phase enzyme assay, fluorescent immunoassay, luminescent immunoassay, and metal colloid immunoassay. At this time, a conventionally known non-specific reaction inhibitor (for example, skim milk,
Albumin or a surfactant) may be used in combination.

[0033]

In general, nonspecific reactions in immunological reactions using insoluble carriers are due to hydrophilicity, hydrophobicity, chemical structure, charge, surface or interface free energy, or fine structure of the insoluble carrier itself. Possible impact. On the other hand, PEC is a hydrophilic microdomain structure,
Although it exhibits remarkably diverse properties due to structural changes in surface water or changes in charge balance, it has a high degree of freedom in controlling these physical properties. Therefore, by imparting such controllable physical properties to the insoluble carrier, the factors of the nonspecific reaction can be suppressed. Furthermore, P
ECs also have the property of maintaining the activity of immunologically active substances immobilized on insoluble carriers.

[0034]

EXAMPLES The present invention will be described in more detail below with reference to examples, but these do not limit the scope of the present invention. The average molecular weights described in the following examples are number average molecular weights measured by the vapor pressure drop method. Ab is an abbreviation for antibody. The polymers used in the following examples and their abbreviations are as follows. (A) Cationic polymer 6X: poly [(dimethyliminio) hexamethylene (dimethyliminio) methylene-1,4-phenylenemethylene dichloride (average molecular weight about 10,000) 2X: poly [(dimethyliminio) ethylene (dimethyliminio) ) Methylene-1,4-phenylene methylene dichloride (average molecular weight of about 6000) PVBMA: poly (vinylbenzyltrimethylammonium chloride) (average molecular weight of about 15000) PLL: poly (L-lysine) (average molecular weight of about 3000) Chitosan: (desorption Acetylation degree 100%, average molecular weight about 20
00) (b) Anionic polymer CLA: Acrylic acid / lauryl acrylate random copolymer (acrylic acid content about 80 mol%, average molecular weight about
10000) COA: Random copolymer of acrylic acid / 2-ethylhexyl acrylate (acrylic acid content about 60 mol%, average molecular weight about 8000) PGA: polyglutamic acid (average molecular weight about 4000) Alg: sodium alginate (average molecular weight about 4000)

Example 1: Measurement of α-fetoprotein (1) Preparation of bead-like carrier (beads / Ab / PEC) Cationic polymer 6X was added at a concentration of 173.5 μg / ml (10 ⁻³ M as ion seat; It was adjusted to be 10 ⁻³ UM using a 10 mM phosphate buffer solution (pH 7.0) containing 0.15 M NaCl (hereinafter also referred to as PBS) to prepare a 6 × solution. Further, 165.6Myug the CLA is an anionic polymer / ml of a concentration (10 ^{- 3}
UM) to prepare CLA solution. An anti-human α-fetoprotein F (ab ′) ₂ antibody [anti-human AFP antibody: polystyrene beads (diameter 6.35 mm: Sekisui Chemical Co., Ltd.)]
Yatron Co., Ltd.] solution (8 μg / ml: PBS) 0.5
After adding ml, the mixture was shaken at 56 ° C. for 30 minutes, and then washed with physiological saline three times. A PEC solution was prepared by mixing 0.25 ml of the 6X solution and 0.25 ml of the CLA solution. Add one antibody-sensitized bead to the solution and mix at 56 ° C for 3
After shaking for 0 minutes, the plate was washed 3 times with physiological saline. The beaded carrier thus obtained was stored in physiological saline and used for the measurement described below. (2) Preparation of beaded carrier (beads / PEC + Ab) 0.25 ml of the 6X solution of the above item (1) and CL of the above item (1)
After preparing a PEC solution by mixing 0.25 ml of the A solution, the anti-human AFP antibody solution (4 mg / m 2) of the above (1)
1: PBS) 1 μl (final concentration 8 μg / ml) was added.
To this solution, one polystyrene bead of the above (1) was added, shaken at 56 ° C. for 30 minutes, and then washed three times with physiological saline. The beaded carrier thus obtained was stored in physiological saline and used for the measurement described below. (3) Bead-shaped carrier (beads / cationic polymer + ani)
Preparation of on-polymer + Ab) 0.25 ml of 6X solution of the above (1) and CL of the above (1)
A solution (0.25 ml) and the anti-human AFP antibody (4 mg / ml: PBS) solution (1 μl) described in (1) above were simultaneously added and mixed. To this solution, one polystyrene bead of the above (1) was added, shaken at 56 ° C. for 30 minutes, and then washed three times with physiological saline. The beaded carrier thus obtained was stored in physiological saline and used for the measurement described below.

(4) Bead-shaped carrier (beads / (cations
Preparation of polymer + Ab) + anionic polymer) 1 μl of the anti-human AFP antibody (4 mg / ml: PBS) solution of the preceding paragraph (1) was added to 0.25 ml of the 6X solution of the preceding paragraph (1), and then mixed. One polystyrene bead was added. Further, 0.25 ml of the CLA solution described in (1) above was added, and the mixture was shaken at 56 ° C. for 30 minutes and then washed 3 times with physiological saline. The beaded carrier thus obtained was stored in physiological saline and used for the measurement described below. (5) Bead-shaped carrier (beads / (anion polymer + A
Preparation of b) + cationic polymer) To 0.25 ml of the CLA solution of the above (1), 1 μl of the anti-human AFP antibody (4 mg / ml: PBS) solution of the above (1) was added and mixed, and then polystyrene of the above (1) was added. Beads 1
Added pieces. Further, 0.25 ml of the 6X solution described in (1) above was added, and the mixture was shaken at 56 ° C for 30 minutes, and then washed 3 times with physiological saline. The beaded carrier thus obtained was stored in physiological saline and used for the measurement described below. (6) Preparation of conventional beads (uncoated with PEC) For one polystyrene bead of the above item (1), the anti-human AFP antibody solution (8 μg / ml: PBS) of the above item (1)
0.5 ml was added, the mixture was shaken at 56 ° C. for 30 minutes, and then washed with physiological saline three times. The beaded carrier thus obtained was stored in physiological saline and used for the measurement described below. (7) Measurement of AFP PBS containing 0.1% denatured bovine albumin (hereinafter referred to as d-BS
AFP standard solution [Yatron Co., Ltd.] was diluted with A) to prepare each dilution series of 0 to 40 ng / ml as AFP. One insoluble carrier prepared by the above-mentioned operations (1) to (6) was added to each 400 μl of the dilution series, allowed to stand at 37 ° C. for 1 hour, and then washed twice with physiological saline. A peroxidase-labeled anti-human AFP antibody solution (8 μg /
400 μl of (ml: d-BSA) was added, the mixture was allowed to stand at 37 ° C. for 1 hour, and washed 3 times with physiological saline. The treated insoluble carrier is transferred to a new container, and 0.25% o-
400 μl of 0.1 M citrate buffer (pH 6.0) containing phenylenediamine and 0.015% H ₂ O ₂ was added, and the mixture was left standing at room temperature for 25 minutes to develop color. 1N-HC
Color development was stopped by adding 1 and the absorbance at a wavelength of 492 nm was measured. The results are shown in FIGS. 1 to 5 by comparing the carrier of the above (1) to (5) with the carrier of the above (6).
Shown in. As is clear from FIGS. 1 to 5, it was confirmed that any polystyrene beads treated with PEC had a reduced blank value and increased effective sensitivity (ΔE: absorbance of sample−blank value) as compared with the conventional method. Was done.

Example 2: Measurement of α-fetoprotein As combinations of cationic polymer and anionic polymer, 6X-COA, 2X-CLA, PVBMA-COA, PVBMA-CLA, PLL-PG.
Using the combination of A, 6X-Alg, chitosan-Alg, and chitosan-COA, the PEC was performed in the same manner as in Example 1 (1).
As a result of preparing a coated beaded carrier and measuring the AFP, the polystyrene beads treated with various combinations of PECs have a reduced blank value and S / N as compared with the conventional method.
Good results were shown, such as an increase in the ratio.

Example 3: Measurement of α-fetoprotein
(Difference due to charge balance) (1) PEC coated beaded carrier (beads / PEC / A
Preparation of b) 211.5 μg of the cationic polymer PVBMA /
A PVBMA solution was prepared by adjusting the concentration of PBS (10 ⁻³ UM) with PBS. Further, CLA, which is an anionic polymer, was adjusted to a concentration of 10 ⁻³ UM with PBS in the same manner as in Example 1 (1) to prepare a CLA solution. PVB above
2 ml of MA solution and 1 ml of CLA solution were mixed to prepare a PEC solution having a charge balance of +3.3. This solution 0.
Polystyrene beads 1 used in Example 1 for 5 ml
Add them and shake at 56 ° C for 1 hour, then add 3 times with physiological saline.
Washed twice. The anti-human AFP antibody solution of Example 1 (8 μg / ml: PBS) was added to one PEC-coated beaded carrier.
After adding 0.5 ml, the mixture was allowed to stand at 4 ° C for one day and then washed with physiological saline three times. The prepared insoluble carrier was stored in physiological saline and used for the measurement described below. Similarly, PEC (charge balance +0.9) from 1.2 ml of PVBMA solution and 1 ml of CLA solution, PEC (charge balance ± 0) from 1 ml of PVBMA solution and 1 ml of CLA solution, PVBMA
PEC (charge balance -0.9) from 1 ml of solution and 1.2 ml of CLA solution, and 1 ml of PVBMA solution and CL
Using each solution of PEC (charge balance-3.3) from 2 ml of solution A, each bead was prepared by the same operation as described above. (2) Preparation of beads by conventional method (uncoated with PEC) For one polystyrene bead used in Example 1, the anti-human AFP antibody solution of Example 1 (8 μg / ml: PBS)
After adding 0.5 ml, the mixture was allowed to stand at 4 ° C for one day and then washed with physiological saline three times. The insoluble carrier thus prepared was stored in physiological saline and used for the measurement described below. (3) Measurement of AFP Using each insoluble carrier prepared by the above operation, AFP was measured by the same operation as in Example 1 (7). The results are shown in FIGS. PE with a charge balance of +3.3
The beads prepared by using C (Fig. 6) had the same effective sensitivity (blank value increased) as the conventional method, but the carrier using PEC having other charge balance was compared with the conventional method. It was confirmed that the blank value decreased and the effective sensitivity increased.

Example 4: Inhibitory effect of non-specific reaction derived from serum
As a result, 6X, which is a cationic polymer, was adjusted to a concentration of 173.5 μg / ml (1 × 10 ⁻³ UM) by using PBS with respect to one polystyrene bead used in Example 1 to obtain a 6X solution. did. In addition, C which is an anionic polymer
LA was treated with PBS in the same manner as in Example 1 (1) to a concentration of 10 ⁻³ UM.
To prepare a CLA solution. A 6X solution and a CLA solution were mixed in equal amounts to prepare a PEC solution, and 0.5 PEC solution was added to one polystyrene bead used in Example 1.
Add ml, shake at 56 ° C for 30 minutes, and add 3 ml of saline.
The beads were washed once to prepare a PEC-coated beaded carrier. next,
To 100 μl of human serum, 300 μl of d-BSA and one polystyrene bead prepared by the above operation were added,
After standing still at 37 ° C. for 1 hour, it was washed twice with physiological saline. 400 μl of a peroxidase-labeled anti-goat IgG antibody solution (8 μg / ml) was added thereto, allowed to stand at 37 ° C. for 1 hour, and washed 3 times with physiological saline. The treated insoluble carrier was transferred to a new container, which contained 0.25% o-phenylenediamine and 0.015% H ₂ O ₂ as a color-developing solution.
400 μl of 1M citrate buffer (pH 6.0) was added, and the mixture was allowed to stand at room temperature for 25 minutes to develop color, and 1N-HCl was added.
(3 ml) was added to stop the reaction, and the absorbance at a wavelength of 492 nm was measured. As a control test, the same operation was performed using PEC-uncoated polystyrene beads. The results are shown in Table 1.

[0040]

[Table 1] As shown in the results of Table 1, even under the condition that color should not be originally developed, the PEC uncoated beads cause color development due to interference of substances other than the substance to be detected in serum,
It was confirmed that similar interference was effectively suppressed in the PEC-coated beaded carrier. Among the combinations shown in Examples 1 to 3 above, similar effects were observed in combinations other than the combination of 6X-CLA, and the usefulness of the present invention could be confirmed.

Example 5: of α 1 -antichymotrypsin
Measurement Anti-human α1-antichymotrypsin antibody [Institute of Medical Biology] was digested with pepsin by a known means to prepare anti-human α1-antichymotrypsin F (ab ') ₂ antibody (anti-human α1-ACT antibody). did. To one polystyrene bead [diameter 6.35 mm; Sekisui Chemical Co., Ltd.], the anti-human α ₁ -ACT antibody solution (12.5 μg /
0.5 ml) was added, the mixture was shaken at 37 ° C. for 2 hours to bind the antibody to the beads, and the beads were washed 3 times with physiological saline. 6X, which is a cationic polymer, should be P so that it becomes 1 × 10 ⁻³ UM.
A 6X solution was prepared using BS. Further, PVBMA, which is a cationic polymer, was adjusted to 1 × 10 ⁻³ UM using PBS to prepare a PVBMA solution. Further, COA as an anionic polymer was adjusted to 1 × 10 ⁻³ UM with PBS to prepare a COA solution.
A 6X solution and a COA solution were mixed in equal amounts to prepare a PEC consisting of a combination of 6X-COA, 0.5 ml of which was added to the beads to which the anti-human α ₁ -ACT antibody had been bound, and the mixture was allowed to stand at 37 ° C. for 4 hours. After shaking for a time, the plate was washed 3 times with physiological saline and used for the measurement described below. Similarly, PVBMA solution and CO
An equal amount of the solution A was mixed to prepare a PEC of the PVBMA-COA combination, and the beads were treated by the same operation as described above and used for the measurement described below.

Α ₁ -ACT standard solution [Medical and Biological Laboratories, Inc.] was added to PBS containing 0.1% bovine serum albumin.
(Hereinafter, also referred to as BSA-PBS) and diluted with α ₁ -AC
Each dilution series of 0 to 20 ng / ml was prepared as T. One bead-shaped insoluble carrier prepared by the above operation was added to 400 μl of each dilution column, and the mixture was allowed to stand at 37 ° C. for 1 hour and then washed twice with physiological saline. There, peroxidase-labeled anti-human α ₁ -ACT antibody solution (23 μg / ml)
400 μl was added, and the mixture was allowed to stand at 37 ° C. for 1 hour and washed 3 times with physiological saline. The treated insoluble carrier was transferred to a new container, and 400 μl of 0.1 M citrate buffer solution (pH 6.0) containing 0.25% o-phenylenediamine and 0.015% H ₂ O ₂ as a color developing solution was added. The mixture was allowed to stand at room temperature for 25 minutes for color development, 1N-HCl was added to stop the reaction, and the absorbance at a wavelength of 492 nm was measured. The result is shown in FIG. The blank value was reduced and the effective sensitivity was increased by both PECs.

[0043]

INDUSTRIAL APPLICABILITY The use of the insoluble carrier having at least the surface composed of the polyelectrolyte complex according to the present invention effectively suppresses the nonspecific reaction in the conventional immunological detection method, improves the measurement accuracy, and improves the biological accuracy. It is possible to detect or measure a substance to be inspected, which is contained in a sample only in an extremely small amount, with high accuracy and accuracy.

[Brief description of drawings]

FIG. 1 shows an AFP by EIA using a PEC-coated carrier of the present invention [Example 1 (1)] and a conventional PEC-uncoated carrier.
It is a graph which shows the change of concentration and light absorbency when it measures.

FIG. 2 AFP by EIA using a PEC-coated carrier of the present invention [Example 1 (2)] and a conventional PEC-uncoated carrier.
It is a graph which shows the change of concentration and light absorbency when it measures.

FIG. 3 shows an AFP by EIA using a PEC-coated carrier of the present invention [Example 1 (3)] and a conventional PEC-uncoated carrier.
It is a graph which shows the change of concentration and light absorbency when it measures.

FIG. 4 shows AFP by EIA using a PEC-coated carrier of the present invention [Example 1 (4)] and a conventional PEC-uncoated carrier.
It is a graph which shows the change of concentration and light absorbency when it measures.

FIG. 5: AFP by EIA using a PEC-coated carrier of the present invention [Example 1 (5)] and a conventional PEC-uncoated carrier.
It is a graph which shows the change of concentration and light absorbency when it measures.

FIG. 6 shows changes in concentration and absorbance when AFP was measured by EIA using a PEC-coated carrier of the present invention [Example 3 (1): charge balance = + 3.3] and a conventional PEC-uncoated carrier. It is a graph shown.

FIG. 7 shows changes in concentration and absorbance when AFP was measured by EIA using a PEC-coated carrier of the present invention [Example 3 (1): charge balance = + 0.9] and a conventional PEC-uncoated carrier. It is a graph shown.

FIG. 8 shows changes in concentration and absorbance when AFP was measured by EIA using a PEC-coated carrier of the present invention [Example 3 (1): charge balance = ± 0] and a conventional PEC-uncoated carrier. It is a graph.

FIG. 9 shows changes in concentration and absorbance when AFP was measured by EIA using a PEC-coated carrier of the present invention [Example 3 (1): charge balance = −0.9] and a conventional PEC-uncoated carrier. It is a graph which shows.

FIG. 10: PEC-coated carrier of the present invention [Example 3 (1):
6 is a graph showing changes in concentration and absorbance when AFP was measured by EIA using a charge balance = −3.3] and a conventional PEC uncoated carrier.

FIG. 11: α ₁ -AC by EIA using the PEC-coated carrier of the present invention (Example 5) and a conventional PEC-uncoated carrier.
It is a graph which shows the change of concentration and light absorbency when T is measured.

Claims (5)

[Claims] 1. An insoluble carrier for immunological detection, characterized in that at least the surface is formed of a polyelectrolyte complex. 2. An immunologically active substance capable of immunologically binding to a substance to be detected is immobilized on a surface in a state capable of binding to the substance to be detected, and at least the surface is formed by a polyelectrolyte complex. Is characterized by being
Insoluble carrier. 3. A cationic polyelectrolyte in which the polyelectrolyte complex contains an N ⁺ atom in a repeating unit,
-COO in the repeating unit ^- based, -SO ₃ ^- group, -PO ₃
The insoluble carrier according to claim 1 or 2, which is a polyelectrolyte complex obtained by reacting with an anionic polyelectrolyte containing an H ⁻ group or a —PO ₃ ^{2 —} group. 4. The polymer electrolyte complex comprises an amine compound polymer, a quaternary ammonium compound polymer,
A cationic polyelectrolyte selected from the group consisting of basic amino acid polymers and cationic polysaccharides, and an anionic polyelectrolyte selected from the group consisting of anionic polysaccharides, acidic amino acid polymers and acrylic acid-based polymers. The insoluble carrier according to claim 3, which is a polyelectrolyte complex obtained by reacting with. 5. An immunologically active substance capable of immunologically binding to a substance to be detected is immobilized on the surface in a state capable of binding to the substance to be detected, and at least the surface is formed by a polyelectrolyte complex. Inducing an immunological reaction by contacting an insoluble carrier and a test sample, and detecting a detectable change resulting from the reaction,
A method for immunologically detecting a substance to be detected in a test sample.