JPH10153599A - Agent for preventing nonspecific adsorption of protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable highly sensitive and highly accurate measurement of an immunologically active substance, by causing a specified acrylic copolymer to contain an oxyalkylene group to be adsorbed on an immunologically active substance-immobilized solid phase to prevent nonspecific adsorption of protein. SOLUTION: This nonspecific adsorption preventing agent for protein is obtained by using an acrylic copolymer containing an oxyalkylene group produced by polymerizing a monomar as given by the formula, which is caused to be adsorbed on an immunologically active substance immobilized solid phase with an immunologically active substance immobilized on a solid phase. In the formula, R<1> represents hydrogen atom or methyl group, R<2> hydrogen atom or a hydrocarbon group having 1-10 carbons, OA an oxyalkylene group having 2-4 carbons and n the integer of 1-1,000 as average added molar number of the oxyalkylene group. To prevent nonspecific adsorption of protein using the adsorption preventing agent obtained, the preventing agent is put to be adsorbed on the surface of material desired to prevent adsorption. The type of the protein is not limited in the prevention of the nonspecific adsorption, and the nonspecific adsorption can be prevented for any protein regardless of the type thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to the adsorption of a labeled antibody to an antibody-immobilized solid phase in the measurement of an immunologically active substance by a sandwich method or the like which is widely used in the field of clinical reagents and the like. Non-specific adsorption) to prevent non-specific adsorption of proteins, such as adsorption of labeled antigen to solid phase on which antigen is immobilized (non-specific adsorption of labeled antigen) or adsorption of protein in sample to solid phase Protein non-specific adsorption inhibitor,
And its uses.

[0002]

2. Description of the Related Art A solid phase method based on a two-site method (sandwich measurement method) is generally used as an immunometric assay widely used in the field of clinical diagnostic agents and the like.
This measurement method uses two types of antibodies (Ab1, Ab) having different epitopes on the substance to be measured (the test substance is represented by Ag).
b2) is used. First, Ab1 is immobilized on the surface of a solid phase / carrier (denoted as SP) made of a synthetic polymer or the like, and Ag is added thereto to bind. Next, a labeled antibody (represented as Ab2 *) is reacted, washed, and free Ab
2 * was removed, and Ab2 * (bound type,
The standard activity of B) is determined. In this case, B
And a standard curve is obtained between the two. From this standard curve, the amount of antigen in the sample is measured. In addition, a method of measuring the amount of antibody in a sample by reversing the antigen and the antibody, that is, using a labeled antigen (Ag1, Ag2 * and A
b). For these labeling substances, isotopes, enzymes, fluorescent substances or luminescent substances are used.

One of the main factors affecting the sensitivity of such a sandwich method is nonspecific adsorption of a labeled antibody to an antibody-bound solid phase or nonspecific adsorption of a labeled antigen to an antigen-bound solid phase. Such non-specific adsorption depends on the properties of the labeling substance used for labeling.For example, in the case of non-specific adsorption of an enzyme-labeled antibody, non-specific adsorption of an alkaline phosphatase-labeled antibody, a glucose oxidase-labeled antibody, and a peroxidase-labeled antibody, 1 / 300,000,000 of the amount added
And the non-specific adsorption of the β-D-galactosidase-labeled antibody is 1/2000 {Medical Shoin, “Enzyme Immunoassay”, pp. 158-163 (1987)}.
These non-specific adsorptions cause a decrease in sensitivity and a lack of reproducibility in the sandwich method.

Heretofore, in order to prevent such non-specific adsorption, a method in which an immunoassay is performed using a weakly acidic buffer solution of, for example, pH 5 to 6, or an extra protein binding site on a solid phase after Ab1 is adsorbed. Is known using ovalbumin, bovine serum albumin, fetal bovine serum or normal serum. Also, a non-specific adsorption inhibitor obtained by dissolving milk protein in a buffer containing an organic acid as a main component and sterilizing the same is known (Japanese Patent Application Laid-Open No. 1-217266).

[0005] However, the above-mentioned weak acid operation or blocking method with various proteins does not have sufficient ability to prevent non-specific adsorption of proteins, and thus has a superior ability to prevent non-specific adsorption of proteins in fields such as clinical diagnosis. It has been desired to develop an inhibitor and an immunologically active substance-immobilized solid phase that has been subjected to a protein nonspecific adsorption prevention treatment. In addition, when commercially available proteins such as ovalbumin, bovine serum albumin (abbreviated as BSA), fetal bovine serum, and milk protein are used,
There is a problem in that immunoglobulins, enzymes, hormones, and the like often enter the measurement system, adversely affect the reaction and cause errors in analytical values.

[0006]

SUMMARY OF THE INVENTION The first object of the present invention has no influence on a measurement system, that is, it is limited to a labeling substance such as an isotope, an enzyme, a fluorescent substance or a chemiluminescent substance, and a substance to be measured. Without labeling, adsorption of the labeled antibody to the antibody-immobilized solid phase (non-specific adsorption of the labeled antibody), adsorption of the labeled antigen to the antigen-immobilized solid phase (non-specific adsorption of the labeled antigen), or the An object of the present invention is to provide a protein nonspecific adsorption inhibitor capable of preventing nonspecific adsorption of proteins such as adsorption to a solid phase.

A second object of the present invention is to provide an immunological activity capable of preventing non-specific adsorption of a protein by using the above-mentioned adsorption inhibitor to measure a target substance with high sensitivity and high accuracy. It is an object of the present invention to provide a solid phase immobilized with an adsorption inhibitor and an immunologically active substance for measuring substances.

[0008] A third object of the present invention is to propose a method for preventing non-specific protein adsorption, which can easily prevent non-specific adsorption of protein to a solid phase on which an immunologically active substance is immobilized. .

A fourth object of the present invention is to provide an immobilization method capable of stably maintaining the immunological activity of an immunologically active substance immobilized on an immunologically active substance-immobilized solid phase for a long period of time. It is to propose a method for stabilizing an immunologically active substance.

A fifth object of the present invention is to provide a method for measuring an immunologically active substance which can prevent a non-specific adsorption of a protein and can measure a target substance with high sensitivity and high accuracy. is there.

[0011]

Means for Solving the Problems The present inventors have made intensive studies in view of the above problems, and as a result, have found that the above problems can be solved by using a specific polymer, and have completed the present invention. . That is, the present invention provides the following protein non-specific adsorption inhibitor, solid phase immobilized with the adsorption inhibitor immunologically active substance, method for preventing protein non-specific adsorption using the same, stabilization of the immobilized immunologically active substance. And a method for measuring an immunologically active substance. (1) General formula (1)

Embedded image (Wherein, R ¹ is a hydrogen atom or a methyl group, R ² is a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms, and OA is a carbon atom having 2 to 2 carbon atoms.
The oxyalkylene group of 4 and n are integers of 1 to 1000 in the average number of moles of the oxyalkylene group added. A non-protein specific adsorption inhibitor comprising an oxyalkylene group-containing acrylic polymer obtained by polymerizing the monomer represented by the formula (1). (2) An immunologically active substance-immobilized solid phase in which an immunologically active substance is immobilized on a solid phase, wherein the protein non-specific adsorption inhibitor according to the above (1) is adsorbed. A solid phase on which an adsorption inhibitor is adsorbed for measuring an immunologically active substance. (3) A protein non-specific, characterized in that the protein non-specific adsorption inhibitor according to (1) is adsorbed on the immunologically active substance-immobilized solid phase in which the immunologically active substance is immobilized on the solid phase. Specific adsorption prevention method. (4) Immobilization characterized by adsorbing the protein non-specific adsorption inhibitor described in (1) above to the immunologically active substance-immobilized solid phase in which the immunologically active substance is immobilized on the solid phase. A method for stabilizing an immunologically active substance. (5) The immunologically active substance immobilized on the solid phase is contacted with the immunologically active substance to be measured and the antigen-antibody reaction is carried out on the immobilized immunologically immobilized solid phase. And forming a complex between the active substance and the analyte, and then contacting the labeled immunologically active substance labeled with the labeling substance, and causing an antigen-antibody reaction to perform the immobilized immunologically active substance-the analyte and the labeled immunology. The immunologically active substance-immobilized solid phase, wherein the immunologically active substance-immobilized solid phase is formed by forming a complex with the immunologically active substance. A method for measuring an immunologically active substance, which comprises using the solid phase on which the immunologically active substance adsorbed by the adsorption inhibitor according to the above is immobilized.

In the present invention, “(meth) acryl” means “acryl” and / or “methacryl”. “Immunologically active substance” means “antigen” and / or “antibody”.

In the general formula (1), specific examples of the hydrocarbon group having 1 to 10 carbon atoms represented by R ² include a methyl group, an ethyl group, an n-propyl group, an isopropyl group,
C1-C10 linear or branched alkyl groups such as n-butyl group, isobutyl group, s-butyl group, t-butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, etc. And so on.

The oxyalkylene group represented by OA in the general formula (1) is an oxyalkylene group having 2 to 4 carbon atoms, such as an oxyethylene group, an oxypropylene group, an oxytrimethylene group, and an oxy-1-ethylethylene group. ,
Examples thereof include an oxy-1,2-dimethylethylene group and an oxytetramethylene group.

In the general formula (1), n is 1 to 1000;
It is preferably an integer of 2 to 300, more preferably 4 to 50. If n exceeds 1000, the viscosity becomes high and the production becomes difficult. When n is 2 or more, the types of the oxyalkylene groups may be the same or different. In the latter case, they may be added randomly or in blocks. When the types of the oxyalkylene groups are different, the proportion of the oxyethylene groups in all oxyalkylene groups is preferably 50 mol% or more from the viewpoint of solubility in water.

In order for the oxyalkylene group-containing acrylic polymer obtained by polymerizing the monomer represented by the general formula (1) as a constituent unit to be easily dissolved in a buffer solution or the like, n should be from 4 to 50. More preferred. If the carbon number of R ^{2 in} the monomer represented by the general formula (1) exceeds 10, for example, the solubility of the oxyalkylene group-containing acrylic polymer in a buffer solution or the like becomes low, which is not preferable.

Specific examples of the monomer represented by the general formula (1) used in the present invention include, for example, polyoxyethylene mono (meth) acrylate, methoxypolyoxyethylene mono (meth) acrylate, and octyloxy polyoxyethylene mono. (Meth) acrylate, polyoxyethylene polyoxypropylene mono (meth) acnolate,
Methoxy polyoxyethylene polyoxypropylene mono (meth) acrylate, octyloxy polyoxyethylene polyoxypropylene mono (meth) acrylate,
And polyoxyethylene polyoxytetramethylene (random adduct) mono (meth) acrylate. These can be used alone or in combination of two or more.

The oxyalkylene group-containing acrylic polymer used in the present invention may be a homopolymer or a copolymer of the monomer represented by the general formula (1). It may be a copolymer of the represented monomer and another copolymerizable monomer. The content of the monomer represented by the general formula (1) constituting the oxyalkylene group-containing acrylic polymer is 1 to 100 mol%, preferably 5 to 100 mol in the oxyalkylene group-containing acrylic polymer. % Is desirable. When the content is less than 1 mol%, it is difficult to prevent nonspecific adsorption of proteins, which is not preferable. The oxyalkylene group-containing acrylic polymer also has a number average molecular weight of usually 1,000 to 1,000,000, preferably 2 depending on the polymerization temperature, the amount of polymerization initiator used, and the use of a polymerization degree regulator. 2,000 to 500,000 polymers or copolymers.

In order to prepare the oxyalkylene group-containing acrylic polymer, a monomer represented by the general formula (1) may be homopolymerized by a conventionally known method, for example, in the presence of a known polymerization initiator. Alternatively, it can be obtained by a method of copolymerizing the monomer represented by the general formula (1) with another copolymerizable monomer component.

Examples of the monomer copolymerizable with the monomer represented by the general formula (1) include n-butyl (meth) acrylate, methyl (meth) acrylate, ethyl (meth) acrylate, Alkyl (meth) acrylates such as butyl (meth) acrylate, pentyl (meth) acrylate, hexyl (meth) acrylate, heptyl (meth) acrylate, octyl (meth) acrylate, and tridecine (meth) acrylate (Meth) acrylic acid; styrene-based monomers such as styrene, α-methylstyrene, methyl-substituted styrene, and chloro-substituted styrene; halogen-based monomers such as vinyl chloride and vinylidene chloride; ethylene, propylene, isobutylene, and the like Unsaturated hydrocarbon monomers; vinyl ester monomers such as vinyl acetate and vinyl propionate;
Vinyl ether monomers such as ethyl vinyl ether and n-butyl vinyl ether; diethyl itaconate, di-
Polycarboxylic acid ester monomers such as n-butyl itaconate and the like can be mentioned. Among these, (meth) acrylates, styrene and the like are preferable.

The polymerization initiator is not particularly limited as long as it is a usual radical polymerization initiator. For example, 2,2'-azobisisobutyronitrile, benzoyl peroxide, diisopropylperoxydicarbonate,
t-butylperoxy-2-ethylhexanoate, t
And persulfates such as -butylperoxypivalate, t-butylperoxydiisobutyrate, ammonium persulfate and potassium persulfate. Further, a redox-based polymerization accelerator may be used. The amount of the polymerization initiator used is desirably 0.01 to 10 parts by weight, preferably 0.1 to 5 parts by weight, based on 100 parts by weight of all the monomers.

The polymerization is preferably carried out at 30 to 80 ° C., preferably at 40 to 70 ° C., for 2 to 72 hours. At this time, a solvent may be used in order to carry out the polymerization reaction more smoothly. Such solvents include, for example, water,
Examples thereof include methanol, ethanol, propanol, t-butanol, benzene, toluene, dimethylformamide, tetrahydrofuran, chloroform and a mixture thereof.

The protein nonspecific adsorption inhibitor of the present invention contains the oxyalkylene group-containing acrylic polymer obtained as described above, and may be composed of only the oxyalkylene group-containing acrylic polymer. , And this polymer in water, saline, phosphate buffer, acetate buffer,
It may be a solution or suspension dissolved or suspended in a buffer such as a carbonate buffer and a citrate buffer.

In order to prevent non-specific adsorption of a protein using the protein non-specific adsorption inhibitor of the present invention, the protein of the present invention is placed on the surface of the substance to be prevented from adsorbing (hereinafter sometimes referred to as a carrier). It can be carried out by adsorbing a non-specific adsorption inhibitor.

The above-mentioned adsorption can be carried out by bringing the carrier into contact with the protein non-specific adsorption inhibitor of the present invention. Usually, the reaction can be carried out by bringing a carrier into contact with a solution or suspension of a protein non-specific adsorption inhibitor (hereinafter sometimes referred to as a solution containing an adsorption inhibitor). The contact can be carried out by any method such as immersing the carrier in a liquid containing an adsorption inhibitor. The concentration of the oxyalkylene group-containing acrylic polymer in the anti-adsorption agent-containing liquid is preferably 0.00001 to 10% by weight, and more preferably 0.0001 to 5% by weight. Usually, the protein non-specific adsorption inhibitor can be adsorbed on the surface of the carrier by contacting at a temperature of 0 to 55 ° C. for 1 minute to 72 hours.

The material of the carrier is not particularly limited, and resins such as polystyrene, polyvinyl chloride, polypropylene, and polymethyl methacrylate; nitrocellulose, cellulose,
Organic polymers such as methylcellulose; and inorganic materials such as metal, glass, ceramic, and silicon rubber. Also, the shape of the carrier is not particularly limited, and examples thereof include a plate shape, a film shape, and a particle shape.

The type of protein for which nonspecific adsorption is prevented by the protein nonspecific adsorption inhibitor of the present invention is not particularly limited, and nonspecific adsorption of any type of protein is prevented. .

The adsorption inhibitor for measuring an immunologically active substance of the present invention, the immobilized solid phase on which an immunologically active substance is immobilized (hereinafter sometimes abbreviated as a solid phase for measuring an immunologically active substance) may be used for a sandwich assay. The protein non-specific adsorption inhibitor is adsorbed on an immunologically active substance-immobilized solid phase conventionally used in an immunologically active substance measurement method such as a method.

The immunologically active substance-immobilized solid phase is obtained by immobilizing an immunologically active substance on a solid phase comprising a carrier. The material of the carrier is not particularly limited, and those conventionally used can be used, for example, polystyrene, polyvinyl chloride, polypropylene, polymethyl methacrylate,
Organic carriers such as nitrocellulose, cellulose, and methylcellulose; inorganic carriers such as metal, glass, ceramics, and silicone rubber can be used. The shape is not particularly limited, and examples thereof include a test tube, a titer plate, a latex, a filter, a film, and fine particles.

The immunologically active substance immobilized on the solid phase is not particularly limited, and specific examples thereof include the following immunologically active substances. C-reactive protein (CRP), rheumatoid factor (R
F), plasma proteins such as transferrin, or antibodies against these plasma proteins. Thyroid stimulating hormone (TSH), triiodothyronine (T ₃ ), thyroxine (T ₄ ), thyroxine binding protein (TBG), thyroglobulin, insulin, estriol (E ₃ ), chorionic gonadotropin (HCG),
Hormones such as human placental lactogen (HPL), or antibodies against these hormones. Tumor-related substances such as carcinoembryonic antigen (CEA), β ₂ -microglobulin, α-fetoprotein (AFP), and antibodies against these tumor-related substances. HB _S antigen, HB _S antibody, HB _e antigen, the antigen or antibody of viral hepatitis such as HB _e antibody or antigen or an antibody to an antigen or antibody of these viruses hepatitis. Antibodies or antigens against various biological components such as viruses such as mumps, herpes, measles, rubella and cytomegalo, and anti-AIDS antibodies. Antibodies to various drugs such as phenobarbital, acetaminophenone, salicylic acid, and cyclosporine. Enzymes or antibodies against enzymes. Note that an antigen for the immobilized antibody or an antibody for the immobilized antigen is the immunologically active substance (test substance) of the measurement object.

The solid phase for measuring an immunologically active substance of the present invention comprises:
The protein non-specific adsorption inhibitor is adsorbed on the immunologically active substance-immobilized solid phase. The above-mentioned adsorption can be carried out by the method already described. Hereinafter, the case of adsorption to a solid phase on which an immunologically active substance is immobilized will be described in detail.

For the above-mentioned adsorption, a solution or suspension of the oxyalkylene group-containing acrylic polymer (solution containing an adsorption inhibitor) can be used, and specific examples thereof include a phosphate buffer, an acetate buffer, and a carbonate buffer. , A citrate buffer, various solutions or suspensions dissolved or suspended in a medium such as physiological saline. An organic solvent such as dimethylsulfoxide, tetrahydrofuran or N, N-dimethylformamide may be added to these solutions or suspensions in an amount of 0.01 to 20% by weight. Preferable examples include a solution dissolved in a phosphate buffer or various physiological saline.

The concentration of the oxyalkylene group-containing acrylic polymer in the above solution or suspension is from 0.00001 to 10
%, Preferably 0.0001 to 5% by weight, which has high ability to prevent nonspecific adsorption and stabilize the immobilized immunologically active substance. 0.00001 polymer concentration
When the amount is lower than 10% by weight, the stability of the immobilized immunologically active substance is deteriorated, which is not preferable. When the concentration of the polymer is higher than 10% by weight, the viscosity of the polymer becomes high, which is not preferable.

In preparing the solid phase for measuring an immunologically active substance of the present invention, after binding an antibody or an antigen to the surface of the solid phase, a solution containing an adsorption inhibitor is added, and then the mixture is incubated or incubated. The solution containing the anti-adsorption agent may be removed without performing, but it is preferable to incubate. The above incubation time depends on the concentration of the anti-adsorption agent-containing solution, the incubation temperature, and the like, but is from 1 minute to 7 minutes.
It is preferably 2 hours, preferably 30 minutes to 48 hours, at which a sufficient effect of preventing nonspecific adsorption and stabilizing effect of the immobilized immunologically active substance can be obtained.

If the incubation time is too short, a sufficient effect cannot be obtained. The incubation temperature is desirably 0 to 55 ° C, preferably 4 to 40 ° C which does not affect the immunological activity of the immobilized immunologically active substance.
If the incubation temperature is lower than 0 ° C., the incubation time becomes longer and rapid measurement becomes difficult, which is not preferable. If it is higher than 55 ° C., the stability of the immobilized immunologically active substance is deteriorated, which is not preferable.

The method of preserving the solid phase for measuring an immunologically active substance of the present invention is not particularly limited, and a preservation method such as leaving, sealing, freezing, or sealing after freeze-drying can be employed. It is preferable to employ a storage method such as sealing or freeze-drying, which shows the stabilizing effect of the immunologically active substance, and the like.

The solid phase for measuring an immunologically active substance of the present invention can be widely used in all fields and methods, for example, in clinical test, immunology, biochemistry, molecular biology, and the like. Enzyme immunoassay (ELIS
A), radioimmunoassay (RIA), or immunological assay such as Western blotting can be suitably used.

The immunologically active substance measuring method of the present invention is different from the conventional immunologically active substance measuring method using the immunologically active substance-immobilized solid phase in that the conventional immunologically active substance-immobilized solid phase is used. Instead of using the solid phase for immunologically active substance measurement of the present invention, the method for measuring an immunologically active substance by a solid phase method called a so-called two-site method or a sandwich method. This is a method using a solid phase for measurement of biologically active substances.

That is, in the method for measuring an immunologically active substance of the present invention, an immunologically active substance to be measured is brought into contact with the above-mentioned solid phase for immunologically active substance measurement, and an antigen-antibody reaction is carried out. A complex of the biologically active substance and the analyte (an immobilized immunologically active substance-analyte) and then contacting the labeled immunologically active substance labeled with a labeling substance, Reacting to form a complex of the complex and the labeled immunologically active substance (immobilized immunologically active substance-measured substance-labeled immunologically active substance complex), and labeling in this complex This is a method of measuring a measurement target using a substance as an index.

The immunologically active substance to be measured in the measuring method of the present invention is not particularly limited, and examples thereof include those similar to those exemplified as the immunologically active substance to be immobilized. As the labeling substance, a conventionally used labeling substance such as an enzyme, an isotope, a fluorescent substance or a luminescent substance can be used without particular limitation, and those conventionally used as a labeled immunologically active substance can be used. Can be used as is.

The antigen-antibody reaction can be carried out under the same conditions as in the past, and the reaction temperature is usually 2 to 55 ° C., preferably 10 to 55 ° C.
~ 35 ° C, reaction time is 3 minutes ~ 24 hours, preferably 1
For 5 minutes to 12 hours, the pH of the reaction system is desirably 3 to 10, preferably 5 to 8. As the reaction medium, the same medium as the above-mentioned medium used for preparing the solid phase for measuring an immunologically active substance can be used.

The measurement of the object to be measured can be performed by a known method using the labeling substance as an index and according to the labeling substance. For example, when the labeling substance is an enzyme, the enzyme activity is measured, when the labeling substance is an isotope, the amount of radioactivity is measured, and when the labeling substance is a fluorescent substance or a luminescent substance, the measurement is performed by measuring the amount of light. it can.

Since the measurement method of the present invention uses a solid phase for measuring an immunologically active substance in which non-specific adsorption of a protein is prevented, adsorption of a labeled antibody to an antibody-immobilized solid phase, Non-specific adsorption of proteins, such as adsorption of a protein on an antigen-immobilized solid phase and adsorption of a protein in a sample to a solid phase, can be prevented, and thus measurement can be performed with high sensitivity and high accuracy.

Specific methods for preparing the solid phase for measuring an immunologically active substance of the present invention and methods for measuring an immunologically active substance using an antigen-antibody reaction are various as described above. A specific description will be given of a substance measurement method using an enzyme activity measurement using a general polystyrene titer plate as an example. For example, by performing the following steps (1) to (3), the amount of the measurement target in the sample can be measured with high sensitivity and high accuracy.

A solution containing an antibody that specifically reacts with an object to be measured is added to a carrier, incubated at 4 ° C. for 12 hours, and then washed three times with physiological saline to immobilize the immunologically active substance. Prepare a solid phase. A solution containing 0.01% by weight of the oxyalkylene group-containing acrylic polymer is added to the immunologically active substance-immobilized solid phase, and the mixture is incubated at 4 ° C. for 12 hours, and then contains the oxyalkylene group-containing acrylic polymer. By removing the solution, a solid phase for measuring an immunologically active substance is prepared. To the above-mentioned solid phase for immunologically active substance measurement, a solution (standard solution) containing an analyte having a known concentration and a solution (analyte) containing an unknown amount of an analyte are separately added, and the solution is added.
The immobilized immunologically active substance and the analyte are subjected to an antigen-antibody reaction by incubating at 2 ° C. for 2 hours to form an immobilized immunologically active substance-analyte complex.
Thereafter, washing is performed three times with physiological saline. Next, a solution containing an enzyme-labeled antibody that specifically reacts with the measurement target is added, and the mixture is incubated at 25 ° C. for 2 hours to cause an antigen-antibody reaction between the measurement target and the enzyme-labeled antibody. A substance-measurement-enzyme-labeled antibody complex is formed. Thereafter, washing is performed three times with physiological saline. The enzyme activity of the immobilized immunologically active substance-measured object-enzyme-labeled antibody complex is measured, and the enzyme activity in the sample is compared with the enzyme activity in the standard solution.

The method for preventing non-specific protein adsorption of the present invention comprises:
The protein non-specific adsorption inhibitor of the present invention is adsorbed on the immunologically active substance-immobilized solid phase in which the immunologically active substance is immobilized on the solid phase, thereby allowing non-specific adsorption of the protein to the solid phase. It is a method to prevent. This adsorption preventing method can easily prevent nonspecific adsorption of a protein which causes an error in a measured value in a method for measuring an immunologically active substance or the like.

The method for stabilizing an immobilized immunologically active substance of the present invention comprises the step of immobilizing the immunologically active substance on a solid phase and immobilizing the immunologically active substance on a solid phase. This is a method of adsorbing an anti-adsorption agent to stabilize the immobilized immunologically active substance. Since this stabilization method can stably maintain the immunological activity of the immobilized immunologically active substance for a long period of time, it is highly sensitive and highly accurate in a method for measuring an immunologically active substance. In addition to enabling measurement, it is also possible to prepare a solid phase in advance, which simplifies and speeds up measurement.

As the solid phase for measuring an immunologically active substance used in the above step, a solid phase prepared immediately before an antigen-antibody reaction with an object to be measured can be used, or a previously prepared solid phase can be stored by the above-mentioned storage method. You can also use what you have set. Even when the preserved one is used, the activity of the immobilized immunologically active substance is stably maintained in a high state, so that the measurement can be performed with high sensitivity and high accuracy.

In the above step, since the nonspecific adsorption of the protein not involved in the antigen-antibody reaction is prevented by the protein nonspecific adsorption inhibitor of the present invention, the measurement can be performed with high sensitivity and high accuracy. Can be.

[0050]

The protein non-specific adsorption inhibitor of the present invention comprises:
Since it contains an oxyalkylene group-containing acrylic polymer obtained by polymerizing a specific monomer, it is possible to prevent specific adsorption of proteins with excellent adsorption prevention ability, for example, to a solid phase on which an antibody is immobilized on a labeled antibody. Non-specific adsorption of proteins, such as adsorption of labeled antigen, adsorption of labeled antigen to the solid phase on which the antigen is immobilized, and adsorption of protein in the sample to the solid phase, without affecting the measurement system, with excellent adsorption prevention ability Can be prevented.

The adsorption inhibitor of the present invention for measuring an immunologically active substance is immobilized on the solid phase on which the immunologically active substance is immobilized.
Non-specific adsorption of proteins can be prevented, and the target substance can be measured with high sensitivity and high accuracy.

The method for preventing non-specific protein adsorption of the present invention comprises:
Since the adsorption inhibitor is adsorbed to the immunologically active substance-immobilized solid phase, nonspecific adsorption of proteins can be easily prevented.

In the method for stabilizing an immobilized immunologically active substance of the present invention, the immobilized immunologically active substance is immobilized because the adsorption inhibitor is adsorbed on the solid phase on which the immunologically active substance is immobilized. Can be stably maintained for a long time.

In the method for measuring an immunologically active substance of the present invention, since the solid phase on which the immunologically active substance is adsorbed with the adsorption inhibitor is used, non-specific adsorption of proteins can be carried out without affecting the measurement system. In this way, measurement can be performed with high sensitivity and high accuracy.

[0055]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail by way of examples. Table 1 shows the monomers represented by the formula (1) used in Examples and Comparative Examples.

[0056]

[Table 1]

Synthesis Example 1-1 << Synthesis of Monomer >> 2-liter capacity was placed in a stainless steel pressure vessel.
130 g (1 mol) of hydroxyethyl methacrylate,
Hydroquinone monomethyl ether 0.1 as a polymerization inhibitor.
Take 18g, boron trifluoride 2.2g, pressure 0.1 ~
The atmosphere was replaced with nitrogen three times at 5 atm. Then, the reaction was carried out at a reaction temperature of 30 to 40 ° C., at a pressure of 5 atm or less, for a reaction time of 8 hours while dropping 158.4 g (3.6 mol) of ethylene oxide from the measuring tank, and then aged at the same temperature for 1 hour. After cooling the reaction vessel, return to normal pressure, while blowing nitrogen gas,
Excess ethylene oxide was removed. The contents were transferred to another container, neutralized with about 17.4 g of a 5% by weight sodium hydroxide solution, and then dehydrated and filtered to obtain 245 g of a monomer.
As a result of analyzing the monomer, the hydroxyl value was 214 and the saponification value was 213.

Synthesis Example 1-2 << Synthesis of Monomer >> In a 2-liter capacity stainless steel pressure vessel,
130 g (1 mol) of hydroxyethyl methacrylate,
Hydroquinone monomethyl ether 0.1 as a polymerization inhibitor.
Take 66 g, 9.5 g of boron trifluoride, pressure 0.1 ~
The atmosphere was replaced with nitrogen three times at 5 atm. Next, a reaction solution is reacted at a reaction temperature of 30 to 40 ° C., 5 atm or less, and a reaction time of 8 hours while a mixed solution of 475.2 g (10.8 mol) of ethylene oxide and 450 g (6.3 mol) of tetrahydrofuran is dropped from the measuring tank. Then, it was aged at a temperature of 50 ° C. for 1 hour. After cooling the reaction vessel, the pressure was returned to normal pressure, and excess ethylene oxide and tetrahydrofuran were removed while blowing nitrogen gas. The contents were transferred to another container, neutralized with about 75.1 g of a 5% by weight sodium hydroxide solution, and then dehydrated and filtered to obtain 844 g of a monomer. As a result of analyzing the monomer, the hydroxyl value was 63.3 and the saponification value was 63.2.

Synthesis Example 1-3 << Synthesis of Monomer >> In a 2-liter stainless steel pressure vessel, 2-
130 g (1 mol) of hydroxyethyl methacrylate,
Hydroquinone monomethyl ether 0.1 as a polymerization inhibitor.
Take 76 g, 8.6 g of boron trifluoride, pressure 0.1 ~
The atmosphere was replaced with nitrogen three times at 5 atm. Next, a mixture of ethylene oxide 475.2 g (10.8 mol) and propylene oxide 348 g (6.0 mol) was added dropwise from the measuring tank, and the reaction was carried out at a reaction temperature of 30 to 40 ° C., 5 atm or less, and a reaction time of 16 hours. And then aged at the same temperature for 1 hour. After cooling the reaction vessel, the pressure was returned to normal pressure, and excess ethylene oxide and propylene oxide were removed while blowing nitrogen gas. The contents were transferred to another container, neutralized with about 68.0 g of a 5% by weight sodium hydroxide solution, and then dehydrated and filtered to obtain 762 g of a monomer. As a result of analyzing the monomer, the hydroxyl value was 68.8 and the saponification value was 68.7.

Synthesis Example 1-4 << Synthesis of Monomer >> In a 2-liter stainless steel pressure vessel, 2-
130 g (1 mol) of hydroxyethyl methacrylate,
Hydroquinone monomethyl ether 0.1 as a polymerization inhibitor.
Take 76 g, 8.6 g of boron trifluoride, pressure 0.1 ~
The atmosphere was replaced with nitrogen three times at 5 atm. Then, while dropping 475.2 g (10.8 mol) of ethylene oxide from the measuring tank, the reaction was allowed to proceed at a reaction temperature of 30 to 40 ° C., 5 atm or less, for a reaction time of 8 hours, and then aged at the same temperature for 1 hour. In addition, 348 g (6 mol) of propylene oxide from the measuring tank
While dropping, the reaction temperature 30 ~ 40 ℃, 5 atm or less,
The reaction was carried out for a reaction time of 8 hours, and further aged at the same temperature for 1 hour. After cooling the reaction vessel, the pressure was returned to normal pressure, and excess ethylene oxide and propylene oxide were removed while blowing nitrogen gas. Transfer contents to another container, 5% by weight
The mixture was neutralized with about 68.0 g of a sodium hydroxide solution, and then dehydrated and filtered to obtain 762 g of a monomer. As a result of analyzing the monomer, the hydroxyl value was 68.8 and the saponification value was 68.7.

Synthesis Example 1-5 << Synthesis of Monomer >> 130 g (1 mol) of octyl alcohol and potassium hydroxide 1 were placed in a 3-liter stainless steel pressure vessel.
5.6 g was taken and the atmosphere was replaced with nitrogen three times at a pressure of 0.1 to 5 atm. Next, 14789 g of ethylene oxide was measured from the measuring tank.
(33.6 moles) while the reaction temperature was from 120 to 1
The reaction was carried out at 30 ° C., 5 atm or less, for a reaction time of 6 hours, and then aged at the same temperature for 1 hour. Further, while dropping 620.6 g (10.7 mol) of propylene oxide from the measuring tank, the reaction was carried out at a reaction temperature of 105 to 115 ° C., 5 atm or less, and a reaction time of 8 hours, and further aged at the same temperature for 1.5 hours. . After cooling the reaction vessel, the pressure was returned to normal pressure, and excess ethylene oxide and propylene oxide were removed while blowing nitrogen gas. Transfer contents to another container, 35% by weight
Then, the solution was neutralized with about 38 g of hydrochloric acid, followed by dehydration filtration to obtain 2118 g of an octyl alcohol and ethylene oxide propylene oxide block polymer. As a result of analyzing this compound, the hydroxyl value was 26.5.

1059 g (0.5 mol) of the above compound,
Methacrylic acid 77.4 g (0.9 mol), p-toluenesulfonic acid (monohydrate) 47 g, solvent toluene 1110
g was taken in a flask equipped with a water test tube, thermometer cooling tube, and nitrogen blowing tube, and subjected to azeotropic dehydration at 110 to 115 ° C. for esterification for 9 hours. Thereafter, 10% by weight of sodium hydroxide 7
Unreacted methacrylic acid was washed and removed twice using 30 g and a 20% saline mixed solution (500 g) twice. Then, after dehydration and toluene removal were performed under reduced pressure, the mixture was filtered to obtain 930 g of a monomer. As a result of analyzing the monomer, the hydroxyl value was 0 and the saponification value was 25.7.

Synthesis Example 2-1 Under the conditions that the total monomer concentration was 1.0 mol / l and the polymerization initiator was 1 mol% based on the monomer, the polyoxyethylene (4 mol adduct) shown in Table 1 was used. ) Monomethacrylate (hereinafter, referred to as a monomer) was polymerized. That is, 5.25 g (20.0 mmol) of a monomer was weighed into a glass reaction tube for polymerization, and 0.02 g (0.23 g) of 2,2'-azobisisobutyronitrile (AIBN) was added thereto as a polymerization initiator. .
2 mmol) and 20 ml of methanol as a polymerization solvent. After sufficiently replacing the inside of the reaction tube with argon, it was sealed. This was heated to 60 ° C. for 24 hours to carry out a polymerization reaction.

After the reaction mixture was cooled on ice, the polymer was precipitated by adding dropwise to 400 ml of diethyl ether. This was separated by filtration, washed sufficiently with diethyl ether, and dried under reduced pressure to obtain 3.502 g of a white powdery polymer (referred to as a polymer). The conversion was 66.7%. The molecular weight was measured by analyzing the buffer solution of the polymer using gel permeation chromatography (hereinafter abbreviated as GPC). As a result, the number average molecular weight was 73,000 in terms of polyethylene glycol.

Synthesis Example 2-2 Monomer / styrene (hereinafter referred to as St) monomer charge molar ratio: monomer / St = 70/30, total monomer concentration: 1.0 mol / l , The polymerization initiator is 1
The copolymerization was performed under the condition of mol%. That is, 3.67 g (14.0 mmol) of monomer and 0.63 g of St
g (6.0 mmol) in a glass reaction tube for polymerization, and 0.033 g (0.2 mmol) of 2,2'-azobisisobutyronitrile (AIBN) as a polymerization initiator.
l), 20 ml of methanol was added as a polymerization solvent. After sufficiently replacing the inside of the reaction tube with argon, it was sealed. This was heated to 60 ° C. for 24 hours to carry out a polymerization reaction.

After the reaction mixture was cooled with ice, it was added dropwise to 400 ml of diethyl ether to precipitate a polymer. This was separated by filtration, sufficiently washed with diethyl ether, and dried under reduced pressure to obtain 2.801 g of a white powdery copolymer. The polymerization rate was 65.1%. As a result of measuring the molecular weight in the same manner as in Synthesis Example 2-1, the number average molecular weight was 64000 in terms of polyethylene glycol. From the result of elemental analysis, the molar composition ratio was monomer / St = 68.7 / 31.3. This copolymer is referred to as a polymer.

Synthesis Example 2-3 In Synthesis Example 2-2, 3.67 g (14.0 g) of the monomer was used.
12.42 g of the monomer of Table 1 in place of
(14.0 mmol), except that copolymerization was carried out in the same manner as in Synthesis Example 2-2. 8.09 g of a white powder copolymer was obtained from the monomer and styrene. The polymerization rate was 62.0%, and the number average molecular weight was 84,000 in terms of polyethylene glycol based on the result of GPC measurement. As a result of elemental analysis, the molar composition ratio was monomer / St = 64.5 / 35.5. This copolymer is referred to as a polymer.

Synthesis Example 2-4 3.67 g (14.0 g) of the monomer in Synthesis Example 2-2
mmol) and 14.22 g of the monomer in Table 1
(14.0 mmol), except that copolymerization was carried out in the same manner as in Synthesis Example 2-2. 9.80 g of a white powder copolymer was obtained from the monomer and styrene. The polymerization rate was 66.0%, and the number average molecular weight was 69000 in terms of polyethylene glycol based on the result of GPC measurement. As a result of elemental analysis, the molar composition ratio was monomer / St = 68.5 / 31.5. This copolymer is referred to as a polymer.

Synthesis Example 2-5 3.67 g (14.0 g) of the monomer in Synthesis Example 2-2
mmol) and 11.44 g of the monomers in Table 1
(14.0 mmol), except that copolymerization was carried out in the same manner as in Synthesis Example 2-2. 8.38 g of a white powder copolymer was obtained from the monomer and styrene. The polymerization rate is 69.4%
From the result of GPC measurement, the polymer had a number average molecular weight of 72,000 in terms of polyethylene glycol. As a result of elemental analysis, the molar composition ratio was monomer / St = 71.2 / 28.8.
Met. This copolymer is referred to as a polymer.

Synthesis Example 2-6 3.67 g (14.0 g) of the monomer in Synthesis Example 2-2
30.64 g of the monomer shown in Table 1 in place of
(14.0 mmol), except that copolymerization was carried out in the same manner as in Synthesis Example 2-2. From the monomer and styrene, 20.33 g of a white powder copolymer was obtained. The polymerization rate is 65.0.
%, And the number average molecular weight was 91,000 in terms of polyethylene glycol based on the GPC measurement result. As a result of elemental analysis, the molar composition ratio was monomer / St = 72.3 / 27.
It was 7. This copolymer is referred to as a polymer.

[0071]

[Table 2]

Example 1-1 << Preparation of Solid Phase for Measurement of Immunologically Active Substance (1) >> A 10 μg / ml saline solution of anti-mouse antibody was added to a polystyrene titer plate at 100 μl / well, and the mixture was added at 4 ° C. After overnight incubation, the anti-mouse antibody was physically adsorbed to the titer plate. Thereafter, it was washed four times with a physiological saline solution. Next, 300 μl / well of a physiological saline solution containing the polymer of Synthesis Example 2-1 (Mn = 73000) at a concentration of 0.0001% by weight was added, and the mixture was incubated at 4 ° C. overnight, and then the polymer solution was removed.

The amount of the immobilized anti-mouse antibody was determined by adding 200 μl of physiological saline containing 1% -sodium dodecyl sulfate to each of the eight wells of a polystyrene titer plate to release the immobilized anti-mouse antibody. Then, a protein content measuring device (trade name “Micro BC” manufactured by PIERCE)
A Protein Assay Kit ”). Table 3 shows the measurement results.

Example 1-2 << Preparation of Solid Phase for Measurement of Immunologically Active Substance (2) >> Synthetic Example 2 in place of the polymer used in Example 1-1
Example 1-1 was carried out except that the polymer obtained in -2 was used. The amount of the immobilized anti-mouse antibody was measured in the same manner as in Example 1-1. Table 3 shows the measurement results.

Examples 1-3 to 1-6 In place of the polymer used in Example 1-1, Synthesis Example 2-
3, 2-4, the polymer obtained in 2-5 or 2-6,
The measurement was performed in the same manner as in Example 1-1, except that or was used. The results are shown in Table 3.

Comparative Example 1-1 The procedure of Example 1-1 was repeated, except that 1% by weight of bovine serum albumin was used instead of the polymer used in Example 1-1. The amount of the immobilized anti-mouse antibody was measured in the same manner as in Example 1-1. Table 3 shows the measurement results.

[0077]

[Table 3]

From the results in Table 3, it can be seen that there is no difference in the amount of the immobilized anti-mouse antibody immobilized on the titer plate using non-specific adsorption.

Example 2-1 to Example 2-6 << Quantification of Measurement Object (1, 2) >> The solid phase for immunologically active substance measurement prepared in Examples 1-1 to 1-6 was used. , 0 μg / ml, 0.05 μg / ml,
0.1 μg / ml, 0.2 μg / ml, 0.4 μg / m
1, 100 μl / well of mouse antibody physiological saline solution at each concentration of 0.8 μg / ml. Then, after incubating at 25 ° C. for 2 hours, the plate was washed four times with physiological saline. Then, 100 μl of a dilution obtained by diluting a peroxidase-labeled anti-mouse IgG antibody [trade name “peroxidase-labeled-anti-mouse antibody-antibody OPD tablet” manufactured by Wako Pure Chemical Industries, Ltd.] with a physiological saline solution at 10,000 times was used. After adding / well, the mixture was incubated at 25 ° C. for 2 hours. Thereafter, the plate was washed four times with physiological saline.

Next, one tablet of o-phenylenediamine dihydrochloride (trade name "OPD tablet" manufactured by Wako Pure Chemical Industries, Ltd.) was added to a phosphate / citrate buffer solution containing 0.006% hydrogen peroxide. After adding 100 μl / well of the solution dissolved in 12 ml, the mixture was incubated at 25 ° C. for 10 minutes. Then, after adding 50 μl / well of a 2N sulfuric acid solution, a microplate reader (manufactured by Tosoh Corporation, trade name “MPR-
A4i ") was used to measure the absorbance at 492 nm of each well. The number of measurements (n) was 8, and the average value, standard deviation and CV value (%) (= standard deviation ÷ average value × 100) are shown in Tables 4 and 5.

Comparative Example 2-1 Except that the solid phase for immunologically active substance immobilization prepared in Comparative Example 1-1 was used instead of the solid phase for immunologically active substance measurement used in Example 2-1. Was performed in the same manner as in Example 2-1. The measurement results are shown in Table 5.

[0082]

[Table 4]

[0083]

[Table 5]

Example 3-1 to Example 3-6 << Stability Test (1, 2) >> The solid phase for measuring an immunologically active substance prepared in Examples 1-1 to 1-6 was sealed. Thereafter, it was stored at 40 ° C. After 0 day (test start date), 1 week, 2 weeks, 3 weeks, and 4 weeks, 100 µl / well of a 1 µg / ml mouse antibody physiological saline solution was added, followed by incubation at 25 ° C for 2 hours. Thereafter, the plate was washed four times with physiological saline. Then, a peroxidase-labeled anti-mouse IgG antibody [trade name “peroxidase-labeled- manufactured by Wako Pure Chemical Industries, Ltd.”
Anti-mouse antibody-antibody ”] was diluted 10000-fold with physiological saline, and 100 μl / well was added.
Incubated at 5 ° C for 2 hours. Thereafter, the plate was washed four times with physiological saline.

Next, one tablet of o-phenylenediamine dihydrochloride (trade name "OPD tablet" manufactured by Wako Pure Chemical Industries, Ltd.) was added to a phosphate / citrate buffer solution containing 0.006% hydrogen peroxide. After adding 100 μl / well of the solution dissolved in 12 ml, the mixture was incubated at 25 ° C. for 10 minutes. Then, after adding 10 μl / well of a 2N sulfuric acid solution, the absorbance at 492 nm of each well was measured using a microplate reader “MPR-A4i” (manufactured by Tosoh Corporation), and the absorbance at day 0 was defined as 100%. The% after each week was determined. Table 6 shows the measurement results.

Comparative Example 3-1 Except that the solid phase for immunologically active substance immobilization prepared in Comparative Example 1-1 was used instead of the solid phase for immunologically active substance measurement used in Example 3-1. Was performed in the same manner as in Example 3-1. The measurement results are shown in Table 6.

[0087]

[Table 6]

From the results shown in Table 3, the anti-mouse was also observed in Examples 1-1 to 1-6 in which the oxyalkylene group-containing acrylic polymer of the present invention was immobilized, similarly to the bovine serum albumin of Comparative Example 1-1. It can be seen that the antibody was adsorbed. Also,
From the results of Tables 4 and 5, Examples 2-1 to 2-6 show that
0.0 μg / ml, 0.05 μg / ml, 0.1 μg / ml
ml, 0.2μg / ml, 0.4μg / ml, 0.8μ
g / ml, there is a significant difference, 0.05 μg / m
It can be seen that up to 1 can be measured. However, Comparative Example 2-1
Are 0.0 μg / ml, 0.05 μg / ml, 0.1 μg
It can be seen that there is no significant difference in g / ml and measurement cannot be performed.
Furthermore, from the results in Table 6, it can be seen that the immobilized antibodies of Examples 3-1 to 3-6 are clearly stabilized as compared with Comparative Example 3-1.

Claims (5)

[Claims] 1. A compound of the general formula (1) (Wherein, R ¹ is a hydrogen atom or a methyl group, R ² is a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms, and OA is a carbon atom having 2 to 2 carbon atoms.
The oxyalkylene group of 4 and n are integers of 1 to 1000 in the average number of moles of the oxyalkylene group added. A non-protein specific adsorption inhibitor comprising an oxyalkylene group-containing acrylic polymer obtained by polymerizing the monomer represented by the formula (1). 2. An immunologically active substance-immobilized solid phase in which an immunologically active substance is immobilized on a solid phase, wherein the protein non-specific adsorption inhibitor according to claim 1 is adsorbed. A solid phase immobilized with an adsorption inhibitor adsorbed immunologically active substance for measuring an immunologically active substance. 3. A protein comprising the immunologically active substance-immobilized solid phase in which the immunologically active substance is immobilized on the solid phase, wherein the protein-specific non-specific adsorption inhibitor according to claim 1 is adsorbed. Non-specific adsorption prevention method. 4. An immobilization method comprising adsorbing the non-specific protein adsorption inhibitor according to claim 1 onto an immunologically active substance-immobilized solid phase in which an immunologically active substance is immobilized on a solid phase. A method for stabilizing a chemical immunologically active substance. 5. An immunologically active substance-immobilized solid phase in which an immunologically active substance is immobilized on a solid phase, and an immunologically active substance to be measured is brought into contact with the solid phase, and an antigen-antibody reaction is performed to immobilize the immunologically active substance. An immunologically active substance-analyte complex is formed, and then a labeled immunologically active substance labeled with a labeling substance is contacted, and an antigen-antibody reaction is performed to immobilize the immunologically active substance-an analyte-label. In an immunologically active substance measuring method for forming an immunologically active substance complex and measuring an object to be measured using a labeling substance in the complex as an index, the immunologically active substance-immobilized solid phase is claimed. 2. A method for measuring an immunologically active substance, which comprises using the solid phase immobilized with the adsorption inhibitor and the immunologically active substance according to 2.