JPH10114800A - Polymer adsorbed immunologically active substance immobilizing stationary phase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject solid phase useful for clinical diagnosis, capable of suppressing nonspecific adsorption and carrying out measurement excellent in sensitivity and reproducibility, by adsorbing a phosphorylcholine group- containing polymer on a stationary phase prepared by immobilizing an immunologically active substance to a carrier. SOLUTION: A phosphorylcholine group-containing polymer of the formula (R<1> is a 1-10C hydrocarbon group) composed of a polymer obtained by polymerizing a polymerizable component containing 2-methacryloyloxyethyl-2'-(trimethyl ammonio)ethyl phosphate is adsorbed on an immunologically active substance immobilized stationary phase prepared by immobilizing an immunologically active substance (e.g. antibody) to a carrier (e.g. titer plate made of polystyrene) to give the objective polymer absorbing immunologically active substance immobilized stationary phase capable of carrying out measurement excellent in sensitivity and reproducibility while preventing nonspecific adsorption to an immunologically active substance-immobilized stationary phase useful in a two- site method (sandwich measurement) widely used in the field of clinical diagnostic.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a polymer-adsorbed immunologically active substance-immobilized solid phase, a method for measuring an immunologically active substance,
The present invention relates to a method for preventing nonspecific adsorption and a method for stabilizing immobilized immunologically active substances. More specifically, in a sandwich method widely used in the field of clinical reagents and the like, adsorption of a labeled antibody to an antibody-bound solid phase (non-specific adsorption of a labeled antibody), adsorption of a labeled antigen to an antigen-bound solid phase (Non-specific adsorption of labeled antigen) or polymer-adsorbed immunologically active substance-immobilized solid phase suitable for preventing non-specific adsorption of proteins such as adsorption of proteins in a sample to a solid phase; The present invention relates to a method for measuring an immunologically active substance, a method for preventing nonspecific adsorption, and a method for stabilizing a substance having immobilized immunological activity.

[0002]

2. Description of the Related Art Immunometric assays widely used in the field of clinical diagnostic agents and the like are generally known as two-s
A solid phase method based on the item method (sandwich measurement method) is used. This measurement method is based on the substance to be measured (test substance;
Ag) having two different epitopes (Ab1,
Ab2) is used. First, Ab1 is immobilized on the surface of a solid phase (SP) made of a synthetic polymer or the like, and then Ag is added thereto and bound. Next, after reacting with a labeled antibody (Ab2 *), washing is performed to remove free Ab2 *, and the standard activity of Ab2 * (bound type, B) bound to the solid phase is measured. In this case, B increases according to the amount of Ag, 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 an antibody in a sample by inverting an antigen and an antibody, that is, using a labeled antigen is also used (measured using Ag1, Ag2 * and Ab). For these labeling substances, isotopes, enzymes, fluorescent substances, luminescent substances and the like are used.

One of the main factors affecting the sensitivity of the 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 nature 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, or a peroxidase-labeled antibody Is 1 / 300,000,000 of the added amount, and the non-specific adsorption of the β-D-galactosidase-labeled antibody is 1/2000 (Medical Books, Enzyme Immunoassay, pp. 158 ~, 1989). Year). These non-specific adsorptions cause a decrease in sensitivity and a decrease in reproducibility in the sandwich method.

Conventionally, the following methods (1) to (3) have been known to prevent such non-specific adsorption. (1) a method in which an immunoassay is carried out with a weakly acidic buffer solution having a pH of 5 to 6; (3) a method of dissolving milk protein in a buffer containing an organic acid as a main component and using a non-specific adsorption inhibitor sterilized (JP-A-01-217266) . However, (1)
The operation at weak acidity and the blocking with various proteins in (2) and (3) are not sufficient in preventing non-specific adsorption of proteins, and are superior in preventing non-specific adsorption of proteins in fields such as clinical diagnosis. It has been desired to develop an agent and a solid phase on which an immunologically active substance is immobilized, which has been subjected to a protein nonspecific adsorption prevention treatment. In addition, commercially available proteins such as ovalbumin, bovine serum albumin, fetal bovine serum, and milk proteins often contain contamination with immunoglobulins, enzymes or hormones,
This is a problem because it affects the reaction and causes an error in the analysis value.

[0005]

A first object of the present invention is to measure an immunologically active substance without affecting a measurement system, that is, labeling of an isotope, an enzyme, a fluorescent substance or a chemiluminescent substance. The adsorption of the labeled antibody to the antibody-bound solid phase (non-specific adsorption of the labeled antibody) and the adsorption of the labeled antigen to the antigen-bound solid phase (non-specific adsorption of the labeled antigen) are not limited to the substance and the substance to be measured. ) Or a solid phase on which a polymer-adsorbed immunologically active substance is immobilized, which can analyze the target substance with excellent accuracy by suppressing non-specific protein adsorption such as adsorption of the protein in the sample to the solid phase. To provide.
A second object of the present invention is to prevent non-specific protein adsorption,
An object of the present invention is to provide a method for measuring an immunologically active substance, which can accurately analyze a target substance. A third object of the present invention is to provide a method for preventing non-specific protein adsorption, which can sufficiently suppress non-specific protein adsorption when measuring an immunologically active substance. A fourth object of the present invention is to provide a method for stabilizing an immobilized immunologically active substance capable of stabilizing an immunologically active substance immobilized on an immunologically active substance-immobilized solid phase with time. It is in.

[0006]

According to the present invention, the following inventions (a) to (d) are provided. (A) An immunologically active substance-immobilized solid phase obtained by immobilizing an immunologically active substance on a carrier has the following formula (1) (wherein, R ¹ represents a hydrocarbon group having 1 to 10 carbon atoms) Polymer adsorption immunology obtained by adsorbing a polymer obtained by polymerizing a polymer containing a phosphorylcholine group-containing polymer, preferably 2-methacryloyloxyethyl-2 ′-(trimethylammonio) ethyl phosphate, represented by the formula: Active substance immobilized solid phase.

[0007]

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(B) A method for measuring an immunologically active substance using an antigen-antibody reaction, wherein the solid phase on which the polymer-adsorbed immunologically active substance is immobilized is used as the solid phase on which the immunologically active substance is immobilized. Method for measuring an immunologically active substance. (C) In the measurement of an immunologically active substance using an antigen-antibody reaction, it is determined that a labeled antibody, a labeled antigen, a protein in a sample, or a mixture thereof is non-specifically adsorbed to the immunologically active substance-immobilized solid phase. In the prevention, a method for preventing nonspecific adsorption, wherein the solid phase on which the polymer-adsorbed immunologically active substance is immobilized is used as the solid phase on which the immunologically active substance is immobilized. (D) Immobilizing the immunologically active substance In stabilizing the immunologically active substance immobilized on the solid phase, the immunologically active substance is immobilized on a carrier and then expressed by the above formula (1). A method for stabilizing an immobilized immunologically active substance, comprising adsorbing a phosphorylcholine group-containing polymer.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The polymer-adsorbed immunologically active substance-immobilized solid phase of the present invention comprises an immunologically active substance immobilized on the immunologically active substance-immobilized solid phase, The immunologically active substance to be measured when the immunologically active substance is measured by the antigen-antibody reaction is not particularly limited, but includes, for example, the following. C-reactive protein (CRP), rheumatoid factor (R
F), plasma proteins such as transferrin or antibodies against these plasma proteins, thyroid stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4), thyroxine binding protein (TBG), thyroglobulin, insulin, estriol (E3 ), Chorionic gonadotrobin (HC
G), 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), or these tumor-related substances Antigens or antibodies against viral hepatitis such as antibodies, HBS antigens, HBS antibodies, HBe antigens, HBe antibodies, or antibodies or antigens against these viral hepatitis antigens or antibodies, viruses such as mumps, herpes, measles, rubella, cytomegalo, etc. Antibodies or antigens against various biological components such as AIDS antibodies, antibodies against various drugs such as phenobarbital, acetaminophenone, salicylic acid, cyclosporin, enzymes or antibodies against enzymes. In addition, an antigen against the immobilized antibody or an antibody against the immobilized antigen can be used as the immunologically active substance (test substance) to be measured.

[0010] In the solid phase immobilized with the polymer-adsorbed immunologically active substance of the present invention, the material and shape of the carrier are not particularly limited.
Synthetic resins such as polyvinyl chloride, polypropylene, (meth) acrylic resin, and polymethyl methacrylate; cellulose derivatives such as nitrocellulose, cellulose, and methylcellulose; and inorganic substances such as metal, ceramic, glass, and silicone rubber. Examples of the shape include a test tube, a titer plate, a latex, a filter, a film, and fine particles.

In the present invention, the phosphorylcholine (hereinafter abbreviated as PC) group-containing polymer adsorbed on the solid phase immobilized on the immunologically active substance is a polymer having a PC group represented by the above formula (1). And a polymer obtained by polymerizing a polymerization component containing a monomer having a PC group. Examples of the monomer having a PC group include 2- (meth) acryloyloxyethyl-2 ′-(trimethylammonio) ethyl phosphate and 2- (meth) acryloyloxyethyl-2 ′
-(Triethylammonio) ethyl phosphate, 2-
(Meth) acryloyloxyethyl-2 ′-(tripropylammonio) ethyl phosphate, 2- (meth) acryloyloxyethyl-2 ′-(tributylammonio) ethyl phosphate, 2- (meth) acryloyloxyethyl-2 ′ -(Trioctylammonio) ethyl phosphate and the like. Especially from the point of availability etc.
2-methacryloyloxyethyl-2 ′-(trimethylammonio) ethyl phosphate {= 2-methacryloyloxyethyl phosphorylcholine (hereinafter abbreviated as MPC)} is preferred. The PC group-containing polymer used in the present invention is a homopolymer of a monomer having a PC group, or a copolymer of a monomer having a PC group and another copolymerizable vinyl monomer. May be. P in PC group-containing polymer
The C group content is 1 to 100 with respect to the PC group-containing polymer.
Mol% is preferable, and particularly preferably 5 to 10 mol%. If the content is less than 1 mol%, it is difficult to prevent non-specific adsorption, which is not preferable. The PC group-containing polymer also varies depending on the polymerization temperature, the amount of the polymerization initiator used, the use of the polymerization degree regulator, and the like, but preferably has a number average molecular weight (Mn) of 1,000 to 1,000,000, particularly preferably. It is a polymer of 2,000 to 500,000.

Other vinyl monomers copolymerizable with the monomer having a PC group include, for example, methyl (meth) acrylate, ethyl (meth) acrylate, and n-butyl (meth) acrylate. , Isobutyl (meth) acrylate,
(Meth) acrylates such as pentyl (meth) acrylate, hexyl (meth) acrylate, heptyl (meth) acrylate, octyl (meth) acrylate, tridecyl (meth) acrylate, and 2-hydroxyethyl methacrylate; Meth) acrylates; styrene monomers such as styrene, α-methylstyrene, methyl-substituted styrene, chloro-substituted styrene, etc .; substituted or unsubstituted hydrocarbons such as vinyl chloride, vinylidene chloride, ethylene, propylene, isobutylene, etc. Monomer, vinyl ester monomers such as vinyl acetate and vinyl propionate; vinyl ether monomers such as ethyl vinyl ether and n-butyl vinyl ether; and diethyl itaconate and di-n-butyl itaconate. Particularly preferably, methacrylic acid ester, styrene and the like can be preferably mentioned.

In order to prepare a PC group-containing polymer, a polymer containing the above-mentioned monomer having a PC group is polymerized by a usual polymerization method such as radical polymerization using a polymerization initiator. be able to.

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-butylperoxypivalate, t-butylperoxydiisobutyrate,
Persulfate, persulfuric acid-bisulfite and the like can be mentioned. The amount of the polymerization initiator to be used is preferably 0.01 to 10 parts by weight, particularly preferably 0.1 to 5 parts by weight, based on 100 parts by weight of all monomers used.

The polymerization is preferably carried out at 30 to 80 ° C., particularly 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. Examples of the solvent include water, methanol, ethanol, propanol, t-butanol, benzene, toluene, dimethylformamide, tetrahydrofuran, chloroform and mixtures thereof. And the like.

To prepare the solid phase on which the polymer-adsorbed immunologically active substance of the present invention is immobilized, the immunologically active substance is immobilized on the carrier by, for example, incubation or the like. The phase can be obtained by, for example, adding a solution containing the PC group-containing polymer to the phase and adsorbing the PC group-containing polymer. The solution containing the PC group-containing polymer may be a suspension or a solution, preferably a phosphate buffer, an acetate buffer, a carbonate buffer, a citrate buffer, various physiological saline, and the like. A solution or suspension is mentioned. An organic solvent such as dimethylsulfoxide, tetrahydrofuran, N, N-dimethylformamide and the like can be added to these liquids in an amount of 0.01 to 20% by weight. Particularly preferably, a dissolving solution such as a phosphate buffer solution and various physiological salines can be used. The concentration of the PC group-containing polymer in the solution containing the PC group-containing polymer is preferably 0.00001 to 10% by weight, and in particular, non-specific adsorption of the protein to the solid phase is prevented and the immobilization is performed. 0.0001 to 5% by weight is preferable so that the ability to stabilize the immunologically active substance can be significantly improved. In order to adsorb the PC group-containing polymer to the immunologically active substance-immobilized solid phase, after binding the immunologically active substance to the surface of the carrier, the solution containing the PC group-containing polymer is added and kept as it is. Alternatively, it can be carried out by incubating for a predetermined time after the addition, and then removing the solution containing the residual PC group-containing polymer. The incubation time from the addition of the solution containing the PC group-containing polymer to the removal thereof depends on the concentration of the solution containing the PC group-containing polymer or the incubation temperature, but is preferably 1 minute to 72 hours. In particular, in the method for measuring an immunologically active substance using an antigen-antibody reaction, an effect of sufficiently preventing non-specific adsorption of a protein to a solid phase is imparted, and the stability of the immobilized immunologically active substance is stabilized. 30 to improve the
Minutes to 48 hours are preferred. If the incubation time is less than 1 minute, the desired effect may not be obtained. The incubation temperature is preferably from 0 to 55 [deg.] C, especially from 4 to 4 without affecting the immunological activity of the immobilized immunologically active substance.
0 ° C. is preferred. The amount of the PC group-containing polymer adsorbed on the solid phase is not particularly limited, but is preferably 10 n
g / 48 well to 10000 ng / 48 well, particularly preferably contributing to the stabilization of the immobilized immunologically active substance,
The viscosity of the polymer solution to be added is low and easy to handle, 100 ng
/ 48 wells to 1000 ng / 48 wells. If it is less than 10 ng / 48 well, the immobilized immunologically active substance may be insufficiently stabilized, and
If it exceeds ng / 48 wells, it is necessary to increase the concentration of the polymer solution to be added or to lengthen the time from the addition of the polymer solution to the removal thereof. If the concentration of the polymer solution is increased, the viscosity is also increased and handling becomes difficult, and if the time is extended, rapid measurement becomes difficult, which is not preferable.

Since the polymer-adsorbed immunologically active substance-immobilized solid phase of the present invention has a PC group-containing polymer adsorbed thereon, the immobilized immunologically active substance upon storage has good stability. is there. The method for preserving the solid phase immobilized with the polymer-adsorbed immunologically active substance thus prepared is not particularly limited, but is preferably left as it is, sealed, frozen, or sealed after freeze-drying, and particularly preferably. In order to further improve the stabilizing effect of the immobilized immunologically active substance, it is preferable to seal or freeze-dry before storing.

The polymer-adsorbed immunologically active substance-immobilized solid phase of the present invention can be used in various fields and methods, for example, in research fields such as clinical examination, immunology, biochemistry, and molecular biology. In particular, enzyme immunoassay (ELIS
A), a radioimmunoassay (RIA), or an immunological assay such as Western blotting can be frequently used.

A specific method for preparing the solid phase immobilized with the polymer-adsorbed immunologically active substance of the present invention and a method for measuring an immunologically active substance using an antigen-antibody reaction are described, for example, by using a polystyrene titer plate as a carrier. The following is a description of the case.

(1) First, a solution containing an antibody that specifically reacts with an object to be measured is added to a polystyrene titer plate, and incubated under desired conditions such as 4 ° C. and 12 hours. Wash twice to prepare an immunologically active substance-immobilized solid phase. (2) Next, a PC group-containing polymer such as an MPC polymer
A solution containing 1% by weight was added to the solid phase on which the immunologically active substance was immobilized, and the mixture was incubated under desired conditions such as 4 ° C. and 12 hours. The biologically active substance-immobilized solid phase is prepared. (3) Subsequently, 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 under predetermined conditions such as 25 ° C. and 2 hours. Incubate to form an antigen-antibody reaction between the immobilized immunologically active substance and the analyte to form an immobilized immunologically active substance-analyte complex, and then wash several times with physiological saline. I do. (4) A solution containing an enzyme-labeled antibody that specifically reacts with the measurement target is added, and the mixture is incubated under desired conditions at 25 ° C. for 2 hours, and the measurement target and the enzyme-labeled antibody are allowed to undergo an antigen-antibody reaction. Then, an immobilized immunologically active substance-analyte-to-enzyme-labeled antibody complex is formed, followed by washing with physiological saline several times. (5) Measure the enzyme activity of the immobilized immunologically active substance-measurement object-enzyme-labeled antibody complex, and compare the enzyme activity in the sample with the enzyme activity in the standard solution. The physical quantity can be obtained. In such a measurement method, by using a polymer-adsorbed immunologically active substance-immobilized solid phase to which a PC group-containing polymer is adsorbed,
Non-specific adsorption of proteins to the solid phase is prevented.

[0021]

EFFECTS OF THE INVENTION The solid phase on which the polymer-adsorbed immunologically active substance of the present invention is immobilized contains a PC group-containing polymer free from contamination with enzymes, hormones and the like. Non-specific adsorption of proteins is low in the method for measuring active substances. In addition, the immobilized immunologically active substance is stable for a long period of time, so that highly sensitive and accurate analysis can be performed.

[0022]

The present invention will be described in more detail with reference to the following examples. Synthesis Example 1: Synthesis of MPC polymer MPC 5.9 such that the total monomer concentration is 1.0 mol / l and the amount of polymerization initiator is 1 mol% with respect to the monomer.
05 g (0.02 mol) was weighed into a polymerization glass reaction tube, and 0.0328 g of 2,2′-azobisisobutyronitrile (hereinafter abbreviated as AIBN) was added thereto as a polymerization initiator.
(0.2 mmol) and 20 ml of methanol as a polymerization solvent. After sufficiently replacing the inside of the reaction tube with argon, it was sealed. Next, the polymerization reaction was performed by heating to 50 ° C. for 24 hours. After cooling the reaction mixture on ice, the polymer was precipitated by dropwise addition to 400 ml of diethyl ether. The precipitate was separated by filtration, sufficiently washed with diethyl ether, and dried under reduced pressure to obtain 3.691 g of a white powdery polymer (referred to as polymer A). The yield of the polymer was 62.5%. The molecular weight was measured by analyzing the phosphate buffer solution of the polymer using GPC (gel permeation chromatography), and as a result, it was 68,000 in terms of polyethylene glycol.

Synthesis Example 2: MPC-methacrylic acid-n-butyl
Synthesis of Chill (hereinafter abbreviated as BMA) copolymer MPC / B
MA = 40/60, total monomer concentration 1.0 mol / l,
And 1.435 g (4.9 mmol) of MPC, BM, so that the amount of the polymerization initiator is 1 mol% with respect to the monomer.
A 2.153 g (15.1 mmol) was weighed into a glass reaction tube for polymerization, and AIBN 0.0
328 g (0.2 mmol) and 20 ml of methanol as a polymerization solvent were added. After sufficiently replacing the inside of the reaction tube with argon, it was sealed. Next, the polymerization reaction was performed by heating to 60 ° C. for 24 hours. After cooling the reaction mixture on ice, the polymer was precipitated by dropwise addition to 400 ml of diethyl ether. The precipitate was separated by filtration, sufficiently washed with diethyl ether, and dried under reduced pressure to obtain 2.019 g of a white powdery polymer (hereinafter, referred to as polymer B). The yield of the polymer was 65.3%. The molecular weight was measured by analyzing a solution of the polymer in tetrahydrofuran using GPC.
00. From the results of elemental analysis, the molar composition
C / BMA = 38.5 / 61.5.

Synthesis Example 3 A copolymer (hereinafter referred to as polymer C) was prepared according to Synthesis Example 2 using methyl methacrylate (abbreviated as MMA) instead of BMA.
) Was synthesized. The molar composition and molecular weight of the obtained copolymer were MPC / MMA = 34.4 / 65.6, M
n = 69000.

Synthesis Example 4 Styrene (hereinafter abbreviated as St) was used instead of BMA.
A copolymer (referred to as polymer D) was synthesized according to Synthesis Example 2. The molar composition and molecular weight of the resulting copolymer are
C / St = 38.5 / 61.5 and Mn = 26000.

Synthesis Example 5 A copolymer (hereinafter referred to as polymer E) was synthesized according to Synthesis Example 2 using 2-hydroxyethylmethyl methacrylate (hereinafter abbreviated as HEMA) instead of BMA. The molar composition and molecular weight of the obtained copolymer were determined by MPC / HEM.
A = 21.5 / 78.5 and Mn = 32000.

Table 1 shows the yields, molar compositions and molecular weights of the monomers used in Synthesis Examples 1 to 5 and the obtained copolymers.

[0028]

[Table 1]

Example 1-1: Polymer-adsorbed immunologically active substance
Preparation of Solid Phase Immobilized Solid Solution 10 μl / ml of an anti-mouse antibody physiological saline solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a polystyrene titer plate at 100 μl / well, and incubated at 4 ° C. overnight, physically. After the adsorption, washing was performed four times with a physiological saline solution. Next, the polymer B0.
001% by weight of a physiological saline solution was added at 300 μl / well, and the mixture was incubated at 4 ° C. overnight. Thereafter, the copolymer solution was removed to prepare a solid phase on which a polymer-adsorbed immunologically active substance was immobilized. The amount of anti-mouse antibody immobilized by adsorption was determined by adding 200 μl of physiological saline containing 1% -sodium dodecyl sulfate to 10 wells of a polystyrene titer plate to release the immobilized anti-mouse antibody. Product name “Micro BCA” manufactured by PIERCE
It was measured using "Protein Assay Kit". Table 2 shows the measurement results. Further, the amount of the adsorbed polymer was measured using a 48-well “Phospholipid B-Test Wako” (trade name, manufactured by Wako Pure Chemical Industries, Ltd.). Table 3 shows the measurement results.

Examples 1-2 and 1-3: Polymer adsorption immunity
Preparation of biologically active substance-immobilized solid phase Example 1 was repeated except that polymers C and D synthesized in Synthesis Examples 3 and 4 were used instead of Polymer B synthesized in Synthesis Example 2.
In the same manner as in Example 1, a solid phase on which a polymer-adsorbed immunologically active substance was immobilized was obtained, and each measurement was performed. The results are shown in Tables 2 and 3.

Comparative Example 1-1 Immobilization of a polymer-adsorbed immunologically active substance in the same manner as in Example 1-1 except that 1% by weight of bovine serum albumin was used instead of the polymer B synthesized in Synthesis Example 2. An immobilized solid phase was obtained. The amount of anti-mouse antibody immobilized by adsorption was the same as in Example 1-1, and the amount of bovine serum albumin adsorbed was 200 μl / well in 10 wells as in the case of measuring the amount of anti-mouse antibody.
% -Sodium dodecyl sulfate is added to each of the physiological saline to exfoliate the immobilized anti-mouse antibody and bovine serum albumin. The product name is “Micro” manufactured by PIERCE.
The amount of the adsorbed protein was determined using "BCA Protein Assay Kit", and the amount of the immobilized anti-mouse antibody was subtracted from the value. The results are shown in Tables 2 and 3.

Example 1-4: Polymer-adsorbed immunologically active substance
Preparation of solid phase immobilized in glass test tube (diameter: 12 mm, height: 75 mm)
After 0.5 ml of aminopropyltriethoxysilane was added, the mixture was incubated at room temperature for 20 minutes, and then washed four times with physiological saline. Next, 0.5 ml of physiological saline containing 2.5% by weight of glutaraldehyde was added, and the mixture was incubated at room temperature for 2 hours, and then washed twice with physiological saline. Next, 0.5 ml of a 10 μl / ml saline solution of an anti-mouse antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was incubated at room temperature for 2 hours.
Washing was performed twice. Next, 1 ml of a physiological saline solution containing 0.01% by weight of the polymer A synthesized in Synthesis Example 1 was added,
After incubating at room temperature for 30 minutes, the polymer solution was removed to obtain a polymer-adsorbed immunologically active substance-immobilized solid phase. The amount of immobilized anti-mouse antibody was determined by measuring the amount of unreacted anti-mouse antibody by the trade name “Micro BC” manufactured by PIERCE.
A Protein Assay Kit "was used to determine the difference between the total amount of added anti-mouse antibody and the amount of unreacted anti-mouse antibody. Table 2 shows the measurement results. Also,
The amount of the adsorbed MPC polymer was measured using 20 test tubes using “Phospholipid B-Test Wako” (trade name, manufactured by Wako Pure Chemical Industries, Ltd.). Table 3 shows the measurement results.

Example 1-5: Polymer-adsorbed immunologically active substance
Preparation of a solid phase immobilized with a polymer In the same manner as in Example 1-4 except that the polymer E synthesized in Synthesis Example 5 was used instead of the polymer A used in Example 1-4, An active substance-immobilized solid phase was obtained, and each measurement was performed. Tables 2 and 3 show the measurement results.

Comparative Example 1-2 Polymer adsorption immunological activity in the same manner as in Example 1-4 except that 1% by weight of bovine serum albumin was used in place of the polymer A used in Example 1-4. A substance-immobilized solid phase was obtained. The amount of solidified anti-mouse antibody was determined in the same manner as in Example 1-4, and the amount of bovine serum albumin was determined from the difference between the amount of bovine serum albumin added and the amount of unadsorbed bovine serum albumin. Tables 2 and 3 show the measurement results.

[0035]

[Table 2]

[0036]

[Table 3]

Examples 2-1 to 2-3: Measurement of an object to be measured Solid phase immobilized with the polymer-adsorbed immunologically active substance prepared in Examples 1-1, 1-2 and 1-3. 0 μg /
ml, 0.05 μg / ml, 0.1 μg / ml, 0.2
After adding 100 μl / well of a physiological saline solution of mouse antibody (manufactured by Wako Pure Chemical Industries, Ltd.) at each concentration of μg / ml, 0.4 μg / ml and 0.8 μg / ml, the mixture was incubated at 25 ° C. for 2 hours. Then, it was washed four times with physiological saline. Then, peroxidase labeling-anti-mouse antibody-antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was
After diluting 2-fold and adding 100 μl / well, 25
C., incubated for 2 hours, followed by 4 washes with saline. Then, the product name “O” manufactured by Wako Pure Chemical Industries, Ltd.
One PD tablet (o-femylenediamine) at 0.006
Phosphate / citrate buffer 12 containing wt% hydrogen peroxide
After adding 50 μl / well of the solution dissolved in ml,
After incubating at 25 ° C. for 10 minutes and subsequently adding a 2N sulfuric acid solution at 100 μl / well, a microplate reader “MPR-A41” (trade name) manufactured by Tosoh Corporation is used.
Was used to measure the absorbance at 492 nm of each well.
The number of measurements (n) is 8, and the average value, standard deviation, and CV value (%) {(average value / standard deviation) × 100} are shown in Table 4.

Examples 2-4 and 2-5: Measurement of object to be measured
The polymer-adsorbed immunologically active substance-immobilized solid phase prepared in Example 1-4 and Example 1-5 was loaded with 0 μg / ml, 0.05
μg / ml, 0.1 μg / ml, 0.2 μg / ml,
Mouse antibody physiological saline solution (manufactured by Wako Pure Chemical Industries, Ltd.) of each concentration of 0.4 μg / ml and 0.8 μg / ml 0.5 m
After adding 1 to the test tube, the mixture was incubated at 25 ° C. for 2 hours, and then washed four times with physiological saline. Next, the peroxidase-labeled anti-mouse antibody-antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted 20,000-fold with physiological saline to give 0.1%.
After addition to a 5 ml test tube, the mixture was incubated at 25 ° C. for 2 hours, and then washed four times with physiological saline. Next, one tablet of “OPD tablet” manufactured by Wako Pure Chemical Industries, Ltd.
12m phosphate / citrate buffer containing 2% hydrogen peroxide
After adding 0.5 ml of the solution dissolved in
Incubated at 5 ° C for 10 minutes. Subsequently, after adding 0.5 ml of 2N sulfuric acid solution to the test tube, using a spectrophotometer manufactured by JASCO Corporation, trade name "Ubest-50",
The absorbance at 492 nm of each test tube was measured. The number of measurements (n) was 5, and the average value, standard deviation, and CV value (%) are shown in Table 4.

Comparative Example 2-1 In place of the solid phase immobilized with the polymer-adsorbed immunologically active substance prepared in Example 1-1 and used in Example 2-1, Comparative Example 1 was used.
Each measurement was performed in the same manner as in Example 2-1 except that the immunologically active substance-immobilized solid phase prepared in -1 was used. Table 5 shows the measurement results.

Comparative Example 2-2 Comparative Example 1 was used in place of the polymer-adsorbed immunologically active substance-immobilized solid phase prepared in Example 1-4, which was used in Example 2-4.
Each measurement was performed in the same manner as in Example 2-4, except that the solid phase immobilized with the immunologically active substance prepared in -2 was used. Table 5 shows the measurement results.

[0041]

[Table 4]

[0042]

[Table 5]

Examples 3-1 to 3-3: Stability test The solid phase immobilized with the polymer-adsorbed immunologically active substance prepared in Examples 1-1, 1-2 and 1-3 was used. After sealing in an aluminum laminated polyethylene bag, it was stored at 40 ° C. 0 days later (test start date), 1 week later, 2 weeks later, 3 days later
After one week and four weeks, 50 μl / well of a 1 μg / ml mouse antibody physiological saline solution was added, incubated at 25 ° C. for 2 hours, and washed four times with physiological saline.
Next, a peroxidase-labeled anti-mouse antibody-antibody (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted 10000-fold with physiological saline, and added at 100 μl / well.
Incubate for hours, followed by 4 washes with saline. Next, the product name "OPD tablet" 1 manufactured by Wako Pure Chemical Industries, Ltd.
A solution obtained by dissolving the tablets in 12 ml of a phosphate / citrate buffer containing 0.006% hydrogen peroxide was added at 100 μl / well, followed by incubation at 25 ° C. for 10 minutes. Next, after adding 100 μl / well of a 2N sulfuric acid solution, a microplate reader “MPR-A” manufactured by Tosoh Corporation was added.
Using “41” (trade name), the absorbance at 492 nm of each well was measured, and the absorbance at day 0 was defined as 100%, and the percent after each week was determined. Table 6 shows the measurement results.

Examples 3-4 and 3-5: Stability test The polymer-adsorbed immunologically active substance-immobilized solid phase prepared in Examples 1-4 and 1-5 was sealed, and then sealed at 40 ° C. saved. 0 days later (test start date), 1 week later, 2 weeks later,
After 3 weeks and 4 weeks, 0.5 ml of a 1 μg / ml mouse antibody physiological saline solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the test tube, followed by incubation at 25 ° C. for 2 hours.
Subsequently, it was washed four times with physiological saline. Next, peroxidase labeling-anti-mouse antibody-antibody (Wako Pure Chemical Industries, Ltd.)
20,000 times diluted with physiological saline, and 0.5 ml
Was added to the test tube, followed by incubation at 25 ° C. for 2 hours, followed by washing four times with physiological saline. After adding 0.5 ml of a solution obtained by dissolving one OPD tablet (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) in 12 ml of a phosphate / citrate buffer containing 0.006% hydrogen peroxide to a test tube. , 25 ° C, 1
Incubate for 0 min, 0.5 ml of 2N sulfuric acid solution
Was added to the test tubes, and 492n of each test tube was measured using a spectrophotometer manufactured by JASCO Corporation, trade name “Ubest-50”.
The absorbance at m was measured, and the absorbance at day 0 was determined as 100%, and the% at each week was determined. Table 6 shows the measurement results.

Comparative Example 3-1 Instead of the solid phase immobilized with the polymer-adsorbed immunologically active substance prepared in Example 1-1 and used in Example 3-1, Comparative Example 1 was used.
The measurement was carried out in the same manner as in Example 3-1 except that the immunologically active substance-immobilized solid phase prepared in -1 was used. Table 6 shows the measurement results.

Comparative Example 3-2 Instead of the solid phase immobilized with the polymer-adsorbed immunologically active substance prepared in Example 1-4 and used in Example 3-4, Comparative Example 1 was used.
The measurement was carried out in the same manner as in Example 3-4, except that the immunologically active substance-immobilized solid phase prepared in -2 was used. Table 6 shows the measurement results.

[0047]

[Table 6]

From the above results, it can be seen from the results in Table 2 that the anti-mouse antibody was immobilized on the immobilized solid phase by using the solid phase immobilized with the polymer-adsorbed immunologically active substance of the present invention. Also, from the results in Table 3, the amount of the polymer of the present invention and the BSA of the comparative example adsorbed thereafter is 600 to 765 ng.
It can be seen that / 48 wells are adsorbed. Furthermore, from the results of Tables 4 and 5, it can be seen that the measurement can be performed with high sensitivity even at a lower concentration of the antibody. In this case, the stability of the immobilized immunologically active substance is found to be excellent.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Shujiro Sakaki 2-20-3, Kasuga, Tsukuba, Ibaraki Prefecture (72) Inventor Kenshiro Shuto 3-7-1, Hanahata, Tsukuba, Ibaraki Prefecture (72) Inventor Satoshi Yamada Ibaraki 2-20-3, Kasuga, Tsukuba, Japan (72) Inventor Kazuo Matsuyama 2-17-14, Kasuga, Tsukuba, Ibaraki (72) Inventor Norio Nakabayashi 5-6-20, Koganehara, Matsudo, Chiba (72) Inventor Kazuhiko Ishihara 3-16-37 Josuihoncho, Kodaira City, Tokyo

Claims (5)

[Claims] 1. An immunologically active substance-immobilized solid phase obtained by immobilizing an immunologically active substance on a carrier has the following formula (1) wherein R ¹ is a hydrocarbon group having 1 to 10 carbon atoms. Shown) adsorbed a phosphorylcholine group-containing polymer represented by
Solid phase immobilized with polymer-adsorbed immunologically active substance. Embedded image 2. The phosphorylcholine group-containing polymer according to claim 1, wherein the polymer is 2-methacryloyloxyethyl-2′-.
The polymer-adsorbed immunologically active substance-immobilized solid phase according to claim 1, which is a polymer obtained by polymerizing a polymerization component containing (trimethylammonio) ethyl phosphate. 3. The method for measuring an immunologically active substance using an antigen-antibody reaction, wherein the solid phase on which the immunologically active substance is immobilized is the solid phase on which the polymer-adsorbed immunologically active substance is immobilized according to claim 1 or 2. A method for measuring an immunologically active substance, characterized by using: 4. In the measurement of an immunologically active substance using an antigen-antibody reaction, a labeled antibody, a labeled antigen, a protein in a sample, or a mixture thereof is non-specifically adsorbed to a solid phase on which the immunologically active substance is immobilized. 3. A method for preventing non-specific adsorption, comprising using the solid phase on which the polymer-adsorbed immunologically active substance is immobilized according to claim 1 or 2 as the solid phase on which the immunologically active substance is immobilized. . 5. In stabilizing the immunologically active substance immobilized on the solid phase on which the immunologically active substance is immobilized, the immunologically active substance is immobilized on a carrier, and then the following formula (1) ( Wherein R ¹ represents a hydrocarbon group having 1 to 10 carbon atoms), wherein the immobilized immunologically active substance is stabilized. Embedded image