JP2008191129A - Blocking agent, probe-bound particle, and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a blocking agent manufacturable easily, having a sufficient noise reduction effect, a probe-bound particle and its manufacturing method. <P>SOLUTION: This blocking agent comprises a water-soluble polymer having a functional group shown by the following expression (1): -C(=O)OCH<SB>2</SB>CH(OH)CH<SB>2</SB>(OH). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a blocking agent applicable to, for example, the surface of immunodiagnostic particles, probe-binding particles, and a method for producing the same.

  In recent years, for the purpose of early detection of diseases, etc., it has been required to increase the sensitivity of tests, and improving the sensitivity of diagnostic agents has become a major issue. In diagnostic agents using a solid phase such as magnetic particles, in order to improve sensitivity, a method using enzyme color development as a detection method is being switched to a method using fluorescence or chemiluminescence that can obtain higher sensitivity. With the development of these detection techniques, it is theoretically said that it has reached a level at which even the presence of a single molecule of a test substance can be detected, but in reality, sufficient sensitivity has not been obtained. The reason for this is that in the diagnosis of detecting specific substances in the presence of biomolecules such as serum, coexisting biomolecules, secondary antibodies, luminescent substrates, etc. adsorb non-specifically to the solid phase, resulting in noise. Increasing the sensitivity is hindering. For this reason, in immunodiagnostic measurement, albumin, casein, gelatin are usually used to reduce the decrease in sensitivity due to non-specific adsorption of substances other than substances that specifically bind to the solid surface used for the immune reaction. By using a biological material such as a blocking agent, non-specific adsorption is suppressed and noise is reduced.

  However, even if the blocking operation by the conventional method is performed, non-specific adsorption still remains, and when a biologically derived blocking agent is used, there is a problem of biological contamination represented by BSE. Development of a high-performance blocking agent is desired.

As a blocking agent by chemical synthesis, polymers having polyoxyethylene have been proposed in Japanese Patent Application Laid-Open No. 11-287802 and Japanese Patent No. 34079797, but the noise reduction effect was insufficient.
JP 11-287802 A Japanese Patent No. 34079797

  An object of the present invention is to provide a chemically synthesized blocking agent, probe-binding particles, and a method for producing the same, which are easy to produce and have a sufficient noise reduction effect.

  In order to solve this problem, the present inventors have found a synthesis method that can be easily produced as a blocking agent, and further, by treating the immunodiagnostic particle with the blocking agent, the particle can be signaled. The inventors have found that the enhancing effect is exhibited and completed the present invention.

The blocking agent according to one embodiment of the present invention is
It consists of a water-soluble polymer having a functional group represented by the following formula (1).
_{-C (= O) OCH 2 CH} (OH) CH 2 (OH) ····· (1)

  In the blocking agent, the water-soluble polymer can be obtained by copolymerizing 50 to 99 parts by mass of 2,3-dihydroxypropyl (meth) acrylate and 50 to 1 part by mass of other monomers.

A method for producing probe-bound particles according to an aspect of the present invention includes:
Binding a probe to the surface of the particle;
Treating the particles to which the probe is bound, using the blocking agent.

  In the probe-bonded particle manufacturing method, in the step of bonding the probe, the particle may have at least one group selected from a carboxyl group, an active ester group, a tosyl group, an amino group, and an epoxy group. .

  In the method for producing probe-bonded particles, the particles may be magnetic particles.

The probe binding particle which concerns on 1 aspect of this invention has a functional group shown by following formula (1).
_{-C (= O) OCH 2 CH} (OH) CH 2 (OH) ····· (1)

  The probe-coupled particles according to one embodiment of the present invention are obtained by the above-described method for producing probe-coupled particles.

  The probe binding particle according to one embodiment of the present invention has the blocking agent on the surface.

  The above-mentioned blocking agent is easy to produce and can be produced by chemical synthesis, so there is no possibility of biological contamination, and the noise reducing effect is higher than that of conventionally used blocking agents.

  Moreover, the said blocking agent can express a signal enhancement effect, when it uses for the particle | grains (for example, magnetic particle) used for an immunodiagnosis.

  Hereinafter, a blocking agent, probe-binding particles, and a production method thereof according to an embodiment of the present invention will be described.

1. Blocking agent and probe-binding particles and production method thereof 1.1. Structure of blocking agent The blocking agent according to one embodiment of the present invention comprises a water-soluble polymer having a functional group represented by the following formula (1).
_{-C (= O) OCH 2 CH} (OH) CH 2 (OH) ····· (1)

  The amount of the functional group represented by the above formula (1) contained in the water-soluble polymer is preferably 1 mmol / g or more, more preferably 2 mmol / g or more, and most preferably 3 to 6 mmol / g. When the amount of the functional group is less than 1 mmol / g, the noise reduction effect may be inferior.

  In addition to the functional group represented by the above formula (1), the water-soluble polymer includes a hydroxyl group other than the hydroxyl group contained in the functional group represented by the above formula (1), an alkoxyalkyl group, a polyoxyethylene group, a carboxyl group, and a carbonyl group. Hydrophilic groups such as groups, sulfone groups, primary to quaternary amino groups, and amide groups; and hydrophobic groups such as alkyl groups, aryl groups, and carboxylic acid alkyl ester groups may be included as side chains.

  The molecular weight of the water-soluble polymer is preferably 2,000 or more, more preferably 5,000 or more, and most preferably 10,000 to 100,000.

  The water-soluble polymer preferably has a functional group represented by the above formula (1) in the side chain. In this case, examples of the main chain of the water-soluble polymer include polyethylene, polyether, polyamide, polyester, polycarbonate and the like, and since the production is easy, the preferable main chain is polyethylene.

  Examples of the water-soluble polymer include a polymer of 2,3-dihydroxypropyl (meth) acrylate (hereinafter also referred to as “glycerol (meth) acrylate”), and the co-polymerization of glycerol (meth) acrylate and other monomers. Polymer, glycerol vinyl benzoate polymer, glycerol vinyl benzoate and other monomer copolymer, glycerol glutamate polymer, glycerol glutamate and other monomer copolymer (polyamide), hyaluronic acid glycerol ester derivative And a copolymer (polyether) of a glycerol ester derivative of hyaluronic acid and other monomers, and a copolymer of glycerol (meth) acrylate and other monomers is preferable.

1.2. Production of Blocking Agent The blocking agent according to this embodiment can be produced by polymerizing monomers such as glycerol (meth) acrylate, glycerol vinylbenzoate, glycerol glutamate, and glycerol ester of hyaluronic acid. At this time, copolymerization with other monomers is also preferable.

  Among them, as a polymerization method of a glycerol (meth) acrylate copolymer preferable as a blocking agent according to the present embodiment, for example, (1) a method of copolymerizing glycerol (meth) acrylate and other monomers, (2) glycidyl ( The method of copolymerizing a meth) acrylate and another monomer, and hydrolyzing a glycidyl group etc. are mentioned, More preferably, it is the method of (1).

  In the polymerization method of (1), a preferable monomer ratio is 50 to 99 parts by mass of glycerol (meth) acrylate and 50 to 1 part by mass of another monomer, and a more preferable monomer ratio is 60 to 99 parts of glycerol (meth) acrylate. It is 98 mass parts and 40-2 mass parts of other monomers.

  As other monomers, monomers having a hydroxyl group such as hydroxyethyl (meth) acrylate and allyl alcohol; monomers having an alkoxyalkyl group such as methoxyethyl (meth) acrylate and diethylene glycol ethyl ether (meth) acrylate; polyethylene glycol (meth) Monomers having a polyoxyethylene group such as acrylic ester and polyethylene glycol di (meth) acrylic ester; Monomers having a carboxyl group such as acrylic acid, methacrylic acid, itaconic acid, unmatched maleic acid, crotonic acid; diacetone acrylamide, acrolein, Monomers having a carbonyl group such as formylstyrene; styrenesulfonic acid, isoprenesulfonic acid, 2-acrylamido-2-methylpropanesulfur Monomers having sulfone groups such as acid; monomers having primary to quaternary amino groups such as allylamine, aminostyrene, N, N-dimethylaminopropylacrylamide, and methyl chloride quaternary salt of N, N-dimethylaminopropylacrylamide Monomers having amide groups such as (meth) acrylamide and N, N-dimethyl (meth) acrylamide; methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, lauryl (meth) acrylate And hydrophobic monomers such as cyclohexyl (meth) acrylate, isobornyl (meth) acrylate, benzyl (meth) acrylate, and styrene.

  Among these monomers, by copolymerizing a monomer having an amino group, it becomes possible to perform a Coulomb bond with the anionic solid phase surface, thereby enhancing the blocking effect. Further, by copolymerizing a hydrophobic monomer, it becomes possible to form a hydrophobic bond with the hydrophobic solid phase surface, thereby enhancing the blocking effect.

  The blocking agent according to the present embodiment is added with these monomers and, if necessary, a polymerization initiator, a surfactant, an electrolyte, a molecular weight regulator, and the like, which are auxiliary materials, if necessary, water, alcohol, THF, It can be obtained by polymerization in a solvent such as DMF or a mixture thereof. The polymerization time is preferably 1 to 24 hours, and the polymerization temperature is preferably 0 to 120 ° C.

  As a polymerization initiator, 2,2′-azobis (2-methylpropionamidine) dihydrochloride (“V-50” manufactured by Wako Pure Chemical Industries, Ltd.), 2,2′-azobis (1-imino-1- By using a cationic polymerization initiator such as pyrrolidino-2-ethylpropane) dihydrochloride (“VA-067” manufactured by Wako Pure Chemical Industries, Ltd.), the blocking agent should be coulomb bonded to the anionic solid phase surface. And the blocking effect can be enhanced. The amount of the polymerization initiator used is preferably 0.02 to 5 parts by mass with respect to 100 parts by mass of the total amount of monomers.

1.3. Use of blocking agent and probe-binding particles and production thereof The blocking agent according to this embodiment further suppresses non-specific adsorption by substituting albumin, casein, gelatin and the like used in conventional immunodiagnostic measurement, Noise can be reduced.

  For example, in the plate method, after binding a probe such as an antibody to the plate, the blocking agent according to this embodiment can be added to treat the plate surface.

  Moreover, the blocking agent which concerns on this embodiment can be used conveniently for manufacture of probe coupling | bonding particle | grains, for example. The method for producing probe-bound particles according to the present embodiment includes a step of binding a probe to the surface of the particle and a step of processing the particles to which the probe is bound using the blocking agent according to the present embodiment.

  Here, the process of processing the particle | grains with which the probe was couple | bonded using the blocking agent which concerns on this embodiment can be performed by making the blocking agent which concerns on this embodiment contact the particle | grain surface for a predetermined time, for example. . Thereby, nonspecific adsorption | suction on the particle | grain surface can be suppressed and noise can be reduced. Moreover, this process can be performed in the state which disperse | distributed the particle | grains in the solution of the blocking agent which concerns on this embodiment, for example.

  In this case, the particle to which the probe is bonded preferably has at least one group selected from a carboxyl group, an active ester group, a tosyl group, an amino group, and an epoxy group on the surface (the reason will be described later). . In this case, the particles are preferably magnetic particles.

  More specifically, for example, in an immunochromatography method, after binding a probe such as an antibody to a colored particle, the blocking agent according to this embodiment is added to treat the surface of the colored particle, thereby binding the probe-bound particle. Can be obtained. Further, for example, in an assay method such as EIA, CLIA, CLEIA, etc., after binding a probe such as an antibody to the surface of the magnetic particle, the blocking agent according to this embodiment is added to treat the surface of the magnetic particle. Thus, probe-bound particles can be obtained.

  As explained above, the blocking agent according to the present embodiment comprises a water-soluble polymer having a functional group represented by the above formula (1), thereby suppressing non-specific adsorption and reducing noise. Can do.

  When the particles having at least the surface of an active group such as a tosyl group or an epoxy group (for example, magnetic particles) are treated using the blocking agent according to the present embodiment, the hydroxyl group in the blocking agent and the active group on the particle surface Since the covalent bond is formed and the orientation of the immunodiagnostic antibody is improved, the effect of enhancing the signal appears. Similarly, a method of covalently bonding a blocking agent having an amino group to particles having an active group such as a carboxyl group or an active ester group on the surface, or a particle having an active group such as an amino group on the surface. Thus, a method of covalently binding a blocking agent having an active ester group is also effective as a method for enhancing the signal. Further, since the elimination of the blocking agent can be suppressed, the test reagent is extremely stable with respect to buffers containing a surfactant or the like.

  The carrier in which the blocking agent according to this embodiment exhibits a particularly good effect is particles having a carboxyl group (for example, magnetic particles). As a preferable treatment method, there is a method in which particles having a carboxyl group are converted into active esters with water-soluble carbodiimide or the like, and after binding an immunodiagnostic probe, the particles are treated with a blocking agent (signal enhancer) according to this embodiment.

  The probe binding particles according to this embodiment can be obtained by the above-described method for manufacturing probe binding particles. For example, the probe binding particle according to the present embodiment can have the above-described blocking agent on the surface. That is, the probe binding particle according to the present embodiment has a functional group represented by the above formula (1).

2. Examples Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

2.1. Example 1
Glycerol methacrylate (95 g) and methyl methacrylate (5 g) were dissolved in a mixed solvent of 167 g of water and 333 g of isopropanol, and placed in a separable flask equipped with a stirrer. While blowing nitrogen into this, the temperature was raised to 70 ° C., 0.2 g of 2,2′-azobis (2-methylpropionamidine) dihydrochloride was added, polymerization was continued for 4 hours, and the temperature was further raised to 80 ° C. After aging for 2 hours, the mixture was cooled to room temperature. The obtained copolymer solution was purified by dialysis and further freeze-dried to obtain 82 g of the blocking agent (A-1) of this example. The concentration of the functional group of the formula (1) contained in A-1 corresponds to 5.9 mmol / g. The molecular weight distribution by liquid chromatography was a broad peak with a molecular weight of 12,000 as the main peak.

  1-Ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (manufactured by Dojin Chemical Co., Ltd.) was dispersed in an aqueous dispersion having a solid content concentration of 1% in which 1 mg of magnetic particles having a carboxyl group (trade name “MAG1101”, manufactured by JSR) was dispersed. ) The carboxyl group was activated by adding an aqueous solution and rotating and stirring at room temperature for 2 hours. After separating this magnetically and discarding the supernatant, 10 μg of an antibody against human α-fetoprotein (hereinafter referred to as “AFP”) which is a tumor marker (hereinafter referred to as “anti-AFP antibody”, manufactured by Cosmo Bio Inc.) The mixture was reacted for 15 hours at room temperature. After the reaction, 125 μL of a 0.4% aqueous solution of blocking agent (A-1) was added, and the mixture was further stirred at room temperature for 1 hour. This was magnetically separated, washed repeatedly with a cleaning solution (containing 25 mmol / L Tris-HCl, pH 7.4, 0.01% Tween 20), diluted with a cleaning solution to a particle concentration of 0.5%, and used as a primary probe. Protein-bound particles (immunoassay particles) bound with anti-AFP antibodies were obtained. Take 10 μl of the resulting immunological particle dispersion (corresponding to 50 μg of particles) in a test tube and mix with 50 μl of a standard specimen of AFP antigen (manufactured by Nippon Biotest) diluted to 100 ng / mL with fetal calf serum (FCS). And allowed to react at 37 ° C. for 10 minutes. After separating the particles by magnetic separation and removing the supernatant, an anti-AFP antibody labeled with alkaline phosphatase (hereinafter referred to as “ALP”) as a secondary antibody (manufactured by Fujirebio Inc., attached to Lumipulse AFP-N) Using reagent) 40 μl was added and allowed to react at 37 ° C. for 10 minutes. Next, after magnetic separation and removal of the supernatant, the particles obtained by repeating washing three times with PBS were dispersed in 50 μl of 0.01% Tween 20, and transferred to a new tube. After adding 100 μl of ALP substrate solution (Lumipulse substrate solution: manufactured by Fujirebio Inc.) and reacting at 37 ° C. for 10 minutes, signal intensity was obtained by measuring the amount of chemiluminescence. A chemiluminescence measuring apparatus (trade name “Lumat LB9507”) manufactured by Bertol Japan KK was used for the measurement of the chemiluminescence amount. As a result, the signal intensity was 126,377 RIU.

  In addition, instead of 50 μl of the standard sample of AFP antigen diluted to 100 ng / mL with fetal calf serum (FCS), the amount of chemiluminescence should be measured in the same manner as above except that 50 μl of FCS not containing AFP antigen is used. The noise intensity obtained by was 65 RIU.

2.2. Comparative Example 1
A blocking agent having no functional group represented by the above formula (1) was obtained in the same manner as in Example 1 except that polyethylene glycol methacrylate was used instead of glycerol methacrylate. The molecular weight distribution of the blocking agent obtained in this Comparative Example by liquid chromatography was a broad peak with a molecular weight of 24,000 as the main peak. Further, the magnetic particles were processed in the same manner as in Example 1 using the blocking agent obtained in this Comparative Example, and the signal intensity and noise intensity were determined. As a result, the signal intensity was 109,033 RIU, and the noise intensity was 103 RIU.

2.3. Comparative Example 2
In Example 1, except that bovine serum albumin was used instead of the blocking agent (A-1), the magnetic particles were processed in the same manner as in Example 1 to determine the signal intensity and noise intensity. As a result, the signal intensity was 97,625 RIU, and the noise intensity was 85 RIU.

2.4. Comparative Example 3
In Example 1, except that the blocking agent (A-1) was not used, the magnetic particles were processed in the same manner as in Example 1 to determine signal intensity and noise intensity. As a result, the signal intensity was 94,753 RIU, and the noise intensity was 121 RIU.

Claims (8)

A blocking agent comprising a water-soluble polymer having a functional group represented by the following formula (1).
_{-C (= O) OCH 2 CH} (OH) CH 2 (OH) ····· (1) In claim 1,
The water-soluble polymer is a blocking agent obtained by copolymerizing 50 to 99 parts by mass of 2,3-dihydroxypropyl (meth) acrylate and 50 to 1 parts by mass of other monomers. Binding a probe to the surface of the particle;
Treating the particles to which the probe is bound with the blocking agent according to claim 1 or 2,
A method for producing probe-bound particles, comprising: In claim 3,
In the step of binding the probe, the particle has at least one group selected from a carboxyl group, an active ester group, a tosyl group, an amino group, and an epoxy group. In claim 3 or 4,
The method for producing probe-bonded particles, wherein the particles are magnetic particles. A probe-binding particle having a functional group represented by the following formula (1).
_{-C (= O) OCH 2 CH} (OH) CH 2 (OH) ····· (1)   Probe-bound particles obtained by the method for producing probe-bound particles according to claim 3.   Probe binding particles having the blocking agent according to claim 1 or 2 on a surface thereof.