WO2010082681A1 - Immuno-latex particles and process for producing same - Google Patents
Immuno-latex particles and process for producing same Download PDFInfo
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- WO2010082681A1 WO2010082681A1 PCT/JP2010/050661 JP2010050661W WO2010082681A1 WO 2010082681 A1 WO2010082681 A1 WO 2010082681A1 JP 2010050661 W JP2010050661 W JP 2010050661W WO 2010082681 A1 WO2010082681 A1 WO 2010082681A1
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- C08J3/26—Crosslinking, e.g. vulcanising, of macromolecules of latex
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- the present invention relates to an immunolatex particle having a surface on which a mixed polyethylene glycol and an antibody are co-immobilized and a method for producing the same.
- Latex particles coated with appropriate antibodies (or antigens) have been used extensively in research and diagnostic methods for the quantification of a wide variety of biomolecules in biological fluids (see, for example, Non-Patent Document 1).
- the coating of latex particles with antigen or antibody uses physical adsorption, and the covalent binding of antigen or antibody to latex particles stabilizes the formed protein-supported particles over time. It is known to improve analysis performance as compared with it.
- latex particles having an amino group, chloromethyl group or acetal group as a functional group that can be covalently bonded to an amino acid residue of a protein as a surface functional group share, for example, an anti-ferritin antibody via the functional group.
- an anti-ferritin antibody via the functional group.
- Such a latex / antibody complex which is a type for immunodiagnosis, can be rapidly aggregated by antibody-antigen interaction and used in a turbidimetric or nephrometric assay indicating the presence of the antigen. Therefore, it is very important to avoid so-called non-specific aggregation due to factors other than antigen and latex / antibody complex.
- Such antibody-bound latex particles can be used to detect the corresponding antigens as they are, but if the sample to be measured is serum or the like, it is nonspecific due to other serum proteins coexisting with the analyte antigen. Aggregation may occur.
- bovine serum albumin (BSA) is used as a blocking agent for producing latex / antibody complex particles.
- the inventors including a part of the present inventors, when forming latex particles, as a further monomer together with the monomer for forming the particles Then, a macromer having two types of polyethylene glycol chains having different chain lengths was emulsion-polymerized in an aqueous medium to propose latex particles whose latex particle surface can be coated with mobile polyethylene glycol chains in an aqueous medium (patent) Reference 1).
- the particles are intended to immobilize molecules that can specifically bind to biomolecules such as antibodies via functional groups present at the non-polymerizable ends of the polyethylene glycol.
- the inventors including a part of the present inventors also mixed polyethylene glycol on the surface of gold particles, the surface of semiconductor nanoparticles, the surface of magnetic particles, the surface of silica particles, and the latex particles containing any one of these particles.
- Patent Document 2 a particle in which non-covalent bonds are bonded through physical bonds or chemical bonds.
- Patent Document 3 an antibody / mixed polyethylene glycol co-immobilized particle in which mixed polyethylene glycol is covalently bonded to the surface of carboxyl-activated magnetic particles is also provided (Non-patent Document 3).
- latex particles prepared using a macromer having a mixed polyethylene glycol chain disclosed in Patent Document 1 can provide immune particles in which molecules that specifically bind to biomolecules such as antibodies are stably immobilized. It has been found that it is not always easy to carry antibody molecules and the like at a sufficient density.
- BSA as a blocking agent
- the blocking effect and the effect of dispersing particles in an aqueous medium may be limited due to the protein characteristics of BSA itself. Therefore, in the present invention, the dispersibility of the particles is highly improved, various non-specific interactions between the particles are reduced to the limit, the reactivity with the antigen is improved, and high-sensitivity and high-performance immunity is achieved.
- An object is to provide diagnostic latex particles.
- the antibody is expected to be in a naked state by binding to the non-fixed end of the polyethylene glycol chain fixed on the surface of the gold particle or the like of Patent Document 2 above It was confirmed that the antibody binding properties are not inferior to those of Furthermore, surprisingly, antibodies co-immobilized via covalent bonds with mixed polyethylene glycol on the latex particle surface are more thermally stable than antibodies that are normally free or not co-immobilized with mixed polyethylene glycol. It was also confirmed that the antibody still has a high level of binding properties to the antigen, which is inherent to the antibody, while the resistance to high salt concentration is increased.
- immune latex particles in which mixed polyethylene glycol and antibody or antigen are randomly co-immobilized on the surface.
- the mixed polyethylene glycol in the latex particle is composed of a long chain polyethylene glycol molecule group and a short chain polyethylene glycol molecule group, and the co-immobilization corresponds to the functional group on the latex particle surface and the mixed polyethylene glycol and the antibody or antigen, respectively. This is accomplished by the formation of a covalent bond through the functional group.
- a method for producing the above-described immune latex particle the latex particle having a functional group capable of reacting with an amino group or a carboxyl group of an amino acid residue of a protein, an antibody or an antigen,
- the antibody or antigen is covalently bound to the latex particle by contacting the functional group with the amino group or carboxyl group under a condition that can form a covalent bond by reaction, and then the antibody or antigen thus obtained is covalently bound.
- a method for producing the latex particles comprising the step of bringing the latex particles into contact with a mixed polyethylene glycol having an amino group or a carboxyl group at one end under the above conditions to further covalently bond the mixed polyethylene glycol to the latex particles. Is done.
- a method for detecting an analyte a molecule or structure capable of specifically binding to an antibody or an antigen immobilized on a latex particle, an antigen, for example, a cell or a cell lysate
- an analyte a molecule or structure capable of specifically binding to an antibody or an antigen immobilized on a latex particle, an antigen, for example, a cell or a cell lysate
- the step of causing the latex particles to exist in the sample containing the analyte, and the conditions under which the analyte and the antibody or antigen immobilized on the latex particles can bind Incubating (usually 30 seconds to 10 minutes) a sample containing an analyte in which latex particles are present (usually between 20 and 30 ° C.) to aggregate latex particles, and the degree of latex particle aggregation Is provided as a measure of the presence of the analyte.
- the degree of aggregation may be evaluated by visual observation, a general spectroscope, or an automatic measurement device based on the principle of spectroscopic detection existing in some clinical sites.
- Latex particles are fine particles that form an emulsion dispersed in water in a colloidal form, and mean particles that can be used in an immunoaggregation assay. It can be used without being limited by the type and manufacturing method.
- the monomers used include styrene, styrene-butadiene and acrylonitrile-butadiene, and the like, as well as monomers capable of forming a synthetic rubber latex derived from derivatives of these monomers, and acrylic, vinyl acetate, It is produced by emulsion polymerization using a mixed system of monomers selected from the group consisting of so-called resin latex or a combination of one or more monomers derived from derivatives of these monomers and vinyl chlorides, etc. Latex particles that can be used in the present invention are included. Of these, those based on styrene are preferably used.
- styrene Based on styrene means that units derived from styrene or its derivatives (eg, chloromethylstyrene, divinylbenzene) occupy at least 50% or more of the total particle weight.
- the functional group on the surface of the latex particles is a monomer having the above-mentioned monomers and a corresponding functional group such as a carboxyl group, amino group, chloromethyl group, or acetal group, such as acrylic acid, methacrylic acid, aminoethyl methacrylate.
- Chloromethylstyrene, acrolein diethyl acetal, and the like may be introduced by copolymerization, and if necessary, such functional groups may be converted to other functional groups and supported after formation of the particles.
- a functional group on the latex surface a functional group having one end of mixed polyethylene glycol (hereinafter, polyethylene glycol may be abbreviated as PEG) co-immobilized on the surface, and the amino acid residue of the antibody or protein antigen Formation of a covalent bond with a functional group of each group is, for example, a carboxyl group and an amino group, a carboxyl group and a hydroxyl group, an amino group and an amino group via a glutaraldehyde bridge, a chloromethyl group and an amino group, or an acetal group, respectively.
- the functional group on the surface of the latex particle may be subjected to an activation reaction known per se in the art.
- the optimum conditions for using these combinations vary depending on the characteristics of the functional group at the end of the PEG to be immobilized. For example, in the case of PEG having a polyamine having a positive charge as the terminal functional group, the functional group on the surface of the latex particle is used. It is preferable to select such that is a negatively charged carboxyl group.
- the other end of PEG is terminated with a hydroxyl group that does not adversely affect the covalent bond forming reaction between the functional groups of PEG and the latex surface, or an optionally substituted C 1 -C 6 alkoxy group. It may be.
- the substituent can be an acetal moiety, a hydroxyl group, a mono or di C 1 -C 6 alkyl substituted amino group, a halogen atom, and the like.
- the latex particle has a functional group on the surface of the particle which is not limited, but is introduced at intervals of at least 63 cm 2 / functional group, and the average particle size of the particle is 50 nm to 150 ⁇ m, preferably 100 nm. Those having a thickness of ⁇ 800 nm, more preferably 100 nm to 500 nm can be advantageously used.
- commercially available latex particles for example, AJ26 COOH-Clean manufactured by Ikerlat (spain) can also be used.
- the mixed polyethylene glycol that is co-immobilized on the surface of the latex particles is composed of a long chain polyethylene glycol molecule group having a number average molecular weight of polyethylene glycol part of 2000 to 8000 g / mol and a short chain polyethylene glycol molecule group having 1500 to 4000 g / mol, and A polyethylene glycol derivative having a difference in average molecular weight between the long chain polyethylene glycol molecule group and the short chain polyethylene glycol molecule group of at least 3000 g / mol, preferably PEG having a polyamine introduced at the reaction terminal with the latex particle is advantageously used. it can.
- the antibody or antigen co-immobilized on the particle surface is preferably a monoclonal or polyclonal antibody from the viewpoint of reaction characteristics or stability of the antibody molecule.
- antibodies that can be classified into antibodies having an isoelectric point of neutral to basic (7 or more) can be preferably used.
- the long-chain polyethylene glycol molecule group and the short-chain polyethylene glycol molecule group of the mixed polyethylene glycol are present in a molar ratio (when charged) of 1: 5 to 5: 1, preferably 3: 5 to 2: 5.
- the amount of the antibody co-immobilized with the polyethylene glycol molecule group varies generally depending on the conditions, but is generally about 350 to 1200 particles / latex particles.
- the above-described immune latex particles may be produced by any method.
- the latex particle having a functional group capable of reacting with the amino group or carboxyl group of the amino acid residue of the protein constituting the antibody on the surface, the antibody or antigen, the functional group and the amino group or carboxyl group can react with each other under conditions that allow the reaction to form a covalent bond, if necessary, after the carboxyl group has been activated esterified by a conventional method, and then under conditions that do not adversely affect the binding activity of the antibody or antigen.
- the antibody or antigen is covalently bonded to the latex particle, and then the latex particle to which the antibody or antigen thus obtained is covalently bonded and a long chain polyethylene glycol molecule group of mixed polyethylene glycol having an amino group or a carboxyl group at one end are combined. After contacting under the above conditions, short chain polyethylene glycol molecules are In contact, the mixture of polyethylene glycol can be obtained by the method for producing the latex particles comprising the step of allowed to further covalently bonded to the latex particles.
- the immunolatex particles of the present invention include, but are not limited to, biological fluids that may contain biomolecules as analytes, such as blood, saliva, urine, spinal fluid, cells, cell debris, etc.
- the analyte in the sample can be detected or quantified without being adversely affected by contaminating proteins or the like.
- the antibody immobilized on the particles can be used stably even at a temperature at which the antibody is inherently advantageous, generally at a temperature exceeding room temperature (about 25 ° C.) by several tens of degrees.
- the immune latex particles of the present invention can be used without losing the antibody characteristics even when the fluid is higher in salt concentration than those derived from the living body.
- FIG. 1 is a schematic diagram of the surface of the immune latex particle surface of the present invention.
- FIG. 2 shows the LAmP complex (black and white squares in FIG. 2 a and b, respectively) and the LAP complex (black and white triangles and white in FIG. 2 a and b, respectively), according to Example 2. It is a graph display which shows the result of the coupling
- FIG. 3 is a graphical representation showing the results of measuring ferritin in a phosphate buffer according to Example 3.
- 4 is a graphical representation showing the results of measuring ferritin in fetal bovine serum (FBS) according to Example 3.
- FBS fetal bovine serum
- EDC 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride
- the amino functional group of the antibody was reacted with the activated carboxyl group of the particle surface to immobilize the antibody on the surface of the latex particle.
- the final concentration of latex particles was 0.1% w / v.
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Abstract
Immuno-latex particles comprising a mixed polyethylene glycol and an antibody or antigen both randomly co-immobilized on the surfaces thereof via covalent bonds. The latex particles can be used effectively in immunoassays.
Description
本発明は、混合ポリエチレングリコールと抗体が共固定化された表面を所有する免疫ラテックス粒子及びその製造方法に関する。
The present invention relates to an immunolatex particle having a surface on which a mixed polyethylene glycol and an antibody are co-immobilized and a method for producing the same.
適当な抗体（または抗原）で被覆したラテックス粒子は生物学的流体における多種多様な生体分子の定量に関する研究及び診断法で広範囲にわたって利用されてきた（例えば、非特許文献１参照）。このようなラテックス粒子が良好な分析感度を示す抗原抗体反応の向上した免疫アッセイの適用範囲を拡大してきた。また、ラテックス粒子の抗原または抗体の被覆は物理的吸着を利用するよりラテックス粒子への抗原または抗体の共有結合が、形成されたタンパク質担持粒子を経時的により安定にすることから、物理的吸着に比べて分析性能を向上することが知られている。このような観点からタンパク質のアミノ酸残基と共有結合できる官能基としてアミノ基、クロロメチル基またはアセタール基を表面官能基として有するラテックス粒子が、該官能基を介して、例えば、抗フェリチン抗体を共有結合せしめ、血清フェリチンの定量に必要な高感度を達成するために提案されている（例えば、非特許文献２）。
このような免疫診断用の一種であるラテックス／抗体複合体は、抗体−抗原の相互作用により迅速に凝集し、抗原の存在を示す比濁アッセイ（ｔｕｒｂｉｄｉｍｅｔｒｉｃ ｏｒ ｎｅｐｈｅｌｏｍｅｔｒｉｃ ａｓｓａｙ）に使用できる。したがって、抗原以外の因子とラテックス／抗体複合体による、所謂、非特異凝集を避けることが非常に重要である。このような抗体結合ラテックス粒子はそのまま対応する抗原を検出するのに使用できるが、使用条件、例えば、測定対象サンプルが血清等である場合、被検体抗原と共存する他の血清タンパク質等により非特異凝集が起こる場合がある。このような非特異反応を、ブロックするためにブロッキング剤として牛血清アルブミン（ＢＳＡ）がラテックス／抗体複合体粒子作製に利用されている。
また、上記のような非特異凝集が生じるのを防ぐために、本発明者の一部を含む発明者等は、ラテックス粒子を形成する際に、該粒子を形成するためのモノマーとともに、さらなるモノマーとして、鎖長の異なる２種のポリエチレングリコール鎖を有するマクロマーを、水性媒体中で乳化重合せしめて、ラテックス粒子表面が水性媒体中で可動性のポリエチレングリコール鎖で被覆され得るラテックス粒子を提案した（特許文献１）。この粒子では、該ポリエチレングリコールの非重合性末端に存在する官能基を介して抗体等の生体分子と特異的に結合し得る分子を固定することが企図されている。そして、かような抗体分子は該ポリエチレングリコール鎖の末端に存在するので、可動性のポリエチレングリコール鎖に覆われることなく裸の状態で存在することが予測されることから、該抗体本来の活性を奏し易いこと、一方、ラテックス粒子表面を覆うポリエチレングリコール鎖が該粒子への抗原以外の測定サンプル中に存在するタンパク質等の非特異結合を防止できることが示唆されている。
なお、ラテックス粒子表面でないが、金、半導体または磁性体の表面における上記の如き非特異結合を防止するものとして、該表面へ混合ポリエチレングリコールを物理的または化学的に結合せしめた生体分子の測定用チップが提案されている。また、本発明者の一部を含む発明者等も、金粒子表面、半導体ナノ粒子表面、磁性体粒子表面、シリカ粒子表面、およびこれらの粒子のいずれか一種を含むラテックス粒子表面に混合ポリエチレングリコールを物理結合または化学結合を介して非共有結合的結合せしめた粒子も提案した（特許文献２）。さらにカルボキシル活性化磁性粒子の表面に混合ポリエチレングリコールを共有結合せしめた抗体／混合ポリエチレングリコールの共固定された粒子も提供されている（非特許文献３）。
しかしながら、前者のブロッキング剤として牛血清アルブミンを使用する系では、ラテックス／抗体複合体の分散安定性や免疫診断能力（信号／ノイズ比）が必ずしも十分ではなく、さらなる改善の余地がある。一方、後者のポリエチレングリコールマクロマーに由来する単位をラッテックス粒子中に組み込む系では、夾雑タンパク質等の非特異結合が低減された、物理的に安定な免疫ラッテックス粒子が提供できるものの、それらの作製が必ずしも容易でないし、ラッテックス粒子表面に結合せしめる抗体または抗原の種類によっては、それらを介する特異的結合能が低下することもしばしば起こる。 Latex particles coated with appropriate antibodies (or antigens) have been used extensively in research and diagnostic methods for the quantification of a wide variety of biomolecules in biological fluids (see, for example, Non-Patent Document 1). The scope of application of immunoassays with improved antigen-antibody reaction, in which such latex particles exhibit good analytical sensitivity, has been expanded. In addition, the coating of latex particles with antigen or antibody uses physical adsorption, and the covalent binding of antigen or antibody to latex particles stabilizes the formed protein-supported particles over time. It is known to improve analysis performance as compared with it. From this point of view, latex particles having an amino group, chloromethyl group or acetal group as a functional group that can be covalently bonded to an amino acid residue of a protein as a surface functional group share, for example, an anti-ferritin antibody via the functional group. It has been proposed to achieve high sensitivity necessary for quantification of serum ferritin (for example, Non-Patent Document 2).
Such a latex / antibody complex, which is a type for immunodiagnosis, can be rapidly aggregated by antibody-antigen interaction and used in a turbidimetric or nephrometric assay indicating the presence of the antigen. Therefore, it is very important to avoid so-called non-specific aggregation due to factors other than antigen and latex / antibody complex. Such antibody-bound latex particles can be used to detect the corresponding antigens as they are, but if the sample to be measured is serum or the like, it is nonspecific due to other serum proteins coexisting with the analyte antigen. Aggregation may occur. In order to block such non-specific reactions, bovine serum albumin (BSA) is used as a blocking agent for producing latex / antibody complex particles.
In addition, in order to prevent the occurrence of non-specific aggregation as described above, the inventors including a part of the present inventors, when forming latex particles, as a further monomer together with the monomer for forming the particles Then, a macromer having two types of polyethylene glycol chains having different chain lengths was emulsion-polymerized in an aqueous medium to propose latex particles whose latex particle surface can be coated with mobile polyethylene glycol chains in an aqueous medium (patent) Reference 1). The particles are intended to immobilize molecules that can specifically bind to biomolecules such as antibodies via functional groups present at the non-polymerizable ends of the polyethylene glycol. Since such an antibody molecule is present at the end of the polyethylene glycol chain, it is predicted that the antibody molecule exists in a naked state without being covered by the mobile polyethylene glycol chain. On the other hand, it has been suggested that the polyethylene glycol chain covering the latex particle surface can prevent non-specific binding of proteins and the like present in the measurement sample other than the antigen to the particle.
For measurement of biomolecules that are not latex particle surfaces but have physical or chemical binding of mixed polyethylene glycol to the surface to prevent non-specific binding as described above on the surface of gold, semiconductor or magnetic material. Tips have been proposed. Further, the inventors including a part of the present inventors also mixed polyethylene glycol on the surface of gold particles, the surface of semiconductor nanoparticles, the surface of magnetic particles, the surface of silica particles, and the latex particles containing any one of these particles. There has also been proposed a particle in which non-covalent bonds are bonded through physical bonds or chemical bonds (Patent Document 2). Further, an antibody / mixed polyethylene glycol co-immobilized particle in which mixed polyethylene glycol is covalently bonded to the surface of carboxyl-activated magnetic particles is also provided (Non-patent Document 3).
However, in the former system using bovine serum albumin as a blocking agent, the dispersion stability and immunodiagnostic ability (signal / noise ratio) of the latex / antibody complex are not always sufficient, and there is room for further improvement. On the other hand, a system in which a unit derived from the latter polyethylene glycol macromer is incorporated into a latex particle can provide physically stable immune latex particles with reduced non-specific binding of contaminating proteins, etc. Depending on the type of antibody or antigen bound to the surface of the latex particle, it is not easy, and the specific binding ability through them often decreases.
このような免疫診断用の一種であるラテックス／抗体複合体は、抗体−抗原の相互作用により迅速に凝集し、抗原の存在を示す比濁アッセイ（ｔｕｒｂｉｄｉｍｅｔｒｉｃ ｏｒ ｎｅｐｈｅｌｏｍｅｔｒｉｃ ａｓｓａｙ）に使用できる。したがって、抗原以外の因子とラテックス／抗体複合体による、所謂、非特異凝集を避けることが非常に重要である。このような抗体結合ラテックス粒子はそのまま対応する抗原を検出するのに使用できるが、使用条件、例えば、測定対象サンプルが血清等である場合、被検体抗原と共存する他の血清タンパク質等により非特異凝集が起こる場合がある。このような非特異反応を、ブロックするためにブロッキング剤として牛血清アルブミン（ＢＳＡ）がラテックス／抗体複合体粒子作製に利用されている。
また、上記のような非特異凝集が生じるのを防ぐために、本発明者の一部を含む発明者等は、ラテックス粒子を形成する際に、該粒子を形成するためのモノマーとともに、さらなるモノマーとして、鎖長の異なる２種のポリエチレングリコール鎖を有するマクロマーを、水性媒体中で乳化重合せしめて、ラテックス粒子表面が水性媒体中で可動性のポリエチレングリコール鎖で被覆され得るラテックス粒子を提案した（特許文献１）。この粒子では、該ポリエチレングリコールの非重合性末端に存在する官能基を介して抗体等の生体分子と特異的に結合し得る分子を固定することが企図されている。そして、かような抗体分子は該ポリエチレングリコール鎖の末端に存在するので、可動性のポリエチレングリコール鎖に覆われることなく裸の状態で存在することが予測されることから、該抗体本来の活性を奏し易いこと、一方、ラテックス粒子表面を覆うポリエチレングリコール鎖が該粒子への抗原以外の測定サンプル中に存在するタンパク質等の非特異結合を防止できることが示唆されている。
なお、ラテックス粒子表面でないが、金、半導体または磁性体の表面における上記の如き非特異結合を防止するものとして、該表面へ混合ポリエチレングリコールを物理的または化学的に結合せしめた生体分子の測定用チップが提案されている。また、本発明者の一部を含む発明者等も、金粒子表面、半導体ナノ粒子表面、磁性体粒子表面、シリカ粒子表面、およびこれらの粒子のいずれか一種を含むラテックス粒子表面に混合ポリエチレングリコールを物理結合または化学結合を介して非共有結合的結合せしめた粒子も提案した（特許文献２）。さらにカルボキシル活性化磁性粒子の表面に混合ポリエチレングリコールを共有結合せしめた抗体／混合ポリエチレングリコールの共固定された粒子も提供されている（非特許文献３）。
しかしながら、前者のブロッキング剤として牛血清アルブミンを使用する系では、ラテックス／抗体複合体の分散安定性や免疫診断能力（信号／ノイズ比）が必ずしも十分ではなく、さらなる改善の余地がある。一方、後者のポリエチレングリコールマクロマーに由来する単位をラッテックス粒子中に組み込む系では、夾雑タンパク質等の非特異結合が低減された、物理的に安定な免疫ラッテックス粒子が提供できるものの、それらの作製が必ずしも容易でないし、ラッテックス粒子表面に結合せしめる抗体または抗原の種類によっては、それらを介する特異的結合能が低下することもしばしば起こる。 Latex particles coated with appropriate antibodies (or antigens) have been used extensively in research and diagnostic methods for the quantification of a wide variety of biomolecules in biological fluids (see, for example, Non-Patent Document 1). The scope of application of immunoassays with improved antigen-antibody reaction, in which such latex particles exhibit good analytical sensitivity, has been expanded. In addition, the coating of latex particles with antigen or antibody uses physical adsorption, and the covalent binding of antigen or antibody to latex particles stabilizes the formed protein-supported particles over time. It is known to improve analysis performance as compared with it. From this point of view, latex particles having an amino group, chloromethyl group or acetal group as a functional group that can be covalently bonded to an amino acid residue of a protein as a surface functional group share, for example, an anti-ferritin antibody via the functional group. It has been proposed to achieve high sensitivity necessary for quantification of serum ferritin (for example, Non-Patent Document 2).
Such a latex / antibody complex, which is a type for immunodiagnosis, can be rapidly aggregated by antibody-antigen interaction and used in a turbidimetric or nephrometric assay indicating the presence of the antigen. Therefore, it is very important to avoid so-called non-specific aggregation due to factors other than antigen and latex / antibody complex. Such antibody-bound latex particles can be used to detect the corresponding antigens as they are, but if the sample to be measured is serum or the like, it is nonspecific due to other serum proteins coexisting with the analyte antigen. Aggregation may occur. In order to block such non-specific reactions, bovine serum albumin (BSA) is used as a blocking agent for producing latex / antibody complex particles.
In addition, in order to prevent the occurrence of non-specific aggregation as described above, the inventors including a part of the present inventors, when forming latex particles, as a further monomer together with the monomer for forming the particles Then, a macromer having two types of polyethylene glycol chains having different chain lengths was emulsion-polymerized in an aqueous medium to propose latex particles whose latex particle surface can be coated with mobile polyethylene glycol chains in an aqueous medium (patent) Reference 1). The particles are intended to immobilize molecules that can specifically bind to biomolecules such as antibodies via functional groups present at the non-polymerizable ends of the polyethylene glycol. Since such an antibody molecule is present at the end of the polyethylene glycol chain, it is predicted that the antibody molecule exists in a naked state without being covered by the mobile polyethylene glycol chain. On the other hand, it has been suggested that the polyethylene glycol chain covering the latex particle surface can prevent non-specific binding of proteins and the like present in the measurement sample other than the antigen to the particle.
For measurement of biomolecules that are not latex particle surfaces but have physical or chemical binding of mixed polyethylene glycol to the surface to prevent non-specific binding as described above on the surface of gold, semiconductor or magnetic material. Tips have been proposed. Further, the inventors including a part of the present inventors also mixed polyethylene glycol on the surface of gold particles, the surface of semiconductor nanoparticles, the surface of magnetic particles, the surface of silica particles, and the latex particles containing any one of these particles. There has also been proposed a particle in which non-covalent bonds are bonded through physical bonds or chemical bonds (Patent Document 2). Further, an antibody / mixed polyethylene glycol co-immobilized particle in which mixed polyethylene glycol is covalently bonded to the surface of carboxyl-activated magnetic particles is also provided (Non-patent Document 3).
However, in the former system using bovine serum albumin as a blocking agent, the dispersion stability and immunodiagnostic ability (signal / noise ratio) of the latex / antibody complex are not always sufficient, and there is room for further improvement. On the other hand, a system in which a unit derived from the latter polyethylene glycol macromer is incorporated into a latex particle can provide physically stable immune latex particles with reduced non-specific binding of contaminating proteins, etc. Depending on the type of antibody or antigen bound to the surface of the latex particle, it is not easy, and the specific binding ability through them often decreases.
上述したように、特許文献１に開示された混合ポリエチレングリコール鎖を有するマクロマーを用いて作製したラテックス粒子は、さらに抗体等の生体分子と特異結合する分子を安定に固定した免疫粒子を提供できるものの、十分な密度に抗体分子等を担持させることが必ずしも容易ではないことが判明した。また、ブロッキング剤としてＢＳＡを使用するラテックス粒子の系では、ＢＳＡ自体のタンパク質の特徴に起因してブロッキング効果と粒子を水性媒体中に分散させる効果が制限される場合がある。
したがって、本発明では、粒子の分散性を高度に向上させ、粒子間の様々な非特異的相互作用を極限まで低下させるとともに、抗原との反応性を向上させ、しかも高感度・高性能な免疫診断ラテックス粒子を提供することを目的とする。
このような目的を達成すべく、新規なラテックスを作製し、それらの特性について検討してきたところ、上記非特許文献３に記載された表面の修飾された磁性粒子に比べてよりフレキシブルな構造を有することが予測されるラテックス粒子でも、生体分子と特異的に結合する分子、抗体または抗原等と混合ポリエチレングリコールを該粒子表面へ効果的に共固定できるこことがここに見出された。
さらに、理論に拘束されるものでないが、抗体または抗原等と混合ポリエチレングリコールを該粒子表面に共固定した場合には、抗体または抗原、特に、タンパク質性の抗原、例えば、抗体はポリエチレングリコール鎖によりランダムに取り囲まれることが予測されるにもかかわらず、上記特許文献２の金粒子等の表面に固定されたポリエチレングリコール鎖の非固定末端に結合し、裸の状態にあることが予測される抗体に比べて、勝るとも劣らない抗体の結合特性を示すことが確認された。さらに、驚くべきことに、ラテックス粒子表面に混合ポリエチレングリコールとともに共有結合を介して共固定された抗体は、通常遊離の状態または混合ポリエチレングリコールと共固定されていない状態にある抗体に比べて熱安定性、高塩濃度に対する抵抗性等が高まる一方で、抗体が本来有している抗原への結合特性を依然として高度に保持している、ことも確認された。
こうして、本発明によれば、混合ポリエチレングリコールと抗体または抗原がランダムに表面に共固定された免疫ラテックス粒子が提供される。該ラッテックス粒子における、混合ポリエチレングリコールは長鎖ポリエチレングリコール分子群と短鎖ポリエチレングリコール分子群からなり、そして前記共固定が、それぞれラテックス粒子表面上の官能基と混合ポリエチレングリコールおよび抗体または抗原の対応する官能基を介する共有結合の形成により達成される。
また、別の態様の本発明として、上記免疫ラテックス粒子の製造方法であって、タンパク質のアミノ酸残基のアミノ基またはカルボキシル基と反応できる官能基を表面に有するラテックス粒子と抗体または抗原を、該官能基と該アミノ基またはカルボキシル基が反応して共有結合を形成できる条件下で接触させて該抗体または抗原を該ラテックス粒子に共有結合せしめ、次いで、こうして得られた抗体または抗原が共有結合したラテックス粒子と一末端にアミノ基またはカルボキシル基を有する混合ポリエチレングリコールを前記条件下で接触させて該混合ポリエチレングリコールを該ラテックス粒子にさらに共有結合せしめる工程を含んでなる上記ラテックス粒子の製造方法も提供される。
さらに別の態様の本発明として、上記ラテックス粒子を用いる被検体（ラッテックス粒子に固定された抗体または抗原に特異的に結合しうる分子、構造物、例えば、細胞若しくは細胞破砕物等）の検出方法も提供される。各要素については後述するものもあるが、具体的には、被検体を含有するサンプル中に上記ラテックス粒子を存在させるステップ、被検体とラテックス粒子に固定された抗体または抗原が結合し得る条件下（通常、２０~３０℃の間）でラテックス粒子が存在する被検体を含有するサンプルをインキュベート（通常、３０秒~１０分）して、ラテックス粒子を凝集させるステップ、およびラテックス粒子の凝集の程度を被検体が存在する尺度として評価するステップを含んで成る、被検体の検出方法が提供される。凝集の程度は、目視、もしくは、一般的な分光器、さらには一部臨床現場に存在する分光検出を原理とする自動測定装置などにより、評価すればよい。
発明の詳細な記述
以下、本発明で使用する技術用語または技術内容についてより具体的に説明する。本発明または本明細書で使用する用語は特記しない限り、当該技術分野で普通に用いられている意味内容を表示するものとして理解されねばならない。
ラテックス粒子は、コロイド状に水中に分散した乳濁液を形成する微粒子であって、免疫凝集アッセイで用いることのできる粒子を意味し、上記の本発明の目的に沿うものである限り、使用モノマーの種類、製造方法により制限されることなく使用できる。限定されるものでないが、使用モノマーとしては、スチレン、スチレン−ブタジエンおよびアクリロニトリル−ブタジエン等、並びにこれらのモノマーの誘導体に由来する合成ゴムラテックスを形成できるモノマー、さらには、アクリル系、酢酸ビニール系、および塩化ビニル系等、並びにこれらのモノマーの誘導体に由来する、所謂、樹脂ラッテックスからなる群より選ばれるモノマーの混合系またはそれらの一種以上のモノマーの組合せ物を用いて、乳化重合法により製造される粒子が本発明で用いことのできるラテックス粒子が包含される。これらのうち、スチレンをベースとするものが好ましく用いられる。スチレンをベースとするとは、スチレンまたはその誘導体（例えば、クロロメチルスチレン、ジビニルベンゼン）に由来する単位が、総粒子重量当り少なくとも５０％以上占めることを意味する。
該ラテックス粒子表面の官能基は、上記に挙げたモノマーと、相当する官能基、例えば、カルボキシル基、アミノ基、クロロメチル基、アセタール基を有するモノマー、例えば、アクリル酸、メタクリル酸、アミノエチルメタクリレート、クロロメチルスチレン、アクロレインジエチルアセタール等を共重合させることにより導入し、必要があれば、かような官能基を粒子の形成後に別の官能基に転換せしめて担持させることもできる。このようなラテックス表面上の官能基と、該表面に共固定する混合ポリエチレングリコール（以下、ポリエチレングリコールをＰＥＧと略称する場合がある）の一末端に有する官能基および抗体もしくはタンパク質性抗原のアミノ酸残基の官能基との共有結合の形成は、それぞれ、例えば、カルボキシル基とアミノ基、カルボキシル基とヒドロキシル基、アミノ基とグルタールアルデヒド架橋を介するアミノ基、クロロメチル基とアミノ基、またはアセタール基（アセタールの酸加水分解を介するアルデヒド基もしくはホルミル基）とアミノ基、アミノ基とカルボキシル基、ヒドロキシル基とカルボキシル基との組み合わせにより形成できる。かような組み合わせによる共有結合の形成に先立ち、必要があれば、ラテックス粒子表面上の官能基をそれ自体当該技術分野で周知の活性化反応に供してもよい。
これらの組み合わせの使用の最適条件は、固定されるＰＥＧ末端の官能基の特性により変動するが、例えば、正電荷を有するポリアミンを末端官能基に有するＰＥＧの場合には、ラテックス粒子表面の官能基がマイナス電荷を有するカルボキシル基となるように選択するのがよい。また、ＰＥＧのもう一方の末端は、ＰＥＧとラテックス表面のそれぞれの官能基の共有結合形成反応に悪影響を及ぼさないヒドロキシル基または置換されていてもよいＣ１−Ｃ６アルコキシ基で末端が形成されていてもよい。Ｃ１−Ｃ６アコキシ基が置換されている場合の置換基はアセタール部分、ヒドロキシル基、モノもしくはジＣ１−Ｃ６アルキル置換アミノ基、ハロゲン原子等であることができる。
ラテックス粒子は、該粒子表面の官能基が、限定されるものでないが、少なくとも６３Å２／官能基以下の間隔で導入されたものであり、該粒子の平均粒経が５０ｎｍ~１５０μｍ、好ましくは１００ｎｍ~８００ｎｍ、より好ましくは１００ｎｍ~５００ｎｍであるものが有利に使用できる。また、このような基準を参考に、市販のラテックス粒子、例えば、Ｉｋｅｒｌａｔ（ｓｐａｉｎ）社製のＡＪ２６ ＣＯＯＨ−Ｃｌｅａｎを用いることもできる。
該ラテックス粒子表面に共固定させる混合ポリエチレングリコールは、ポリエチレングリコール部の数平均分子量が２０００~８０００ｇ／ｍｏｌの長鎖ポリエチレングリコール分子群と１５００~４０００ｇ／ｍｏｌの短鎖ポリエチレングリコール分子群からなり、かつ、長鎖ポリエチレングリコール分子群と短鎖ポリエチレングリコール分子群の平均分子量の差が少なくとも３０００ｇ／ｍｏｌである、ポリエチレングリコール誘導体、ラテックス粒子との反応末端に好ましくはポリアミンが導入されたＰＥＧが有利に使用できる。一方、該粒子表面に共固定される抗体または抗原は、モノクローナルもしくはポリクローナル抗体を使用するのが、抗体分子の反応特性または安定性の観点から好ましい。限定されるものでないが、等電点は中性~塩基性（７以上）を有する抗体に分類できる抗体が好ましく使用できる。
混合ポリエチレングリコールの長鎖ポリエチレングリコール分子群と短鎖ポリエチレングリコール分子群がモル比（仕込時）で、１：５~５：１、好ましくは３：５~２：５で存在する。ポリエチレングリコール分子群と共固定される抗体の量は、条件により好適な量は変動するが、一般的に３５０から１２００個／ラテックス粒子、程度である。（これらは、作製した粒子の分散安定性および反応活性の変化を観測することにより確認することができる。）
上記の免疫ラテックス粒子は、いかなる方法によって製造方法したものであってもよい。しかし、都合よくは、抗体を構成するタンパク質のアミノ酸残基のアミノ基またはカルボキシル基と反応できる官能基を表面に有する上記ラテックス粒子と、抗体または抗原を、該官能基と該アミノ基またはカルボキシル基が反応して共有結合を形成できる条件下、必要があれば、常法によりカルボキシル基を活性エステル化した後、抗体または抗原の結合活性に悪影響を及ばさない条件下、で相互に接触させて該抗体または抗原を該ラテックス粒子に共有結合せしめ、次いで、こうして得られた抗体または抗原が共有結合したラテックス粒子と一末端にアミノ基またはカルボキシル基を有する混合ポリエチレングリコールの長鎖ポリエチレングリコール分子群を前記条件下で接触させた後、短鎖ポリエチレングリコール分子群を前記条件下で接触させて、該混合ポリエチレングリコールを該ラテックス粒子にさらに共有結合せしめる工程を含んでなるラテックス粒子の製造方法により取得できる。当業者であれば、上記の条件は、後述する実施例を参考に、必要により適宜改変した条件を設定することができるであろう。
本発明の免疫ラテックス粒子は、限定されるものでないが、被検体として生体分子を含む可能性のある生物学的流体、例えば、血液、唾液、尿、髄液、細胞、細胞破砕物、等のサンプル中の被検体を、夾雑タンパク質等に悪影響を受けることなく、検出または定量できる。加えて、該粒子に固定された抗体が本来有利の状態できる温度、一般に、室温（約２５℃）を数十度越えた温度でも安定に使用できるとの効果を奏する。本発明の免疫ラテックス粒子は、さらに、前記流体が生体に由来するものより、さらに高塩濃度であっても、抗体の特性を失うことなく、使用できる。
理論により拘束されるものでないが、当該粒子の表面の概念図の略図を示す図１から理解できるように、短鎖ポリエチレングリコール鎖と長鎖ポリエチレングリコール鎖とが密に抗体を取り囲むことにより、該抗体分子の方向性が規制されるために抗体本来の特異的結合性を保持したまま、一方では、高温または高塩濃度等の外的環境の影響を低減できるものと理解できる。
また、限定されるものでないが、該免疫ラテックス粒子に抗体として抗フェリチン抗体を担持せしめると、特有の構造（例えば、鉄を含む分子量４６万のタンパク質）を有するフェリチンですら、極めて高感度（１０−１００ｎｇ／ｍｌ）で選択的に検出することができる。これは、水性媒体中で高い可動性を示し、血清タンパク質等を排斥することが知られているポリエチレングリコールが密に抗フェリチン抗体を取り囲んでいることが予測されることを考慮すると、驚くべきことである。 As described above, latex particles prepared using a macromer having a mixed polyethylene glycol chain disclosed in Patent Document 1 can provide immune particles in which molecules that specifically bind to biomolecules such as antibodies are stably immobilized. It has been found that it is not always easy to carry antibody molecules and the like at a sufficient density. In addition, in a latex particle system using BSA as a blocking agent, the blocking effect and the effect of dispersing particles in an aqueous medium may be limited due to the protein characteristics of BSA itself.
Therefore, in the present invention, the dispersibility of the particles is highly improved, various non-specific interactions between the particles are reduced to the limit, the reactivity with the antigen is improved, and high-sensitivity and high-performance immunity is achieved. An object is to provide diagnostic latex particles.
In order to achieve such an object, new latexes have been prepared and their properties have been studied. As a result, they have a more flexible structure than the surface-modified magnetic particles described in Non-Patent Document 3 above. It has now been found that even latex particles that are predicted to be capable of effectively co-immobilizing mixed polyethylene glycol on the particle surface with molecules, antibodies or antigens that specifically bind to biomolecules.
Furthermore, although not bound by theory, when a mixed polyethylene glycol and an antibody or an antigen are co-immobilized on the particle surface, the antibody or antigen, particularly a protein antigen, for example, the antibody is bound by a polyethylene glycol chain. Despite being expected to be surrounded at random, the antibody is expected to be in a naked state by binding to the non-fixed end of the polyethylene glycol chain fixed on the surface of the gold particle or the like ofPatent Document 2 above It was confirmed that the antibody binding properties are not inferior to those of Furthermore, surprisingly, antibodies co-immobilized via covalent bonds with mixed polyethylene glycol on the latex particle surface are more thermally stable than antibodies that are normally free or not co-immobilized with mixed polyethylene glycol. It was also confirmed that the antibody still has a high level of binding properties to the antigen, which is inherent to the antibody, while the resistance to high salt concentration is increased.
Thus, according to the present invention, there are provided immune latex particles in which mixed polyethylene glycol and antibody or antigen are randomly co-immobilized on the surface. The mixed polyethylene glycol in the latex particle is composed of a long chain polyethylene glycol molecule group and a short chain polyethylene glycol molecule group, and the co-immobilization corresponds to the functional group on the latex particle surface and the mixed polyethylene glycol and the antibody or antigen, respectively. This is accomplished by the formation of a covalent bond through the functional group.
Further, as another aspect of the present invention, there is provided a method for producing the above-described immune latex particle, the latex particle having a functional group capable of reacting with an amino group or a carboxyl group of an amino acid residue of a protein, an antibody or an antigen, The antibody or antigen is covalently bound to the latex particle by contacting the functional group with the amino group or carboxyl group under a condition that can form a covalent bond by reaction, and then the antibody or antigen thus obtained is covalently bound. There is also provided a method for producing the latex particles, comprising the step of bringing the latex particles into contact with a mixed polyethylene glycol having an amino group or a carboxyl group at one end under the above conditions to further covalently bond the mixed polyethylene glycol to the latex particles. Is done.
As another aspect of the present invention, there is provided a method for detecting an analyte (a molecule or structure capable of specifically binding to an antibody or an antigen immobilized on a latex particle, an antigen, for example, a cell or a cell lysate) using the latex particle. Is also provided. Each element is described later. Specifically, the step of causing the latex particles to exist in the sample containing the analyte, and the conditions under which the analyte and the antibody or antigen immobilized on the latex particles can bind. Incubating (usually 30 seconds to 10 minutes) a sample containing an analyte in which latex particles are present (usually between 20 and 30 ° C.) to aggregate latex particles, and the degree of latex particle aggregation Is provided as a measure of the presence of the analyte. The degree of aggregation may be evaluated by visual observation, a general spectroscope, or an automatic measurement device based on the principle of spectroscopic detection existing in some clinical sites.
DETAILED DESCRIPTION OF THE INVENTION The technical terms or technical contents used in the present invention will be described more specifically below. Unless otherwise stated, terms used in the present invention or the present specification should be understood as indicating meanings commonly used in the art.
Latex particles are fine particles that form an emulsion dispersed in water in a colloidal form, and mean particles that can be used in an immunoaggregation assay. It can be used without being limited by the type and manufacturing method. Although not limited, the monomers used include styrene, styrene-butadiene and acrylonitrile-butadiene, and the like, as well as monomers capable of forming a synthetic rubber latex derived from derivatives of these monomers, and acrylic, vinyl acetate, It is produced by emulsion polymerization using a mixed system of monomers selected from the group consisting of so-called resin latex or a combination of one or more monomers derived from derivatives of these monomers and vinyl chlorides, etc. Latex particles that can be used in the present invention are included. Of these, those based on styrene are preferably used. Based on styrene means that units derived from styrene or its derivatives (eg, chloromethylstyrene, divinylbenzene) occupy at least 50% or more of the total particle weight.
The functional group on the surface of the latex particles is a monomer having the above-mentioned monomers and a corresponding functional group such as a carboxyl group, amino group, chloromethyl group, or acetal group, such as acrylic acid, methacrylic acid, aminoethyl methacrylate. Chloromethylstyrene, acrolein diethyl acetal, and the like may be introduced by copolymerization, and if necessary, such functional groups may be converted to other functional groups and supported after formation of the particles. Such a functional group on the latex surface, a functional group having one end of mixed polyethylene glycol (hereinafter, polyethylene glycol may be abbreviated as PEG) co-immobilized on the surface, and the amino acid residue of the antibody or protein antigen Formation of a covalent bond with a functional group of each group is, for example, a carboxyl group and an amino group, a carboxyl group and a hydroxyl group, an amino group and an amino group via a glutaraldehyde bridge, a chloromethyl group and an amino group, or an acetal group, respectively. (Aldehyde group or formyl group via acid hydrolysis of acetal) and an amino group, an amino group and a carboxyl group, or a combination of a hydroxyl group and a carboxyl group. Prior to the formation of the covalent bond by such a combination, if necessary, the functional group on the surface of the latex particle may be subjected to an activation reaction known per se in the art.
The optimum conditions for using these combinations vary depending on the characteristics of the functional group at the end of the PEG to be immobilized. For example, in the case of PEG having a polyamine having a positive charge as the terminal functional group, the functional group on the surface of the latex particle is used. It is preferable to select such that is a negatively charged carboxyl group. In addition, the other end of PEG is terminated with a hydroxyl group that does not adversely affect the covalent bond forming reaction between the functional groups of PEG and the latex surface, or an optionally substituted C 1 -C 6 alkoxy group. It may be. When the C 1 -C 6 acoxy group is substituted, the substituent can be an acetal moiety, a hydroxyl group, a mono or di C 1 -C 6 alkyl substituted amino group, a halogen atom, and the like.
The latex particle has a functional group on the surface of the particle which is not limited, but is introduced at intervals of at least 63 cm 2 / functional group, and the average particle size of the particle is 50 nm to 150 μm, preferably 100 nm. Those having a thickness of ˜800 nm, more preferably 100 nm to 500 nm can be advantageously used. In addition, referring to such a standard, commercially available latex particles, for example, AJ26 COOH-Clean manufactured by Ikerlat (spain) can also be used.
The mixed polyethylene glycol that is co-immobilized on the surface of the latex particles is composed of a long chain polyethylene glycol molecule group having a number average molecular weight of polyethylene glycol part of 2000 to 8000 g / mol and a short chain polyethylene glycol molecule group having 1500 to 4000 g / mol, and A polyethylene glycol derivative having a difference in average molecular weight between the long chain polyethylene glycol molecule group and the short chain polyethylene glycol molecule group of at least 3000 g / mol, preferably PEG having a polyamine introduced at the reaction terminal with the latex particle is advantageously used. it can. On the other hand, the antibody or antigen co-immobilized on the particle surface is preferably a monoclonal or polyclonal antibody from the viewpoint of reaction characteristics or stability of the antibody molecule. Although not limited, antibodies that can be classified into antibodies having an isoelectric point of neutral to basic (7 or more) can be preferably used.
The long-chain polyethylene glycol molecule group and the short-chain polyethylene glycol molecule group of the mixed polyethylene glycol are present in a molar ratio (when charged) of 1: 5 to 5: 1, preferably 3: 5 to 2: 5. The amount of the antibody co-immobilized with the polyethylene glycol molecule group varies generally depending on the conditions, but is generally about 350 to 1200 particles / latex particles. (These can be confirmed by observing changes in dispersion stability and reaction activity of the produced particles.)
The above-described immune latex particles may be produced by any method. However, conveniently, the latex particle having a functional group capable of reacting with the amino group or carboxyl group of the amino acid residue of the protein constituting the antibody on the surface, the antibody or antigen, the functional group and the amino group or carboxyl group. Can react with each other under conditions that allow the reaction to form a covalent bond, if necessary, after the carboxyl group has been activated esterified by a conventional method, and then under conditions that do not adversely affect the binding activity of the antibody or antigen. The antibody or antigen is covalently bonded to the latex particle, and then the latex particle to which the antibody or antigen thus obtained is covalently bonded and a long chain polyethylene glycol molecule group of mixed polyethylene glycol having an amino group or a carboxyl group at one end are combined. After contacting under the above conditions, short chain polyethylene glycol molecules are In contact, the mixture of polyethylene glycol can be obtained by the method for producing the latex particles comprising the step of allowed to further covalently bonded to the latex particles. Those skilled in the art will be able to set the above-mentioned conditions appropriately modified with reference to the examples described later.
The immunolatex particles of the present invention include, but are not limited to, biological fluids that may contain biomolecules as analytes, such as blood, saliva, urine, spinal fluid, cells, cell debris, etc. The analyte in the sample can be detected or quantified without being adversely affected by contaminating proteins or the like. In addition, there is an effect that the antibody immobilized on the particles can be used stably even at a temperature at which the antibody is inherently advantageous, generally at a temperature exceeding room temperature (about 25 ° C.) by several tens of degrees. Furthermore, the immune latex particles of the present invention can be used without losing the antibody characteristics even when the fluid is higher in salt concentration than those derived from the living body.
Although not bound by theory, as can be understood from FIG. 1, which shows a schematic diagram of the surface of the particle, the short-chain polyethylene glycol chain and the long-chain polyethylene glycol chain closely surround the antibody, Since the directionality of the antibody molecule is regulated, it can be understood that the influence of the external environment such as high temperature or high salt concentration can be reduced while maintaining the specific binding property inherent to the antibody.
In addition, although not limited thereto, when an anti-ferritin antibody is supported as an antibody on the immune latex particle, even ferritin having a specific structure (for example, a protein having a molecular weight of 460,000) has extremely high sensitivity (10 −100 ng / ml). This is surprising considering that polyethylene glycol, which is highly mobile in aqueous media and is known to excrete serum proteins etc., is closely surrounding anti-ferritin antibodies. It is.
したがって、本発明では、粒子の分散性を高度に向上させ、粒子間の様々な非特異的相互作用を極限まで低下させるとともに、抗原との反応性を向上させ、しかも高感度・高性能な免疫診断ラテックス粒子を提供することを目的とする。
このような目的を達成すべく、新規なラテックスを作製し、それらの特性について検討してきたところ、上記非特許文献３に記載された表面の修飾された磁性粒子に比べてよりフレキシブルな構造を有することが予測されるラテックス粒子でも、生体分子と特異的に結合する分子、抗体または抗原等と混合ポリエチレングリコールを該粒子表面へ効果的に共固定できるこことがここに見出された。
さらに、理論に拘束されるものでないが、抗体または抗原等と混合ポリエチレングリコールを該粒子表面に共固定した場合には、抗体または抗原、特に、タンパク質性の抗原、例えば、抗体はポリエチレングリコール鎖によりランダムに取り囲まれることが予測されるにもかかわらず、上記特許文献２の金粒子等の表面に固定されたポリエチレングリコール鎖の非固定末端に結合し、裸の状態にあることが予測される抗体に比べて、勝るとも劣らない抗体の結合特性を示すことが確認された。さらに、驚くべきことに、ラテックス粒子表面に混合ポリエチレングリコールとともに共有結合を介して共固定された抗体は、通常遊離の状態または混合ポリエチレングリコールと共固定されていない状態にある抗体に比べて熱安定性、高塩濃度に対する抵抗性等が高まる一方で、抗体が本来有している抗原への結合特性を依然として高度に保持している、ことも確認された。
こうして、本発明によれば、混合ポリエチレングリコールと抗体または抗原がランダムに表面に共固定された免疫ラテックス粒子が提供される。該ラッテックス粒子における、混合ポリエチレングリコールは長鎖ポリエチレングリコール分子群と短鎖ポリエチレングリコール分子群からなり、そして前記共固定が、それぞれラテックス粒子表面上の官能基と混合ポリエチレングリコールおよび抗体または抗原の対応する官能基を介する共有結合の形成により達成される。
また、別の態様の本発明として、上記免疫ラテックス粒子の製造方法であって、タンパク質のアミノ酸残基のアミノ基またはカルボキシル基と反応できる官能基を表面に有するラテックス粒子と抗体または抗原を、該官能基と該アミノ基またはカルボキシル基が反応して共有結合を形成できる条件下で接触させて該抗体または抗原を該ラテックス粒子に共有結合せしめ、次いで、こうして得られた抗体または抗原が共有結合したラテックス粒子と一末端にアミノ基またはカルボキシル基を有する混合ポリエチレングリコールを前記条件下で接触させて該混合ポリエチレングリコールを該ラテックス粒子にさらに共有結合せしめる工程を含んでなる上記ラテックス粒子の製造方法も提供される。
さらに別の態様の本発明として、上記ラテックス粒子を用いる被検体（ラッテックス粒子に固定された抗体または抗原に特異的に結合しうる分子、構造物、例えば、細胞若しくは細胞破砕物等）の検出方法も提供される。各要素については後述するものもあるが、具体的には、被検体を含有するサンプル中に上記ラテックス粒子を存在させるステップ、被検体とラテックス粒子に固定された抗体または抗原が結合し得る条件下（通常、２０~３０℃の間）でラテックス粒子が存在する被検体を含有するサンプルをインキュベート（通常、３０秒~１０分）して、ラテックス粒子を凝集させるステップ、およびラテックス粒子の凝集の程度を被検体が存在する尺度として評価するステップを含んで成る、被検体の検出方法が提供される。凝集の程度は、目視、もしくは、一般的な分光器、さらには一部臨床現場に存在する分光検出を原理とする自動測定装置などにより、評価すればよい。
発明の詳細な記述
以下、本発明で使用する技術用語または技術内容についてより具体的に説明する。本発明または本明細書で使用する用語は特記しない限り、当該技術分野で普通に用いられている意味内容を表示するものとして理解されねばならない。
ラテックス粒子は、コロイド状に水中に分散した乳濁液を形成する微粒子であって、免疫凝集アッセイで用いることのできる粒子を意味し、上記の本発明の目的に沿うものである限り、使用モノマーの種類、製造方法により制限されることなく使用できる。限定されるものでないが、使用モノマーとしては、スチレン、スチレン−ブタジエンおよびアクリロニトリル−ブタジエン等、並びにこれらのモノマーの誘導体に由来する合成ゴムラテックスを形成できるモノマー、さらには、アクリル系、酢酸ビニール系、および塩化ビニル系等、並びにこれらのモノマーの誘導体に由来する、所謂、樹脂ラッテックスからなる群より選ばれるモノマーの混合系またはそれらの一種以上のモノマーの組合せ物を用いて、乳化重合法により製造される粒子が本発明で用いことのできるラテックス粒子が包含される。これらのうち、スチレンをベースとするものが好ましく用いられる。スチレンをベースとするとは、スチレンまたはその誘導体（例えば、クロロメチルスチレン、ジビニルベンゼン）に由来する単位が、総粒子重量当り少なくとも５０％以上占めることを意味する。
該ラテックス粒子表面の官能基は、上記に挙げたモノマーと、相当する官能基、例えば、カルボキシル基、アミノ基、クロロメチル基、アセタール基を有するモノマー、例えば、アクリル酸、メタクリル酸、アミノエチルメタクリレート、クロロメチルスチレン、アクロレインジエチルアセタール等を共重合させることにより導入し、必要があれば、かような官能基を粒子の形成後に別の官能基に転換せしめて担持させることもできる。このようなラテックス表面上の官能基と、該表面に共固定する混合ポリエチレングリコール（以下、ポリエチレングリコールをＰＥＧと略称する場合がある）の一末端に有する官能基および抗体もしくはタンパク質性抗原のアミノ酸残基の官能基との共有結合の形成は、それぞれ、例えば、カルボキシル基とアミノ基、カルボキシル基とヒドロキシル基、アミノ基とグルタールアルデヒド架橋を介するアミノ基、クロロメチル基とアミノ基、またはアセタール基（アセタールの酸加水分解を介するアルデヒド基もしくはホルミル基）とアミノ基、アミノ基とカルボキシル基、ヒドロキシル基とカルボキシル基との組み合わせにより形成できる。かような組み合わせによる共有結合の形成に先立ち、必要があれば、ラテックス粒子表面上の官能基をそれ自体当該技術分野で周知の活性化反応に供してもよい。
これらの組み合わせの使用の最適条件は、固定されるＰＥＧ末端の官能基の特性により変動するが、例えば、正電荷を有するポリアミンを末端官能基に有するＰＥＧの場合には、ラテックス粒子表面の官能基がマイナス電荷を有するカルボキシル基となるように選択するのがよい。また、ＰＥＧのもう一方の末端は、ＰＥＧとラテックス表面のそれぞれの官能基の共有結合形成反応に悪影響を及ぼさないヒドロキシル基または置換されていてもよいＣ１−Ｃ６アルコキシ基で末端が形成されていてもよい。Ｃ１−Ｃ６アコキシ基が置換されている場合の置換基はアセタール部分、ヒドロキシル基、モノもしくはジＣ１−Ｃ６アルキル置換アミノ基、ハロゲン原子等であることができる。
ラテックス粒子は、該粒子表面の官能基が、限定されるものでないが、少なくとも６３Å２／官能基以下の間隔で導入されたものであり、該粒子の平均粒経が５０ｎｍ~１５０μｍ、好ましくは１００ｎｍ~８００ｎｍ、より好ましくは１００ｎｍ~５００ｎｍであるものが有利に使用できる。また、このような基準を参考に、市販のラテックス粒子、例えば、Ｉｋｅｒｌａｔ（ｓｐａｉｎ）社製のＡＪ２６ ＣＯＯＨ−Ｃｌｅａｎを用いることもできる。
該ラテックス粒子表面に共固定させる混合ポリエチレングリコールは、ポリエチレングリコール部の数平均分子量が２０００~８０００ｇ／ｍｏｌの長鎖ポリエチレングリコール分子群と１５００~４０００ｇ／ｍｏｌの短鎖ポリエチレングリコール分子群からなり、かつ、長鎖ポリエチレングリコール分子群と短鎖ポリエチレングリコール分子群の平均分子量の差が少なくとも３０００ｇ／ｍｏｌである、ポリエチレングリコール誘導体、ラテックス粒子との反応末端に好ましくはポリアミンが導入されたＰＥＧが有利に使用できる。一方、該粒子表面に共固定される抗体または抗原は、モノクローナルもしくはポリクローナル抗体を使用するのが、抗体分子の反応特性または安定性の観点から好ましい。限定されるものでないが、等電点は中性~塩基性（７以上）を有する抗体に分類できる抗体が好ましく使用できる。
混合ポリエチレングリコールの長鎖ポリエチレングリコール分子群と短鎖ポリエチレングリコール分子群がモル比（仕込時）で、１：５~５：１、好ましくは３：５~２：５で存在する。ポリエチレングリコール分子群と共固定される抗体の量は、条件により好適な量は変動するが、一般的に３５０から１２００個／ラテックス粒子、程度である。（これらは、作製した粒子の分散安定性および反応活性の変化を観測することにより確認することができる。）
上記の免疫ラテックス粒子は、いかなる方法によって製造方法したものであってもよい。しかし、都合よくは、抗体を構成するタンパク質のアミノ酸残基のアミノ基またはカルボキシル基と反応できる官能基を表面に有する上記ラテックス粒子と、抗体または抗原を、該官能基と該アミノ基またはカルボキシル基が反応して共有結合を形成できる条件下、必要があれば、常法によりカルボキシル基を活性エステル化した後、抗体または抗原の結合活性に悪影響を及ばさない条件下、で相互に接触させて該抗体または抗原を該ラテックス粒子に共有結合せしめ、次いで、こうして得られた抗体または抗原が共有結合したラテックス粒子と一末端にアミノ基またはカルボキシル基を有する混合ポリエチレングリコールの長鎖ポリエチレングリコール分子群を前記条件下で接触させた後、短鎖ポリエチレングリコール分子群を前記条件下で接触させて、該混合ポリエチレングリコールを該ラテックス粒子にさらに共有結合せしめる工程を含んでなるラテックス粒子の製造方法により取得できる。当業者であれば、上記の条件は、後述する実施例を参考に、必要により適宜改変した条件を設定することができるであろう。
本発明の免疫ラテックス粒子は、限定されるものでないが、被検体として生体分子を含む可能性のある生物学的流体、例えば、血液、唾液、尿、髄液、細胞、細胞破砕物、等のサンプル中の被検体を、夾雑タンパク質等に悪影響を受けることなく、検出または定量できる。加えて、該粒子に固定された抗体が本来有利の状態できる温度、一般に、室温（約２５℃）を数十度越えた温度でも安定に使用できるとの効果を奏する。本発明の免疫ラテックス粒子は、さらに、前記流体が生体に由来するものより、さらに高塩濃度であっても、抗体の特性を失うことなく、使用できる。
理論により拘束されるものでないが、当該粒子の表面の概念図の略図を示す図１から理解できるように、短鎖ポリエチレングリコール鎖と長鎖ポリエチレングリコール鎖とが密に抗体を取り囲むことにより、該抗体分子の方向性が規制されるために抗体本来の特異的結合性を保持したまま、一方では、高温または高塩濃度等の外的環境の影響を低減できるものと理解できる。
また、限定されるものでないが、該免疫ラテックス粒子に抗体として抗フェリチン抗体を担持せしめると、特有の構造（例えば、鉄を含む分子量４６万のタンパク質）を有するフェリチンですら、極めて高感度（１０−１００ｎｇ／ｍｌ）で選択的に検出することができる。これは、水性媒体中で高い可動性を示し、血清タンパク質等を排斥することが知られているポリエチレングリコールが密に抗フェリチン抗体を取り囲んでいることが予測されることを考慮すると、驚くべきことである。 As described above, latex particles prepared using a macromer having a mixed polyethylene glycol chain disclosed in Patent Document 1 can provide immune particles in which molecules that specifically bind to biomolecules such as antibodies are stably immobilized. It has been found that it is not always easy to carry antibody molecules and the like at a sufficient density. In addition, in a latex particle system using BSA as a blocking agent, the blocking effect and the effect of dispersing particles in an aqueous medium may be limited due to the protein characteristics of BSA itself.
Therefore, in the present invention, the dispersibility of the particles is highly improved, various non-specific interactions between the particles are reduced to the limit, the reactivity with the antigen is improved, and high-sensitivity and high-performance immunity is achieved. An object is to provide diagnostic latex particles.
In order to achieve such an object, new latexes have been prepared and their properties have been studied. As a result, they have a more flexible structure than the surface-modified magnetic particles described in Non-Patent Document 3 above. It has now been found that even latex particles that are predicted to be capable of effectively co-immobilizing mixed polyethylene glycol on the particle surface with molecules, antibodies or antigens that specifically bind to biomolecules.
Furthermore, although not bound by theory, when a mixed polyethylene glycol and an antibody or an antigen are co-immobilized on the particle surface, the antibody or antigen, particularly a protein antigen, for example, the antibody is bound by a polyethylene glycol chain. Despite being expected to be surrounded at random, the antibody is expected to be in a naked state by binding to the non-fixed end of the polyethylene glycol chain fixed on the surface of the gold particle or the like of
Thus, according to the present invention, there are provided immune latex particles in which mixed polyethylene glycol and antibody or antigen are randomly co-immobilized on the surface. The mixed polyethylene glycol in the latex particle is composed of a long chain polyethylene glycol molecule group and a short chain polyethylene glycol molecule group, and the co-immobilization corresponds to the functional group on the latex particle surface and the mixed polyethylene glycol and the antibody or antigen, respectively. This is accomplished by the formation of a covalent bond through the functional group.
Further, as another aspect of the present invention, there is provided a method for producing the above-described immune latex particle, the latex particle having a functional group capable of reacting with an amino group or a carboxyl group of an amino acid residue of a protein, an antibody or an antigen, The antibody or antigen is covalently bound to the latex particle by contacting the functional group with the amino group or carboxyl group under a condition that can form a covalent bond by reaction, and then the antibody or antigen thus obtained is covalently bound. There is also provided a method for producing the latex particles, comprising the step of bringing the latex particles into contact with a mixed polyethylene glycol having an amino group or a carboxyl group at one end under the above conditions to further covalently bond the mixed polyethylene glycol to the latex particles. Is done.
As another aspect of the present invention, there is provided a method for detecting an analyte (a molecule or structure capable of specifically binding to an antibody or an antigen immobilized on a latex particle, an antigen, for example, a cell or a cell lysate) using the latex particle. Is also provided. Each element is described later. Specifically, the step of causing the latex particles to exist in the sample containing the analyte, and the conditions under which the analyte and the antibody or antigen immobilized on the latex particles can bind. Incubating (usually 30 seconds to 10 minutes) a sample containing an analyte in which latex particles are present (usually between 20 and 30 ° C.) to aggregate latex particles, and the degree of latex particle aggregation Is provided as a measure of the presence of the analyte. The degree of aggregation may be evaluated by visual observation, a general spectroscope, or an automatic measurement device based on the principle of spectroscopic detection existing in some clinical sites.
DETAILED DESCRIPTION OF THE INVENTION The technical terms or technical contents used in the present invention will be described more specifically below. Unless otherwise stated, terms used in the present invention or the present specification should be understood as indicating meanings commonly used in the art.
Latex particles are fine particles that form an emulsion dispersed in water in a colloidal form, and mean particles that can be used in an immunoaggregation assay. It can be used without being limited by the type and manufacturing method. Although not limited, the monomers used include styrene, styrene-butadiene and acrylonitrile-butadiene, and the like, as well as monomers capable of forming a synthetic rubber latex derived from derivatives of these monomers, and acrylic, vinyl acetate, It is produced by emulsion polymerization using a mixed system of monomers selected from the group consisting of so-called resin latex or a combination of one or more monomers derived from derivatives of these monomers and vinyl chlorides, etc. Latex particles that can be used in the present invention are included. Of these, those based on styrene are preferably used. Based on styrene means that units derived from styrene or its derivatives (eg, chloromethylstyrene, divinylbenzene) occupy at least 50% or more of the total particle weight.
The functional group on the surface of the latex particles is a monomer having the above-mentioned monomers and a corresponding functional group such as a carboxyl group, amino group, chloromethyl group, or acetal group, such as acrylic acid, methacrylic acid, aminoethyl methacrylate. Chloromethylstyrene, acrolein diethyl acetal, and the like may be introduced by copolymerization, and if necessary, such functional groups may be converted to other functional groups and supported after formation of the particles. Such a functional group on the latex surface, a functional group having one end of mixed polyethylene glycol (hereinafter, polyethylene glycol may be abbreviated as PEG) co-immobilized on the surface, and the amino acid residue of the antibody or protein antigen Formation of a covalent bond with a functional group of each group is, for example, a carboxyl group and an amino group, a carboxyl group and a hydroxyl group, an amino group and an amino group via a glutaraldehyde bridge, a chloromethyl group and an amino group, or an acetal group, respectively. (Aldehyde group or formyl group via acid hydrolysis of acetal) and an amino group, an amino group and a carboxyl group, or a combination of a hydroxyl group and a carboxyl group. Prior to the formation of the covalent bond by such a combination, if necessary, the functional group on the surface of the latex particle may be subjected to an activation reaction known per se in the art.
The optimum conditions for using these combinations vary depending on the characteristics of the functional group at the end of the PEG to be immobilized. For example, in the case of PEG having a polyamine having a positive charge as the terminal functional group, the functional group on the surface of the latex particle is used. It is preferable to select such that is a negatively charged carboxyl group. In addition, the other end of PEG is terminated with a hydroxyl group that does not adversely affect the covalent bond forming reaction between the functional groups of PEG and the latex surface, or an optionally substituted C 1 -C 6 alkoxy group. It may be. When the C 1 -C 6 acoxy group is substituted, the substituent can be an acetal moiety, a hydroxyl group, a mono or di C 1 -C 6 alkyl substituted amino group, a halogen atom, and the like.
The latex particle has a functional group on the surface of the particle which is not limited, but is introduced at intervals of at least 63 cm 2 / functional group, and the average particle size of the particle is 50 nm to 150 μm, preferably 100 nm. Those having a thickness of ˜800 nm, more preferably 100 nm to 500 nm can be advantageously used. In addition, referring to such a standard, commercially available latex particles, for example, AJ26 COOH-Clean manufactured by Ikerlat (spain) can also be used.
The mixed polyethylene glycol that is co-immobilized on the surface of the latex particles is composed of a long chain polyethylene glycol molecule group having a number average molecular weight of polyethylene glycol part of 2000 to 8000 g / mol and a short chain polyethylene glycol molecule group having 1500 to 4000 g / mol, and A polyethylene glycol derivative having a difference in average molecular weight between the long chain polyethylene glycol molecule group and the short chain polyethylene glycol molecule group of at least 3000 g / mol, preferably PEG having a polyamine introduced at the reaction terminal with the latex particle is advantageously used. it can. On the other hand, the antibody or antigen co-immobilized on the particle surface is preferably a monoclonal or polyclonal antibody from the viewpoint of reaction characteristics or stability of the antibody molecule. Although not limited, antibodies that can be classified into antibodies having an isoelectric point of neutral to basic (7 or more) can be preferably used.
The long-chain polyethylene glycol molecule group and the short-chain polyethylene glycol molecule group of the mixed polyethylene glycol are present in a molar ratio (when charged) of 1: 5 to 5: 1, preferably 3: 5 to 2: 5. The amount of the antibody co-immobilized with the polyethylene glycol molecule group varies generally depending on the conditions, but is generally about 350 to 1200 particles / latex particles. (These can be confirmed by observing changes in dispersion stability and reaction activity of the produced particles.)
The above-described immune latex particles may be produced by any method. However, conveniently, the latex particle having a functional group capable of reacting with the amino group or carboxyl group of the amino acid residue of the protein constituting the antibody on the surface, the antibody or antigen, the functional group and the amino group or carboxyl group. Can react with each other under conditions that allow the reaction to form a covalent bond, if necessary, after the carboxyl group has been activated esterified by a conventional method, and then under conditions that do not adversely affect the binding activity of the antibody or antigen. The antibody or antigen is covalently bonded to the latex particle, and then the latex particle to which the antibody or antigen thus obtained is covalently bonded and a long chain polyethylene glycol molecule group of mixed polyethylene glycol having an amino group or a carboxyl group at one end are combined. After contacting under the above conditions, short chain polyethylene glycol molecules are In contact, the mixture of polyethylene glycol can be obtained by the method for producing the latex particles comprising the step of allowed to further covalently bonded to the latex particles. Those skilled in the art will be able to set the above-mentioned conditions appropriately modified with reference to the examples described later.
The immunolatex particles of the present invention include, but are not limited to, biological fluids that may contain biomolecules as analytes, such as blood, saliva, urine, spinal fluid, cells, cell debris, etc. The analyte in the sample can be detected or quantified without being adversely affected by contaminating proteins or the like. In addition, there is an effect that the antibody immobilized on the particles can be used stably even at a temperature at which the antibody is inherently advantageous, generally at a temperature exceeding room temperature (about 25 ° C.) by several tens of degrees. Furthermore, the immune latex particles of the present invention can be used without losing the antibody characteristics even when the fluid is higher in salt concentration than those derived from the living body.
Although not bound by theory, as can be understood from FIG. 1, which shows a schematic diagram of the surface of the particle, the short-chain polyethylene glycol chain and the long-chain polyethylene glycol chain closely surround the antibody, Since the directionality of the antibody molecule is regulated, it can be understood that the influence of the external environment such as high temperature or high salt concentration can be reduced while maintaining the specific binding property inherent to the antibody.
In addition, although not limited thereto, when an anti-ferritin antibody is supported as an antibody on the immune latex particle, even ferritin having a specific structure (for example, a protein having a molecular weight of 460,000) has extremely high sensitivity (10 −100 ng / ml). This is surprising considering that polyethylene glycol, which is highly mobile in aqueous media and is known to excrete serum proteins etc., is closely surrounding anti-ferritin antibodies. It is.
図１は本発明の免疫ラテックス粒子表面の概念図の略図である。
図２は実施例２に係る、ＬＡｍＰ複合体（図２のａおよびｂ中、それぞれ、黒塗り四角または白抜き四角）およびＬＡＰ複合体（図２のａおよびｂ中、それぞれ黒塗り三角および白抜き三角）の濃度の変動するフェリチンとの結合の結果を示すグラフ表示である。
図３は実施例３に係る、リン酸緩衝液中でフェリチンを測定した結果を示すグラフ表示である。
図４は実施例３に係る、牛胎児血清（ＦＢＳ）中でフェリチンを測定した結果を示すグラフ表示である。 FIG. 1 is a schematic diagram of the surface of the immune latex particle surface of the present invention.
FIG. 2 shows the LAmP complex (black and white squares in FIG. 2 a and b, respectively) and the LAP complex (black and white triangles and white in FIG. 2 a and b, respectively), according to Example 2. It is a graph display which shows the result of the coupling | bonding with the ferritin from which the density | concentration of a (triangular triangle) fluctuates.
FIG. 3 is a graphical representation showing the results of measuring ferritin in a phosphate buffer according to Example 3.
4 is a graphical representation showing the results of measuring ferritin in fetal bovine serum (FBS) according to Example 3. FIG.
図２は実施例２に係る、ＬＡｍＰ複合体（図２のａおよびｂ中、それぞれ、黒塗り四角または白抜き四角）およびＬＡＰ複合体（図２のａおよびｂ中、それぞれ黒塗り三角および白抜き三角）の濃度の変動するフェリチンとの結合の結果を示すグラフ表示である。
図３は実施例３に係る、リン酸緩衝液中でフェリチンを測定した結果を示すグラフ表示である。
図４は実施例３に係る、牛胎児血清（ＦＢＳ）中でフェリチンを測定した結果を示すグラフ表示である。 FIG. 1 is a schematic diagram of the surface of the immune latex particle surface of the present invention.
FIG. 2 shows the LAmP complex (black and white squares in FIG. 2 a and b, respectively) and the LAP complex (black and white triangles and white in FIG. 2 a and b, respectively), according to Example 2. It is a graph display which shows the result of the coupling | bonding with the ferritin from which the density | concentration of a (triangular triangle) fluctuates.
FIG. 3 is a graphical representation showing the results of measuring ferritin in a phosphate buffer according to Example 3.
4 is a graphical representation showing the results of measuring ferritin in fetal bovine serum (FBS) according to Example 3. FIG.
以下、本発明の実施の形態について、詳細に説明する。
Hereinafter, embodiments of the present invention will be described in detail.
免疫ラテックス粒子の作製：
表面にカルボキシル基を有するラテックス粒子（Ｂｉｏｋｉｔ Ｓ．Ａ．社製、ＡＪ２６ ＣＯＯＨ−Ｃｌｅａｎ（登録商標）；３０μｌ；１０％ ｗ／ｖ）と１６７μｌのＮａＨ２ＰＯ４水溶液（１０ｍＭ，ｐＨ＝４．８）を混ぜた後、１−エチル−３−［３−ジメチルアミノプロピル］カルボジイミド塩酸（ＥＤＣ、３μｌ、１２．５ｍｇ／ｍｌ、ＥＤＣと粒子表面のカルボキシル基のモル比＝１：１）の活性エステルの水溶液を加え、２５℃で２０分間撹拌（撹拌速度：１０００ｒｐｍ）させることでラテックス粒子の表面のカルボキシル基を活性化した。こうして得られた混合溶液を１００μｌのリン酸緩衝液（１０ｍＭ，ｐＨ＝７．４）で希釈した後、混合溶液から５０μｌを分取後、これに対して２７５μｌのリン酸緩衝液（１０ｍＭ，ｐＨ＝７．４）と１０μｌの抗体溶液（ａｎｔｉ−ｆｅｒｒｉｔｉｎ（Ｂｉｏｋｉｔ Ｓ．Ａ．社から入手）、０．５１６−１．７１ｍｇ／ｍｌ、ｐＨ＝７．４）を添加し、２５℃で６０分間撹拌せる（撹拌速度＝１０００ｒｐｍ）ことで、抗体に有するアミノ官能基と活性化した粒子表面のカルボキシル基と反応させ、抗体をラテックス粒子の表面に固定した。このようにして作製したラテックス／抗体（以下、ＬＡと略記する場合あり）複合体を３００μｌ取り、α−メトキシ−ポリ（エチレングリコール）−ペンタエチレンヘキサミン（Ｎ６−ＰＥＧ−５ｋ、０．３％ ｗ／ｖ、ｐＨ＝７．４）を含むリン酸緩衝液１５０μｌを加え、２５℃で３０分間撹拌（撹拌速度＝１０００ｒｐｍ）することでＮ６−ＰＥＧ−５ｋを粒子表面上に固定化し（末端のアミノ基と活性化した粒子表面のカルボキシル基との反応）、ラテックス／抗体／ＰＥＧの複合体（以下、ＬＡＰと略記する場合あり）が得られた。また別に、３００μｌのＬＡ複合体を７５μｌのＮ６−ＰＥＧ−５ｋ（０．６％ ｗ／ｖ、ｐＨ＝７．４）に入れて同様に反応させた後、更に鎖長の短いＰＥＧ、Ｎ６−ＰＥＧ−２ｋ（０．４％ ｗ／ｖ、ｐＨ＝７．４、５ｋ／２ｋ＝１：０．６７）を入れて同様に反応させることで、ラテックス／抗体／混合ＰＥＧ鎖の複合体（以下、ＬＡｍＰと略記する場合あり）が得られた。ラテックス粒子の最終濃度は０．１％ ｗ／ｖであった。
ＢＳＡをブロッキング剤として用いたＬＡＢ（ｌａｔｅｘ／ａｎｔｉｂｏｄｙ／ＢＳＡ）複合体は、上記ＬＡＰの作製法と同じで、単にＮ６−ＰＥＧ−５ｋの代わりにＢＳＡ（０．０４％ ｗ／ｖ、ｐＨ＝７．４）を使用することで得た。
Preparation of immune latex particles:
Latex particles having a carboxyl group on the surface (manufactured by Biokit SA, AJ26 COOH-Clean (registered trademark); 30 μl; 10% w / v) and 167 μl of NaH 2 PO 4 aqueous solution (10 mM, pH = 4.8) ), And then an active ester of 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, 3 μl, 12.5 mg / ml, molar ratio of EDC to carboxyl group on the particle surface = 1: 1) Was added and stirred at 25 ° C. for 20 minutes (stirring speed: 1000 rpm) to activate the carboxyl groups on the surface of the latex particles. The mixed solution thus obtained was diluted with 100 μl of phosphate buffer (10 mM, pH = 7.4), 50 μl was taken from the mixed solution, and 275 μl of phosphate buffer (10 mM, pH) = 7.4) and 10 μl of antibody solution (anti-ferritin (obtained from Biokit SA), 0.516-1.71 mg / ml, pH = 7.4) and added at 25 ° C. for 60 minutes By stirring (stirring speed = 1000 rpm), the amino functional group of the antibody was reacted with the activated carboxyl group of the particle surface to immobilize the antibody on the surface of the latex particle. 300 μl of the latex / antibody (hereinafter sometimes abbreviated as LA) complex prepared in this way was taken out and α-methoxy-poly (ethylene glycol) -pentaethylenehexamine (N6-PEG-5k, 0.3% w / V, pH = 7.4) is added, and N6-PEG-5k is immobilized on the particle surface by stirring at 25 ° C. for 30 minutes (stirring speed = 1000 rpm) (terminal amino acid). Reaction with carboxyl groups on the activated particle surface), a latex / antibody / PEG complex (hereinafter sometimes abbreviated as LAP) was obtained. Separately, 300 μl of the LA complex was placed in 75 μl of N6-PEG-5k (0.6% w / v, pH = 7.4) and reacted in the same manner, and then PEG, N6- By adding PEG-2k (0.4% w / v, pH = 7.4, 5k / 2k = 1: 0.67) and reacting in the same manner, a latex / antibody / mixed PEG chain complex (hereinafter referred to as “the complex”) , Sometimes abbreviated as LAmP). The final concentration of latex particles was 0.1% w / v.
The LAB (latex / antibody / BSA) complex using BSA as a blocking agent is the same as the method for preparing LAP described above, and simply BSA (0.04% w / v, pH = 7 instead of N6-PEG-5k). 4) was used.
表面にカルボキシル基を有するラテックス粒子（Ｂｉｏｋｉｔ Ｓ．Ａ．社製、ＡＪ２６ ＣＯＯＨ−Ｃｌｅａｎ（登録商標）；３０μｌ；１０％ ｗ／ｖ）と１６７μｌのＮａＨ２ＰＯ４水溶液（１０ｍＭ，ｐＨ＝４．８）を混ぜた後、１−エチル−３−［３−ジメチルアミノプロピル］カルボジイミド塩酸（ＥＤＣ、３μｌ、１２．５ｍｇ／ｍｌ、ＥＤＣと粒子表面のカルボキシル基のモル比＝１：１）の活性エステルの水溶液を加え、２５℃で２０分間撹拌（撹拌速度：１０００ｒｐｍ）させることでラテックス粒子の表面のカルボキシル基を活性化した。こうして得られた混合溶液を１００μｌのリン酸緩衝液（１０ｍＭ，ｐＨ＝７．４）で希釈した後、混合溶液から５０μｌを分取後、これに対して２７５μｌのリン酸緩衝液（１０ｍＭ，ｐＨ＝７．４）と１０μｌの抗体溶液（ａｎｔｉ−ｆｅｒｒｉｔｉｎ（Ｂｉｏｋｉｔ Ｓ．Ａ．社から入手）、０．５１６−１．７１ｍｇ／ｍｌ、ｐＨ＝７．４）を添加し、２５℃で６０分間撹拌せる（撹拌速度＝１０００ｒｐｍ）ことで、抗体に有するアミノ官能基と活性化した粒子表面のカルボキシル基と反応させ、抗体をラテックス粒子の表面に固定した。このようにして作製したラテックス／抗体（以下、ＬＡと略記する場合あり）複合体を３００μｌ取り、α−メトキシ−ポリ（エチレングリコール）−ペンタエチレンヘキサミン（Ｎ６−ＰＥＧ−５ｋ、０．３％ ｗ／ｖ、ｐＨ＝７．４）を含むリン酸緩衝液１５０μｌを加え、２５℃で３０分間撹拌（撹拌速度＝１０００ｒｐｍ）することでＮ６−ＰＥＧ−５ｋを粒子表面上に固定化し（末端のアミノ基と活性化した粒子表面のカルボキシル基との反応）、ラテックス／抗体／ＰＥＧの複合体（以下、ＬＡＰと略記する場合あり）が得られた。また別に、３００μｌのＬＡ複合体を７５μｌのＮ６−ＰＥＧ−５ｋ（０．６％ ｗ／ｖ、ｐＨ＝７．４）に入れて同様に反応させた後、更に鎖長の短いＰＥＧ、Ｎ６−ＰＥＧ−２ｋ（０．４％ ｗ／ｖ、ｐＨ＝７．４、５ｋ／２ｋ＝１：０．６７）を入れて同様に反応させることで、ラテックス／抗体／混合ＰＥＧ鎖の複合体（以下、ＬＡｍＰと略記する場合あり）が得られた。ラテックス粒子の最終濃度は０．１％ ｗ／ｖであった。
ＢＳＡをブロッキング剤として用いたＬＡＢ（ｌａｔｅｘ／ａｎｔｉｂｏｄｙ／ＢＳＡ）複合体は、上記ＬＡＰの作製法と同じで、単にＮ６−ＰＥＧ−５ｋの代わりにＢＳＡ（０．０４％ ｗ／ｖ、ｐＨ＝７．４）を使用することで得た。
Latex particles having a carboxyl group on the surface (manufactured by Biokit SA, AJ26 COOH-Clean (registered trademark); 30 μl; 10% w / v) and 167 μl of NaH 2 PO 4 aqueous solution (10 mM, pH = 4.8) ), And then an active ester of 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, 3 μl, 12.5 mg / ml, molar ratio of EDC to carboxyl group on the particle surface = 1: 1) Was added and stirred at 25 ° C. for 20 minutes (stirring speed: 1000 rpm) to activate the carboxyl groups on the surface of the latex particles. The mixed solution thus obtained was diluted with 100 μl of phosphate buffer (10 mM, pH = 7.4), 50 μl was taken from the mixed solution, and 275 μl of phosphate buffer (10 mM, pH) = 7.4) and 10 μl of antibody solution (anti-ferritin (obtained from Biokit SA), 0.516-1.71 mg / ml, pH = 7.4) and added at 25 ° C. for 60 minutes By stirring (stirring speed = 1000 rpm), the amino functional group of the antibody was reacted with the activated carboxyl group of the particle surface to immobilize the antibody on the surface of the latex particle. 300 μl of the latex / antibody (hereinafter sometimes abbreviated as LA) complex prepared in this way was taken out and α-methoxy-poly (ethylene glycol) -pentaethylenehexamine (N6-PEG-5k, 0.3% w / V, pH = 7.4) is added, and N6-PEG-5k is immobilized on the particle surface by stirring at 25 ° C. for 30 minutes (stirring speed = 1000 rpm) (terminal amino acid). Reaction with carboxyl groups on the activated particle surface), a latex / antibody / PEG complex (hereinafter sometimes abbreviated as LAP) was obtained. Separately, 300 μl of the LA complex was placed in 75 μl of N6-PEG-5k (0.6% w / v, pH = 7.4) and reacted in the same manner, and then PEG, N6- By adding PEG-2k (0.4% w / v, pH = 7.4, 5k / 2k = 1: 0.67) and reacting in the same manner, a latex / antibody / mixed PEG chain complex (hereinafter referred to as “the complex”) , Sometimes abbreviated as LAmP). The final concentration of latex particles was 0.1% w / v.
The LAB (latex / antibody / BSA) complex using BSA as a blocking agent is the same as the method for preparing LAP described above, and simply BSA (0.04% w / v, pH = 7 instead of N6-PEG-5k). 4) was used.
長鎖ＰＥＧ修飾粒子と混合ＰＥＧ修飾粒子のフェリチン検出能の評価と比較：
実施例１にて作製した粒子のうち、０．５１６ｍｇ／ｍｌの抗体濃度とＮ６−ＰＥＧ−５ｋのみを用いて作製した粒子（ＬＡＰ）と、０．５１６ｍｇ／ｍｌの抗体濃度と長鎖のＮ６−ＰＥＧ−５ｋおよび短鎖のＮ６−ＰＥＧ−２ｋを用いて作製した粒子（ＬＡｍＰ）の、リン酸バッファー水溶液（ＰＢ）中と血清（ＦＢＳ）中におけるフェリチン応答能を調査した（結果を図２に示す）。この検討に先立ち、ＰＥＧ鎖を表面に修飾してない抗体固定化ラテックス粒子の粒子分散性を検討したところ、ＰＢ中における分散状態を維持することができず、抗原添加に伴う優位な凝集応答は示さなかった。これに対し、上述したＬＡＰとＬＡｍＰは、バッファー水溶液中および血清中において高い分散安定性を示すと同時に、１０−１００ｎｇ／ｍｌのフェリチン溶液を添加した際に、直線性のある非常に高い抗原応答能を示すことがあきらかとなった。また、ＰＢならびにＦＢＳ中において、ＬＡｍＰ複合体はＬＡＰ複合体よりも高いフェリチン検出能を有することわかる（図２のａおよびｂ参照）。
以上の結果は、長鎖ＰＥＧ単独を修飾するよりも、長鎖／短鎖の混合ＰＥＧを粒子表面に修飾したほうが高感度な検出能を有するラテックス粒子が作製可能であることを示す。 Evaluation and comparison of long-chain PEG modified particles and mixed PEG modified particles for ferritin detection ability:
Among the particles prepared in Example 1, 0.516 mg / ml antibody concentration and particles (LAP) prepared using only N6-PEG-5k, 0.516 mg / ml antibody concentration and long chain N6 -The ferritin-responsive ability of particles (LAmP) prepared using PEG-5k and short-chain N6-PEG-2k in phosphate buffer aqueous solution (PB) and serum (FBS) was investigated (results shown in FIG. 2). To show). Prior to this study, we examined the particle dispersibility of antibody-immobilized latex particles whose PEG chains were not modified on the surface. As a result, the dispersion state in PB could not be maintained, and the dominant aggregation response accompanying the addition of antigen was Not shown. In contrast, LAP and LAmP described above exhibit high dispersion stability in aqueous buffer solution and serum, and at the same time, when a 10-100 ng / ml ferritin solution is added, the linear response is very high. It became clear to show Noh. In addition, in PB and FBS, it can be seen that the LAmP complex has higher ferritin detection ability than the LAP complex (see a and b in FIG. 2).
The above results indicate that it is possible to produce latex particles with high sensitivity by modifying the long-chain / short-chain mixed PEG on the particle surface rather than modifying the long-chain PEG alone.
実施例１にて作製した粒子のうち、０．５１６ｍｇ／ｍｌの抗体濃度とＮ６−ＰＥＧ−５ｋのみを用いて作製した粒子（ＬＡＰ）と、０．５１６ｍｇ／ｍｌの抗体濃度と長鎖のＮ６−ＰＥＧ−５ｋおよび短鎖のＮ６−ＰＥＧ−２ｋを用いて作製した粒子（ＬＡｍＰ）の、リン酸バッファー水溶液（ＰＢ）中と血清（ＦＢＳ）中におけるフェリチン応答能を調査した（結果を図２に示す）。この検討に先立ち、ＰＥＧ鎖を表面に修飾してない抗体固定化ラテックス粒子の粒子分散性を検討したところ、ＰＢ中における分散状態を維持することができず、抗原添加に伴う優位な凝集応答は示さなかった。これに対し、上述したＬＡＰとＬＡｍＰは、バッファー水溶液中および血清中において高い分散安定性を示すと同時に、１０−１００ｎｇ／ｍｌのフェリチン溶液を添加した際に、直線性のある非常に高い抗原応答能を示すことがあきらかとなった。また、ＰＢならびにＦＢＳ中において、ＬＡｍＰ複合体はＬＡＰ複合体よりも高いフェリチン検出能を有することわかる（図２のａおよびｂ参照）。
以上の結果は、長鎖ＰＥＧ単独を修飾するよりも、長鎖／短鎖の混合ＰＥＧを粒子表面に修飾したほうが高感度な検出能を有するラテックス粒子が作製可能であることを示す。 Evaluation and comparison of long-chain PEG modified particles and mixed PEG modified particles for ferritin detection ability:
Among the particles prepared in Example 1, 0.516 mg / ml antibody concentration and particles (LAP) prepared using only N6-PEG-5k, 0.516 mg / ml antibody concentration and long chain N6 -The ferritin-responsive ability of particles (LAmP) prepared using PEG-5k and short-chain N6-PEG-2k in phosphate buffer aqueous solution (PB) and serum (FBS) was investigated (results shown in FIG. 2). To show). Prior to this study, we examined the particle dispersibility of antibody-immobilized latex particles whose PEG chains were not modified on the surface. As a result, the dispersion state in PB could not be maintained, and the dominant aggregation response accompanying the addition of antigen was Not shown. In contrast, LAP and LAmP described above exhibit high dispersion stability in aqueous buffer solution and serum, and at the same time, when a 10-100 ng / ml ferritin solution is added, the linear response is very high. It became clear to show Noh. In addition, in PB and FBS, it can be seen that the LAmP complex has higher ferritin detection ability than the LAP complex (see a and b in FIG. 2).
The above results indicate that it is possible to produce latex particles with high sensitivity by modifying the long-chain / short-chain mixed PEG on the particle surface rather than modifying the long-chain PEG alone.
抗体固定化量を変化させて作製したラテックス粒子のフェリチン検出能の評価と比較：
実施例１にて作製した粒子、０．５１６ｍｇ／ｍｌ、１．１３ｍｇ／ｍｌ、１．７１ｍｇ／ｍｌの抗体濃度と長鎖のＮ６−ＰＥＧ−５ｋおよび短鎖のＮ６−ＰＥＧ−２ｋを用いて作製した粒子（ＬＡｍＰ０．４５、ＬＡｍＰ１．０、ＬＡｍＰ１．５、）のＰＢ中におけるフェリチン検出能を評価した（結果を図３ａに示す）。結果として、抗フェリチン抗体を最も多く固定化した粒子（抗体濃度１．７１ｍｇ／ｍｌを用いて作製）が最も高いフェリチン検出能を示すことが明らかとなった。
コントロールとして、従来ブロッキング剤として汎用されている牛血清アルブミンを用いて抗体固定化ラテックス粒子を作製し、その検出能をＬＡｍＰと比較した。実施例１に記載した同様の操作により、０．５１６ｍｇ／ｍｌ、１．１３ｍｇ／ｍｌ、１．７１ｍｇ／ｍｌの抗体濃度を用いて作製したラテックス／抗体（以下、ＬＡと略記する場合あり）複合体を３００μｌ取り、ＢＳＡ（０．０４％ ｗ／ｖ、ｐＨ＝７．４）を含むリン酸緩衝液１５０μｌを加え、２５℃で３０分間撹拌（撹拌速度＝１０００ｒｐｍ）することでＢＳＡを粒子表面上に固定化し（ＢＳＡのアミノ基と活性化した粒子表面のカルボキシル基との反応）、ラテックス／抗体／ＢＳＡの複合体（以下、ＬＡＢと略記する場合あり）が得られた。ＬＡＢのＰＢ中におけるフェリチン検出能を評価したところ、抗フェリチン抗体を最も多く固定化した粒子（抗体濃度１．７１ｍｇ／ｍｌを用いて作製）が最も高いフェリチン検出能を示すことが明らかとなった。
ＬＡｍＰとＬＡＢを比較した場合、混合ＰＥＧをブロッキング剤として修飾したＬＡｍＰの方が高いフェリチン検出能を有することがわかる（図３のａ，ｂ参照）。特に、ＦＢＳ中にて同様の評価、比較を行ったところ、ＬＡＢ粒子は固定した抗体の量にかかわらず反応活性がほぼ観察されないことに対して（図４ｄ）、ＬＡｍＰ粒子は高いフェリチン検出能を示した（図４ｃ）。 Evaluation and comparison of ferritin detection ability of latex particles prepared by changing the amount of immobilized antibody:
Using the particles prepared in Example 1, 0.516 mg / ml, 1.13 mg / ml, 1.71 mg / ml antibody concentration and long chain N6-PEG-5k and short chain N6-PEG-2k The ferritin detection ability in PB of the produced particles (LAmP0.45, LAmP1.0, LAmP1.5) was evaluated (results are shown in FIG. 3a). As a result, it was revealed that the most immobilized anti-ferritin antibody particles (produced using an antibody concentration of 1.71 mg / ml) showed the highest ferritin detection ability.
As a control, antibody-immobilized latex particles were prepared using bovine serum albumin, which has been widely used as a blocking agent in the past, and its detection ability was compared with LAmP. Latex / antibody (hereinafter sometimes abbreviated as LA) composite prepared using antibody concentrations of 0.516 mg / ml, 1.13 mg / ml and 1.71 mg / ml by the same procedure described in Example 1. Take 300 μl of the body, add 150 μl of phosphate buffer containing BSA (0.04% w / v, pH = 7.4), and stir at 25 ° C. for 30 minutes (stirring speed = 1000 rpm). Immobilized above (reaction between the amino group of BSA and the carboxyl group of the activated particle surface), a latex / antibody / BSA complex (hereinafter sometimes abbreviated as LAB) was obtained. Evaluation of ferritin detection ability in PB of LAB revealed that the most immobilized anti-ferritin antibody particles (prepared using an antibody concentration of 1.71 mg / ml) showed the highest ferritin detection ability. .
When LAmP and LAB are compared, it can be seen that LAmP modified with mixed PEG as a blocking agent has higher ferritin detection ability (see a and b in FIG. 3). In particular, when the same evaluation and comparison were performed in FBS, LAB particles showed almost no reaction activity regardless of the amount of immobilized antibody (FIG. 4d), whereas LAmP particles had high ferritin detection ability. Shown (Figure 4c).
実施例１にて作製した粒子、０．５１６ｍｇ／ｍｌ、１．１３ｍｇ／ｍｌ、１．７１ｍｇ／ｍｌの抗体濃度と長鎖のＮ６−ＰＥＧ−５ｋおよび短鎖のＮ６−ＰＥＧ−２ｋを用いて作製した粒子（ＬＡｍＰ０．４５、ＬＡｍＰ１．０、ＬＡｍＰ１．５、）のＰＢ中におけるフェリチン検出能を評価した（結果を図３ａに示す）。結果として、抗フェリチン抗体を最も多く固定化した粒子（抗体濃度１．７１ｍｇ／ｍｌを用いて作製）が最も高いフェリチン検出能を示すことが明らかとなった。
コントロールとして、従来ブロッキング剤として汎用されている牛血清アルブミンを用いて抗体固定化ラテックス粒子を作製し、その検出能をＬＡｍＰと比較した。実施例１に記載した同様の操作により、０．５１６ｍｇ／ｍｌ、１．１３ｍｇ／ｍｌ、１．７１ｍｇ／ｍｌの抗体濃度を用いて作製したラテックス／抗体（以下、ＬＡと略記する場合あり）複合体を３００μｌ取り、ＢＳＡ（０．０４％ ｗ／ｖ、ｐＨ＝７．４）を含むリン酸緩衝液１５０μｌを加え、２５℃で３０分間撹拌（撹拌速度＝１０００ｒｐｍ）することでＢＳＡを粒子表面上に固定化し（ＢＳＡのアミノ基と活性化した粒子表面のカルボキシル基との反応）、ラテックス／抗体／ＢＳＡの複合体（以下、ＬＡＢと略記する場合あり）が得られた。ＬＡＢのＰＢ中におけるフェリチン検出能を評価したところ、抗フェリチン抗体を最も多く固定化した粒子（抗体濃度１．７１ｍｇ／ｍｌを用いて作製）が最も高いフェリチン検出能を示すことが明らかとなった。
ＬＡｍＰとＬＡＢを比較した場合、混合ＰＥＧをブロッキング剤として修飾したＬＡｍＰの方が高いフェリチン検出能を有することがわかる（図３のａ，ｂ参照）。特に、ＦＢＳ中にて同様の評価、比較を行ったところ、ＬＡＢ粒子は固定した抗体の量にかかわらず反応活性がほぼ観察されないことに対して（図４ｄ）、ＬＡｍＰ粒子は高いフェリチン検出能を示した（図４ｃ）。 Evaluation and comparison of ferritin detection ability of latex particles prepared by changing the amount of immobilized antibody:
Using the particles prepared in Example 1, 0.516 mg / ml, 1.13 mg / ml, 1.71 mg / ml antibody concentration and long chain N6-PEG-5k and short chain N6-PEG-2k The ferritin detection ability in PB of the produced particles (LAmP0.45, LAmP1.0, LAmP1.5) was evaluated (results are shown in FIG. 3a). As a result, it was revealed that the most immobilized anti-ferritin antibody particles (produced using an antibody concentration of 1.71 mg / ml) showed the highest ferritin detection ability.
As a control, antibody-immobilized latex particles were prepared using bovine serum albumin, which has been widely used as a blocking agent in the past, and its detection ability was compared with LAmP. Latex / antibody (hereinafter sometimes abbreviated as LA) composite prepared using antibody concentrations of 0.516 mg / ml, 1.13 mg / ml and 1.71 mg / ml by the same procedure described in Example 1. Take 300 μl of the body, add 150 μl of phosphate buffer containing BSA (0.04% w / v, pH = 7.4), and stir at 25 ° C. for 30 minutes (stirring speed = 1000 rpm). Immobilized above (reaction between the amino group of BSA and the carboxyl group of the activated particle surface), a latex / antibody / BSA complex (hereinafter sometimes abbreviated as LAB) was obtained. Evaluation of ferritin detection ability in PB of LAB revealed that the most immobilized anti-ferritin antibody particles (prepared using an antibody concentration of 1.71 mg / ml) showed the highest ferritin detection ability. .
When LAmP and LAB are compared, it can be seen that LAmP modified with mixed PEG as a blocking agent has higher ferritin detection ability (see a and b in FIG. 3). In particular, when the same evaluation and comparison were performed in FBS, LAB particles showed almost no reaction activity regardless of the amount of immobilized antibody (FIG. 4d), whereas LAmP particles had high ferritin detection ability. Shown (Figure 4c).
Claims (11)
- 混合ポリエチレングリコールと抗体または抗原がランダムに表面に共固定された免疫ラテックス粒子であって、混合ポリエチレングリコールが長鎖ポリエチレングリコール分子群と短鎖ポリエチレングリコール分子群からなり、そして前記共固定が、それぞれラテックス粒子表面上の官能基と混合ポリエチレングリコールおよび抗体または抗原の対応する官能基を介する共有結合の形成により達成される、上記ラテックス粒子。 Immune latex particles in which mixed polyethylene glycol and antibody or antigen are randomly co-immobilized on the surface, wherein the mixed polyethylene glycol consists of a group of long-chain polyethylene glycol molecules and a group of short-chain polyethylene glycol molecules, Latex particles as described above, achieved by the formation of covalent bonds via functional groups on the surface of the latex particles and the corresponding functional groups of the mixed polyethylene glycol and antibody or antigen.
- ラテックス粒子がポリスチレンをベースとし、かつ該粒子表面の官能基と混合ポリエチレングリコールおよび抗体の対応する官能基が、それぞれ、カルボキシル基とアミノ基、カルボキシル基とヒドロキシル基、アミノ基とグルタールアルデヒド架橋を介するアミノ基、クロロメチル基とアミノ基、またはアセタール基とアミノ基、アミノ基とカルボキシル基、ヒドロキシル基とカルボキシル基、である、請求項１記載のラテックス粒子。 Latex particles are based on polystyrene, and the functional groups on the particle surface and mixed polyethylene glycol and the corresponding functional groups of the antibody have carboxyl groups and amino groups, carboxyl groups and hydroxyl groups, amino groups and glutaraldehyde crosslinks, respectively. The latex particles according to claim 1, which are an amino group, a chloromethyl group and an amino group, an acetal group and an amino group, an amino group and a carboxyl group, or a hydroxyl group and a carboxyl group.
- 抗体または抗原が抗体であり、ラテックス粒子表面の官能基がカルボキシル基である、請求項１または２記載のラテックス粒子。 The latex particle according to claim 1 or 2, wherein the antibody or antigen is an antibody, and the functional group on the surface of the latex particle is a carboxyl group.
- ラテックス粒子表面の官能基が、少なくとも６３Å２／官能基以下の間隔で導入されたものであり、該粒子の平均粒経が５０ｎｍ~１５０μｍである、請求項１~３のいずれかに記載のラテックス粒子。 The latex according to any one of claims 1 to 3, wherein the functional groups on the surface of the latex particles are introduced at intervals of at least 63 cm 2 / functional group or less, and the average particle size of the particles is 50 nm to 150 µm. particle.
- 混合ポリエチレングリコールがポリエチレングリコール部の数平均分子量が２０００~８０００ｇ／ｍｏｌの長鎖ポリエチレングリコール分子群と１５００~４０００ｇ／ｍｏｌの短鎖ポリエチレングリコール分子群からなり、かつ、長鎖ポリエチレングリコール分子群と短鎖ポリエチレングリコール分子群の平均分子量の差が少なくとも３０００ｇ／ｍｏｌである、請求項１~４のいずれかに記載のラテックス粒子。 The mixed polyethylene glycol is composed of a long chain polyethylene glycol molecule group having a polyethylene glycol part number average molecular weight of 2000 to 8000 g / mol and a short chain polyethylene glycol molecule group having 1500 to 4000 g / mol. The latex particles according to any one of claims 1 to 4, wherein the difference in average molecular weight of the chain polyethylene glycol molecule group is at least 3000 g / mol.
- 混合ポリエチレングリコールがα−メトキシ−ポリ（エチレングリコール）−ペンタエチレンヘキサミンに由来し、ポリ（エチレングリコール）が約５０００ダルトンの分子量を有するものと約２０００ダルトンの分子量を有するものを含んで成り、前者と後者が３：５~２：５のモル比で存在する、請求項１~４のいずれかに記載のラテックス粒子。 The mixed polyethylene glycol is derived from α-methoxy-poly (ethylene glycol) -pentaethylenehexamine, the poly (ethylene glycol) comprising those having a molecular weight of about 5000 daltons and those having a molecular weight of about 2000 daltons, the former The latex particles according to any one of claims 1 to 4, wherein the latter and the latter are present in a molar ratio of 3: 5 to 2: 5.
- 抗体または抗原が抗体であって、抗フェリチン抗体である、請求項６記載のラテックス粒子。 The latex particle according to claim 6, wherein the antibody or antigen is an antibody and is an anti-ferritin antibody.
- ラテックス粒子が、ＡＪ２６ ＣＯＯＨ−Ｃｌｅａｎ（登録商標）である、請求項７記載の粒子。 The particles according to claim 7, wherein the latex particles are AJ26 COOH-Clean (registered trademark).
- 請求項１記載の免疫ラテックス粒子の製造方法であって、タンパク質のアミノ酸残基のアミノ基またはカルボキシル基と反応できる官能基を表面に有するラテックス粒子と抗体または抗原を、該官能基と該アミノ基またはカルボキシル基が反応して共有結合を形成できる条件下で接触させて該抗体または抗原を該ラテックス粒子に共有結合せしめ、次いで、こうして得られた抗体または抗原が共有結合したラテックス粒子と一末端にアミノ基またはカルボキシル基を有する混合ポリエチレングリコールを前記条件下で接触させて該混合ポリエチレングリコールを該ラテックス粒子にさらに共有結合せしめる工程を含んでなり、かつ、混合ポリエチレングリコールが長鎖ポリエチレングリコール分子群と短鎖ポリエチレングリコール分子群からなる、上記ラテックス粒子の製造方法。 The method for producing immune latex particles according to claim 1, wherein the latex particles having a functional group capable of reacting with an amino group or a carboxyl group of an amino acid residue of a protein, an antibody or an antigen, the functional group and the amino group. Alternatively, the antibody or antigen is covalently bound to the latex particle by contact under a condition in which a carboxyl group can react to form a covalent bond, and then the antibody or antigen thus obtained is covalently bound to the latex particle at one end. Contacting the mixed polyethylene glycol having an amino group or a carboxyl group under the above conditions to further covalently bond the mixed polyethylene glycol to the latex particles, and the mixed polyethylene glycol is a group of long-chain polyethylene glycol molecules. Is it a short-chain polyethylene glycol molecule group? Made, method of producing the latex particles.
- 被検体を含有するサンプル中に請求項１~８のいずれかに記載のラテックス粒子を存在させるステップ、
被検体とラテックス粒子に固定された抗体または抗原が結合し得る条件下でラテックス粒子が存在する被検体を含有するサンプルをインキュベートして、ラテックス粒子を凝集させるステップ、および
ラテックス粒子の凝集の程度を被検体が存在する尺度として評価するステップを含んで成る、被検体の検出方法。 A step of causing the latex particles according to any one of claims 1 to 8 to be present in a sample containing an analyte;
Incubating the sample containing the analyte in the presence of latex particles under conditions where the analyte and the antibody or antigen immobilized on the latex particles can bind, agglutinate the latex particles, and the degree of aggregation of the latex particles. A method for detecting an object comprising the step of evaluating as a measure of the presence of the object. - 被検体がフェリチンであり、抗体が抗フェリチン抗体である、請求項１０記載の方法。 The method according to claim 10, wherein the subject is ferritin and the antibody is an anti-ferritin antibody.
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