JP2005181110A - Kit for analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a kit for analysis, capable of preventing sensitivity decline by preventing interception of a bonding reaction between a low molecular ligand and a bonding partner, even if the labeling particle is a giant molecule. <P>SOLUTION: This kit for analysis includes (1) (a) a spacer group, having a reactive terminal group bondable to a primary specific partner to be bonded specifically to an analysis object material or bondable to a secondary specific partner to be bonded specifically to the primary specific partner to be bonded specifically to the analysis object material, (b) an aggregate including the low molecular ligand bonded to the opposite side end to the reactive terminal group of the spacer group, (2) (a) a bonding partner to be bonded specifically to the low molecular ligand, and (b) a marker containing the labeling particle bonded to the bonding partner. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an analysis kit. “Analysis” in the present specification includes “detection” for determining the presence / absence of an analysis target substance and “measurement” for quantitatively or semi-quantitatively determining the amount of the analysis target substance.

In vivo, biotin, which is one of the substances that catalyze the carbon fixation reaction, binds strongly to avidin mainly in egg white. Therefore, the combination of biotin and avidin is an immunoassay or hybridization assay. Widely used in measurement systems. A reagent in which N-hydroxysuccinimide (NHS) is bound to biotin is commercially available in order to bind a protein (for example, an antibody or an antigen) or a nucleic acid probe that specifically binds to an analysis target substance to biotin. Since NHS can bind to an amino group under mild conditions, it can easily bind a protein or a nucleic acid probe into which an amino group has been introduced and biotin.
On the other hand, since a labeling reagent in which avidin and a label are bound easily binds to a biotinylated compound, when constructing an immunoassay system, only prepare a reagent in which a tag antibody is biotinylated for various measurement items. When constructing a hybridization assay system, it is only necessary to prepare a biotinylated reagent for a nucleic acid probe, which is useful as a universal reagent. The combination of such a low molecular ligand (biotin) and its binding partner (avidin) is not limited to the biotin-avidin combination, but also a biotin-antibiotin antibody, a digoxin-antidigoxin antibody, and a dinitrophenyl group-antidinitrophenyl. A low molecular ligand such as a group antibody is bound to an antibody against the analyte (in a hybridization assay, a nucleic acid probe against the analyte), and a binding partner that can specifically bind to the low molecular ligand is used as a labeling reagent. Many combinations have been developed as universal reagents.

  In order to increase the sensitivity of immunoassays, the labeling reagents have been improved. In particular, when a fluorescent substance or a luminescent substance is labeled, attempts have been made to improve detection sensitivity by binding a large amount of fluorescent substance or luminescent substance to a protein such as an antibody or avidin used as a labeling reagent. In recent years, in the method of labeling fluorescent substances and luminescent substances by covalently binding to amino groups of proteins such as antibodies and avidin, the number of labeled molecules that can be bound to one molecule of antibody or avidin is limited. A method of binding a large amount of a fluorescent substance or a luminescent substance to a macromolecule (for example, thyroglobulin or a synthetic polymer) and binding a protein such as an antibody against the substance to be analyzed has been developed. On the other hand, in the method using an enzyme as a label, the enzyme molecule is cross-linked with a reagent such as glutaraldehyde to produce an enzyme polymer, and a protein such as an antibody or avidin is bound to the enzyme polymer. Methods have been developed to bind many enzyme molecules and proteins such as antibodies to the molecule. Similar to the immunoassay, a method using a macromolecule as a labeling reagent has been developed as an attempt to analyze DNA and RNA with high sensitivity.

In recent years, macromolecules used for labeling reagents have been actively designed, and molecules having a diameter of more than 20 nm have been developed. The present inventors also developed a label in which a fluorescent substance is enclosed in a styrene core of hydrophilic nanoparticles (diameter = 65 nm, 106.7 nm, and 167 nm) having a styrene core and a surface covered with polyethylene glycol. The immunoassay has been successfully improved in sensitivity (Patent Document 1).
WO02 / 097436A1 pamphlet

The measurement system using a macromolecule as a labeling reagent can bind a large amount of a labeling substance such as a fluorescent substance, a luminescent substance, or an enzyme to a protein such as an antibody or avidin, and can amplify a signal. Sensitivity analysis can be performed. However, in universal reagents using a combination of small molecule ligands (eg biotin) and binding partners (eg avidin) that specifically bind to them, the use of such macrolabeled molecules is often the The size of the steric hindrance was a steric hindrance, which caused a decrease in detection sensitivity in immunoassays and hybridization assays. Therefore, it has been desired to develop an analysis method that does not cause a decrease in detection sensitivity in a measurement system using a combination of a low molecular ligand (for example, biotin) and a binding partner (for example, avidin) that specifically binds to them. .
The present inventor is specific for an analyte in a universal reagent that uses a combination of a small molecule ligand (eg, biotin) and a binding partner (eg, avidin) that specifically binds to them, and a macrolabel molecule. As a result of intensive studies on the method of binding low molecular weight ligands and antibodies or nucleic acid probes that bind to the antibody, and the binding method of binding partners to macrolabeled molecules, the length between the low molecular ligand and the antibody or nucleic acid probe is more than a certain length. By inserting the spacer molecule, it was found that analysis can be performed with unexpectedly high sensitivity without being affected by steric hindrance due to the size of the giant labeled molecule.
The present invention is based on these findings.

Therefore, the present invention
(1) (a) For a primary specific partner that can bind to a primary specific partner that specifically binds to an analyte or that specifically binds to an analyte A spacer group having a reactive end group capable of binding to a secondary specific partner that specifically binds, and (b) a small molecule ligand bound to the end of the spacer group opposite to the reactive end group And (2) (a) a binding partner that specifically binds to the low molecular ligand, and (b) a label that includes labeled particles bound to the binding partner. Relates to the kit.

In a preferred embodiment of the analytical kit of the present invention, the labeled particles are fluorescent rare earth metal complex-encapsulated core-shell type particles.
In another preferred embodiment of the analytical kit of the present invention, the diameter of the labeled particles is 40 nm or more.
In still another preferred embodiment of the analytical kit of the present invention, the spacer group is a chain compound group having a length of 1 nm or more.
In still another preferred embodiment of the analytical kit of the present invention, the small molecule ligand is biotin, and the binding partner that specifically binds to the small molecule ligand is avidin or streptavidin, or the small molecule ligand is a physiologically active substance. The binding partner that specifically binds to the low molecular ligand is an antibody against the physiologically active substance or a fragment thereof, or the low molecular ligand is a physiologically active substance and specifically binds to the low molecular ligand. The binding partner is a receptor for the physiologically active substance or a fragment thereof.

The present invention also provides:
(1) (a) It specifically binds to a primary specific partner that specifically binds to the analyte, or a primary specific partner that specifically binds to the analyte. A secondary specific partner, (b) a spacer group bound to the primary specific partner or secondary specific partner via a reactive end group, and (c) the reactive end group of the spacer group A conjugate comprising a small molecule ligand bound to the opposite end of the membrane, and (2) (a) a binding partner that specifically binds to the small molecule ligand, and (b) a labeled particle that binds to the binding partner The present invention also relates to an analysis kit characterized by comprising a labeling substance.

  According to the analysis kit of the present invention, a combination of a conjugate (1) containing a low molecular ligand and a label (2) containing a labeled particle bound to a binding partner that specifically binds to the low molecular ligand. When constructing a measurement system, since the conjugate (1) containing a low molecular ligand has a spacer group, the binding reaction between the low molecular ligand and the binding partner is hindered even when the labeled particle is a macromolecule. In the immunoassay and hybridization assay, it is possible to prevent a decrease in sensitivity due to the use of a large target molecule, and it is possible to achieve a significant increase in sensitivity particularly at a low concentration.

The analysis kit according to the present invention is as described above.
(1) a conjugate containing a low molecular weight ligand, and (2) a label containing a labeled particle bound to the binding partner.
The combination of the low molecular ligand contained on the conjugate (1) side and the binding partner specifically bound to the low molecular ligand on the label (2) side is a conventionally known universal reagent. For example, a combination of biotin (small molecule ligand) -avidin (binding partner) or biotin (small molecule ligand) -streptavidin (binding partner) can be mentioned.

  Further, the low molecular ligand is a physiologically active substance, and an antibody against the physiologically active substance or a fragment thereof can be used as a binding partner that specifically binds to the low molecular ligand. Examples of such combinations include a combination of biotin (small molecule ligand) -anti-biotin antibody or fragment thereof (binding partner), digoxin (small molecule ligand) -anti-digoxin antibody or fragment thereof (binding partner), or A combination of a dinitrophenyl group (low molecular ligand) -anti-dinitrophenyl group antibody or a fragment thereof (binding partner) can be mentioned. The antibody fragment is a fragment containing a site that specifically binds to the physiologically active substance.

  Furthermore, a low molecular ligand is a physiologically active substance, and a receptor for the physiologically active substance or a fragment thereof can be used as a binding partner that specifically binds to the low molecular ligand. Examples of such combinations include estrogen (low molecular ligand) -estrogen receptor or a fragment thereof (receptor). The receptor fragment is a fragment containing a site that specifically binds to the physiologically active substance.

The first aspect of the conjugate (1) comprising the low molecular ligand is as follows:
(A1) A spacer group capable of binding to a primary specific partner that specifically binds to the analyte, or (a2) a primary specific partner that specifically binds to the analyte. A spacer group having a reactive end group capable of binding to a secondary specific partner that specifically binds, and (b) a small molecule ligand bound to the end of the spacer group opposite to the reactive end group (Hereinafter, the conjugate of this embodiment may be referred to as an “unbound conjugate”).

A second embodiment of the conjugate (1) comprising the low molecular ligand is as follows:
(A1) A primary specific partner that specifically binds to the analyte, or (a2) that specifically binds to a primary specific partner that specifically binds to the analyte. Secondary specific partners,
(B) a spacer group bound to the primary specific partner (a1) or the secondary specific partner (a2) via a reactive end group; and (c) the reactive end group of the spacer group; Includes a small molecule ligand bound to the opposite end (hereinafter, the conjugate of this embodiment may be referred to as a “bound conjugate”).

  The primary specific partner that specifically binds to the analyte is an immunological partner that specifically binds immunologically to the analyte when the analyte is a protein. For example, when the analyte is an antigen, the primary specific partner is an antibody, and when the analyte is an antibody, the primary specific partner is an antigen. When the analyte is a nucleic acid (for example, DNA or RNA), the primary specific partner is a nucleic acid (for example, DNA or RNA) that can hybridize thereto. Alternatively, a receptor for a physiologically active substance can be used as a primary specific partner for a physiologically active substance as an analyte, and conversely, a primary specific for a receptor as an analyte. As an active partner, a physiologically active substance that is a specific ligand of the receptor can also be used.

  The secondary specific partner that specifically binds to the primary specific partner is, for example, a substance to be analyzed that is an antigen, and immunology to the antigen as the primary specific partner. When an antibody that specifically binds specifically is used, an antibody that binds immunologically specifically to the primary specific partner can be used as the secondary specific partner.

  In the conjugate, the spacer group has a reactive end group at one end and a low molecular weight ligand at the other end. Said reactive end group is ready to bind to a primary specific partner or a secondary specific partner (in the case of unbound conjugates), or a primary specific partner or 2 It is bound to the next specific partner (in the case of a bound conjugate). The other end is bound to a small molecule ligand. Thus, the spacer group can be, for example, a succinimide group, an isothiocyanate group, a chlorosulfonyl group, a maleimide group, an amino group, or a hydrazide group as a reactive end group. The succinimide group, isothiocyanate group, or chlorosulfonyl group can be reacted with an amino group. The maleimide group can be reacted with, for example, an SH group, and the chlorosulfonyl group can be reacted with a phenolic hydroxyl group. The amino group can be reacted with a phosphate group or a carboxyl group, and the hydrazide group can be reacted with a saccharide.

In the spacer group, the divalent intermediate chain portion located between the reactive end group and the low molecular weight ligand can have an arbitrary structure, for example, substituted or unsubstituted, saturated, partially saturated Or an unsaturated aliphatic group (a nitrogen atom, an oxygen atom, a sulfur atom, a cycloaliphatic group, and / or an aromatic group may be interposed). Specifically, for example, the formula (I):
R ¹ — [— (CH ₂ ) _m —A—] _n — (CH ₂ ) _p —R ² (I)
[Wherein, R ¹ is a group capable of binding to a reactive terminal group, R ² is a group capable of binding to a small molecule ligand, and A is —CH ₂ —, —N—, —O— , -S-, -NHCO-, a substituted or unsubstituted phenylene group, or a substituted or unsubstituted cycloalkyl group having 3 to 8 carbon atoms, and m, n, and p are each independently 0 or It is an integer greater than or equal to 1]
The compound represented by these can be mentioned. In the compound represented by the formula (I), one or more hydrogen atoms in the main chain thereof are substituted with a substituent [for example, methyl group, oxo group (═O), amino group, or carboxyl group]. You can also.

In addition, when n in Formula (I) is 2 or more, _{m in} [— (CH ₂ ) _m —A—] which is a repeating unit can be independently selected for each repeating unit, and the same It can be a number or a different number. Similarly, when n in formula (I) is 2 or more, A in the repeating unit [— (CH ₂ ) _m —A—] can be independently selected for each repeating unit, They can be the same group or different groups. Moreover, it is preferable that m, n, and p in the said formula (I) are values from which the molecular length of the compound represented by the formula (I) is 1 nm to 30 nm.

  The length of the spacer group is not particularly limited, but is preferably 1 nm or more, more preferably 1.5 nm or more, and further preferably 2 nm or more. The upper limit of the spacer group is not particularly limited, but is preferably about 30 nm. When the spacer group length is less than 1 nm, it may be difficult to obtain the effects of the present invention, and when it exceeds 30 nm, synthesis may be difficult.

  The binding reaction with the primary specific partner or the secondary specific partner (in the case of “bound conjugate”) uses, for example, an amino group in the primary specific partner or secondary specific partner molecule. Can be carried out by covalent bonding.

で表されるスルホ−ＮＨＳ−ＬＣ−ＬＣ−ビオチン［sulfosuccinimidyl-6’-(biotinamido)-6-hexanamido hexanoate；分子長＝２．２４ｎｍ］、
式：

で表されるＮＨＳ−ＬＣ−ＬＣ−ビオチン［succinimidyl-6’-(biotinamido)-6-hexanamido hexanoate；分子長＝３．０５ｎｍ］、
式：

で表されるＮＨＳ−ＬＣ−ビオチン［succinimidyl-6-(biotinamido) hexanoate；分子長＝２．２４ｎｍ］、
式：

で表されるＮＨＳ−ＰＣ−ＬＣ−ビオチン［photocleavable biotin, NHS ester；分子長＝３．００ｎｍ］、
式：

で表されるＰＥＯ−マレイミド−ビオチン［(+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine；分子長＝２．９１ｎｍ］、
式：

で表されるビオチン−ＢＭＣＣ［1-biotinamido-4-[4’-(maleimidomethyl)cyclohexane-carboxamido] butane；分子長＝３．２６ｎｍ］、
式：

で表されるビオチン−ＰＥＯ−アミン［(+)-biotinyl-3,6-dioxaoctanediamine；分子長＝２．０４０ｎｍ］、又は
式：

で表されるビオチン−ＰＥＯ−ＬＣ−アミン［(+)-biotinyl-3,6,9-dioxaundecanediamine；分子長＝２．２９ｎｍ］
（以上、PIERCE社）などを挙げることができる。 As an example of the conjugate (1) that can be used in the present invention, when the low molecular ligand is biotin, for example,
formula:

Sulfo-NHS-LC-LC-biotin [sulfosuccinimidyl-6 ′-(biotinamido) -6-hexanamido hexanoate; molecular length = 2.24 nm] represented by
formula:

NHS-LC-LC-biotin [succinimidyl-6 ′-(biotinamido) -6-hexanamido hexanoate; molecular length = 3.05 nm] represented by
formula:

NHS-LC-biotin represented by the formula [succinimidyl-6- (biotinamido) hexanoate; molecular length = 2.24 nm],
formula:

NHS-PC-LC-biotin [photocleavable biotin, NHS ester; molecular length = 3.00 nm] represented by
formula:

PEO-maleimido-biotin represented by the formula [(+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine; molecular length = 2.91 nm],
formula:

Biotin-BMCC [1-biotinamido-4- [4 ′-(maleimidomethyl) cyclohexane-carboxamido] butane; molecular length = 3.26 nm],
formula:

Biotin-PEO-amine [(+)-biotinyl-3,6-dioxaoctanediamine; molecular length = 2.040 nm] represented by the formula:

Biotin-PEO-LC-amine [(+)-biotinyl-3,6,9-dioxaundecanediamine; molecular length = 2.29 nm]
(Above, PIERCE).

As described above, the label (2) contained in the analysis kit of the present invention is as follows.
(A) a binding partner that specifically binds to the small molecule ligand; and (b) a labeled particle that binds to the binding partner. The size of the labeled particles is not limited, but when the diameter of the labeled particles is 40 nm or more, an excellent effect can be obtained by binding a spacer group to the low molecular ligand. When the diameter of the label particles is particularly 60 nm or more, a remarkable effect can be obtained. The upper limit of the diameter of the labeled particle is not particularly limited, but when it is about 1000 nm, the mass and volume of the labeled particle are affected, and the binding between the analyte and the primary specific partner, or the primary specific partner and May cause dissociation of binding with a secondary specific partner.

  The analysis kit of the present invention is preferably used as a known time-resolved fluorescence measurement kit using, for example, fluorescent rare earth metal complex-encapsulated core-shell type particles as the labeling particles. In the time-resolved fluorescence measurement method, a known fluorescent rare earth metal complex can be used as a label. For example, a fluorescent rare earth metal complex-encapsulated core-shell type particle can be used as a label. The fluorescent rare earth metal complex-encapsulated core-shell type particle has a core-shell having a water-soluble polymer compound brush having a reactive functional group on the surface thereof on the surface of a core portion mainly composed of a water-insoluble polymer compound. Type particles, and a fluorescent rare earth metal complex is encapsulated in a core portion thereof.

Hereinafter, the fluorescent rare earth metal complex encapsulated core-shell type particles used in the time-resolved fluorescence measurement method will be described.
The core-shell type particle in which the fluorescent rare earth metal complex can be encapsulated in the core portion is, for example,
(A) a core part mainly composed of a water-insoluble polymer compound; and (b) a shell part mainly comprising a water-soluble polymer compound having a reactive functional group and covering the surface of the core part in a brush shape. Become.

  The water-insoluble polymer compound that can be used to form the core portion is a polymer compound that does not dissolve in water, and when the core portion is formed, a fluorescent rare earth metal complex is encapsulated therein. The polymer compound is not particularly limited as long as the polymer compound can be used. Examples of the water-insoluble polymer compound include hydrophobic polymers [eg, polystyrene, polymethyl methacrylate, poly (2-hydroxyethyl methacrylate), poly (N-isopropylacrylamide) polyisoprene, polyvinyl chloride, polylactic acid. , Polylactone, polylactam, etc.], a water-soluble polymer (eg, polyvinyl alcohol, polyacrylamide, polydimethylacrylamide, polyallylamine, polyacrylic acid, etc.) cross-linked gel, or a water-insoluble natural polymer compound (eg, gelatin or Polysaccharides).

  The core portion may be substantially formed of only one type of the water-insoluble polymer compound or may be substantially formed of a combination of two or more types of the water-insoluble polymer compound.

  The shape of the core part is not particularly limited, but is generally approximately spherical or approximately elliptical. Further, the dimension of the core part is not particularly limited and can be appropriately changed according to the use, but the diameter of the substantially spherical core part is generally about 5 nm to 500 nm.

  The water-soluble polymer compound that can be used to form the shell portion is a linear polymer compound that can be bonded to the surface of the core portion at one end (bonding end), and As long as it has a reactive functional group at one end (free end) or a reactive functional group can be introduced and can be arranged in a brush shape on the surface of the core portion, in particular It is not limited. In this specification, the “shell part covering the surface of the core part in a brush shape” means that the shell part is composed of a number of linear water-soluble polymer compounds, and each of the linear water-soluble polymer compounds is one of them. In the reaction solution in which the other free end is reacted with at least the water-soluble polymer compound and the binding partner, the reaction solution is thread-like or rod-like in the reaction solution system. It means that it protrudes from the surface. In addition, at the free ends of these brush-like water-soluble polymer compound chains, there are reactive functional groups that can bind to the binding partners, respectively, so that the outermost shell of the shell portion is composed of a large number of reactive functional groups. Covered.

  Examples of the water-soluble polymer compound include polyethylene glycol (PEG), polyvinyl alcohol, polyvinyl pyrrolidone, polyamino acid, polyacrylic acid, polydimethylaminoethyl methacrylate, and polyallylamine. PEG or polyvinyl Alcohol is preferred.

  The water-soluble polymer compound chains constituting the shell part are substantially formed from only one kind of the water-soluble polymer compounds described above, or the water-soluble polymer compound chains are mutually connected. Can be formed substantially from a combination of two or more different water-insoluble polymer compounds. In addition, the lengths of the respective chains do not have to be the same, and even when each water-soluble polymer compound chain is formed of only one type of water-soluble polymer compound, the length of one type is different. It can be formed substantially only from a chain, or can be formed substantially from a combination of two or more types of chains. Furthermore, even when each water-soluble polymer compound chain is substantially formed from a combination of two or more different water-insoluble polymer compounds, it is also possible to select and combine different chain lengths. .

  It is preferable that the water-soluble polymer compound chain constituting the shell portion substantially completely covers the entire surface of the core portion. In addition, each water-soluble polymer compound chain constituting the shell portion does not need to have substantially the same length, but is approximately spherical or approximately elliptical depending on the free end of each water-soluble polymer compound chain. A shell partial shell surface is preferably formed.

  The ratio between the diameter of the core portion and the thickness of the shell portion can be appropriately changed according to the application. For example, the diameter of the core portion can be, for example, 5 nm to 500 nm, and the thickness of the shell portion can be, for example, 5 nm to 500 nm.

  The reactive functional group present at the free end of the water-soluble polymer compound chain is the other end (free end) before the one end (bonding end) of the water-soluble polymer compound is bonded to the core surface. Alternatively, after one end (bonding end) of the water-soluble polymer compound is bonded to the surface of the core portion, it can be introduced into the other end (free end). Any of these reactive functional groups is stable in water (or an aqueous solvent), and can react with a binding partner when using the fluorescent rare earth metal complex-encapsulated core-shell type particle as a labeling substance. As long as it is a functional group, it is not particularly limited, and examples thereof include an aldehyde group, a carboxyl group, a mercapto group, an amino group, a maleimide group, a vinyl sulfone group, and a methanesulfonyl group. Group, carboxyl group or maleimide group is preferred.

  In addition, in order to adjust the amount of binding partner introduced when using fluorescent-rare earth metal complex-encapsulated core-shell type particles as labeled particles, a water-soluble polymer compound chain having no reactive functional group present at the free end It is also possible to mix (for example, a water-soluble polymer compound chain having a hydrogen atom, a methylene group, a hydroxyl group, or the like at a free end) at an arbitrary ratio. In this case, in order not to interfere with the reaction between the binding partner to be introduced and the functional group present at the free end, the length of the water-soluble polymer compound chain not having the reactive functional group is set to the water-soluble high molecular weight having the reactive functional group. It is desirable to make it shorter than the molecular compound chain.

As a method for preparing core-shell type particles having a water-soluble polymer compound brush having a reactive functional group on the surface layer on the surface of a core portion mainly composed of a water-insoluble polymer compound, there have been reported so far. Various known methods can be applied. As the known method, for example,
(1) (a) A block copolymer (parent-hydrophobic block copolymer) in which a hydrophilic segment having a reactive functional group and a hydrophobic segment are combined, and (b) a hydrophobic polymer are mixed. Emulsion method of preparing particles;
(2) A dispersion polymerization method in which a hydrophobic monomer is polymerized using a water-soluble polymer macromonomer having a reactive functional group as a dispersant; or (3) a method of introducing a water-soluble polymer compound brush on the surface of hydrogel particles, etc. Can be mentioned.

  Examples of the method for synthesizing the hydrophilic-hydrophobic block copolymer used in the emulsion method (1) and the method for synthesizing the water-soluble polymer macromonomer having a reactive functional group used in the dispersion polymerization method (2) include: It is possible to use a method (for example, WO96 / 33233, WO99 / 571743, JP11-322917, or JP2001-324507) already developed by the present inventor and its collaborators. it can.

［式中、Ｒ^１Ａ及びＲ^２Ａは、独立して、炭素数１〜１０のアルコキシ基、アリールオキシ基、又はアリール−（炭素数１〜３のアルキルオキシ）基であるか、あるいは、Ｒ^１Ａ及びＲ^２Ａは、一緒になって、炭素数１〜６のアルキル基で置換されていてもよい式：
−Ｏ−ＣＨ（Ｒ’）−ＣＨ−Ｏ−
（ここでＲ’は水素原子又は炭素数１〜６のアルキル基である）
で表されるエチレンジオキシであるか、あるいは、Ｒ^１Ａ及びＲ^２Ａは、一緒になって、オキシ（＝Ｏ）であり、
Ｌは、式：
−ＣＨ（Ｒ^３Ａ）−Ｏ−ＣＯ−ＣＨ（Ｒ^４Ａ）−
又は
−（ＣＨ_２）_ｒ−
で表される２価の基であり、ここで、Ｒ^３Ａ及びＲ^４Ａは、独立して、水素原子、炭素数１〜１０のアルキル基、アリール基、又はアリール−（炭素数１〜３のアルキルオキシ）基であり、ｒは２〜５の整数であり、ｍ_Ａは２〜１０，０００の整数であり、ｎ_Ａは２〜１０，０００の整数であり、ｐ_Ａは１〜５の整数であり、ｑは０又は１〜２０の整数であり、そして、Ｚ_Ａは、ｑが０であるとき、水素原子、アルカリ金属、アセチル基、アクリロイル基、メタクリロイル基、シンナモイル基、ｐ−トルエンスルホニル基、２−メルカプトプロピオニル基、２−アミノプロピオニル基、アリル基、又はビニルベンジル基であり、ｑが１〜２０の整数であるとき、炭素数１〜６のアルコキシカルボニル基、カルボキシルメルカプト基、又はアミノ基である］
で表されるヘトロテレケリックブロックコポリマーを挙げることができる。前記の式（ＩＡ）で表されるヘトロテレケリックブロックコポリマーは、例えば、ＷＯ９６／３３２３３号公報に記載の製造方法により調製することができる。 Examples of the compound that can be used as the parent-hydrophobic block copolymer or the water-soluble polymer macromonomer include, for example, the formula (IA) described in WO96 / 33233:

[Wherein, R ^1A and R ^2A are independently an alkoxy group having 1 to 10 carbon atoms, an aryloxy group, or an aryl- (alkyloxy having 1 to 3 carbon atoms) group, or R ^1A And R ^2A together may be substituted with an alkyl group having 1 to 6 carbon atoms:
-O-CH (R ')-CH-O-
(Where R ′ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)
Or R ^1A and R ^{2A taken} together are oxy (═O),
L is the formula:
—CH (R ^3A ) —O—CO—CH (R ^4A ) —
Or - _{(CH 2) r} -
In which R ^3A and R ^4A are independently a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an aryl group, or aryl- (having 1 to 3 carbon atoms). alkyl) group, r is integer from 2 to 5, _{m a} is an integer of 2 to 10,000, _{n a} is an integer of 2 to 10,000, _{p a} is from 1 to 5 is an integer, q is 0 or an integer of 1 to 20, and, _{Z a} when q is 0, a hydrogen atom, an alkali metal, an acetyl group, an acryloyl group, methacryloyl group, cinnamoyl group, p- toluene A sulfonyl group, a 2-mercaptopropionyl group, a 2-aminopropionyl group, an allyl group, or a vinylbenzyl group, and when q is an integer of 1 to 20, an alkoxycarbonyl group having 1 to 6 carbon atoms, a carboxyl mercapto group, Or Is an amino group]
And a heterotelechelic block copolymer represented by the formula: The heterotelechelic block copolymer represented by the above formula (IA) can be prepared, for example, by the production method described in WO96 / 33233.

（式中、Ａ’及びＢ’は、相互に独立して、有機シリル型のアミノ保護基を表すか、あるいは、それらが結合する窒素原子と一緒になって、４〜７員のジシラ−アザシクロヘテロ環式環を形成することのできる有機シリル型のアミノ保護基であり、
Ｙは、水素原子、アルカリ金属、又は適当な反応によりアルカリ金属に代えて導入可能な有機基であり、
Ｒは水素原子又は炭素数１〜６のアルキル基であり、
ｎ_Ｂは１〜２０，０００の整数であり、そして、
ｍ_Ｂは０〜２０，０００の整数である）
で表されるポリオキシエチレン誘導体を挙げることができる。前記の式（ＩＢ）で表されるポリオキシエチレン誘導体は、例えば、ＷＯ９９／５７１７４３号公報に記載の製造方法により調製することができる。 Examples of the compound that can be used as the above-mentioned parent-hydrophobic block copolymer or water-soluble polymer macromonomer include the formula (IB) described in WO99 / 571743:

Wherein A ′ and B ′, independently of one another, represent an organosilyl-type amino protecting group, or together with the nitrogen atom to which they are attached, a 4-7 membered disila-aza An organosilyl-type amino protecting group capable of forming a cycloheterocyclic ring;
Y is a hydrogen atom, an alkali metal, or an organic group that can be introduced in place of the alkali metal by an appropriate reaction,
R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms,
n _B is an integer from 1 to 20,000, and
m _B is an integer of 0 to 20,000)
The polyoxyethylene derivative represented by these can be mentioned. The polyoxyethylene derivative represented by the formula (IB) can be prepared, for example, by the production method described in WO99 / 571743.

（式中、Ｒ^１Ｃ、Ｒ^２Ｃ、及びＲ^３Ｃは、相互に独立して、直鎖若しくは分岐のアルキル基又はアラルキル基を表し、
Ｚ_Ｃは、水素原子、アクリロイル基、メタクリロイル基、ビニルベンジル基、アリル基、パラトルエンスルホニル基、モノ−若しくはジ−低級アルキル置換アミノ基、カルボキシル基若しくはそのエステル基を有するアルキル基、アルデヒド基若しくはそのアセタール基を有するアルキル基、及びアルカリ金属からなる群より選ばれ、
ｍ_Ｃは０又は１であり、
ｎ_Ｃは０〜２０，０００の整数であり、そして、
ｐ_Ｃは正数２又は３であるが、
但し、ｍ_Ｃとｎ_Ｃは同時に０とならない）
で表される有機シリルスルフィド基含有化合物又はポリオキシエチレン誘導体を挙げることができる。前記の式（ＩＣ）で表される有機シリルスルフィド基含有化合物又はポリオキシエチレン誘導体は、例えば、特開平１１−３２２９１７号公報に記載の製造方法により調製することができる。 Examples of the compound that can be used as the above-mentioned parent-hydrophobic block copolymer or water-soluble polymer macromonomer include, for example, the formula (IC) described in JP-A-11-322917:

(Wherein R ^1C , R ^2C and R ^3C each independently represent a linear or branched alkyl group or an aralkyl group,
Z _C represents a hydrogen atom, an acryloyl group, a methacryloyl group, a vinylbenzyl group, allyl group, p-toluenesulfonyl group, mono- - or di - lower alkyl-substituted amino group, an alkyl group having a carboxyl group or its ester group, or aldehyde group Selected from the group consisting of an alkyl group having an acetal group and an alkali metal,
m _C is 0 or 1,
n _C is an integer from 0 to 20,000, and
p _C is a positive number 2 or 3,
However, m _C and n _C are not 0 at the same time)
An organic silyl sulfide group-containing compound or a polyoxyethylene derivative represented by The organic silyl sulfide group-containing compound or polyoxyethylene derivative represented by the above formula (IC) can be prepared, for example, by the production method described in JP-A-11-322917.

のユニットを表し、Ｒ^１は、水素原子又は低級アルキル基であり、Ｒ^２は、水素原子、メチル基、１−メチルエチル基、１−メチルプロピル基、２−メチルプロピル基、２−ベンジルオキシカルボニルエチル基、又はベンジルオキシカルボニルメチル基であり、Ｌ_１は、式：
−（ＣＨ_２）ｑ_Ｄ−Ｏ−
（ここで、ｑ_Ｄは１〜６の整数である）の連結基を表し、Ｌ_２は、原子価結合又は式：
−ＣＨ_２ＣＨ_２ＮＨ−
の連結基を表し、Ｌ_３は、カルボニル基、メチレン基、

の基を表し、Ｒは、水素原子又はメチル基を表し、ｍ_Ｄは１０〜２０，０００の整数であり、そして、ｎ_Ｄは０〜１０，０００の整数である］
で表されるホモポリマー又はブロックコポリマーを挙げることができる。なお、官能基Ａは、特に限定されるものではないが、例えば、アルデヒド基（−ＣＨＯ）、アミノ基（−ＮＨ_２）、メルカプト基（−ＳＨ）、水酸基（−ＯＨ）、カルボキシル基（−ＣＯＯＨ）を挙げることができる。前記の式（ＩＤ）で表されるホモポリマー又はブロックコポリマーは、例えば、特開２００１−３２４５０７号公報に記載の製造方法により調製することができる。 Furthermore, examples of the compound that can be used as the above-mentioned parent-hydrophobic block copolymer or water-soluble polymer macromonomer include, for example, the formula (ID) described in JP-A No. 2001-324507:
_{A-L 1 - (CH 2} CH 2 O) m D -L 2 - (B) n D -L 3 -CR = CH 2 (ID)
[Wherein A represents a functional group and B represents

R ¹ is a hydrogen atom or a lower alkyl group, and R ² is a hydrogen atom, a methyl group, a 1-methylethyl group, a 1-methylpropyl group, a 2-methylpropyl group, 2-benzyloxy. A carbonylethyl group or a benzyloxycarbonylmethyl group, and L ₁ is a group represented by the formula:
- _{(CH 2)} q D -O-
Wherein q _D is an integer from 1 to 6, and L ₂ is a valence bond or a formula:
—CH ₂ CH ₂ NH—
L ₃ represents a carbonyl group, a methylene group,

R represents a hydrogen atom or a methyl group, m _D is an integer of 10 to 20,000, and n _D is an integer of 0 to 10,000]
The homopolymer or block copolymer represented by these can be mentioned. The functional group A is not particularly limited. For example, the functional group A is an aldehyde group (—CHO), an amino group (—NH ₂ ), a mercapto group (—SH), a hydroxyl group (—OH), a carboxyl group (— COOH). The homopolymer or block copolymer represented by the formula (ID) can be prepared, for example, by the production method described in JP-A-2001-324507.

  According to the emulsion method (1) of core-shell type particles, each compound represented by the formula (IA), formula (IB), formula (IC), or formula (ID) (ie, parent-hydrophobic) The core-shell type particles (that is, the particles before encapsulating the fluorescent rare earth metal complex) can be prepared by mixing the type block copolymer) and the hydrophobic polymer.

  Further, according to the dispersion polymerization method (2) of the core-shell type particles, each compound represented by the formula (IA), formula (IB), formula (IC), or formula (ID) (that is, By polymerizing a hydrophobic monomer using a water-soluble polymer macromonomer) as a dispersant, core-shell type particles (that is, particles before encapsulating the fluorescent rare earth metal complex) can be prepared.

Further, according to the dispersion polymerization method (2) of the core-shell type particles, by using a water-soluble polymer macromonomer as a dispersant, and then suspending the fluorescent rare earth metal complex and the hydrophobic monomer, Encapsulation of the fluorescent rare earth metal complex and preparation of the core-shell type particles can be performed simultaneously. Further, for example, as the group Z _A in the above formula (IA), the group Y in the formula (IB), or the group Z _C in the formula (IC), a carboxyl group which is a functional group capable of forming a complex with a fluorescent rare earth metal, It is also possible to prepare a core-shell type particle by encapsulating a fluorescent rare earth metal complex in which a β-diketone ligand or a ferroin-based ligand is introduced to stabilize the fluorescent rare earth metal complex.

  The fluorescent rare earth metal complex that can be used in the time-resolved fluorescence measurement analyzes the signal from the outside of the core-shell type particle (including detection and measurement) even when encapsulated in the core part of the core-shell type particle. For example, lanthanoid (eg, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium Or lutetium), yttrium, or scandium, and the like. Lanthanoid complexes are preferable, and europium, samarium, terbium, or dysprosium complexes are more preferable.

  The diameter of the core-shell type particles encapsulating the fluorescent rare earth metal complex can also be appropriately changed according to the use, but in the case of a roughly spherical particle, it can be about 10 nm to 1 mm.

  Fluorescent rare earth metal complex-encapsulated core-shell type particles that can be used in the time-resolved fluorescence measurement method are prepared by, for example, a known production method (for example, the production method described in International Publication No. WO02 / 097436). Can do.

  For example, in the production method described in International Publication No. WO02 / 097436, for example, core-shell type particles (that is, particles before encapsulating a fluorescent rare earth metal complex) prepared by various known methods described above are used. In order to encapsulate the fluorescent rare earth metal complex, first, an organic solvent (for example, acetone or toluene) that swells the water-insoluble polymer compound that forms the core portion of the core-shell type particle is included in a certain ratio. The core-shell type particles and the fluorescent rare earth metal complex are immersed in the solution. By the immersion, the water-insoluble polymer compound swells, and the fluorescent rare earth metal complex is taken into the core portion with the swelling. Subsequently, when the organic solvent is removed from the mixture, the water-insoluble polymer compound contracts, but the hydrophobic fluorescent rare earth metal complex cannot escape from the core part and is encapsulated in the core part. . If desired, a fluorescent rare earth metal complex-encapsulated core-shell type particle can be obtained by removing the fluorescent rare earth metal complex that has not been incorporated into the core portion.

  In the production method, the method for removing the organic solvent from the mixture is not particularly limited. For example, the method of evaporating the organic solvent by evaporation or the like, or the method of shrinking in a non-solvent ( For example, a method in which a solution containing an organic solvent is replaced with a solution not containing the organic solvent) can be used.

  In addition, the ratio of the organic solvent in the organic solvent-containing solution used to swell the core portion may swell the water-insoluble polymer compound to such an extent that the fluorescent rare earth metal complex is incorporated into the water-insoluble polymer compound. Although it will not specifically limit as long as it is a ratio which can be performed, For example, it can be 40-60 (vol / vol)%.

  In the labeled body (2) used in the analysis kit of the present invention, the binding partner can be bound as an introduced substance to the reactive functional group present on the surface of the fluorescent rare earth metal complex-encapsulated core-shell type particle. it can. The binding partner and the reactive functional group present on the surface of the fluorescent rare earth metal complex-encapsulated core-shell type particle may be selected from the known binding methods and the types of the introduced product and the reactive functional group. It can be selected as appropriate according to the conditions. For example, when the reactive functional group is an aldehyde group, it can be bound by forming a Schiff base with the amino group of the binding partner.

  Moreover, when a reactive functional group is a carboxyl group, it can couple | bond with the amino group of a binding partner using a condensing agent (for example, carbodiimide etc.). Alternatively, the carboxy group can be activated by succinimide or maleimide in advance, and can be bound by mixing with the binding partner in that state.

  As the core-shell type particles encapsulating the fluorescent rare earth metal complex, the various particles described so far can be used. However, a large amount of the europium chelate having a long lifetime fluorescence is contained in the styrene portion as the core, and the external particle The surface has hydrophilic properties such as hydrophilic polyethylene glycol (PEG) in the form of a brush, and has the property that the binding partner can be bound to the functional group at the PEG end by chemical modification while keeping the activity. High functional particles are preferred.

  When this particle is irradiated with excitation light having a wavelength characteristic in the vicinity of 350 nm as a pulse, and a fluorescence having a wavelength characteristic at 615 nm is measured after a period of time in microseconds, a high S / N ratio is not affected by surrounding fluorescence. Can be detected. Further, these particles do not require a solution at the time of observation, and can be measured in a dry state like a fluorescent substance. One europium chelate has a limit in measurement sensitivity, but this reagent in which a number of europium chelate molecules are encapsulated in particles can be measured with higher sensitivity than a detection system using an enzyme and a luminescent substrate.

  An object that can be analyzed by the analysis kit according to the present invention (that is, an analyte) is not particularly limited as long as a specific binding partner exists, but for example, a protein (for example, an antigen) , Antibody, receptor, etc.), nucleic acid (eg, DNA or RNA), or a biologically active chemical substance. Further, the test sample that can be analyzed with the analysis kit according to the present invention is not particularly limited as long as it is a sample that may contain the analysis target substance, and in particular, a biological sample such as blood, serum, plasma, Examples thereof include urine, spinal fluid, and cell or tissue disruption fluid.

The analysis kit of the present invention can be used in any analysis method. For example, it can be used in an analysis method including the following steps.
(1) a step of immobilizing a specific binding partner that specifically binds to a substance to be analyzed on the surface of an insoluble carrier;
(2) a step of bringing a test sample possibly containing an analyte into contact with an insoluble carrier on which the specific binding partner is immobilized and supported;
(3) Immobilizing and carrying the specific binding partner, and bringing the insoluble carrier into contact with the test sample,
(A) a primary specific partner that specifically binds to the analyte, (b) a spacer group that binds to the primary specific partner via a reactive end group, and (c) the spacer Contacting a conjugate comprising a bound small molecule ligand bound to the end of the group opposite the reactive end group;
(4) removing from the insoluble carrier an unreacted conjugate with the analyte to be bound to the specific binding partner immobilized on the insoluble carrier;
(5) Immobilizing and carrying the specific binding partner, contacting with a test sample, and further contacting the binding body with an insoluble carrier,
(A) contacting a labeled body comprising a binding partner that specifically binds to the small molecule ligand, and (b) a labeled particle bound to the binding partner;
(6) removing from the insoluble carrier an unreacted label that is unreacted with the conjugate further bound to the analyte to be bound to the specific binding partner immobilized on the insoluble carrier;
(7) including a step of analyzing a signal derived from the label of the label further bound to the conjugate further bound to the analyte to be analyzed bound to the specific binding partner immobilized on the insoluble carrier. An analysis method characterized by

Moreover, the analysis kit of the present invention can be used, for example, in an analysis method including the following steps.
(1) a step of bringing an insoluble carrier into contact with a test sample that may contain an analyte, and immobilizing the analyte on the insoluble carrier;
(2) a step of bringing a primary specific partner that specifically binds to the analyte into contact with an insoluble carrier on which the analyte is immobilized and supported;
(3) To the insoluble carrier in which the analyte is immobilized and supported and brought into contact with the primary specific partner,
(A) a secondary specific partner that specifically binds to the primary specific partner, (b) a spacer group bound to the secondary specific partner via a reactive end group, and (c) ) Contacting a conjugate comprising a bound small molecule ligand attached to the end of the spacer group opposite the reactive end group;
(4) removing the unreacted conjugate from the primary specific partner bound to the analyte to be analyzed immobilized on the insoluble carrier from the insoluble carrier;
(5) The insoluble carrier immobilized on the analyte, contacted with a primary specific partner, and further contacted with the conjugate;
(A) contacting a labeled body comprising a binding partner that specifically binds to the small molecule ligand, and (b) a labeled particle bound to the binding partner;
(6) removing from the insoluble carrier an unreacted label that is unbound with the primary specific partner bound to the analyte to be immobilized immobilized on the insoluble carrier;
(7) analyzing the signal derived from the label of the label further bound to the conjugate further bound to the primary specific partner bound to the analyte to be analyzed immobilized on the insoluble carrier; An analysis method characterized by comprising.

For example, a case where a two-step sandwich immunoassay is performed using the analysis kit of the present invention will be described below.
First, a protein that can bind to an analyte (eg, antigen A) (eg, antibody B) is immobilized on a reaction vessel, and a test that may contain the analyte (eg, antigen A) The sample is brought into contact with the sample, and the substance to be analyzed (for example, antigen A) is bound onto the reaction vessel via the protein (for example, antibody B). After washing away unreacted analyte (e.g., antigen A), the bound conjugate, i.e.,
(A) a primary specific partner (eg, antibody C) that specifically binds to the analyte (eg, antigen A),
(B) a spacer group bound to the primary specific partner (eg, antibody C) via a reactive end group, and (c) bound to the end of the spacer group opposite to the reactive end group. Bound small molecule ligand (eg biotin)
And binding the analyte to be analyzed (for example, antigen A) bound to the reaction vessel with the primary specific partner (for example, antibody C), A bound conjugate is bound onto the reaction vessel via the substance to be analyzed (for example, antigen A). Subsequently, after washing away unreacted conjugated conjugate, the label, that is,
(A) contacting with a labeled body comprising a binding partner (for example, avidin) that specifically binds to the small molecule ligand (for example, biotin), and (b) a labeled particle bound to the binding partner (for example, avidin). The low molecular weight ligand (for example, biotin) of the binding type bound to the reaction container is bound to the binding partner (for example, avidin) of the label, and the label is bound to the reaction container. . At this time, since the binding conjugate includes a spacer group, the binding between the low molecular ligand (for example, biotin) and the binding partner (for example, avidin) is not hindered by steric hindrance. Subsequently, after the unreacted label is washed away, the signal of the labeled particles is analyzed by, for example, time-resolved fluorescence measurement.

  In addition to the two-step sandwich immunoassay described above, the present invention can be applied to any immunoassay such as a one-step sandwich immunoassay, a delayed one-step immunoassay, a direct competitive immunoassay, or an indirect competitive immunoassay. When the analyte is an antigen, the primary specific partner is an antibody. Conversely, when the analyte is an antibody, the primary specific partner is an antigen. Furthermore, in the case of a receptor binding assay, if the analyte is a receptor, the primary specific partner is a ligand, and conversely if the analyte is a ligand, the primary specific partner is the receptor. Become. In the hybridization assay, when the analyte is DNA or RNA, the primary specific partner is a probe having a sequence that hybridizes in a complementary manner.

  The analysis kit of the present invention can be applied to any reaction vessel, and can be applied to, for example, a microtitration plate, a styrene ball, a microbead, a ferrite bead, or a glass plate. It can also be applied to a reaction system in which a low molecular ligand in a liquid that does not require a solid phase for the reaction and a labeled body including a labeled particle bound to a binding partner that specifically binds to the low molecular ligand. .

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention. In the following examples, biotin as a small molecule ligand, streptavidin (SA) as a binding partner, and europium (Eu) complex encapsulated nanoparticles (diameters: 167 nm and 65 nm) as labeling particles are used. went. In the following examples, “NHS” is N-hydroxysuccinimide, and “NHS-LC-LC-biotin” is succinimidyl-6 ′-(biotinamide) -6-hexaneamidohexanoate [ “LC-SPDP” is succinimidyl-3- (2-pyridyldithio) hexanoate [Succinimidyl-3- (2-pyridyldithio) hexanoate].

Example 1: Immunoassay of milk allergen (1) Binding of biotin to anti-human IgE mouse monoclonal antibody (IgG) 1 mg of anti-human IgE mouse monoclonal antibody was dissolved in 1 mL of 0.1 M phosphate buffer (pH 8.0). 10 μL of NHS-biotin (molecular length: 1.35 nm) adjusted to 4.26 mg / mL with dimethylformamide, or NHS-LC-LC-biotin (molecular length: 3.05 nm) adjusted to 7.1 mg / mL with dimethylformamide ) 10 μL was added to the antibody solution and reacted at room temperature for 1 hour. Unreacted biotin reagent was removed with a Sephadex G-25 column (1.5 cm × 12 cm; eluent, 20 mM phosphate buffer (pH 7.0) containing 0.15 M NaCl, and two types of biotin-conjugated antibody fractions. Got the minute.

(2) Binding of SA to Eu complex encapsulated nanoparticles (a) Preparation of maleimide group-introduced Eu complex encapsulated nanoparticles Suspension of Eu complex encapsulated nanoparticles (diameter: 167 nm) whose surface is covered with amino groups in distilled water To 1.4 mL of the solution (5.18 mg / mL), 22 μL of 4/3 M phosphate buffer (pH 7.0) was added and stirred. To the resulting suspension, 66 μL of a bi-crosslinking reagent [N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate; SMCC] adjusted to 50 mg / mL with dimethylformamide was added and brought to room temperature. By shaking for 1 hour under light shielding, the amino group on the particle surface was converted to a maleimide group. Subsequently, the precipitate was removed by centrifugation at 14,000 rpm for 10 minutes, and further filtered through a membrane filter having a pore size of 0.2 μm. The filtrate (1.4 mL) was added to a Sepharose CL-6B column [1.5 cm × 90 cm; eluent = 50 mM phosphate buffer (pH 7.0) containing 1 mM-EDTA; flow rate = 20 mL / min; fraction = 2 mL / Fraction] was added to obtain a nanoparticle fraction encapsulating a maleimide group-introduced Eu complex.

(B) Preparation of SH group-introduced SA LC- adjusted to 20 mg / mL with dimethylformamide in 1 mL of SA (Chemicon) adjusted to 0.95 mg / mL with 0.1 M phosphate buffer (pH 8.0) 6.75 μL of SPDP (Pierce) was added and shaken at room temperature for 1 hour. Next, 200 μL of dithiothreitol adjusted to 250 mM with 50 mM acetate buffer (pH 4.2) was added and shaken at room temperature for 30 minutes. The total amount of the reaction solution was applied to a Sephadex G-25 column [1.5 cm × 12 cm; eluent = 0.1 M phosphate buffer (pH 7.0) containing 1 mM-EDTA] to obtain an SH group-introduced SA fraction. It was.

(C) Preparation of SA-bonded Eu complex-encapsulated nanoparticles Maleimide group-introduced Eu complex-encapsulated nanoparticles (3.8 mg / 3.57 mL) suspended in 50 mM phosphate buffer (pH 7.0) containing 1 mM-EDTA; The SH group-introduced SA (0.863 mg / 3 mL) dissolved in the same buffer as above was mixed, and mixed by inverting at room temperature for 18 hours under light shielding. In order to block the maleimide group remaining on the particles, 50 μL of 2-mercaptoethylamine prepared to 1 mg / mL with distilled water was added to the reaction solution, and mixed by inverting at room temperature for 1 hour under light shielding. The reaction solution was applied to a Sepharose CL-6B column (1.5 cm × 90 cm; eluent = 50 mM Tris-HCl (pH 7.8) containing 0.15 M NaCl; flow rate = 20 mL / min, fraction = 2 mL / fraction). Thus, a nanoparticle fraction encapsulating SA-bound Eu complex was obtained.

(D) Preparation of anti-human IgE mouse monoclonal antibody-bound Eu complex encapsulated nanoparticles Maleimide group-introduced Eu complex encapsulated nanoparticles suspended in 50 mM phosphate buffer (pH 7.0) containing 1 mM-EDTA (3.8 mg / 3.57 mL) and anti-human IgE mouse monoclonal antibody Fab ′ (0.662 mg / 3 mL) dissolved in the same buffer as above were mixed, and mixed by inversion at room temperature for 18 hours under light shielding. In order to block the maleimide group remaining on the particles, 50 μL of 2-mercaptoethylamine prepared to 1 mg / mL with distilled water was added to the reaction solution, and mixed by inverting at room temperature for 1 hour under light shielding. The reaction solution was applied to a Sepharose CL-6B column [1.5 cm × 90 cm; eluent = 50 mM Tris-HCl (pH 7.8) containing 0.15M-4NaCl; flow rate = 20 mL / min, fraction = 2 mL / fraction]. Application was performed to obtain a nanoparticle fraction encapsulating anti-human IgE Fab′-conjugated Eu complex.

(3) Preparation of allergen-immobilized microplate Milk protein was selected as the allergen. 50 μL of the milk protein solution was placed in a 96-well plate (manufactured by NUNC; FluoroNUN module plate / maxisorp) and allowed to stand overnight at 4 ° C. After removing the solution by suction, 200 μL of a blocking solution (50 mM sodium bicarbonate aqueous solution containing 1% BSA and 2% sucrose) was added, and the mixture was allowed to stand at 37 ° C. for 2 hours. Allergen-immobilized wells were stored at 4 ° C. until used for immunoassays.

(4) Two-step sandwich immunoassay for allergen-specific IgE antibody A positive sample and a negative sample each containing anti-milk human IgE antibody were added to a milk allergen-immobilized well from which the blocking solution had been removed in an amount of 100 μL, respectively, at 37 ° C. for 1 hour. Reacted with shaking. The wells were washed twice with 300 μL of a washing solution [20 mM phosphate buffer (pH 7.0) containing 0.1% Tween-20], and then a conjugate solution [containing 0.2% BSA and 0.15 M NaCl]. 100 μL of biotin-conjugated anti-human IgE mouse monoclonal antibody solution (antibody amount: 50 ng / test) prepared with 20 mM phosphate buffer (pH 7.0)] was added, and the mixture was shaken at 37 ° C. for 1 hour. After washing twice with 300 μL of the washing solution, the prepared SA-bound Eu complex-encapsulated nanoparticle suspension (total fluorescence: 3 million counts / 50 μL / test) was added to the conjugate solution, and the mixture was incubated at 37 ° C. for 1 hour. Reacted with shaking. After washing with 300 μL of the washing solution four times, the fluorescence intensity of the Eu complex-encapsulated nanoparticles bound on the wells by the antigen-antibody reaction was measured with a fluorometer (manufactured by Wallac; DELFIA fluorometer).

  As a measurement system that does not use biotin-SA in the reaction system, an anti-human IgE-Fab′-conjugated Eu prepared with a conjugate lysate was prepared in the above-described amnoassay procedure, by washing the milk allergen-immobilized well with the specimen. A complex-encapsulated nanoparticle suspension (total fluorescence: 3 million counts / 50 μL / test) was added, and the mixture was shaken at 37 ° C. for 1 hour. After washing with 300 μL of the washing solution four times, the fluorescence intensity of the Eu complex-encapsulated nanoparticles bound on the wells by the antigen-antibody reaction was measured with a fluorometer (manufactured by Wallac; DELFIA fluorometer).

(5) Results of Milk Allergen-Specific IgE Antibody Immunoassay FIG. 1 shows the results of measuring milk allergen-specific IgE antibodies using biotin-conjugated antibodies and SA-conjugated nanoparticles. The measured values of the milk allergen negative samples showed no difference between a biotin-conjugated antibody with a short spacer (molecular length: 1.35 nm) and a biotin-conjugated antibody with a long spacer (molecular length: 3.05 nm). On the other hand, in the positive sample, the measured value of the biotin-conjugated antibody with a long spacer was about 7 times higher than the measured value of the bound antibody with a short spacer. From these results, it is clear that the SA bound on the Eu complex encapsulated nanoparticles, which are giant labeled particles, can easily react with biotin by introducing a spacer between biotin and the antibody. It became.

  On the other hand, FIG. 2 shows the results of measuring milk allergen-specific IgE antibodies using anti-human IgE-Fab′-conjugated Eu complex-encapsulated nanoparticles as labeled particles without using a combination of biotin and SA in the measurement system. The result of immunoassay performed on the same negative sample and positive sample with the anti-human IgE-Fab′-conjugated Eu complex-encapsulated nanoparticles was the result shown in FIG. 1 with a long spacer (3.05 nm) biotin binding The measured values almost coincided with the measured values using the antibody and the SA-conjugated Eu complex-encapsulated nanoparticles. Therefore, by introducing a spacer (3.05 nm) between biotin and the antibody, the SA bound on the particle is not affected by the size of the macromolecule (Eu complex encapsulated nanoparticle). It was proved more strongly that they could be combined.

Example 2: AFP immunoassay (1) Binding of biotin to anti-AFP rabbit polyclonal antibody (F (ab ′) ₂ ) 1 mg of anti-AFP rabbit polyclonal antibody was dissolved in 1 mL of 0.1 M phosphate buffer (pH 8.0). did. 10 μL of NHS-biotin (molecular length: 1.35 nm) adjusted to 2.4 mg / mL with dimethylformamide, or NHS-LC-LC-biotin (molecular length: 3.05 nm) adjusted to 4.0 mg / mL with dimethylformamide ) 10 μL was added to the suspension and allowed to react for 1 hour at room temperature. Unreacted biotin reagent was removed with a Sephadex G-25 column [1.5 cm × 12 cm; eluent = 20 mM phosphate buffer (pH 7.0) containing 0.15 M NaCl], and two types of biotin-conjugated antibodies A fraction was obtained.

(2) Binding of SA to Eu complex-encapsulated nanoparticles 0.2 M carbonate buffer to 25 μL of distilled water suspension (186 mg / mL) of Eu complex-encapsulated nanoparticles (diameter: 65 nm) whose particle surfaces are covered with aldehyde groups After adding 250 μL of liquid (pH 9.5) and mixing, 185 μL of SA adjusted to 5 mg / mL with 0.15 M NaCl was added. Thereto, polyethylene glycol 6000 adjusted to 40% with distilled water was added in an amount of 25 μL and stirred, then 15 μL of an aqueous NaCNBH ₃ solution adjusted to 300 mg / mL was added, and the mixture was mixed by inversion at 37 ° C. for 18 hours. The reaction solution was applied to a Sephacryl S-300HR column [1 cm × 45 cm; eluent = 50 mM Tris-HCl buffer (pH 7.8); flow rate = 10 mL / min; fraction = 1 mL / fraction], and SA-bound Eu complex An encapsulated nanoparticle fraction was obtained.

(3) Preparation of anti-AFP mouse monoclonal antibody immobilized microplate Anti-AFP adjusted to 3 μg / mL in 96-well plate (manufactured by NUNK; FluoroNUN module plate maxi soap) with 0.1M carbonate buffer (pH 9.6) 50 μL of AFP mouse monoclonal antibody solution was added and allowed to stand at 4 ° C. overnight. After removing the solution by suction, 200 μL of a blocking solution (50 mM sodium bicarbonate aqueous solution containing 1% BSA and 2% sucrose) was added, and the mixture was allowed to stand at 37 ° C. for 2 hours. Antibody-immobilized wells were stored at 4 ° C. until used for immunoassays.

(4) AFP two-step sandwich immunoassay method A dilution series prepared from human serum not containing AFP and human serum not containing AFP was added to each anti-AFP antibody-immobilized well from which the blocking solution had been removed in an amount of 100 μL. The reaction was shaken at 1 ° C. for 1 hour. The wells were washed twice with 300 μL of a washing solution [20 mM phosphate buffer (pH 7.0) containing 0.1% Tween-20], and then a conjugate solution [containing 0.2% BSA and 0.15 M NaCl]. 100 μL of a biotin-conjugated anti-AFP rabbit polyclonal antibody solution (antibody amount: 50 ng / test) prepared with 20 mM phosphate buffer (pH 7.0)] was added, and the mixture was shaken at 37 ° C. for 1 hour. After washing twice with 300 μL of the washing solution, the prepared SA-bound Eu complex-encapsulated nanoparticle suspension (total fluorescence: 3 million counts / 50 μL / test) was added to the conjugate solution, and the mixture was incubated at 37 ° C. for 1 hour. Reacted with shaking. After washing with 300 μL of the washing solution four times, the fluorescence intensity of the Eu complex-encapsulated nanoparticles bound on the wells by the antigen-antibody reaction was measured with a fluorometer (manufactured by Wallac; DELFIA fluorometer).

(5) Results of AFP immunoassay FIG. 3 shows the results of an AFP two-step sandwich immunoassay using Eu complex-encapsulated nanoparticles with a particle size of 65 nm and a biotin-conjugated anti-AFP antibody. In the biotin-conjugated anti-AFP antibody, the reactivity using a biotin-conjugated antibody having a long spacer (molecular length: 3.05 nm) between biotin and the anti-AFP antibody is similar to that of a biotin-conjugated antibody having a short spacer (molecular length: 1.35 nm). Compared to the reactivity, it was about 6 times. Therefore, even when giant particles having a particle size of 65 nm are used, it is clear that SA bound on the giant particles can easily react with biotin by lengthening the spacer of the biotin-conjugated antibody. It was.

  The analysis kit according to the present invention does not interfere with the binding reaction between a low molecular ligand and a binding partner even when the labeled particle is a macromolecule, and is based on the use of a macro target molecule in an immunoassay or a hybridization assay. A decrease in sensitivity can be prevented, and a significant improvement in sensitivity can be achieved particularly at low concentrations.

It is a graph which shows the result of having measured the milk allergen specific IgE antibody using the biotin binding antibody and the SA binding Eu complex enclosure nanoparticle (diameter: 167 nm). It is a graph which shows the result of having measured the milk allergen specific IgE antibody using the anti-human IgE Fab 'coupling | bonding Eu complex enclosure nanoparticle (diameter: 167 nm) as a labeling substance. It is a graph which shows the result of having measured AFP using the biotin coupling | bonding antibody and the SA coupling | bonding Eu complex enclosure nanoparticle (diameter: 65 nm).

Claims (9)

(1) (a) For a primary specific partner that can bind to a primary specific partner that specifically binds to an analyte or that specifically binds to an analyte A spacer group having a reactive end group capable of binding to a secondary specific partner that specifically binds, and (b) a small molecule ligand bound to the end of the spacer group opposite to the reactive end group And (2) (a) a binding partner that specifically binds to the low molecular ligand, and (b) a label that includes labeled particles bound to the binding partner. For kit.   The analytical kit according to claim 1, wherein the labeling particles are core-shell type particles encapsulating a fluorescent rare earth metal complex.   The kit for analysis according to claim 1 or 2, wherein the labeling particle has a diameter of 40 nm or more.   The analytical kit according to any one of claims 1 to 3, wherein the spacer group is a chain compound group having a length of 1 nm or more.   The analytical kit according to any one of claims 1 to 4, wherein the low molecular ligand is biotin, and the binding partner that specifically binds to the low molecular ligand is avidin or streptavidin.   The analysis according to any one of claims 1 to 4, wherein the small molecule ligand is a physiologically active substance, and the binding partner that specifically binds to the small molecule ligand is an antibody against the physiologically active substance or a fragment thereof. For kit.   The analysis according to any one of claims 1 to 4, wherein the small molecule ligand is a physiologically active substance, and the binding partner that specifically binds to the small molecule ligand is a receptor for the physiologically active substance or a fragment thereof. For kit.   The analytical kit according to any one of claims 1 to 7, which is a time-resolved fluorescence measurement method. (1) (a) Binding specifically to a primary specific partner that specifically binds to an analyte or a primary specific partner that specifically binds to an analyte A secondary specific partner, (b) a spacer group bound to the primary specific partner or secondary specific partner via a reactive end group, and (c) the reactive end group of the spacer group A conjugate comprising a small molecule ligand bound to the opposite end of the membrane, and (2) (a) a binding partner that specifically binds to the small molecule ligand, and (b) a labeled particle that binds to the binding partner A kit for analysis, comprising a labeling substance.

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