JP2003344413A - Sensitivity enhancer for immunochromatography measurement method, measurement method and instrument for measurement method - Google Patents

Abstract

(57) [Problem] To provide a substance capable of obtaining a stronger sensitivity enhancement effect as compared with a conventionally known immunochromatographic sensitivity enhancer using a substance selected from syphilis-related antibodies as a measurement target substance. (1) The following general formula [1] (Wherein R ^{1 to} R ³ each independently represents a hydrogen atom or an alkyl group optionally having a hydroxyl group, and R ⁴ represents an alkylene group). A sensitivity enhancer for an immunochromatographic assay using a substance selected from syphilis-related antibodies, comprising: (2)
An immunochromatographic measurement method using a substance selected from syphilis-related antibodies as an object of measurement, wherein an antigen-antibody reaction is carried out in the presence of a polymer having a group represented by the general formula [1] in its side chain. (3) An instrument for an immunochromatographic measurement method comprising a substance selected from syphilis-related antibodies, comprising the sensitivity enhancer of (1).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a sensitivity enhancer in an immunochromatographic assay using an syphilis-related antibody as a substance to be measured, and an immunochromatography using the syphilis-related antibody as a substance to be measured in the presence of the sensitivity enhancer. The present invention relates to a measurement method and an instrument for immunochromatography measurement, which comprises the sensitizer for syphilis and contains the sensitivity enhancer.

[0002]

2. Description of the Related Art The immunochromatographic method is a method that combines a chromatographic method utilizing the capillary phenomenon of a porous membrane and an immunological method, and is a quick and simple method for immunoassay without using a special instrument. It is a method that can be performed and is used for detection of various antigens and antibodies.

However, this method has a low sensitivity accuracy in spite of the fact that it can be easily measured, and it may be judged as a false negative even if a positive sample is used, and an immunochromatographic measurement method with higher sensitivity has been desired.

[0004]

SUMMARY OF THE INVENTION A first object of the present invention is to provide a sensitivity enhancer for immunochromatography, which is more accurate than conventional ones. A second object of the present invention is to provide a measuring method using the sensitivity enhancer for immunochromatography. Still further, a third object of the present invention is to provide an instrument for immunochromatographic assay containing the above-mentioned sensitivity enhancer.

[0005]

The present inventors have made the present invention as a result of intensive studies in view of such a current situation. That is, the present inventors, in an immunological assay method using a syphilis-related antibody as a substance to be measured, compared with known sensitivity enhancers, are keenly researched to find a compound having a stronger sensitivity enhancing effect. As a result, they have found that a polymer represented by the above general formula [1] and having a group having a phosphobetaine structure in its side chain has desired performance, and completed the present invention. The following are provided as the means.

(1) The following general formula [1]

[Chemical 7]

(In the formula, R ^{1 to} R ³ each independently represent a hydrogen atom or an alkyl group which may have a hydroxyl group;
⁴ represents an alkylene group. ) A sensitivity enhancer for immunochromatographic assay, which comprises a polymer having a group represented by the formula (1) as a side chain, and a syphilis-related antibody as a measurement target substance.

(2) An immunochromatographic assay method in which an syphilis-related antibody is used as a substance to be measured, characterized in that an antigen-antibody reaction is carried out in the presence of a polymer having a group represented by the general formula [1] in its side chain. .

(3) comprising the sensitivity enhancer of (1),
Instrument for immunochromatographic assay using syphilis-related antibody as the measurement target substance.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the polymer used as a sensitivity enhancer may be any polymer having a group represented by the above general formula [1] in its side chain, and may be a homopolymer or a copolymer. Normally, but not limited to, a molecular weight of 10,0
It is from 00 to 1,000,000, preferably from 10,000 to 500,000, more preferably from 50,000 to 500,000.

More specifically, the following general formula [2]

[Chemical 8]

(In the formula, R ⁵ represents an alkylene group which may have a substituent and may have an oxygen atom in the chain, R ⁶ represents a hydrogen atom or a methyl group, and X represents Represents an oxygen atom or an -NH- group, and R ^{1 to} R ⁴ are preferably the same as those described above).

In the above general formula [1] or [2], R
^The alkyl group of the alkyl group which may have a hydroxyl group represented by ^{1 to} R ³ may be linear, branched or cyclic, and usually has 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms. More preferably, one or more, and more preferably one is mentioned, and specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, se
c-butyl group, tert-butyl group, n-pentyl group, isopentyl group, sec-pentyl group, tert-pentyl group, n-hexyl group, isohexyl group, sec-hexyl group, tert-hexyl group, cyclopropyl group, Examples thereof include a cyclohexyl group and a cyclopentyl group, preferably a methyl group, an ethyl group and the like, more preferably a methyl group and the like.

Examples of the alkyl group having a hydroxyl group include those in which 1 to 2, preferably 1 of the hydrogen atoms of the alkyl group as described above are substituted with a hydroxyl group. Specifically, for example, a hydroxymethyl group. , Hydroxyethyl group, hydroxy-n-propyl group, hydroxyisopropyl group, hydroxy-n-butyl group, hydroxy-isobutyl group, hydroxy-sec-butyl group, hydroxy-tert-butyl group, hydroxy-n-pentyl group, hydroxy -Isopentyl group, hydroxy-sec-pentyl group, hydroxy-tert
-Pentyl group, hydroxy-n-hexyl group, hydroxy-
Examples thereof include an isohexyl group, a hydroxy-sec-hexyl group, a hydroxy-tert-hexyl group, a hydroxy-cyclopropyl group, a hydroxy-cyclohexyl group and a hydroxy-cyclopentyl group, and a hydroxymethyl group and a hydroxyethyl group are preferable.

Examples of the alkylene group represented by R ⁴ include those having 1 to 6 carbon atoms, preferably 2 to 3 carbon atoms, which may be linear, branched or cyclic. Specifically, for example, methylene group, ethylene group, propylene group, trimethylene group, butylene group, 1-ethylethylene group, 2-methyltrimethylene group, 2-ethyltrimethylene group, hexylene group, cyclopropylene group, cyclo Examples thereof include a butylene group, a cyclopentylene group, and a cyclohexylene group, and an ethylene group, a propylene group, a trimethylene group and the like are preferable.

In the alkylene group represented by R ⁵ in the general formula [2], which may have a substituent and may have an oxygen atom in the chain, does not have oxygen. Examples of the alkylene group include, for example, those having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, more preferably 2 to 6 carbon atoms,
These may be linear, branched or cyclic. Specifically, for example, a methylene group, an ethylene group, a propylene group,
Trimethylene group, butylene group, 1-ethylethylene group,
Examples thereof include a 2-methyltrimethylene group, a 2-ethyltrimethylene group, a hexylene group, a cyclopropylene group, a cyclobutylene group, a cyclopentylene group, and a cyclohexylene group. In addition, the substituent has, for example, 1 carbon atom.
To 6, preferably 1 to 3 alkoxyl groups (which may be linear, branched or cyclic. ] More specifically, for example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, n-pentyloxy group, isopentyloxy group. Group, sec-pentyloxy group, tert-pentyloxy group, n-hexyloxy group, isohexyloxy group, sec-hexyloxy group, tert-hexyloxy group,
Cyclopropoxy group, cyclohexyloxy group, cyclopentyloxy group and the like, for example, halogen atom, more specifically, fluorine atom, chlorine atom, bromine atom, iodine atom and the like, preferably ethylene group, propylene group, trimethylene group, butylene. Group etc. Also, if having an oxygen atom in the chain, 1-5 as an oxygen atom, preferably one to three, more specifically _{- (C 2 H 4 O)} n -
C ₂ H ₄ - (wherein, n represents an integer of 1 to 5.), And the like. Among the alkylene groups represented by R ⁵ which may have a substituent and may have an oxygen atom in the chain, ethylene group and propylene group are preferable,
Ethylene groups are more preferred.

As the polymer having a structural unit derived from a monomer having a group represented by the general formula [1] in the side chain according to the present invention, a commercially available polymer may be used, for example, JP-A-10-45794. Japanese Patent Laid-Open No. 2000-2396
Those synthesized according to the method described in Japanese Patent Publication No. 96 etc. may be used.

The constitutional unit based on the monomer represented by the general formula [2] may be any unit having R ^{1 to} R ⁶ as described above, and specifically, for example, the following general formula [5]

[0019]

[Chemical 9]

Examples include constitutional units based on the monomer represented by

When the polymer having a constitutional unit based on the monomer represented by the general formula [2] according to the present invention is a copolymer, as a monomer unit other than the constitutional unit based on the monomer represented by the general formula [2]. Is derived from a monomer selected from acrylic acid or acrylic acid ester, methacrylic acid or methacrylic acid ester, acrylamide or its N-substituted product, methacrylamide or its N-substituted product, or styrene or its derivative.
Two or more kinds of these monomer units may be contained in the copolymer. The ratio of the structural unit based on the monomer represented by the general formula [2] in the copolymer is usually 20% or more and less than 100%, preferably 3%.
It is 0 to 95%, more preferably 30 to 90%.

Alkyl acrylate, aralkyl acrylate and the like are used as the acrylic acid ester as a monomer unit other than the constitutional unit based on the monomer represented by the general formula [2], and as the methacrylic acid ester, alkyl methacrylate and aralkyl methacrylate are used. The N-substituted acrylamide is N-alkylacrylamide or N-aralkylacrylamide, the N-substituted methacrylamide is N-alkylmethacrylamide or N-aralkylmethacrylamide, and the styrene derivative is Examples thereof include α-methylstyrene, styrene having a substituent and α-methylstyrene.

The alkyl group in the above alkyl acrylate, alkyl methacrylate, N-alkyl acrylamide and N-alkyl methacrylamide includes
It may be linear, branched, or cyclic and usually has 1 to 1 carbon atoms.
6, more preferably, 1 to 4, specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert- Butyl group, n-pentyl group, isopentyl group, s
Examples thereof include an ec-pentyl group, a tert-pentyl group, an n-hexyl group, an isohexyl group, a sec-hexyl group, a tert-hexyl group, a cyclopropyl group, a cyclohexyl group and a cyclopentyl group. This alkyl group may have a substituent, and examples of the substituent include a hydroxyl group, a lower alkoxyl group having 1 to 3 carbon atoms, and a trialkylammonio group (as the alkyl group, for example, a methyl group, an ethyl group). Groups having 1 to 3 carbon atoms such as a propyl group, an isopropyl group, an isopropyl group, etc. When the compound has a trialkylammonio group as a substituent, since this substituent is positively charged, a counter anion is usually present. Although bound, examples of such a counter anion include a fluoride ion, a chloride ion, a bromide ion, a halide ion such as an iodide ion, and the like). In addition, examples of the alkyl group having a substituent include the following groups.

[Chemical 10]

(Note that R ⁷ represents an alkyl group having 1 to 3 carbon atoms, and m represents 1 to 100).

Examples of the aralkyl group in aralkyl acrylate, aralkyl methacrylate, N-aralkyl acrylamide and N-aralkyl methacrylamide include those having 7 to 10 carbon atoms, and specific examples include benzyl group and phenyl group. Examples thereof include an ethyl group, a phenylpropyl group and a phenylbutyl group.

The substituent which styrene or α-methylstyrene may have is, for example, linear, branched or cyclic, usually having 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms.
Alkyl group (specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, sec-pentyl group, tert-pentyl group, n-hexyl group, isohexyl group, sec-hexyl group,
tert-hexyl group, cyclopropyl group, cyclohexyl group, cyclopentyl group, etc.), for example, linear, branched, or cyclic aralkyl group having usually 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms (specifically, , For example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, n-pentyloxy group, isopentyloxy group, sec-
Pentyloxy group, tert-pentyloxy group, n-hexyloxy group, isohexyloxy group, sec-hexyloxy group, tert-hexyloxy group, cyclopropoxy group,
Cyclohexyloxy group, cyclopentyloxy group and the like), for example, halogen atom such as fluorine atom, chlorine atom, bromine atom and iodine atom, carboxyl group, hydroxy group, amino group and the like.

Specific examples of the monomer unit other than the constitutional unit based on the monomer represented by the above general formula [2] include, for example, methacrylic acid, methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate,
Dodecyl methacrylate, octadecyl methacrylate, 2-ethylhexyl methacrylate, lauryl methacrylate,
Stearyl methacrylate, 2-trimethylammonioethyl methacrylate, benzyl methacrylate, phenylethyl methacrylate, acrylic acid, methyl acrylate, ethyl acrylate, butyl acrylate, 2-ethylhexyl acrylate, lauryl acrylate, stearyl acrylate , 2-trimethylammonioethyl acrylate, benzyl acrylate, phenylethyl acrylate, acrylamide, N-methylacrylamide, N-ethylacrylamide, N-butylacrylamide, N-2-ethylhexylacrylamide, N-laurylacrylamide, N
-Stearyl acrylamide, N-2-trimethylammonioethyl acrylamide, N-benzyl acrylamide, N-phenyl ethyl acrylamide, methacrylamide, N-methyl methacrylamide, N-ethyl methacrylamide, N-butyl methacrylamide, N-2- Ethylhexyl methacrylamide, N-lauryl methacrylamide, N-stearyl methacrylamide, N-2-trimethylammonioethyl methacrylamide, N-benzyl methacrylamide, N-phenylethyl methacrylamide, styrene, carboxystyrene, hydroxystyrene, aminostyrene , Methylstyrene, ethylstyrene, methoxystyrene, ethoxystyrene, chlorostyrene, bromostyrene, α-methylstyrene, α-methyl-carboxystyrene Α-methyl-hydroxystyrene, α-methyl-aminostyrene, α-methyl-methylstyrene, α-methyl-ethylstyrene, α-methyl-methoxystyrene, α-methyl-ethoxystyrene,
α-Methyl-chlorostyrene, α-methyl-bromostyrene, N, N, N-triethylammonium ethyl methacrylate bromide, N, N, N-trimethylammonium ethyl methacrylate chloride, N, N, -diethyl-N-propylammonium ethyl Examples include those derived from methacrylate bromide, N, N, N-trimethylammonium-2-hydroxypropylmethacrylate chloride (QM), N, N, N-trimethylammonium methylstyrene bromide,
In addition, the following general formula [4]

[0028]

[Chemical 11]

Specific examples include those represented by the formula (wherein m represents 1 to 100).

Among the above, methacrylic acid, stearyl methacrylate, benzyl methacrylate, butyl methacrylate, dodecyl methacrylate, octadecyl methacrylate N, N, N-trimethylammonium-2-hydroxypropylmethacrylate chloride (QM) and the like are used. thing,
Those represented by the general formula [4] are preferable.

The sensitivity enhancer of the present invention comprises a polymer having a group represented by the general formula [1] as a side chain as described above, and preferably a monomer represented by the general formula [2]. A polymer having a structural unit based on The sensitivity enhancer may be used by dissolving it in various reagents or samples used in the immunochromatographic measuring method, or may be used by being carried on an instrument for the immunochromatographic measuring method.
The latter is more preferable because of the ease of operation.

Examples of the immunochromatographic measuring instrument used in the immunochromatographic measuring method of the present invention include (1)
(2) A sample comprising a spreading film, (2) a sample labeling part and a spreading film, wherein the sample labeling part and the spreading film are formed so as to be movable by capillarity, (3) a sample labeling part, a development (4) A sample and a liquid absorbing part, wherein the sample labeling part, the spreading film and the liquid absorbing part are formed to be movable in this order by capillarity, (4) sample dropping part, sample labeling part , A spreading film and a liquid absorbing part, and the sample dropping part, the sample labeling part, the spreading film and the liquid absorbing part are formed so as to be movable in this order by capillarity. .

A measuring section is provided on the developed film,
The measurement part is a portion on which a substance having a binding ability to a measurement target substance (hereinafter, may be abbreviated as a measurement target binding substance) is immobilized and carried, and several types of measurement target substances are simultaneously measured. In such a case, several types of binding substances to be measured are supported and immobilized on the spreading film, for example, in a linear or spot form so that they do not overlap each other.
Further, the sample labeling unit is a labeled binding substance to be measured, which is held in a movable state by a capillary phenomenon, in the case of simultaneously measuring several types of measurement target substances, several types of The labeled binding substance to be measured is held in a movable state by a capillary phenomenon. Further, the sample dropping part is a part formed so as to overlap the spreading film or the sample labeling part for dropping the sample, and the liquid absorbing part is provided in order to facilitate the movement of the sample due to the capillarity. , Represents a site formed so as to overlap the developed film.

Even if a commercially available product is used as the above instrument,
You may use what was produced by the well-known method. When produced by a known method, the base material used in the spreading film may be one usually used in this field, and preferred examples include cellulose, nitrocellulose, nylon and the like. The base material used in the sample labeling part, the sample dropping part, and the liquid absorbing part may be based on these. In addition, so-called blocking treatment may be performed on the developed film in order to prevent the influence of non-specific adsorption on the measurement. Such blocking treatment may be carried out by using a blocking agent which is usually used in this field, and a method which is usually used in this field. Examples of the labeled binding substance to be measured held in the sample labeling unit include those in which the labeling substance is carried on the antigen against the antibody as the measurement target substance. The labeling substance may be any of those usually used in this field, but a labeling substance such as a gold colloid, a selenium colloid, and a colored latex that can give a visually detectable signal is preferable, and particularly, it is Gold colloid is more preferable from the viewpoints of ease of operation and sensitivity. As the labeling substance as described above, a labeling substance prepared by a conventional method may be used, or a commercially available product may be used, and the concentration used may be within the concentration range usually used in this field.

When the sensitivity-enhancing agent of the present invention is used by being carried on an instrument for immunochromatography, the amount thereof is such as the place where it is carried, the type of sensitivity-enhancing agent used, the type of substance to be measured, and the type of substance to be used. Depending on the labeling substance, etc.,
For example, in the case of supporting it on the development membrane, sample labeling part, sample dropping part, etc. of the instrument for immunochromatography, the unit area (cm ² )
The amount of sensitivity enhancer contained per
~ 10 mg, preferably 0.1 μg to 4 mg, more preferably
It is 1 to 800 μg. The supported area varies depending on the type and size of the immunochromatographic measuring instrument used and the amount of the measurement sample, but in the case of the developed membrane of the immunochromatographic measuring instrument, it is usually 10 to 10% of the total area. 60%, preferably 20-30%
1 to 30%, preferably 5 to 15%, in the case of the specimen dropping part,
Usually, it is 5 to 40% of the total area, preferably 10 to 20%. In addition, the site to be carried in the instrument for immunochromatography measurement may be a site that can come into contact with the measurement sample, that is, a site through which the measurement sample passes, and specifically, a development membrane, a development membrane. The above measurement site and the like can be mentioned, and the measurement site is preferable. The method of loading the sensitivity enhancer of the present invention may be any method commonly used in this field, for example, a solution containing the sensitivity enhancer as described above in the spreading film, for example, coating, dropping or spraying. After that, a method of drying it and supporting it by physical adsorption, for example, dissolving the sensitivity enhancer of the present invention in a buffer solution or a washing solution used at the time of preparation of a development film or the like, and preparing a development film using these And the like.

The immunochromatographic assay reagent used in the immunochromatographic assay method according to the present invention contains, in addition to the sensitivity enhancer of the present invention, for example, an antigen against an antibody to be measured or an antigen carrying a labeling substance. It may be contained. The labeling substance used at this time is
The same materials as those used in the above-mentioned immunochromatography measuring instrument can be mentioned, and the concentration, the preparation method and the like may be the same. Furthermore, in the reaction reagent, a buffer (eg Tris buffer, phosphate buffer, veronal buffer, borate buffer, Good's buffer etc.), stabilizer (eg albumin, globulin, water-soluble gelatin, Surfactants, sugars, etc., preservatives (eg salicylic acid, benzoic acid, sodium azide, etc.) and others used in this field, such as inhibiting the stability of coexisting reagents, antigen-antibody You may contain the thing which does not inhibit reaction. Further, the concentration to be used may be within the concentration range usually used in this field.

When the sensitivity enhancer of the present invention is used by dissolving it in various reagents or samples, its concentration is usually 0.1 to 20 w / v%, preferably 0.1 to 20 w / v% as a concentration at which an antigen-antibody reaction is carried out. It is used so as to be 10 w / v%, more preferably 0.1 to 5 w / v%.

To carry out the immunochromatographic assay method of the present invention, the polymer having the side chain of the group represented by the general formula [1] according to the present invention is coexisted in the concentration reagent as described above, or Alternatively, except that the polymer having a group represented by the general formula [1] in the side chain according to the present invention is carried on the above-mentioned instrument for concentration immunochromatography, various reagents used in the immunochromatographic measurement method known per se are used, It may be carried out according to a method known per se.

The substance to be measured by the immunochromatographic assay method of the present invention is an anti-phospholipid antibody, an anti-treponemal antibody or other syphilis-related antibody.

The sensitivity-enhancing agent of the present invention used in the measuring method of the present invention exhibits enhanced sensitivity in any substance to be measured, but the preferred substance varies depending on the substance to be measured. For example, when the substance to be measured is an antiphospholipid antibody,
For example, a polymer comprising a constitutional unit based on the monomer represented by the general formula [2], a polymer comprising a constitutional unit based on the monomer represented by the general formula [2] and a monomer unit derived from butyl methacrylate, the above general formula A polymer comprising a structural unit based on the monomer represented by [2] and a monomer unit derived from benzyl methacrylate, a structural unit based on the monomer represented by the general formula [2] and represented by the general formula [4]. Preferable examples include a polymer composed of a monomer-based constitutional unit. Among them, for example, a polymer composed of a monomer represented by the general formula [2], a polymer composed of a monomer represented by the general formula [2]. And a polymer comprising a monomer unit derived from butyl methacrylate, and a structural unit based on the monomer represented by the general formula [2]. Serial Formula polymers consisting of structural units based on a monomer represented by [4] can be mentioned as being preferred. Also, for example, if the substance to be measured is
In the case of a TP (Treponema Pallidum) antibody, for example, a polymer comprising a constitutional unit based on the monomer represented by the general formula [2], a constitutional unit based on the monomer represented by the general formula [2] and butyl methacrylate-derived Polymer consisting of monomer unit, constitutional unit based on monomer represented by general formula [2] and polymer consisting of monomer units derived from octadecyl methacrylate, constitutional unit based on monomer represented by general formula [2] and methacryl Polymers composed of acid-derived monomer units, polymers composed of monomer units represented by the general formula [2] and monomer units derived from benzyl methacrylate, and structures composed of monomer represented by the general formula [2] Preferable examples include polymers having a unit and a structural unit based on the monomer represented by the general formula [4]. Among them, for example, a polymer composed of a structural unit based on the monomer represented by the general formula [2], a polymer composed of a structural unit based on the monomer represented by the general formula [2] and a monomer unit derived from butyl methacrylate, A polymer comprising a structural unit based on the monomer represented by the general formula [2] and a monomer unit derived from benzyl methacrylate, a structural unit based on the monomer represented by the general formula [2] and represented by the general formula [4] More preferred are polymers and the like composed of structural units based on the above-mentioned monomers.

Further, the instrument for immunochromatographic assay of the present invention comprises a polymer having a group represented by the above general formula [1] in a side chain, a sample dropping part, a sample labeling part, a developing film (measuring part).
Such as a sample supported on a place through which the sample passes, such as dropping and spreading, and preferably a polymer having a side chain having a group represented by the above general formula [1] added to the spreading film, the above general formula Examples thereof include those in which the polymer having the group represented by [1] in the side chain is added to the sample labeling part, and those in which the polymer having the group represented by the above general formula [1] in the side chain is added to the measurement part. Preferred embodiments of the components of the device, specific examples,
The concentration to be used and the like are as described in those used in the immunochromatographic measurement method.

The present invention will be described below with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Further, the MPC polymer in Examples and Comparative Examples represents a polymer synthesized from the following one or two kinds of monomers according to the method described in JP 2001-228149 A, for example. PMPC: 2- (methacryloyloxy) ethyl-2'-
(Trimethylammonio) ethyl phosphate (hereinafter M
Abbreviated as PC monomer. ), A polymer having a molecular weight of 1030 × 10 ³ PMB82: a copolymer of MPC monomer and butyl methacrylate, a molecular weight of 600 × 10 ³ , the composition ratio of MPC monomer and butyl methacrylate; 8: 2 PMB82-1M: MPC monomer And butyl methacrylate copolymer, molecular weight: 1210 × 10 ³ , MPC
Composition ratio of monomer to butyl methacrylate; 8: 2 PMB37: Copolymer composed of MPC monomer and butyl methacrylate, molecular weight; 93 × 10 ³ , composition ratio of MPC monomer and butyl methacrylate; 3: 7 PME ₄ 91: Copolymer consisting of MPC monomer and monomethyl ether polyethylene glycol methacrylate (m = 4 monomer in the general formula [4]), molecular weight: 501 × 10 ³ , composition ratio of MPC monomer and monomethyl ether polyethylene glycol methacrylate; 9: 1 PME ₄ 73: MPC monomer copolymer consisting of a monomethyl ether of polyethylene glycol methacrylate (monomer of the general formula in [4] m = 4), molecular weight; 138 × 10 ^3, MPC monomer and monomethyl ether of polyethylene glycol methacrylate Total _{composition; 7: 3 PMC 18 91:} MPC monomer and polymer consisting of octadecyl methacrylate, molecular weight; 130 × 10 ^3, MPC monomer composition ratio of octadecyl _{methacrylate; 9: 1 PMC 18 82:} MPC monomer and methacrylic acid Polymer composed of octadecyl, molecular weight; 43 × 10 ³ , composition ratio of MPC monomer and octadecyl methacrylate; 8: 2 PMC ₁₂ 82: Polymer composed of MPC monomer and dodecyl methacrylate, molecular weight; 184 × 10 ³ , MPC monomer And the composition ratio of octadecyl methacrylate; 8: 2 PMBz82: Polymer composed of MPC monomer and benzyl methacrylate, molecular weight; 240 × 10 ³ , composition ratio of MPC monomer and octadecyl methacrylate; 8: 2

Example 1 (1) TP (Treponema Pallidum, Treponema pallidum)
Labeling of Antigen and Phospholipid Antigen First, 8 μl of TP antigen (1.0 mg / ml) was dispensed into a centrifuge tube, and then 5 ml of gold colloidal suspension having an average particle size of 35 nm and A _max = 1.2 was poured into the tube at room temperature for 30 minutes. Centrifuge (800
The colloidal gold labeled TP antigen was recovered by performing 0 G for 15 minutes.
The TP antigen is a known recombinant antigen preparation method (Infe
ction and Immunity, volume 54, p500-506, Michael
V. Norgard) was used.

Phospholipid antigen solution is 5 mg / ml cardiolipin (derived from bovine heart, manufactured by Sigma) ethanol solution, 10 mg / m
l Phosphatidylcholine (from egg yolk, manufactured by Kewpie) 2 ml and 0.5 ml of ethanol solution (weight ratio 2:
1) Each mixture was prepared and used.

Pretreated gold colloidal suspension (average particle size 35 nm, A _max = 20.0) 0.8 m in 0.25 ml of the prepared phospholipid antigen solution
l was added and mixed by inversion at room temperature for 2 hours. After that, add 30 ml of blocking solution (50 mM morpholinoethanesulfonic acid (MES) buffer containing 1% BSA and 2% sucrose), leave it for 15 minutes, and centrifuge (8000G, 15 minutes) to collect gold colloid-labeled phospholipid antigen. Recovered.

(2) Preparation of specimen dropping part First, a glass fiber sheet was masked with 25 mM phosphate buffer containing 0.5% BSA and 1% sucrose for 15 minutes, and further 1% poly (1-vinylpyrrolidonecovinyl) was used. Acetate) [poly (1-vinylpyrrolidone-co-vinyl acetate),
Hereinafter, abbreviated as poly-PA. (Manufactured by Aldrich)] and 1
It was washed with a 25 mM phosphate buffer solution containing% sucrose and dried to make a sample dropping part.

(3) Preparation of specimen labeling part The colloidal gold-labeled phospholipid antigen prepared in (1) has A _max = 1.3, and the gold colloid-labeled phospholipid antigen has A _max = 1.4.
The sample dropping part was impregnated with 500 μl per 5 mm × 200 mm of 50 mM MES buffer solution containing 1% poly-PA and 1% sucrose, which was added so as to obtain a sample labeling part.

(4) Preparation of spreading film A mixture of 40 μl of recombinant TP antigen (1.0 mg / ml) and 20 μl of 250 mM phosphate buffer containing 10% sucrose was used as a TP antigen solution.

Also, 125 mg of a 5 mg / ml cardiolipin (derived from beef heart, manufactured by Sigma) ethanol solution, 10 mg / ml phosphatidylcholine (derived from egg yolk, manufactured by NOF Corporation), 250 μl and 20 mg / ml cholesterol (sum) A phospholipid antigen solution consisting of 125 μl of an ethanol solution manufactured by Kojunkaku Co., Ltd. was distilled off with an evaporator to form a lipid thin film. After vacuum drying for 2 hours, 100 mM was applied to the lipid thin film.
500 μl of borate buffer was added and suspended, and the suspension was further sonicated to obtain a liposome suspension. Then, the TP antigen solution and the liposome suspension were each applied to a nylon membrane (20 m
m × 200mm) to another part with 0.25μl / cm wire coating (200mm),
It was dried at 50 ° C for 5 minutes. Then, it was immersed in 50 mM MES buffer containing 1% BSA and 2% sucrose for 15 minutes. further,
After washing twice with 50 mM MES buffer containing 1% Poly-PA and 2% sucrose, and then drying at 50 ° C for 15 minutes, anti-TP
The developed membrane was equipped with a measurement unit for simultaneous detection of antibody and antiphospholipid antibody.

(5) Assembling the measuring instrument The polyethylene terephthalate (PET) sheet (8 cm × 20 cm) laminated with the double-sided tape was attached with the spreading film prepared in (4) at a position 1.2 cm from the lower end, and the spreading film was applied. The glass fiber sheet (4c
m × 20 cm) were pasted together. Next, attach the sample labeling part so that it overlaps the bottom edge of the developed film by 1 mm, and finally attach the sample dropping part with PE.
A sheet with a specimen labeling portion laminated on the lower end of the T sheet and cut into a 4 mm width was used as an instrument for detecting TP antibody / phospholipid antibody. The obtained device was stored at room temperature under dehumidifying conditions. A schematic diagram is shown in FIG. In addition, in FIG. 1, each numeral indicates the following respectively.

1: Support 2: Specimen dropping part 3: Specimen labeling part (portion holding labeled phospholipid antigen and labeled syphilis antigen) 4: Development membrane 5: Supporting part to which phospholipid antigen is immobilized (first) Measurement part) 6: TP antigen carrying part (second measurement part) 7: Liquid absorption part

(6) Specimen 50 in which 0.5 to 3% of various concentrations of MPC polymers shown in Table 1 were added to the specimen dropping part of the test tool prepared in the measurement (5)
-100 μl was added dropwise and left for 15 minutes. After that, the anti-TP antibody / anti-phospholipid antibody was measured with or without the measurement line.
The results are shown in Table 1. In the table, + means colored, + w means lightly colored, ± means colored, and x means not colored.

Comparative Example 1 An experiment was conducted in the same manner as in Example 1 except that polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA) or polyethylene glycol (PEG) was added instead of adding the MPC polymer to the sample. The results are also shown in Table 1.

[0055]

[Table 1]

From these results, although the effect is slightly different depending on the type of MPC polymer, by adding various MPC polymers to the sample, the enhancement of the determination line in the determination part of the anti-TP antibody and / or the antiphospholipid antibody was recognized. It was found that weak positive specimens which could not be detected in the sample without MPC polymer added can be detected.

On the other hand, even if PVP, PVA or PEG was added, a weakly positive sample could not be detected.

Example 2 Further, the following experiment was conducted by changing the place where the MPC polymer was added. (1) When MPC polymer was added to the cleaning liquid used for preparing the sample dropping part When the sample dropping part was prepared in Example 1 (2), 1% poly
-25m containing PA (made by Aldrich) and 1% sucrose
Various MPC polymers listed in Table 1 were added to M phosphate buffer.
A test tool was produced in the same manner as in Example 1 except that 0.5 to 3% was added. The measurement was conducted in the same manner as in Example 1 except that the MPC polymer was not added to the sample.

(2) When the MPC polymer is added to the buffer solution used in the preparation of the sample labeled part When the sample labeled part is prepared in Example 1 (3), the gold colloid labeled TP antigen and the gold colloid labeled phospholipid antigen are added. The various MPs listed in Table 1 were added to 50 mM MES buffer containing 1% poly-PA (manufactured by Aldrich) and 1% sucrose at a predetermined concentration.
A test tool was produced in the same manner as in Example 1 except that 0.5 to 3% of C polymer was added. The measurement was performed in the same manner as in Example 1 except that the MPC polymer was not added to the sample. (3) When MPC polymer was added to the cleaning solution used in the development film preparation. When the development film was prepared in Example 1 (4), 1% poly-PA was used.
50 mM ME containing Aldrich and 2% sucrose
S MP buffer with various concentrations of various MPC polymers listed in Table 1
A test tool was produced in the same manner as in Example 1 except that 0.5 to 3% was added. The measurement was performed in the same manner as in Example 1 except that the MPC polymer was not added to the sample.

As a result of conducting an experiment in which the MPC polymer addition place was changed as described above, the same results as in Table 1 were obtained.

Therefore, it was found that the MPC polymer exerts its sensitivity-enhancing effect not only in the sample but also when it is added to any place where the sample of the instrument for immunochromatographic measurement passes.

[0062]

EFFECTS OF THE INVENTION As is clear from the above description, the present invention is remarkably excellent in effect as compared with the sensitivity enhancer used in the immunochromatographic assay method using a conventionally known syphilis-related antibody as a substance to be measured. The present invention also provides a method for immunochromatography using the sensitivity enhancer, which enables more accurate measurement.

[Brief description of drawings]

FIG. 1 shows a schematic diagram of an instrument for immunochromatographic assay of the present invention.

[Explanation of symbols]

1: Support 2: Specimen dropping part 3: Specimen labeling part (portion holding labeled phospholipid antigen and labeled syphilis antigen) 4: Development membrane 5: Carrying part on which phospholipid antigen is immobilized (first measurement part) 6: TP antigen carrying part (second measuring part) 7: liquid absorbing part

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Masaaki Kida             6-1 Takadacho Amagasaki City, Hyogo Pref.             Within

Claims (14)

[Claims] 1. The following general formula [1]: (In the formula, R ^{1 to} R ³ each independently represent a hydrogen atom or an alkyl group which may have a hydroxyl group, and R ⁴ represents an alkylene group.) A polymer having a side chain. A sensitivity enhancer for immunochromatographic assay, which comprises a syphilis-related antibody as a measurement target substance. 2. The polymer has the following general formula [2]: (In the formula, R ⁵ represents an alkylene group which may have a substituent and may have an oxygen atom in the chain, and R ⁵
Represents a hydrogen atom or a methyl group, X represents an oxygen atom or -N
H-group is shown, and R ^{1 to} R ⁴ are the same as above. The sensitivity enhancing agent according to claim 1, which has a structural unit based on the monomer represented by the formula (1). 3. The polymer has the following general formula [2]: (In the formula, R ^{1 to} R ⁶ and X are the same as above.), A structural unit based on the monomer, acrylic acid or an acrylic acid ester, methacrylic acid or a methacrylic acid ester,
The sensitivity enhancer according to claim 2, which is a copolymer having a constitutional unit based on a monomer selected from acrylamide or an N-substituted product thereof, methacrylamide or an N-substituted product thereof, or styrene or a derivative thereof. 4. A constitutional unit based on the monomer represented by the general formula [2] has the following general formula [3]: A structural unit based on a monomer represented by:
Or the sensitivity enhancer according to 3. 5. The sensitivity enhancer according to claim 3, wherein the acrylic ester is an alkyl acrylate or an aralkyl acrylate. 6. The sensitivity enhancer according to claim 3, wherein the methacrylic acid ester is alkyl methacrylate or aralkyl methacrylate. 7. A monomer unit derived from a methacrylic acid ester is stearyl methacrylate, benzyl methacrylate,
Butyl methacrylate, dodecyl methacrylate, octadecyl methacrylate or N, N, N-trimethylammonium-
Derived from 2-hydroxypropyl methacrylate chloride or represented by the following general formula [4] (In the formula, m represents 1 to 100.) The sensitivity enhancer according to claim 3 or 4, which is a constitutional unit based on a monomer represented by the formula. 8. The sensitivity enhancer according to claim 3, wherein the N-substituted acrylamide is N-alkylacrylamide or N-aralkylacrylamide. 9. The sensitivity enhancer according to claim 3, wherein the N-substituted methacrylamide is N-alkyl methacrylamide or N-aralkyl methacrylamide. 10. A copolymer represented by the following general formula [2]: (In the formula, R ^{1 to} R ⁶ and X are the same as the above.) The ratio of the constituent unit based on the monomer is 20% or more and less than 100%, and the sensitivity enhancement according to any one of claims 2 to 9. Agent. 11. The molecular weight of the polymer is 10,000 to 1,000,00.
The sensitivity enhancer according to any one of claims 1 to 10, which is 0. 12. An immunochromatographic assay method using a syphilis-related antibody as a substance to be assayed, which comprises causing an antigen-antibody reaction in the presence of the sensitivity enhancer according to any one of claims 1 to 11. 13. An instrument for immunochromatographic assay, which comprises the syphilis-related antibody as a substance to be measured, which comprises the sensitivity enhancer according to any one of claims 1 to 11. 14. An instrument for immunochromatographic assay, wherein the sensitivity enhancing agent according to any one of claims 1 to 11 is carried on a measuring section, and an syphilis-related antibody is a substance to be measured.