JP2955405B2 - Immunochromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunochromatography method using dyed synthetic latex particles as particles for binding an antigen or an antibody.

[0002]

2. Description of the Related Art Conventionally, as an immunoassay method for detecting a target substance consisting of a specific antigen or antibody by utilizing a specific reaction by an antigen-antibody, a target substance in a sample is sensitized to fine particles. The agglutination method is a simple immunoassay method in which an antibody or an antigen is bound by an immunoreaction and the aggregation state of the fine particles generated by the binding is a simple immunoassay method, and is a method generally used in that visual judgment is possible. is there.

A radioimmunoassay in which an antibody or antigen labeled with a labeling substance comprising a radioisotope, an enzyme or a fluorescent substance is bound to a substance to be detected in a sample by an immunoreaction, and the bound labeling substance is measured. An enzyme immunoassay or a fluorescence immunoassay is also employed.

[0004] In these immunoassays, a competitive reaction and a sandwich reaction are widely used. Of these,
As a method for measuring a so-called sandwich reaction, an immunochromatography method is known. In a typical example, the following operation is performed to detect a substance to be detected composed of an antigen in a sample.

[0005] By immobilizing fine particles sensitized with an antibody against an antigen to be detected as a solid fine particle on a chromatographic medium, or by immobilizing the antibody itself directly on a chromatographic medium, Prepare a chromatographic medium having
On the other hand, sensitized labeled fine particles are prepared by sensitizing the labeled fine particles with an antibody capable of specifically binding to the substance to be detected. The sensitized labeled microparticles are moved chromatographically together with the sample on a chromatographic medium.

[0006] By the above operation, at the reaction site formed on the chromatographic medium, the immobilized antibody becomes an immobilizing reagent, and the sensitized labeled microparticles specifically bind to the immobilized reagent via the antigen to be detected. As a result, the presence or absence or the amount of the substance to be detected in the sample is measured by visually determining the presence or absence or the degree of the signal generated by the capture of the sensitized labeled fine particles at the reaction site.

In such an immunochromatography method, as fine particles for preparing labeled fine particles, conventionally, colloidal metal particles such as gold, platinum, copper, iron oxide or colloidal metal oxide particles, and colloidal particles such as sulfur have been used. Non-metallic particles and dye particles are used.

[0008]

However, when colloidal metal particles, colloidal metal oxide particles or colloidal nonmetal particles are used as labeling fine particles, the color tone is determined by the preparation conditions and the particle size. As a result, it is not possible to obtain labeled fine particles having a desired clear and deep color tone.

On the other hand, the color tone and color density of the dye particles can be arbitrarily selected, but the dispersion stability in water is low and the dispersion stability is difficult to control, so that sensitization with an antibody or the like is difficult. Is difficult. In addition, even if sensitized, the obtained labeled fine particles are difficult to re-disperse, so it is difficult to move chromatographically.In addition, even when re-dispersed, the particle size distribution is wide, so that chromatographic movement is difficult. This is not preferable in that uniform movement is not achieved uniformly.

As described above, in the case where labeled fine particles composed of colloidal metal particles, colloidal metal oxide particles, colloidal nonmetal particles, or dye particles are used, the signal intensity at the reaction site in the immunochromatography method is desired. There is a problem that it is difficult to set the intensity level, or the appearance of the signal becomes non-uniform, so that accurate visual judgment is difficult and high detection sensitivity cannot be obtained.

[0011] The present invention solves the above-mentioned problems, can perform the intended operation reliably, and has excellent visual judgment.
Accordingly, an object of the present invention is to provide an immunochromatography method capable of obtaining high detection sensitivity.

[0012]

The immunochromatography method of the present invention uses a chromatographic medium having at least one reaction site containing an immobilized reagent capable of binding to a substance to be detected in a sample. The labeled microparticles are moved chromatographically with or subsequently to the sample on the graphing medium, and the sample is brought into contact with the reaction site, whereby the reaction site is detected when the analyte is present in the sample. In the immunochromatography method for detecting the target substance utilizing the fact that the labeled microparticles are specifically bound and captured via the target substance to the immobilized reagent, the labeled microparticles are:
The surface charge obtained by dyeing a latex particle made of a synthetic polymer with an oil-soluble dye is 0.01 to 0.
It is a sensitized colored particle obtained by sensitizing a labeled colored particle of 5 meq / g with a substance capable of binding to the substance to be detected.

[0013] In the above, the labeled coloring particles may be aqueous.
It is preferably obtained by dyeing latex particles in a medium with an emulsion of a solution of an oil-soluble dye in an oily organic solvent.

Hereinafter, the present invention will be specifically described in the case where the substance to be detected is an antigen. However, it goes without saying that the present invention can be applied even when the substance to be detected is an antibody.

The sensitized colored particles used in the immunochromatography method of the present invention are labeled sensitized particles prepared by dyeing latex particles made of a synthetic polymer and sensitized with a substance capable of binding to a substance to be detected. It is.

The labeled colored particles have a surface charge of 0.01 to 0.5 meq / g, preferably 0.0,
1 to 0.3 meq / g, particularly preferably 0.01 to 0.1 meq / g.
It is 2 meq / g. In order to produce labeled colored particles having such a surface charge state, a functional group that gives a negative charge is introduced to the surface of the latex particles in the synthesis reaction of the latex particles. As such a functional group, for example, -COO ^- and -SO ₄ ^- are preferable,
For example, latex particles, by means of a copolymer of styrene and acrylic acid, a copolymer of styrene and methacrylic acid or a copolymer of styrene and itaconic acid, or using persulfate or the like as a polymerization initiator By such means, the above functional groups can be introduced.

It is also possible to apply a negative charge to the surface of the latex particles by means of adding a surfactant to a polymerization reaction system for synthesizing the latex particles.
Latex particles obtained by this means may be destabilized due to the removal of the surfactant on the surface, and may cause agglomeration. Therefore, a preferable dyeing treatment described below is preferably performed. May not be possible. In addition, when sensitizing the labeled colored particles with an antigen or antibody to prepare the sensitized colored particles, sufficient sensitization is inhibited by the surfactant remaining on the surface of the labeled colored particles, and the detection sensitivity is reduced. It may decrease.

The latex particles before dyeing usually have a surface charge of 0.01 to 0.5 meq /
g, preferably 0.01 to 0.3 meq / g, particularly preferably 0.01 to 0.2 meq / g.

The labeled colored particles in the present invention preferably have a particle size in the range of 0.05 to 0.5 μm.
Particularly preferably, it is in the range of 0.05 to 0.3 μm. Labeled colored particles having a particle size larger than 0.5 μm are preferable in that the signal intensity of one obtained sensitized colored particle is increased, but the movement of the sensitized colored particle in the chromatographic medium is slow. . On the other hand, when the particle size is 0.0
If labeled colored particles smaller than 5 μm are used, the resulting sensitized colored particles may have low dispersibility.

The CV value (standard deviation / average value) of the particle diameter of the labeled colored particles is preferably 10% or less, particularly preferably 5% or less, since uniform chromatographic movement can be obtained. However, latex particles composed of a synthetic polymer,
Usually, particles having a sufficiently uniform particle size can be obtained relatively easily.
Therefore, suitable labeled colored particles can be obtained by using such latex particles.

As a preferable treatment method for dyeing, an oil-soluble dye having high solubility in an organic solvent, for example, a solubility in toluene of 1 g / 100 ml or more, preferably 5 g / 100 ml or more.
g / 100 ml or more of an oil-soluble dye dissolved in an organic solvent is finely dispersed in an aqueous medium, for example, in the presence of a surfactant to prepare an oil-in-water dye emulsion. A method of mixing the emulsion with the emulsion of latex particles to be dyed (hereinafter referred to as “dye emulsion method”) can be used.

According to the dye emulsion method, labeled colored particles having a clear and deep color tone can be obtained. Therefore, by repeating such a dyeing treatment method for the same latex particles several times, it is possible to obtain labeled colored particles having a clearer color tone. By using such labeled colored particles, the signal per sensitized colored particle becomes stronger, and even if the amount of the sensitized colored particles used is small, the visual judgment performance is further improved, and the detection in the sample is improved. Substances can be detected with higher sensitivity. In addition, if the amount of the sensitizing colored particles used is small, the ratio of non-specific binding is low, so that the S / N ratio is increased. In this respect, the detection sensitivity can be improved.

Further, according to the dye emulsion method,
Dyeing can be performed without greatly changing the surface state of the latex particles used, and thus labeled colored particles having a surface charge of 0.01 to 0.5 meq / g can be reliably obtained.

As the oil-soluble dye used in the dye emulsion method, an azo dye is preferable because it can be dyed particularly deeply, and quinone dyes such as anthraquinone and naphthoquinone can also be used. Red 23, Solvent Red 27, Solvent Red 111, Solvent Blue 111 and the like, and mixtures thereof.

As another method for dyeing latex particles using an oil-soluble dye, an organic solvent solution of the oil-soluble dye is directly added to the emulsion of the latex particles, and the latex particles are dispersed by utilizing the diffusion of the oil-soluble dye. A dyeing method can also be adopted.

The sharpness of the labeled colored particles used in the present invention is as follows:
When the dried labeled coloring particles are represented by a Munsell dye system, the lightness L ^* is 40 or less, and the saturation C ^* is represented by the following equation 1 ^.
It is preferable that the hue angle H ° is 240 degrees (blue) to 60 degrees (orange), and the lightness L ^* is 35 or less, the saturation C ^* is 55 or more, and the hue angle H ° is 270.
Degrees (blue) to 45 degrees (orange).

[0027]

(Equation 1)

In such a labeled colored particle, the reflectance of the dried labeled colored particle, measured by a spectrophotometer, is the reflectance of red light of 650 nm wavelength and the reflectance of orange 550 nm wavelength light. When the value of the ratio (reflectance at 650 nm / reflectance at 550 nm) is 10 or more, practically good visual judgment performance can be reliably obtained.
Particularly preferred.

The labeled colored particles used in the present invention have high dispersion stability in sensitization for obtaining sensitized colored particles by having a specific surface charge amount, and thus the desired colored particles can be obtained without aggregation. Sensitization can be performed reliably.

In order to further improve the effect of the present invention, the zeta potential on the surface of the labeled colored particles should be -20 to -60 m
V is desirable.

By using such labeled colored particles, the intended operation can be reliably performed in the immunochromatography method, the visual judgment property is excellent, and the object to be detected in the sample can be detected with high sensitivity. it can.

The labeled colored particles obtained as described above are sensitized by a substance that specifically binds to the substance to be detected, whereby sensitized colored particles are prepared.
For example, when the substance to be detected is an antigen, the labeled colored particles may be sensitized by an ordinary method using an antibody against the antigen.

The immunochromatography method of the present invention is typically carried out as follows using the sensitized colored particles obtained as described above.

A means for immobilizing a solution of an antibody as an immobilizing reagent for an antigen to be detected to a chromatographic medium, and an immobilizing reagent comprising solid phase latex particles sensitized by the antibody are applied to a chromatographic medium. A reaction site is formed at an appropriate position on an appropriate chromatographic medium by a means for immobilizing the reaction medium.

The chromatographic medium used here is:
It is necessary that the sensitized colored particles have a pore size larger than the particle size of the sensitized colored particles so that the sensitized colored particles can be chromatographed stably and well and developed sufficiently to reach the reaction site reliably. Specifically, for example, a filter paper made of an inorganic fiber such as glass or silica can be used as a chromatographic medium. A filter paper made of modified cellulose such as nitrocellulose can also be used. However, a filter paper made of simple cellulose is liable to trap sensitized colored particles by cellulose molecules and the like, and the signal may be unclear.

The sensitized colored particles are brought into contact with a sample, and the sensitized colored particles are chromatographed on the above-described chromatographic medium having a reaction site. Specifically, the dispersion liquid of the sensitized colored particles and the sample liquid are mixed, and one end of the chromatographic medium is brought into contact with the mixed liquid so that the mixed liquid reaches the reaction site sufficiently. You just have to expand it. The concentration of the sensitized colored particles in the mixture here is usually 0.0001 to 0.05% by weight.

Thus, when the antigen to be detected is contained in the sample, the antigen in the sample binds to the sensitized colored particles by a specific reaction, and the antigen is immobilized at the reaction site of the chromatographic medium. As a result, the sensitized colored particles are captured at the reaction site.

Instead of bringing the sample into contact with the sensitized colored particles as described above, the sensitized colored particles are previously held and dried between the reaction site of the chromatographic medium and the site of the chromatographic medium where the sample comes into contact. Alternatively, in this state, the sample may be brought into contact with the chromatographic medium, and the sample and the sensitized colored particles may be moved on the chromatographic medium by capillary action. At this time, the sensitized colored particles may be held in another porous material in contact with the chromatographic medium and dried. Also in this case, when the sample contains an antigen to be detected, the sensitized colored particles are captured at the reaction site of the chromatographic medium in the same manner as described above.

Since the sensitized colored particles used here are composed of labeled colored particles, the color of the labeled colored particles appears according to the shape of the reaction site. Therefore, the presence or absence, the amount, and the like of the antigen, which is the substance to be detected, in the sample are detected by visually determining the presence or absence of the coloring signal or the color density.

In the above, various forms are possible. For example, the number of reaction sites formed on the chromatographic medium may be plural, and in this case, the type of the immobilized reagent for each reaction site may be different.

Further, in addition to the original reaction site containing the immobilized reagent capable of binding to the substance to be detected, a confirmation reaction containing the immobilized reagent that does not bind to the substance to be detected but can bind to the sensitized colored particles. A chromatographic medium having sites formed thereon can also be used. In this case, when a substance to be detected is present, a signal appears at the original reaction site.
A signal appears in the confirmation reaction site regardless of the presence or absence of the substance to be detected, so that the absence of the substance to be detected can be positively confirmed. Specifically, when sensitizing a mouse antibody to labeled colored particles, an anti-mouse antibody obtained by immunizing the mouse antibody with another animal is used as an immobilization reagent for forming a reaction site for confirmation. I just need. Then, in the chromatographic medium, if the original reaction site is formed, for example, as a pattern “I” extending in the vertical direction, and the confirmation reaction site is formed, for example, as a pattern “−”, extending in the horizontal direction, the sample is covered in the sample. When an antigen as a detection substance is present, a signal having a “+” pattern appears, while when no antigen is present, a pattern having a “−” pattern appears. Therefore, the presence / absence of the substance to be detected is displayed very clearly by this pattern. In particular, the display of "-" that appears regardless of the presence of the substance to be detected indicates that the sensitized colored particles have reached the reaction site at the same time and the intended immunochromatography has been reliably performed.

[0042]

Hereinafter, embodiments of the present invention will be described.
The present invention is not limited by these. In addition, the surface charge of the latex particles and the labeled colored particles in the following is measured by "Potentiograph E536 type" (manufactured by METTLER GROUP).

Example 1 <Preparation of Labeled Colored Particles (Dye Emulsion Method)> Concentration of red oil-soluble dye “Solvent Red 27” (solubility in toluene at 20 ° C. 8.5 g / 100 ml)
To 1 part by weight of a dye solution consisting of a 5% by weight toluene solution,
5.7 parts by weight of an aqueous solution of sodium dodecyl sulfate having a concentration of 0.25% by weight was added, and an ultrasonic disperser “US300 type”
A red dye emulsion was prepared by dispersing the dye solution using (manufactured by Nippon Seiki Seisakusho).

On the other hand, a blue oil-soluble dye “Solvent Blue 111” (solubility in toluene at 20 ° C. 6.1)
g / 100 ml) to prepare a blue dye emulsion in the same manner as above.

42 g of the above red dye emulsion
Was obtained by soap-free polymerization using 95 parts by weight of styrene as a monomer and 5 parts by weight of methacrylic acid, and potassium persulfate as a polymerization initiator.
m, an emulsion 10 having a solid content of 10% by weight with latex particles having a surface charge of 0.114 meq COO ⁻ / g.
0 g and stirred for 24 hours, then toluene was removed by steam distillation, and sodium dodecyl sulfate and excess dye were removed by washing with distilled water by centrifugal sedimentation.
eqCOO ^- / g was obtained red suspension of the labeled color particles I- a. The same operation was repeated except that the amount of the red dye emulsion was changed to 84 g and 168 g, whereby a suspension of the red labeled coloring particles I- having a surface charge of 0.120 meq COO ⁻ / g and a surface charge of 0 were measured. .126 meqCOO ⁻ / g red labeled colored particles I
-Suspension was obtained.

On the other hand, the above blue dye emulsion was added to 168
g, and the other operations are the same as those for preparing the labeled colored particles I-, and the surface charge is 0.119 meqCOO ⁻ / g.
Of the blue labeled colored particles I-.

Each of the above-mentioned suspensions of the labeled colored particles I- to I- is coated on a glass plate and dried, and the light reflectance is measured using a spectrophotometer “CM-1000” (Minolta Camera Co., Ltd.). Was used for the measurement. Also, lightness L ^* , saturation C ^*
Color difference meter "CR-221 type"
(Minolta Camera Co., Ltd.). Table 1 shows the results
Shown in

[0048]

[Table 1] Note 1) "Ratio of reflectance" in labeled colored particles I- to I-
Is the ratio (650) of the reflectance of light having a wavelength of 650 nm showing red to the reflectance of light having a wavelength of 550 nm showing orange.
nm (reflectance at 550 nm / reflectance at 550 nm) (hereinafter the same in Table 3), and the larger the value of this ratio is, the deeper red is observed. Note 2) The “reflectance ratio” of the labeled colored particles I− is the ratio of the reflectance of light having a wavelength of 450 nm representing blue to the reflectance of light having a wavelength of 550 nm representing orange (reflectance of 450 nm / 550 nm). , And the larger the value of this ratio, the deeper the blue color is observed. Note 3) Indicates lightness in "CIE1976L ^* a ^* b ^* color system solid". The larger the value, the closer to white (the same in Table 3 below). Note 4) Visibility (represented by the above equation 1) in "CIE1976L ^* a ^* b ^* color system chromaticity diagram". The larger this value is, the more vivid it is (the same in Table 3 below). Note 5) Indicates a hue angle, that is, a color tone in the “CIE1976L ^* a ^* b ^* color system chromaticity diagram” (hereinafter the same in Table 3).

<Preparation of sensitized colored particles> Labeled colored particles I-
And 1 ml of a dispersion of labeled colored particles obtained by diluting the suspension of the above with a phosphate buffer (hereinafter referred to as “PBS”) to a solid concentration of 1% by weight, and human chorionic gonadotropin (hereinafter “HCG”). ) Was diluted with PBS so as to have a concentration of 100 µg / ml, and 1 ml of an antibody dilution obtained in an Eppendorf centrifuge tube was shaken at room temperature for 2 hours to sensitize the labeled colored particles I- with the monoclonal antibody. And then washed three times by centrifugation using PBS containing bovine serum albumin (hereinafter referred to as “BSA”) at a concentration of 0.1% by weight, and resuspended to a final volume of 2 ml. A suspension of sensitized colored particles I- was obtained. In addition, labeled colored particles I
Using the suspensions of-, I- and I-, the same operation as described above was performed to obtain suspensions of the sensitized colored particles I- to I-.

<Preparation of Chromatographic Medium> 99.9 parts by weight of styrene and 0.1 of methacrylic acid were used as monomers.
Using a weight part, an emulsion of latex particles obtained by soap-free polymerization using potassium persulfate as a polymerization initiator was diluted with PBS so that the solid content concentration became 0.6% by weight, and 1 ml of the emulsion was diluted with a rabbit antibody against HCG. Was diluted with PBS to a concentration of 100 μg / ml, and 1 ml of a diluted rabbit antibody solution was taken in an Eppendorf centrifuge tube, and shaken at room temperature for 2 hours to sensitize the latex particles with the rabbit antibody. .1% by weight of BS
A solid phase latex particle was prepared by washing with PBS containing A three times by centrifugal sedimentation and resuspending to a final volume of 2 ml. Then, 20 μl of the solid phase latex particles was placed at a position 15 mm from one end of a piece of filter paper made of glass fiber filter paper having a width of 100 mm and a length of 80 mm using an automatic thin-layer chromatographic sampler “Linomart IV” (manufactured by CAMAG). The resulting mixture was applied in a nitrogen atmosphere and dried in a refrigerator to prepare a chromatographic medium having a reaction site.

<Preparation of HCG-Containing Sample> An HCG-containing liquid “Gonatropin 5000” (manufactured by Teikoku Organ Pharmaceutical Co., Ltd.) was diluted with PBS containing 0.1% by weight of BSA to reduce the HCG concentration in 1 ml. 100 mIU, 5
Samples containing 0 mIU and 25 mIU of HCG were prepared.

<Chromatographic treatment> Each suspension of the sensitized colored particles was prepared such that the solid content concentration became 0.0005% by weight.
It is diluted with PBS containing 0.1% by weight of a BSA solution and 0.001% by weight of polyoxyethylene (20) sorbitan monolaurate. One of each of the samples
50 μl or 0.1% by weight of BS as blank
Add 150 μl of PBS containing A, stir the mixture, and immerse the lower end by 5 mm with one end of the side coated with the solid phase latex particles of the above-mentioned chromatographic medium cut to a width of 10 mm downward. The mixture was developed. After 5 minutes, the red signal from the sensitized colored particles at the reaction site was compared visually. Table 2 shows the results of the signal intensities.

[Comparative Example 1] <Preparation of colloidal gold particles> 200 ml of an aqueous solution of gold chloride having a concentration of 0.01% by weight was boiled, and an aqueous solution of sodium citrate having a concentration of 1% by weight was added thereto. From boiling to purple to red to prepare a dispersion of colloidal gold particles A− having an average particle size of 0.03 μm.

<Preparation of Sensitized Labeled Particles> A gold concentration of 0.01
The pH was adjusted to 7.6 by adding a potassium carbonate solution to the dispersion of the above colloidal gold particles A-, which was a weight%, and a monoclonal antibody against HCG was added thereto at a rate of 10 μg per ml of the dispersion of colloidal gold particles. Plus 10
0.1 ml of BSA having a concentration of 30% by weight was added to each ml, and the supernatant was removed by centrifugal sedimentation, and washed three times by centrifugal sedimentation using PBS containing 0.1% by weight of BSA. By re-dispersing, a dispersion of the monoclonal antibody-sensitized gold colloid particles A- was prepared.

<Chromatographic treatment> Immunochromatographic measurement was carried out in the same manner as in Example 1, except that the above-mentioned monoclonal antibody-sensitized gold colloid particles A- were used in place of the sensitized colored particles of Example 1. Table 2 shows the results of the signal intensities.

[0056]

[Table 2] -: No color development at the reaction site is observed. +: Color development at the reaction site is observed. ++: Color development at the reaction site is clearly observed. +++: Color development at the reaction site is strongly observed.

Example 2 Oil-soluble dye “Solvent Red 111” (solubility in toluene at 20 ° C. 1 g /
100 ml) and dissolved in toluene to a concentration of 0.21% by weight to prepare a dye solution. To 100 g of the same emulsion of latex particles as in Example 1, 24 g of the above dye solution was added, and the mixture was stirred for 24 hours. Thereafter, toluene was removed by steam distillation, and the resultant was washed with distilled water by centrifugal sedimentation to obtain a surface charge of 0%. .105me
qCOO ^- / g was obtained a red-labeled colored particles II- suspension of. By repeating the same operation except that the dye solutions having concentrations of 0.42% by weight and 0.84% by weight were used, the suspension of the red labeled coloring particles II- having a surface charge of 0.115 meq COO ⁻ / g was repeated. Suspension and red labeled colored particles with a surface charge of 0.140 meq COO ⁻ / g II
-Suspension was obtained. The labeled colored particles II- to II-
, The light reflectance, lightness L ^* , saturation C ^*, and hue angle H ° were measured in the same manner as in Example 1. Table 3 shows the results.

[0058]

[Table 3]

The sensitized colored particles II- to II- were prepared in the same manner as in Example 1 using the suspensions of the labeled colored particles II- to II-.
A suspension of II- was obtained. These sensitized colored particles II- to II-
Except that each suspension was used in the same manner as in Example 1.
CG immunochromatographic measurement was performed. Table 4 shows the results of the signal intensities.

[0060]

[Table 4] −: No color development at the reaction site is observed ±: Slight color development is observed at the reaction site +: Color development at the reaction site is observed ++: Color development at the reaction site is clearly observed +++: Color development at the reaction site is strongly observed

[Comparative Example 2] A particle size of 0.256 μm, obtained by soap-free polymerization using styrene as a monomer and potassium persulfate as a polymerization initiator, having a surface charge of not more than the detection limit (0.001 meq / g) A suspension of red labeled colored particles III was prepared in the same manner as red labeled colored particles I-, except that an emulsion of latex particles was used, but the surface charge of the labeled colored particles III was below the detection limit. In this case, redispersion during washing by centrifugal sedimentation after sensitization was poor, and aggregates remained. Then, using the obtained labeled colored particles III, HCG was performed in the same manner as in Example 1.
When immunochromatographic measurement was performed, a signal judged as “+” appeared even for a sample containing no HCG, and the signal could not be used for immunochromatographic method.

[0062]

According to the first aspect of the present invention, labeled fine particles are provided.
The latex particles made of synthetic polymer
The surface charge obtained by staining with
標識 0.5 meq / g of the labeled colored particles,
Sensitized colored grains sensitized by substances that can bind to substances
Sensitized colored particles are in a good colored state.
And it shall have good properties, as a result, ensures excellent visual determination property can perform intended immunochromatography operation, therefore it is possible to detect the detection target substance in a sample with high sensitivity. According to the invention of claim 2
If, for example, labeled colored particles, latex particles in an aqueous medium,
Emulsion of solution of oil-soluble dye with oily organic solvent
Because it is obtained by dyeing more,
The signal per particle becomes stronger and the sensitized colored particles
Even if the amount used is small, the visual judgment performance is further improved, and the
It is possible to detect the target substance with higher sensitivity.
You. If the amount of sensitizing colored particles used is small,
S / N ratio is large because the ratio of
In this respect, the detection sensitivity can be improved.
You. The method according to the present invention can be suitably applied to a pregnancy diagnosis.

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tohru Nishihara 1-8-1, Tatsuminishi, Ikuno-ku, Osaka, Osaka Prefecture Rohto Pharmaceutical Co., Ltd. (72) Inventor Masahiko Kinoshita 1-8-1, Tatsuminishi, Ikuno-ku, Osaka, Osaka No. 1 Inside Rohto Pharmaceutical Co., Ltd. (56) References JP-A-2-107965 (JP, A) JP-A 1-276067 (JP, A) JP-A-63-281054 (JP, A) JP-A-58-1983 97656 (JP, A) JP-A-57-168163 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) G01N 33/543 G01N 33/545

Claims (2)

(57) [Claims] 1. Using a chromatographic medium having at least one reaction site containing an immobilized reagent capable of binding to a substance to be detected in a sample, labeled microparticles are added to or together with the sample on the chromatographic medium. Subsequently, while moving chromatographically, the sample is brought into contact with the reaction site, whereby when an analyte is present in the sample, the immobilized reagent at the reaction site passes through the analyte to be detected. In the immunochromatography method for detecting the substance to be detected by utilizing the fact that the labeled fine particles are specifically bound and captured, the labeled fine particles are obtained by dyeing latex particles made of a synthetic polymer with an oil-soluble dye. A substance capable of binding the labeled colored particles having a surface charge amount of 0.01 to 0.5 meq / g negatively charged to the substance to be detected An immunochromatography method characterized in that the particles are sensitized colored particles sensitized by the following method. 2. The method according to claim 1, wherein the labeled colored particles are a latex in an aqueous medium.
Of the solution of the oil-soluble dye with the oily organic solvent.
Characterized by dyeing with Rejon
The immunochromatography method according to claim 1, wherein