Organic colored microparticles, diagnostic reagent kit containing the same, and in vitro diagnosis method

Abstract

Provided are an immunochromatography kit that is highly sensitive and capable of multicoloration, and organic colored microparticles that are ideal as an element of the immunochromatography kit. Organic colored microparticles having an average grain size between 10 and 1,000 nm and a color intensity between 1.0 and 5.0 are prepared using cellulose as the starting material. When the organic colored microparticles are used as a label in an immunochromatography kit, the immunochromatography kit is of a high sensitivity than conventional technology. The immunochromatography kit is also capable of multicoloration and is useful for rapid diagnosis.

Claims

1. Colored cellulose microparticles for use in an immunochromatography kit, wherein the colored cellulose microparticles have an average grain size of 10 nm to 1000 nm and a color intensity of 1.0 to 5.0, wherein 10% by weight to 80% by weight of the colored cellulose microparticles constitute a coloring component which is a reactive dye, and said color intensity being obtained by: preparing a dispersion consisting of the colored cellulose microparticles in water as a dispersion medium at a concentration ranging from 0.01% to 0.1% by weight, measuring the optical absorbance of said dispersion between 400 and 800 nm using a combination of the SV-722 Integrating Sphere and the JASCO-V-650 manufactured by Jasco Corp and an optical path of 10 mm, thereby obtaining a sample absorbance curve, measuring the optical absorbance of the dispersion medium between 400 and 800 nm using said integrating sphere and an optical path of 10 mm, thereby obtaining a background absorbance curve, subtracting the background absorbance curve from the sample absorbance curve, thereby obtaining a corrected absorbance curve, said corrected absorbance curve having a peak with a maximum absorbance value, and dividing the maximum absorbance value of the peak of the corrected absorbance curve by the concentration in % by weight of the dispersion, and then standardized per 0.01% by weight, thereby obtaining the color intensity.

2. The colored cellulose microparticles according to claim 1, wherein a ligand is bound by physical adsorption.

3. The colored cellulose microparticles according to claim 1, having a reactive group.

4. The colored cellulose microparticles according to claim 3, wherein the reactive group has a spacer structure having 3 or more atoms.

5. The colored cellulose microparticles according to claim 3, wherein a ligand is bound to the reactive group by covalent bonding.

6. An immunochromatography kit for detecting a first antigen or antibody as a test substance, the improvement comprising as a label, the colored cellulose microparticles according to any one of claims 1, and 2 to 5, wherein said microparticles are bound to a second antibody or antigen that specifically binds to said first antigen or antibody, and a chromatography substrate coated with a third antibody or antigen that specifically binds to said first antigen or antibody.

7. An immunochromatography kit for detecting a test substance, the improvement comprising as a label, the colored cellulose microparticles according to any one of claims 1, and 2 to 5, wherein said microparticles are bound to a first ligand that specifically binds to said test substance, and a chromatography substrate coated with a second ligand that specifically binds to said test substance.

8. The immunochromatography kit of claim 7, wherein the first ligand and the second ligand are antibodies, antigens, enzymes, genes, hormones, cells, nucleic acids, peptides, or proteins.

Description

EXAMPLES

(1) Although the following provides a detailed explanation of the present invention through examples thereof, the present invention is not limited to these examples.

(2) First, a detailed explanation is provided of a method for measuring organic colored microparticles or a colored microparticle dispersion in the present invention.

(3) All procedures were carried out in an environment at 25 C. unless specifically indicated otherwise.

(4) (1) Particle Size (Particle Diameter) Distribution:

(5) A cellulose microparticle dispersion was measured using the UPA-EX150 Nanotrac Particle Size Analyzer manufactured by Nikkiso Co., Ltd. Unless specifically indicated otherwise, water was used for the liquid in which the cellulose microparticles were dispersed, the cellulose microparticles were measured at a concentration of about 0.1% by weight, and the cumulative number of measurements was 30. In addition, CV values were calculated by dividing the standard deviation in volume grain size distribution as obtained by 30 rounds of measurement by the volume average median diameter.

(6) (2) Color Intensity:

(7) The optical absorbance of cellulose microparticles as well as colored polystyrene latex and gold colloid serving as comparative examples was measured using a combination of the SV-722 Integrating Sphere and the JASCOV-650 manufactured by Jasco Corp. The microparticles were measured at concentrations of 0.01% by weight to 0.1% by weight. Next, values calculated by dividing the maximum value of the absorbance peak (ABS) over a visible light range of 400 nm to 800 nm by the weight percentage of the microparticles were determined in 0.01% by weight increments.

(8) (3) Confirmation of Introduction of Reactive Groups:

(9) A microparticle dispersion introduced with reactive groups was dried to obtain microparticles introduced with reactive groups. The infrared absorption spectrum was measured by the reflection method using the Spectrum 100 Infrared Spectral Analyzer manufactured by Perkin Elmer Co., Ltd., and a comparison was made of the spectra before and after introduction of reactive groups. In the case of carboxyl groups, absorption of free acid of about 1730 cm.sup.1 was confirmed, while in the case of amino groups, absorption of primary amino groups of about 1600 cm.sup.1 was confirmed.

(10) In addition, the M-110-E/H Hydraulic Ultra-high-pressure Homogenizer manufactured by the Microfluidics Corp. was used to break up aggregations of microparticles in the cellulose microparticle dispersion and stained cellulose microparticle dispersion. The treatment pressure at that time was 50 MPa, and the microparticles were passed through the chamber serving as the high-pressure portion of the homogenizer 10 times.

Example 1

Preparation of Microparticles

(11) Linter cellulose was dissolved in a cuprammonium solution followed by diluting with water and ammonia to prepare a cuprammonium solution cellulose solution having a cellulose concentration of 0.37% by weight. The copper concentration of that solution was 0.13% by weight, and the ammonia concentration was 1.00% by weight. Next, a congealing liquid was prepared having a tetrahydrofuran concentration of 90% by weight and water concentration of 10% by weight. 500 g of the preliminarily prepared cuprammonium cellulose solution having a cellulose concentration of 0.37% by weight were then added while slowly stirring 5000 g of the congealing liquid using a magnetic stirrer. After continuing to stir for about 5 seconds, 1000 g of 10% by weight sulfuric acid were added to carry out neutralization and regeneration and obtain 26500 g of a slurry containing the target cellulose microparticles. The resulting slurry was then centrifuged for 10 minutes at a speed of 10000 rpm. The precipitate was removed by decantation, and deionized water was injected and stirred followed by centrifuging again. This procedure was repeated several times until the pH reached 6.0 to 7.0, after which dispersion treatment was carried out with a high-pressure homogenizer to obtain 150 g of a cellulose microparticle dispersion. Furthermore, all of the procedures were carried out in an environment at 25 C.

(12) <Staining of Microparticles>

(13) Next, the cellulose microparticles prepared in the manner described above were stained. 30 g of sodium sulfate and 1 g of Levafix Navy CA Gr. (Registered Trade Mark) manufactured by Dystar GmbH Corp. (to also be referred to as blue dye A) as reactive dye were added to 100 g of the cellulose microparticle dispersion adjusted to a microparticle concentration of 1.0% by weight, followed by heating to 60 C. using a constant temperature bath while stirring. After the temperature reached 60 C., 4 g of sodium carbonate were added followed by staining for 2 hours. Continuing, the resulting crudely stained microparticles were washed with a 5% by weight aqueous solution of sodium hydroxide, recovered by centrifugal separation, washed with pure water and then recovered by centrifugal separation. This series of procedures was defined as one cycle. These procedures were carried out for up to 3 cycles to obtain stained microparticles. The proportion of the dye component was 49% of the weight of the organic colored microparticles.

(14) The results of measuring average grain size and color intensity before and after staining are shown in the following Table 1.

Example 2

(15) Although the unstained cellulose microparticles obtained in Example 1 were stained using the same procedure, the procedure was carried out for a total of 10 cycles to obtain stained microparticles. The results of measuring average particle size and color intensity before and after staining are shown in the following Table 1.

Example 3

(16) Cellulose microparticles and stained cellulose microparticles were obtained using the same method as Example 1 with the exception of using Levafix Rubine CA Gr. (Registered Trade Mark) manufactured by Dystar GmbH Corp. (to also be referred to as red dye B) as a reactive stain to stain the unstained cellulose microparticles obtained in Example 1. The results of measuring average grain size and color intensity before and after staining are shown in the following Table 1.

Example 4

(17) Cellulose microparticles and stained cellulose microparticles were obtained using the same method as Example 1 with the exception of using for congealing a congealing fluid having a tetrahydrofuran concentration of 95% by weight and a water concentration of 5% by weight. The results of measuring average particle size and color intensity before and after staining are shown in the following Table 1.

Example 5

(18) Cellulose microparticles and stained cellulose microparticles were obtained using the same method as Example 1 with the exception of using Levafix Rubine CA Gr. manufactured by Dystar GmbH Corp. (red dye B) as a reactive stain to stain the unstained cellulose microparticles obtained in Example 4. The results of measuring average particle size and color intensity before and after staining are shown in the following Table 1.

Example 6

(19) Cellulose microparticles and stained cellulose microparticles were obtained using the same method as Example 1 with the exception of using for congealing a congealing fluid having an acetone concentration of 26.5% by weight, an ammonia concentration of 0.20% by weight and a water concentration of 73.3% by weight. The results of measuring average particle size and color intensity before and after staining are shown in the following Table 1.

Comparative Example 1

(20) Cellulose microparticles and stained cellulose microparticles were obtained using the same method as Example 1 with the exception of using for congealing a congealing fluid having a tetrahydrofuran concentration of 97% by weight and a water concentration of 3% by weight. The results of measuring average particle size and color intensity before and after staining are shown in the following Table 1.

Example 7

(21) The stained cellulose microparticles obtained in Comparative Example 1 were filtered using a filtration film derived from nitrocellulose having pore size of 0.8 m manufactured by Nihon Millipore K.K. followed by sampling the filtrate. The results of measuring average particle size and color intensity are shown in the following Table 1.

Example 8

(22) Although stained microparticles were obtained by staining the unstained cellulose microparticles obtained in Example 1 using the same procedure as Example 1, only one cycle of the staining procedure was carried out. The results of measuring average particle size and color intensity before and after staining are shown in the following Table 1.

Example 9

(23) Although stained microparticles were obtained by staining the unstained cellulose microparticles obtained in Example 1 using the same procedure as Example 1 with the exception using 0.5 g of Levafix Rubine CA Gr. (Registered Trade Mark) manufactured by Dystar GmbH Corp. (red dye B) for the reactive dye, only one cycle of the staining procedure was carried out. The results of measuring average particle size and color intensity before and after staining are shown in the following Table 1.

Comparative Example 2

(24) Stained microparticles were obtained using the unstained microparticles obtained in Example 1 by carrying out the same procedure as Example 8 with the exception of using 0.2 g of Levafix Navy CA Gr. (Registered Trade Mark) manufactured by Dystar GmbH Corp. (blue dye A) for the reactive dye. The results of measuring average particle size and color intensity before and after staining are shown in the following Table 1.

Comparative Example 3

(25) Stained microparticles were obtained using the unstained microparticles obtained in Example 6 by carrying out the same procedure as Example 8 with the exception of using 0.2 g of Remazol Black B HI-GRAN 150 (Registered Trade Mark) manufactured by Dystar GmbH Corp. (blue dye C) for the reactive dye. The results of measuring average particle size and color intensity before and after staining are shown in the following Table 1.

Comparative Example 4

(26) Color intensity was measured using DS02B (Primary Blue (Registered Trade Mark), average grain size: 0.47 m) manufactured by Bangs Laboratories, Inc. as stained polystyrene latex particles. The results are shown in the following Table 1.

Comparative Example 5

(27) The results of measuring the color intensity of gold colloid particles having an average particle size of 0.04 m are shown in the following Table 1.

(28) TABLE-US-00001 TABLE 1 Uncolored Colored Particles Avg. Avg. particle CV particle CV Dyeing Color size value size value capacity inten- (nm) (%) (nm) (%) Dye (%) sity Ex. 1 248 22 372 30 Blue A 49 2.9 Ex. 2 494 40 Blue A 72 4.1 Ex. 3 391 31 Red B 53 2.8 Ex. 4 484 25 579 43 Blue A 45 3.2 Ex. 5 590 30 Red B 41 2.6 Ex. 6 44 19 62 31 Blue A 39 3.2 Comp. Ex. 1 912 32 1105 51 Blue A 41 3.1 Ex. 7 887 36 2.8 Ex. 8 248 22 269 28 Blue A 20 1.6 Ex. 9 270 25 Red B 13 1.2 Comp. Ex. 2 255 25 Blue A 9 0.8 Comp. Ex. 3 44 19 49 23 Blue C 8 0.6 Comp. Ex. 4 470 22 0.4 Comp. Ex. 5 40 31 2.2
<Performance Evaluation 1>

(29) A performance evaluation was carried out by preparing immunochromatography kits using the stained, colored or chromogenic particles of Examples 1 to 9 and Comparative Examples 1 to 5.

(30) <Preparation of Antibody-Bound Stained Microparticles by Physical Adsorption>

(31) The stained or colored microparticles obtained in Examples 1 to 9 and Comparative Examples 1 to 4 were diluted with a phosphate buffer solution (to be referred to as PBS) to a solid concentration of 1% by weight, 1 ml of the resulting 1% by weight phosphate buffer suspension of stained microparticles and 1 ml of diluted antibody obtained by diluting mouse-derived antibody to human chorionic gonadotropin (to be referred to as hCG) (anti-hCG antibody #504 manufactured by Medix Biochemica Ab) with PBS to a concentration of 100 g/ml were removed into an Eppendorf centrifuge tube and shaken for 2 hours at room temperature, and monoclonal antibody was bound to the stained microparticles, followed by centrifugally washing 3 times using PBS containing bovine serum albumin (BSA) at a concentration of 1% by weight and re-dispersing to a final volume of 2 ml to obtain an antibody-bound stained microparticle dispersion.

(32) <Preparation of Antibody-Bound Gold Colloid>

(33) 200 ml of an aqueous gold chloride solution having a concentration of 0.01% by weight were boiled followed by the addition of aqueous sodium citrate solution having a concentration of 1% by weight thereto and continuing to heat and boil until the color of the solution changed from light yellow to violet-red to prepare a dispersion of the gold colloid particles having an average particle size of 0.04 m indicated in Comparative Example 5. Next, 50 mM potassium dihydrogen phosphate solution was added to the resulting gold colloid dispersion to adjust the pH to 8, followed by adding monoclonal antibody to hCG at a rate of 10 g per 1 ml of gold colloid dispersion, adding 0.1 ml of BSA (bovine serum albumin) having a concentration of 30% by weight to 10 ml thereof, centrifuging, removing the supernatant, washing by centrifuging and precipitating three times using PBS containing BSA at a concentration of 0.1% by weight, and re-dispersing to obtain an antibody-bound gold colloid particle dispersion.

(34) <Preparation of Chromatography Substrate (Membrane)>

(35) A test run antibody was sprayed and imprinted over a width of about 1 mm at a location 7 mm from one end (hereinafter indicating the lower end of a strip, with the other end indicating the upper end of the strip) of a commercially available membrane filter (HA120 manufactured by Nihon Millipore K.K., 25 mm300 mm) using a liquid spraying device perpendicular to the direction of development, or in other words, parallel to the long side of the membrane. More specifically, mouse-derived anti-h subunit antibody (#6601 manufactured by Medix Biochemica Ab) was used for the test run antibody, and a liquid prepared to a concentration of 0.5 mg/ml with PBS was sprayed at 1.0 L/cm. In addition, a control line antibody was sprayed and imprinted over a width of 1 mm at a location 12 mm from the lower end in the same manner. More specifically, rabbit-derived anti-mouse antibody (Z0259 manufactured by Dako Group, Inc.) was used for the control line antibody, and a liquid prepared to a concentration of 0.5 mg/ml with PBS was sprayed at 1.0 L/cm. After spraying each antibody, the substrates were dried for 1 hour followed by blocking using borate buffer solution containing milk casein, washing using Tris-HCl buffer containing sucrose, and fixing overnight at room temperature to prepare a chromatography membrane.

(36) <Preparation of Chromatography Evaluation Samples>

(37) A filter paper absorption pad measuring 20 mm300 mm was contacted with the chromatography membranes using the resulting stained microparticles described in each of the examples and comparative examples at that their respective long sides so that they overlapped over a distance of 5 mm from the upper ends thereof, followed by cutting every 5 mm of width with a guillotine cutter to prepare samples. 60 samples can be obtained based on simple calculation.

(38) <Chromatography Evaluation>

(39) An hCG-containing samples used in a development test were prepared in the manner described below.

(40) hCG was diluted with PBS containing BSA at a concentration of 1% by weight to contain hCG at concentrations of 100, 10 and 0 mIU/ml, respectively. A portion 2 mm from the lower end of the 5 mm wide kit samples obtained as described above was immersed in the sample solutions followed by development of the sample solutions. Ten minutes later, the coloring at the reaction site (label printed portion) on the membrane filter was observed visually. Evaluation criteria consisted of an evaluation of () in the case color was not observed at the test line, (+) in the case color was observed, (++) in the case coloring was clearly visible, and (+++) in the case of observing deep coloring. The evaluation results are shown in the following Table 2.

(41) TABLE-US-00002 TABLE 2 After Staining Avg. Test Line Coloring at Each particle Color Concentration (10 min) size inten- (mIU/ml) (nm) Dye sity 1000 100 10 0 Ex. 1 372 Blue A 2.9 +++ +++ ++ Ex. 2 494 Blue A 4.1 +++ +++ +++ Ex. 3 391 Red B 2.8 +++ ++ ++ Ex. 4 579 Blue A 3.2 +++ ++ ++ Ex. 5 590 Red B 2.6 +++ ++ + Ex. 6 62 Blue A 3.2 ++ ++ + Comp. Ex. 1 1105 Blue A 3.1 ++ ++ ++ + Ex. 7 887 Blue A 2.8 ++ ++ + Ex. 8 269 Blue A 1.6 +++ ++ + Ex. 9 270 Red B 1.2 ++ ++ + Comp. Ex. 2 255 Blue A 0.8 + Comp. Ex. 3 49 Blue C 0.6 Comp. Ex. 4 470 Blue 0.4 ++ + Comp. Ex. 5 40 Red 2.2 +++ + + +

(42) Coloring of the control line was observed in all of the examples and comparative examples. At an hCG concentration of 100 mIU/ml, coloring of the test line was observed in Examples 1 to 9 and Comparative Examples 4 and 5. In addition, at an hCG concentration of 10 mIU/ml or less as well, coloring of the test line was observed in Examples 1 to 9 and Comparative Examples 4 and 5.

(43) In Comparative Example 1, a phenomenon was observed in which apparent sensitivity decreased due to background coloring particularly at high antigen concentrations. In addition, a tendency was observed in which coloring occurred even in the absence of hCG in the samples, namely a tendency towards the occurrence of false positives. In Example 7, when the particles of Comparative Example 1 were used after filtering, although background coloring remained, false positives were no longer observed, thereby indicating that excessively large particle size leads to the occurrence of false positives. An excessively large particle size is unsuitable for diagnostic reagent kits. Since false positives were not observed in Examples 1 to 9, Examples 1 to 9 can be said to have high sensitivity.

(44) On the other hand, in Comparative Examples 2 to 4, coloring was unable to be detected at an hCG concentration of 10 mIU/ml due to low color intensity of the dye. Thus, use of the organic colored microparticles of the present invention can be understood to have high sensitivity in comparison with polystyrene latex.

(45) In addition, in a comparison with the gold colloid of Comparative Example 5, equal or higher sensitivity can be understood to be demonstrated by the inorganic colored microparticles of the present invention. Namely, use of the inorganic colored microparticles of the present invention enables highly sensitive diagnoses with blue color or red color.

(46) <Introduction of Reactive Groups>

(47) Continuing, reactive groups such as carboxyl groups or amino groups were introduced into the stained microparticles obtained in Example 1.

Example 10

(48) Pure water and isopropyl alcohol (Wako Pure Chemical Industries, Ltd., reagent grade) were added to a portion of the blue stained microparticle dispersion obtained in Example 1 to adjust the ratio of isopropyl alcohol to water in the dispersion medium to 85:15 and the microparticle concentration in the dispersion medium to 0.50% by weight. 20 g of the resulting stained cellulose microparticle dispersion were placed in a test tube together with a rotor followed by attaching the test tube to a glass reflux tube. The cellulose microparticle dispersion was heated for 30 minutes in a water bath to a temperature of 50 C. while cooling by refluxing with tap water at about 10 C. Furthermore, heating was carried out while gently stirring using a magnetic stirrer. Subsequently, 74 mg of 40% by weight sodium hydroxide solution were added while stirring followed by continuing to stir for 30 minutes and then adding 216 mg of sodium chloroacetate (Wako Pure Chemical Industries, Ltd.). Carboxyl groups were introduced by continuing to stir and reflux for 3 hours. Three hours later, heating in the water bath was discontinued, a recovery flask was cooled with ice water, and the slurry was cooled after the reaction to a temperature of 20 C. After cooling, 1.0 g of 10% by weight hydrochloric acid was added while continuing to stir, to adjust the pH of the slurry after the reaction to an acidic pH. Dilution by decantation and deionized water was repeated several times using the same centrifuge as that used to wash the microparticles, the pH was adjusted to 6.0 to 7.0, and dispersion treatment was carried out with a high-pressure homogenizer to obtain a carboxylated stained microparticle dispersion. The results of measuring average particle size and color intensity for a portion of the resulting dispersion are shown in the following Table 3.

Example 11

(49) Pure water and acetone (Wako Pure Chemical Industries, Ltd., reagent grade) were added to a portion of the blue stained microparticle dispersion obtained in Example 1 to adjust the ratio of acetone to water in the dispersion medium to 1:1 and the microparticle concentration in the dispersion medium to 1.0% by weight. 10 g of the resulting stained cellulose microparticle dispersion were placed in a test tube together with a rotor followed by attaching the test tube to a glass reflux tube. The cellulose microparticle dispersion was heated for 30 minutes in a water bath to a temperature of 40 C. while cooling by refluxing with tap water at about 10 C. Furthermore, heating was carried out while gently stirring using a magnetic stirrer. Subsequently, 705 mg of 5-hexenoic acid (Wako Pure Chemical Industries, Ltd.), 677 mg of cerium diammonium nitrate (Wako Pure Chemical Industries, Ltd.) and 617 ml of 1 mol/L nitric acid (Wako Pure Chemical Industries, Ltd.) were added. Carboxyl groups were introduced by continuing to stir and reflux for 3 hours. Treatment following the reaction was carried out in the same manner as Example 10 to obtain a carboxylated stained microparticle dispersion. The results of measuring average particle size and color intensity for a portion of the resulting dispersion are shown in the following Table 3.

Example 12

(50) A carboxylated stained microparticle dispersion was obtained using the same method as Example 11 with the exception of using 1654 g of 16-heptadecenoic acid (Wako Pure Chemical Industries, Ltd.) for the reaction agent added to carry out carboxylation. The results of measuring average particle size and color intensity for a portion of the resulting dispersion are shown in the following Table 3.

Example 13

(51) Pure water was added to a portion of the blue stained microparticle dispersion obtained in Example 1 to adjust the microparticle concentration in the dispersion medium to 1.0% by weight. 10 g of the resulting stained cellulose microparticle dispersion were placed in a test tube together with a rotor followed by attaching the test tube to a glass reflux tube. The cellulose microparticle dispersion was heated for 30 minutes in a water bath to a temperature of 35 C. while cooling by refluxing with tap water at about 10 C. Furthermore, heating was carried out while gently stirring using a magnetic stirrer. Subsequently, 571 mg of epichlorhydrin were added, followed by continuing to stir and reflux for 30 minutes to introduce epoxy groups. Subsequently, the temperature of the water bath was raised to 50 C. followed by the addition of 810 g of 6-aminohexanoic acid (Wako Pure Chemical Industries, Ltd.) and continuing to stir and reflux for 1 hour to introduce carboxyl groups. Treatment following the reaction was carried out in the same manner as Example 10 to obtain a carboxylated stained microparticle dispersion. The results of measuring average particle size and color intensity for a portion of the resulting dispersion are shown in the following Table 3.

Example 14

(52) An aminated stained microparticle dispersion was obtained in the same manner as Example 13 with the exception of using 840 g of aqueous ammonia (Wako Pure Chemical Industries, Ltd.) for the reaction agent added to carry out introduction of epoxy groups. The results of measuring average grain size and color intensity for a portion of the resulting dispersion are shown in the following Table 3.

(53) <Confirmation of Reactive Groups with Infrared Spectral Analyzer>

(54) The carboxylated and aminated stained microparticle dispersions obtained in Examples 10 to 14 were dried to prepare carboxylated and aminated stained microparticles, and the introduction of reactive groups was confirmed with an infrared spectral analyzer. Absorption increased at about 1730 cm.sup.1 for the carboxylated stained microparticles and at about 1600 cm.sup.1 for the aminated stained microparticles, thereby confirming successful introduction of reactive groups.

(55) TABLE-US-00003 TABLE 3 Number of Type of Atoms of Average Reactive Spacer particle CV value Color Groups Structure size (nm) (%) intensity Example 10 Carboxyl 1 370 32 2.8 groups Example 11 Carboxyl 5 375 33 2.6 groups Example 12 Carboxyl 16 383 38 2.5 groups Example 13 Carboxyl 9 380 34 2.6 groups Example 14 Amino 3 374 31 2.7 groups
<Performance Evaluation 2>

(56) A performance evaluation was carried out by preparing immunochromatography kits after chemically bonding antibody to the stained microparticles introduced with reactive groups of Examples 10 to 14.

(57) <Preparation of Antibody-Bound Stained Microparticles by Chemical Bonding 1>

(58) A 2-morpholinoethanesulfonate buffer (to be referred to as MES) having a pH of 5.2 and a concentration of 50 mM was prepared using 2-morpholinoethanesulfonic acid (Wako Pure Chemical Industries, Ltd.), sodium hydroxide and pure water, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Wako Pure Chemical Industries, Ltd., to be referred to as carbodiimide) was dissolved in MES buffer and adjusted to a carbodiimide concentration of 20% by weight. After precipitating the carboxylated stained microparticles obtained in Examples 10 to 13 using a centrifuge, the microparticles were re-dispersed in the MES buffer, and the solid concentration was adjusted to a concentration of 1% by weight to obtain carboxylated stained microparticle MES buffer dispersions. 1 g of 20% by weight carbodiimide solution was added to 10 g of the carboxylated stained microparticle MES buffer dispersions and allowed to react for 1 hour in an environment at 25 C. using a constant-temperature shaking water bath, followed by centrifuging for 30 minutes at a speed of 10,000 rpm following completion of the reaction. The precipitate was removed by decantation followed by addition of phosphate buffer and stirring to obtain carbodiimide-activated stained microparticles dispersed in phosphate buffer. Dilution with decantation phosphate buffer solution was repeated three times using the same centrifuge as that used to wash the microparticles to remove unreacted carbodiimide. The resulting carbodiimide-activated stained microparticles were then used to prepare antibody-bound stained microparticles by chemical bonding using the same procedure as that used to prepare antibody-bound stained microparticles by physical absorption.

(59) <Preparation of Antibody-Bound Stained Microparticles by Chemical Bonding 2>

(60) After precipitating the aminated stained microparticle dispersion using a centrifuge, the microparticles were re-dispersed in the aforementioned phosphate buffer and the solid concentration was adjusted to a concentration of 1% by weight to obtain an aminated stained microparticle PBS buffer dispersion. 1 g of 25% by weight glutaraldehyde solution (Wako Pure Chemical Industries, Ltd.) was added to 10 g of the aminated stained microparticle PBS buffer dispersion and allowed to react for 2 hours in an environment at 37 C. using a constant-temperature shaking water bath, followed by centrifuging for 30 minutes at a speed of 10,000 rpm following completion of the reaction. The precipitate was removed by decantation followed by addition of phosphate buffer and stirring to disperse glutaraldehyde-activated stained microparticles in phosphate buffer. Dilution with decantation phosphate buffer solution was repeated three times using the same centrifuge as that used to wash the microparticles to remove unreacted glutaraldehyde. The resulting glutaraldehyde-activated stained microparticles were then used to prepare antibody-bound stained microparticles by chemical bonding using the same procedure as that used to prepare antibody-bound stained microparticles by physical absorption. Unreacted aldehydes were removed by adding 1 g of glycine prior to adding bovine serum albumin at a concentration of 0.1% by weight.

(61) <Chromatography Evaluation>

(62) The antibody-bound stained microparticles by chemical bonding obtained in Examples 10 to 14 and the antibody-bound stained microparticles by physical absorption obtained in Example 1 were evaluated for use as immunochromatography microparticles.

(63) Evaluations were carried out using the same procedure as previously described, and three levels of hCG concentrations were used consisting of 10, 1 and 0 mIU/ml. The evaluation results are shown in the following Table 4.

(64) TABLE-US-00004 TABLE 4 After Staining Test Line Coloring at Each Avg. Color Antibody Concentration (10 min) particle inten- Binding (mIU/ml) size (nm) sity Method 10 1 0 Example 1 372 2.9 Physical ++ adsorption Example 10 370 2.8 Chemical ++ bonding Example 11 375 2.6 Chemical ++ + bonding Example 12 383 2.5 Chemical ++ ++ bonding Example 13 380 2.6 Chemical ++ + bonding Example 14 374 2.7 Chemical ++ + bonding

(65) Coloring was observed for the antibody-bound stained microparticles in which antibody was bound by chemical bonding of Examples 11 to 14 even at an hCG concentration of 1 mIU/ml. In each of these cases, the number of atoms of the spacers of the reactive groups was 3 or more. In contrast, coloring was not observed for the stained microparticles in which antibody was bound by physical adsorption of Example 1 or for the antibody-bound stained microparticles of Example 9 in which the number of atoms of the spacer was 1 at an hCG concentration of 1 mIU/ml. On the basis of these results, the organic colored microparticles of the present invention were determined to be able to support a ligand by chemical bonding.

INDUSTRIAL APPLICABILITY

(66) The organic colored microparticles of the present invention are useful for use as a label for immunodiagnosis and immunochromatography, and can be preferably used in a highly sensitive immunochromatography kit that allows rapid evaluation.