JP2017138271A - Grafted coloring cellulose fine particles - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide chromogenic particles that enable high-sensitivity immunochromatographic diagnosis.SOLUTION: Coloring cellulose fine particles have an average particle diameter of 10-850 nm and a chromogenic intensity of 1.0-10.0, and comprises a bonded polymer comprising a monomer with a reaction active group graft-polymerized with a repeating unit of 2-1000. The repeating unit of the monomer with the reaction active group has a dispersity less than 8. The coloring cellulose fine particles have a sphericity of 10 or less.SELECTED DRAWING: None

Description

  The present invention relates to a grafted colored cellulose fine particle, and a diagnostic kit and in vitro diagnostic using the same.

  In recent years, in the medical field and the food field, a technique that can easily and quickly determine the presence or absence of diseases, pathogenic bacteria, and the like is desired. Various technologies have been developed to meet these needs. Currently, the presence or absence of infection with pathogens such as influenza, pregnancy, cancer markers and myocardial markers, allergic substances in foods and substances that cause food poisoning. Various inspections such as presence / absence can be easily and quickly performed. As these techniques, many measuring methods such as an immunochromatographic measuring method, a fluorescent immunochromatographic measuring method, an enzyme immunoassay method, a latex agglutination measuring method, and a chemiluminescence measuring method have been developed. Among them, the immunochromatographic measurement method that can be judged visually is widely used because it does not require any special equipment or specialized knowledge, and anyone can easily and quickly make a diagnosis. For example, a pregnancy test drug, which is a kind of immunochromatography measurement method, is available at general pharmacies, and is therefore highly versatile for consumers.

The immunochromatographic measurement method (hereinafter referred to as immunochromatography) utilizes an antigen-antibody reaction, and includes a sandwich method and a competitive method. Measurement methods are roughly classified into a lateral flow type in which the detection object is developed in the horizontal direction with respect to the membrane and a flow through type in which the detection object is developed in the vertical direction. From the viewpoint of simplicity, a method of capturing an antigen with an antibody by using a lateral flow type sandwich method is often used. The measurement procedure is as follows.
(1) An antibody that reacts with an antigen to be detected is immobilized on a specific site (test line) on a membrane such as nitrocellulose.
(2) A detection reagent carrying an antibody that reacts with a detection target is prepared on a labeling substance called colored particles, coated on a conjugate pad and dried, and an immunochromatography kit is prepared by combining the membrane and sample pad.
(3) A developing solution in which a specimen to be examined for the presence or absence of an antigen or a specimen obtained by diluting the specimen is dropped on a sample pad, and the presence or absence of color development on the test line is visually observed (or a simple immunochromatographic reader), Determine the presence or absence of antigen.
In general, in many cases of visual determination, it is used as a qualitative analysis for determining only the presence or absence of an antigen (or antibody, protein or DNA) to be examined.

  One of the needs for immunochromatography is to improve analytical sensitivity. The analytical sensitivity is the detection limit concentration of the antigen to be examined. High sensitivity means that the detection limit concentration is low, that is, even if the antigen concentration in the sample is very small. For example, when infectious diseases such as influenza are examined, if the analytical sensitivity of immunochromatography is improved, the presence or absence of infection can be determined even in the early stage of infection when the amount of antigen (virus) is low. Become.

In order to achieve high sensitivity by visual observation, development of colored particles carrying antibodies has been underway. Presently, as color developing particles mainly used in immunochromatography, metal particles such as gold colloid, polymer latex such as polystyrene, and the like can be mentioned. For example, Patent Document 1 below reports on hCG immunochromatography using colloidal gold. Here, there are a physical adsorption type and a chemical bond type as the adsorption method of the antibody and the colored particles. In Patent Document 1, a physical adsorption type gold colloid is used. In the physical adsorption type, the antibody and the colored particles are adsorbed by a physical interaction such as a hydrophobic interaction. On the other hand, the chemical bond type is one in which a carboxyl group or the like which is a reactive group is introduced into the colored particles to form a covalent bond with an amino group or the like in the antibody. In general, it is said that chemically bonded particles are more sensitive than physically adsorbed particles. For example, in Patent Document 2 below, by introducing a reactive group into a gold colloid through a spacer to form a chemically bonded particle, the analytical sensitivity is 1.5 in the hCG system than in the physical adsorption type gold colloid. It is stated to improve by a factor of ~ 2. Patent Document 3 below describes a method for detecting influenza virus using physically adsorbed and chemically bonded latex. Further, in Patent Document 4 below, by using colored cellulose fine particles having a particle size larger than that of gold colloid or latex particles and having high color development strength as color developing particles, the analytical sensitivity is higher than that of gold colloid or latex particles in the hCG system. Has been shown to improve. Furthermore, it has also been reported that when a reactive group such as an amino group or a carboxyl group is introduced into the colored cellulose fine particles to obtain a chemical bond type, the analytical sensitivity is improved as compared with the physical adsorption type colored cellulose fine particles.
However, none of the above methods has reached the sensitivity of fluorescent immunochromatography or chemiluminescence, and development of fine particles for further enhancement of sensitivity is still desired.

JP 2008-197038 A JP 2008-139226 A JP 2007-315883 A International Publication No. 2011-062157

  In view of the present state of the art described above, the problem to be solved by the present invention is to provide colored particles that enable more sensitive diagnosis in immunochromatography.

  As a result of intensive investigations and repeated experiments to solve the above-mentioned problems, the present inventors have found that when colored cellulose fine particles having a large amount of introduction of reactive groups are used as colored particles for immunochromatography, the analytical sensitivity is improved. The headline, the present invention has been reached. That is, the present invention is as follows.

[1] An average particle size of 10 to 850 nm, color development intensity of 1.0 to 10.0, and a polymer having a reactive group active and graft-polymerized with 2 to 1000 repeating units were bonded. The colored cellulose fine particles, wherein the colored cellulose fine particles have a dispersion degree of the repeating unit of the monomer having a reactive group of less than 8 and the sphericity of the colored cellulose fine particles is 10 or less.
[2] The colored cellulose fine particles according to [1], wherein the introduction amount of the reactive group is 0.50 to 30.0 mmol per 1 g of the colored cellulose fine particles.
[3] The colored cellulose fine particles according to [1] or [2], wherein the reactive group is selected from the group consisting of a carboxyl group, an amino group, a phosphoric acid group, and a sulfonic acid group.
[4] The colored cellulose fine particles according to any one of [1] to [3], wherein 10 to 80 wt% of the weight of the colored cellulose fine particles is a coloring component.
[5] The colored cellulose fine particles according to any one of [1] to [4], wherein the coloring component is a dye.
[6] The colored cellulose fine particles according to any one of [1] to [5], wherein a ligand is covalently bonded to the reactive group.
[7] An immunochromatographic diagnostic kit comprising the colored cellulose fine particles according to any one of [1] to [6].

  The analytical sensitivity can be remarkably improved by using the colored cellulose fine particles according to the present invention for immunochromatography. The colored cellulose fine particles according to the present invention can be efficiently bound to an antibody or the like because the amount of reactive active groups introduced is very large by graft polymerization of a monomer having reactive active groups from the fine particles. As a result, it is possible to adjust the direction of antibody binding, the amount of antibody binding, and the like, so that the immunochromatographic analysis sensitivity is improved.

Hereinafter, embodiments will be described in detail.
As described above, Patent Document 4 discloses that the analytical sensitivity is improved by using, for immunochromatography, colored cellulose fine particles having a large particle size and a high color development strength into which a reactive group has been introduced. The target sensitivity has not been reached. The present inventors thought that by introducing a large number of reactive groups into colored cellulose fine particles, antibodies and the like can be efficiently bound to the fine particles. Under such a hypothesis, repeated experiments were carried out, and the development of colored cellulose fine particles having a large amount of reactive active groups introduced was achieved by graft polymerization of a monomer having reactive active groups onto the colored cellulose fine particles. The developed grafted colored cellulose microparticles were used as immunochromatographic coloring particles, and as a result, the sensitivity of the antibody and microparticles was improved, and the analytical sensitivity was successfully improved.

  In one embodiment of the present invention, the average particle size is 10 to 850 nm, the color intensity is 1.0 to 10.0, and the monomer having a reactive group is grafted with 2 to 1000 repeating units. The colored cellulose fine particles to which a polymerized polymer is bonded, wherein the dispersity of the repeating unit of the monomer having a reactive group is less than 8, and the sphericity of the colored cellulose fine particles is 10 or less. Cellulose fine particles.

  The “average particle diameter” of the colored cellulose fine particles in the present embodiment refers to a volume average median diameter measured by a dynamic light scattering method, and the average particle diameter is in the range of 10 to 850 nm. When the average particle diameter is within this range, the surface area of the fine particles is large, so that the test line becomes darker when used as an immunochromatography, that is, the analysis sensitivity is increased. When the average particle diameter is less than 10 nm, the surface area becomes small, and the color intensity per fine particle is lowered. As a result, the analytical sensitivity may be lowered or the fine particles may be aggregated. From the above points, the lower limit of the particle diameter is preferably 40 nm, and more preferably 80 nm. When the fine particle diameter is larger than 850 nm, the pores of the membrane are clogged. As a result, the diagnosis time may become longer, the membrane surface may be colored after the inspection, adversely affect the determination of the inspection result, and the analysis sensitivity may deteriorate. From the above points, the upper limit of the particle diameter is preferably 700 nm, and more preferably 600 nm. In addition, the average particle diameter here is an average value to the last, and a part of particle diameter distribution may remove | deviate from the said range.

  The reason why the volume average is used for the evaluation of the particle size is that too large particles are clogged in the membrane in immunochromatography, but if the volume average is used, the influence of larger particles increases, so even if there are only a few large particles. This is because the impact is reflected. In addition to the volume average, there are various methods for expressing the particle diameter, such as number average and area average. Of course, if the representation is different, the value of the particle diameter also changes, but in this embodiment, volume average is adopted.

  The “color intensity” in the present embodiment is a value that defines the color intensity of the fine particles and is in the range of 1.0 to 10.0. The larger this value, the deeper the color of the fine particles, and the higher the analytical sensitivity when used as an immunochromatography. Of course, the larger the value, the better. Use a darker dye, increase the number of dyeings, connect via some compound as a spacer, increase the amorphous area of the fine particles and make the dye easier to enter, make the fine particles porous For example, it is possible to adopt a method that makes it easier for the dye to enter. However, considering the economy, the upper limit is preferably 7.0, and more preferably 5.0. In addition, the lower the value, the lower the analytical sensitivity when used as an immunochromatography, so the lower limit is preferably 1.5, more preferably 2.0.

  The method for measuring the color intensity is to prepare a pure water dispersion of colored particles of known concentration, measure the visible absorbance using an integrating sphere in the range of 400 to 800 nm with an optical path length of 10 mm, and obtain the peak of the obtained absorbance curve. The value (ABS) is measured, and the obtained value is divided by the weight percentage of the colored particles, and defined as a value converted to an absorbance around 0.01% by weight of the colored particles. For example, when the concentration of the prepared coloring particles is 0.0045% and the peak value of the absorbance curve is 1.0, the coloring intensity is (1 × 0.01) ÷ 0.0045 = 2.2. Become.

  In the present embodiment, the reason for performing the visible absorbance measurement using an integrating sphere for the measurement of the color density of the fine particles is that the color density of the fine particles dispersed in the liquid can be measured most accurately. As a method of measuring the color density of the fine particles, there is a method of measuring a solid obtained by drying the fine particles with a colorimeter or the like, but such a method cannot accurately measure the color depth of the fine particles. . For example, metal colloids and the like have different color tones and maximum wavelengths depending on the particle diameter, and the dry aggregated state cannot accurately reflect the color density of the state dispersed in the liquid. Further, even when the particles are dispersed in the liquid at the same particle concentration, the color density becomes light when aggregation occurs. Furthermore, the reason for using an integrating sphere when performing visible absorbance measurement is to remove the influence of scattering of the particles themselves. Ordinary visible absorbance measurement is a method of measuring transmitted light, and not only the absorption by the coloring component but also the influence of scattering of the fine particles themselves is reflected on the incident light. For example, gold colloids generally used in immunochromatography may have a particle size of 40 nm to 60 nm, and sometimes 100 nm, but in any case, the particle size is small, so there is almost no influence of scattered light. On the other hand, latex particles have a large particle size and are clearly affected by scattered light. For the reasons described above, in the present embodiment, visible absorbance measurement using an integrating sphere is employed in order to more accurately reflect the color density of the particles themselves when the particle diameters and particle materials are different.

  The “reactive group” in the present embodiment is not particularly limited, and a carboxyl group, amino group, phosphoric acid group, sulfonic acid group, hydroxyl group, aldehyde group, thiol group, and the like can be used. From the viewpoints of binding properties with antibodies and workability when binding, those selected from the group consisting of carboxyl groups, amino groups, phosphate groups, and sulfonate groups are particularly preferred. It is also possible to introduce other substituents using the introduced reactive groups. For example, after introducing an amino group, it is possible to introduce a maleimide group from the introduced amino group. Whether or not a reactive group has been introduced can be confirmed by infrared spectroscopy, elemental analysis, fluorescence emission analysis, or the like.

  The “monomer having a reactive group” in the present embodiment is not particularly limited, and acrylic acid, sodium acrylate, methacrylic acid, sodium methacrylate, maleic acid, sodium maleate, β-carboxyethyl acrylate, dimethyl Examples thereof include aminoethyl methacrylate, diethylaminoethyl methacrylate, tertiary butylaminoethyl methacrylate, and vinyl sulfonic acid.

  In the present embodiment, the number of “repeating units of a monomer having a reactive group” indicates how many monomers having a reactive group are polymerized. For example, when ten monomers are polymerized, the number of repeating units is ten. In the present embodiment, there are 2 to 1000 such repeating units. If the number of repeating units of the reactive group is one, the reactive group is buried in the dye layer present on the surface of the fine particles, and the reactivity with the antibody may be deteriorated. Therefore, the lower limit is preferably 5, and more preferably 10. On the other hand, when the number of repeating units is more than 1000, polymer chains generated by graft polymerization become long, so that the polymer chains may be entangled and fine particles may aggregate. When the fine particles are aggregated, the fine particles are likely to be clogged in the membrane when used for immunochromatography, which may cause poor development. Therefore, the upper limit is preferably 800, and more preferably 500. Gel permeation chromatography (GPC) is used as a method for measuring the repeating unit of the monomer having a reactive group. In Patent Document 4, reactive groups are also introduced, but in terms of repeating units, all are one. When the number of repeating units of the monomer having a reactive group is large, more reactive groups are introduced as compared with Patent Document 4, so that the binding efficiency with an antibody or the like is remarkably improved, and an improvement in sensitivity can be expected.

  Moreover, in this embodiment, the dispersion degree of the repeating unit of the monomer which has a reactive group is less than 8.0. The dispersity is a variation in the repeating unit of the monomer having a reactive group for each polymer chain generated by graft polymerization. The closer the dispersity is to 1, the smaller the variation. Therefore, the degree of dispersion is preferably as close to 1 as possible from the viewpoint of immunochromatographic reproducibility. If the degree of dispersion is 8 or more, the number of monomer repeating units having a reactive group varies in the fine particles, so that the reproducibility of the antibody adsorption amount and the reproducibility of the immunochromatography may be significantly reduced. Therefore, the upper limit is preferably 6.0, and more preferably 4.0.

  The “sphericity” in the present embodiment is a value representing the shape of the fine particles, and is calculated from the ratio of the short diameter to the long diameter (long diameter / short diameter) of the colored cellulose fine particles. In the present embodiment, the sphericity is 10 or less. A rod-like, fiber-like or mesh-like one having a sphericity exceeding 10 is not preferred as the colored cellulose fine particles. If this major axis / minor axis exceeds 10, when used for immunochromatographic colored particles, it may not develop, clogging may occur, and the diagnostic time may be prolonged, and the analysis sensitivity may also be reduced. There is also a risk. The upper limit of the average value of the major axis / minor axis of 100 colored cellulose fine particles is preferably 5.0, more preferably 3.0, and still more preferably 2.0. The smaller this value is, the closer the shape of the colored cellulose fine particles is to a true sphere.

  As the “calculation method of sphericity” in the present embodiment, the projected area of fine particles appearing in an electron microscope image is measured, and the circumference of a circle having the same area as that area is represented in the electron microscope image. It can be calculated by the ratio of the actual circumference of the fine particles. In this measurement method, each fine particle is only seen on a plane, but by using an average value obtained by measuring at least 100 fine particles, variation in the observation direction can be taken into consideration, and as a result, the fine particles are three-dimensionally obtained. The degree of the true sphere seen can be shown.

  The “introduced amount of reactive groups” in the present embodiment is the amount of reactive groups introduced per 1 g of colored cellulose fine particles. Such introduction amount is preferably 0.50 to 30.0 mmol / g. As a calculation method, neutralization titration of acid / base or fluorescence emission analysis is used. When the introduction amount is less than 0.50 mmol / g, the binding efficiency with the antibody is lowered, and the analysis sensitivity may be lowered. Therefore, the lower limit is preferably 0.80 mmol / g, and more preferably 1.0 mmol / g. On the other hand, when the amount introduced exceeds 30.0 mmol / g, the weight per fine particle becomes heavy, and the color intensity per fine particle is lowered. When the coloring intensity is lowered, the analytical sensitivity may be lowered when used for colored particles of immunochromatography. Therefore, the upper limit is preferably 20 mmol / g, more preferably 10 mmol / g.

  The “ratio of colored components of colored cellulose fine particles” in the present embodiment refers to the ratio of colored components to the total weight of the colored cellulose fine particles. For example, when 1.0 g of colored cellulose fine particles is composed of 0.2 g of cellulose and 0.8 g of a colored component, the ratio of the colored component is 80 wt%. The ratio of the coloring component of the colored cellulose fine particles is preferably 10 to 80 wt%. When it is within this range, the analytical sensitivity is high when used as an immunochromatography. When the ratio of the coloring component is less than 10 wt%, sufficient color intensity cannot be obtained, and the analytical sensitivity becomes low when used as an immunochromatography. From this viewpoint, the lower limit is preferably 20 wt%, more preferably 30 wt%. Even if the ratio of the coloring component exceeds 80 wt%, there is no particular problem, but the upper limit is preferably 65 wt%, more preferably 70 wt% from the economical viewpoint.

  The “calculation method of the ratio of the coloring component of the colored cellulose fine particles” in the present embodiment can be calculated from a change in weight before and after coloring. In addition, when it is difficult to calculate from the change in weight, an operation of separating the colored component from the fine particles can be performed to isolate and calculate the colored component or fine particles. For example, when cellulose fine particles are dyed with a reactive dye, it can be calculated by cutting the covalent bond between cellulose and dye with acid or alkali and collecting the cellulose fine particles by centrifugation. It can also be calculated by decomposing only cellulose using cellulase. Similarly, the “ratio of cellulose in the colored cellulose fine particles” can also be calculated.

  In the present embodiment, when a dye is used as the coloring component, the “type of dye” is not particularly limited. Dyes such as reactive dyes, direct dyes, metal-containing dyes, acid dyes, basic dyes, disperse dyes, sulfur dyes, vegetable dyes, naphthol dyes, and fluorescent dyes can be used. Of course, any dye may be combined. Among these, a reactive dye that is covalently bonded to a hydroxyl group of cellulose is particularly preferable from the viewpoint of retaining a large amount of dye and the stability.

  In the present embodiment, a ligand can be covalently bound to the reactive group. A “ligand” is a substance having a property of selectively and specifically binding to a specific substance to be examined. Although the kind is not specifically limited, For example, an antibody, an antigen, an enzyme, a gene, a hormone, a nucleic acid, a peptide, protein etc. are mentioned.

  Another embodiment of the present invention is an immunochromatographic diagnostic kit containing the colored cellulose fine particles. The “immunochromatography diagnostic kit” is a method for simply detecting the presence or absence of a test target substance in various specimens. Types of the diagnostic kit include a lateral flow type and a flow through type. Although it will not specifically limit if color developing particles and a sample pad are used, Preferably it is a lateral flow type. Further, among the lateral flow types, there are a dipstick type and a cassette type, but these types are not particularly limited. The configuration of the diagnostic kit is not particularly limited, and any configuration may be used as long as it is generally used in the field. The type of member other than the colored particles is not particularly limited as long as it is used in the art, and includes, for example, a sample pad, a conjugate pad (including antibody-sensitized colored particles), a membrane such as nitrocellulose, an absorption pad, And mount. Further, some of these members may be omitted if necessary.

  The “diagnostic method” using the immunochromatographic diagnostic kit of the present embodiment refers to various diagnoses performed using the immunochromatographic diagnostic kit. The diagnostic object is not particularly limited, and can be used for various diagnostic tests such as human, animal, food, plant, and other environmental tests. In general diagnostic procedures, a sample is collected from the test subject, and if necessary, it is pre-treated, such as extraction and filtration, dripped onto the sample pad, waits for a predetermined time from the start of the test, The diagnosis result is judged from the color development depending on the presence or absence. Of course, the present invention is not limited to this procedure, and the same procedure and principle can also be used for diagnosis. Preferably, extra foreign matters and contaminants can be removed by filtering the specimen sample in advance, so that further shortening of diagnosis time and improvement of diagnosis accuracy can be expected.

  The target that can be diagnosed by the immunochromatography diagnostic kit of the present embodiment is not particularly limited, but specific examples include the following: cancer marker, hormone, infection, autoimmunity, plasma protein, TDM, coagulation / Examples include fibrinolysis, amino acids, peptides, proteins, genes, cells, and the like. More specifically, CEA, AFP, ferritilin, β2 micro, PSA, CA19-9, CA125, BFP, elastase 1, pepsinogen 1 and 2, fecal occult blood, urinary β2 micro, PIVKA-2, urinary BTA, insulin , E3, HCG, HPL, LH, HCV antigen, HBs antigen, HBs antibody, HBc antibody, HBe antigen, HBe antibody, HTLV-1 antibody, HIV antibody, toxoplasma antibody, syphilis, ASO, influenza A antigen, influenza A Antibody, influenza B antigen, influenza B antibody, rota antigen, adenovirus antigen, rota adenovirus antigen, group A streptococci, group B streptococcus, candida antigen, CD fungus, cryptolocus antigen, cholera fungus, meningitis Fungal antigen, granule elastase, Helicobacter pylori antibody, O157 antibody, O157 antigen, Leptospira antibody, Aspergillus antigen, MRSA, RF, total IgE, LE test, CRP, IgG, A, M, IgD, transferrin, urinary albumin, urinary transferrin, myoglobin, C3 / C4, SAA, LP (a), α1-AC, α1-M, haptoglobin, microtransferrin, APR score, FDP, D dimer, plasminogen, AT3, α2PI, PIC, PAI-1, protein C, coagulation factor X3, type IV Collagen, hyaluronic acid, GHbA1c, other various antigens, various antibodies, various viruses, various fungi, various amino acids, various peptides, various proteins, various DNAs, various cells, various allergens, various residual agricultural chemicals, various harmful substances.

  Examples of the method for producing cellulose fine particles, the method for coloring the cellulose fine particles, the method for grafting to the colored cellulose fine particles, the method for producing the immunochromatographic diagnostic kit and the like are illustrated below, but the present invention should not be limited by these examples.

[Method for producing cellulose fine particles]
Cellulose linter is dissolved in a good solvent for cellulose. As the good solvent, a copper ammonia solution prepared by a known method is used. As the coagulation liquid, an organic solvent + water + ammonia mixed system is mainly used. While stirring the coagulation liquid, a previously prepared copper ammonia cellulose solution is added to perform coagulation. Furthermore, the slurry containing the target cellulose fine particle can be obtained by adding and neutralizing and regenerating sulfuric acid. At this time, the slurry is acidic due to the residue of the acid used for the regeneration, and further contains impurities such as ammonium salts generated by neutralization, and therefore it is necessary to perform an operation for purification into a cellulose dispersion composed of cellulose fine particles and a medium. Become. As the purification operation, centrifugation, decantation, and repetition of dilution with a dispersion medium liquid are used. Since the cellulose fine particles in the obtained cellulose fine particle dispersion may be aggregated in the course of the purification operation, in this case, a dispersion treatment by shearing or the like can be performed. A high pressure homogenizer can be used as a means for applying shear.

[Method for coloring cellulose fine particles]
Sodium sulfate and reactive dye are added to the obtained aqueous dispersion of cellulose fine particles, and the temperature is raised to an appropriate temperature in a thermostatic bath while stirring with a magnetic stirrer. After heating, sodium carbonate is added as an alkali to start dyeing. A slurry containing the desired colored cellulose fine particles can be obtained after a predetermined time. At this time, since the slurry is alkaline and further contains sodium sulfate, unreacted dye, etc., it is necessary to refine the slurry to a colored cellulose fine particle dispersion comprising colored cellulose fine particles and a medium. In the same manner as above, purification by centrifugation is performed to obtain a colored cellulose fine particle dispersion. The colored cellulose fine particles in the obtained colored cellulose fine particle dispersion may aggregate in the course of the purification operation, and in this case, a dispersion treatment by shearing or the like can be performed. A high pressure homogenizer can be used as a means for applying shear.

[Grafting method to colored cellulose fine particles]
In the case of graft polymerization of a monomer having a reactive group, the “method of graft polymerization” is not particularly limited. Radiation graft polymerization, reversible non-cleavable chain transfer polymerization (RAFT), radical polymerization via nitroxide, atoms Transfer radical polymerization (ATRP), cellulose cleavage polymerization using hydrogen peroxide or the like, radical dispersion polymerization, or the like can be used. Among these, radical dispersion polymerization in which the reaction proceeds under mild conditions is preferable from the viewpoints of preventing fading and discoloration of coloring components such as dyes, and preventing changes in particle diameter and shape of fine particles. Although the grafting method by radical dispersion polymerization is shown below, of course, it should not be limited to this method.

[Method for preparing immunochromatographic diagnostic kit]
A dispersion of grafted colored cellulose fine particles adjusted to a predetermined concentration is prepared, a buffer solution and an antibody are added, and the mixture is stirred for a certain period of time while adjusting the temperature, and the antibody is bound to the grafted colored cellulose fine particles. After stirring for a fixed time, the colored cellulose fine particles are blocked by adding a blocking agent and stirring for a fixed time while adjusting the temperature. As the blocking agent, various blocking agents can be used according to the composition of the substance to be examined, the specimen, or a solution for diluting it. Casein is particularly preferred for blocking grafted colored cellulose particulates. Centrifugation is performed to wash the grafted colored cellulose fine particles after antibody binding and blocking, and the supernatant liquid containing excess antibody and blocking agent is separated from the precipitated fine particles, and the supernatant liquid is removed by decantation. . A liquid such as a buffer solution is added to the settled fine particles, and if necessary, a dispersion treatment is performed using ultrasonic waves. Washing by a series of operations such as sedimentation by centrifugation, removal of supernatant, and addition of liquid is performed as many times as necessary to prepare a dispersion containing a predetermined concentration of fine particles subjected to antibody adsorption and blocking. Add protein, surfactant, sugars such as sucrose and trehalose to this dispersion as needed, apply a certain amount of the resulting solution to a glass fiber conjugate pad, and dry to prepare the detection reagent containing part. . If necessary, apply a buffer solution, surfactant, protein, reagent for trapping impurities in the sample, antiseptic, antibacterial agent, antioxidant, hygroscopic agent, etc., and dry to prepare a sample pad To do. Further, a membrane made of a nitrocellulose porous membrane in which an antibody is immobilized at a predetermined position and an absorbent pad made of cellulose filter paper for absorbing the specimen are prepared. They are fixed to a sheet having an adhesion site called a backing sheet, and cut into a predetermined size to produce an immunochromatography diagnostic kit.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited only to these examples. Further, all operations not particularly described were performed in an environment of a temperature of 23 ° C. and a relative humidity of 55% RH.

(Measurement of average particle size of colored fine particles)
As a device, a nanotrack particle size distribution measuring device UPA-EX150 (dynamic light scattering type) manufactured by Nikkiso Co., Ltd. was used. As a measurement sample, a sample containing 0.01% by weight of colored fine particles and 99.99% by weight of pure water was used. As measurement conditions, the number of integrations was 30 times, the measurement time per measurement was 30 seconds, and the median diameter was defined as the average particle diameter using a volume average particle size distribution.

[Measurement of coloring intensity of colored fine particles]
As an apparatus, an apparatus in which an integrating sphere unit ISV-722 manufactured by JASCO V-650 was attached to JASCO V-650 manufactured by JASCO Corporation was used. As a sample to be measured, a sample having 0.01% by weight of colored fine particles and 99.99% by weight of pure water was used. The sample was placed in a quartz cell having an optical path length of 10 mm and measured. Among the obtained absorbance peaks, the maximum value (ABS) in the visible light range of 400 to 800 nm was defined as the color intensity.

[Measurement of the number of repeating units of the monomer having a reactive group]
As the apparatus, a high performance liquid chromatograph apparatus Prominence manufactured by Shimadzu Corporation was used. As a sample to be measured, a free initiator added during the grafting reaction of the colored cellulose fine particles was used. The solution after the grafting reaction was collected so that the concentration of the free initiator was 10 mg / ml, and added to 1 ml of 0.05M NaHPO ₄ solution. The sample thus prepared was placed in a 1.5 ml sample bottle and measured. The molecular weight of the free initiator was calculated using a calibration curve prepared using polymethyl methacrylate as a standard. By the following formula (1), the molecular weight (B) of sodium methacrylate was removed from the calculated molecular weight (A) to obtain a repeating unit.
Number of repeating units = A / B (1)
For example, when the calculated molecular weight is 10,000, the repeating unit is 10,000 ÷ 108 = 93. As another method, after adding 20 mL of colored cellulose fine particles grafted on a 20 mL screw tube and 2 mL of 10 wt% sodium hydroxide aqueous solution and stirring at 30 ° C. for 2 hours, the molecular weight is measured and repeated in the same manner as described above. The number of units was calculated.

[Measurement of amount of reactive groups introduced]
As an apparatus, an automatic potentiometric titrator AT-510 manufactured by Kyoto Electronics Co., Ltd. was used. The sample to be measured was obtained by dispersing 50 mg of grafted colored cellulose fine particles in 50 ml of pure water. When the reactive group was an acidic substituent such as a carboxyl group, first, a 0.1 M hydrochloric acid solution was added dropwise until the pH of the sample reached 3. Thereafter, a 0.1 M sodium hydroxide solution was added dropwise until the pH reached 11, and the titer at that time was recorded (A mL). Similarly, the titration amount (B mL) of 0.1 M sodium hydroxide solution of fine particles not grafted was recorded, and the introduction amount was calculated by the following formula (2).
Introduction amount (mmol / g) = 1000 × M × (A−B) / X (2)
{Wherein, X: amount of fine particles used (mg), M: concentration of used sodium hydroxide solution (mol / L)}.
In the case of an alkaline substituent such as an amino group, first, a 0.1 M sodium hydroxide solution is dropped until pH 11 and then a 0.1 M hydrochloric acid solution is dropped until pH 3; Record. The calculation method is the same as in the case of the acidic substituent.
Introduction amount (mmol / g) = 1000 × M × (A−B) / X (3)
{In the formula, A: hydrochloric acid titration amount (mL) of grafted colored cellulose fine particles, B: hydrochloric acid titration amount (mL) of ungrafted colored cellulose fine particles, X: amount of fine particles used (mg), M: used Concentration of hydrochloric acid solution (mol / L)}.
For example, when the drop amount of 0.1 M hydrochloric acid of 50 mg of the fine particles introduced with a carboxyl group is 2.000 ml and the drop amount of 50 mg of the ungrafted fine particles is 1.000 ml, the introduction amount is 1000 × 0 according to formula (3). 0.1 × (2.000-1,000) /50=2.0 mmol / g.

[Calculation of ratio of coloring component of colored fine particles]
Calculation was performed from the weight of the colored fine particles after the predetermined number of coloring operations and the weight of the particles before coloring. For example, 1.0 g of cellulose particles were colored, and when 2.5 g of colored cellulose particles were obtained, the color component was calculated as 2.5 g−1.0 g = 1.5 g. In this case, the ratio of the coloring component is 1.5 g / 2.5 g × 100 = 60.0 wt%. As another method, the calculation was also performed by a method using a device called Multisizer 4 manufactured by Beckman Coulter. As a sample to be measured, 100 mL of a sample having 5.00 wt% colored fine particles and 95.00 wt% pure water was used, and the number of fine particles in the dispersion was calculated (A). Next, the number of fine particles in the dispersion was calculated using 100 mL of a sample containing 5.00 wt% of fine particles before coloring and 95.00 wt% of pure water (B pieces). Next, the sample used for the measurement was dried by heating and drying, and the weight of fine particles in the sample was calculated (colored fine particle weight (g): WA, pre-colored fine particle weight (g): WB). And the ratio of the coloring component was computed by the following formula | equation (4).
Ratio (wt%) = (WA / A−WB / B) / (WA / A) × 100 (4)
For example, when A = 10000, B = 10000, WA = 0.030 g, WB = 0.010 g, the ratio of the coloring component is (0.030 / 10000-0.010 / 10000) according to the formula (4). /(0.030/10000)×100=67 wt%.

[L / D ratio measurement of colored fine particles]
As the apparatus, a scanning electron microscope JSM-6700 manufactured by JEOL Ltd. was used. A sample containing 0.01 wt% of colored fine particles and 99.99 wt% of pure water was dropped onto a mica plate, and after 10 seconds, the colored fine particles were adsorbed on the mica plate, and extra liquid was absorbed and dried by Kimwipe. The obtained mica plate was coated with platinum to prepare a sample for electron microscope measurement. Observation is performed at an acceleration voltage of 1.6 kV and a measurement magnification of 50,000 times, and the required number of images are taken so that the number of particle images becomes 100 or more, and the major axis (L) and minor axis (D) of each particle are measured. Then, an average value (sphericity) of L / D of 100 particles was calculated.

[Method of binding grafted colored cellulose fine particles and antibody]
In a 2 ml Spitz tube, 90 μL of phosphate buffer (100 mM, PH = 6.0), 10 μL of 1 wt% dispersion of grafted colored cellulose fine particles, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride 10 μL of a 1 wt% DMF solution (manufactured by Tokyo Chemical Industry Co., Ltd., D0722) of carbodiimide, manufactured by Tokyo Chemical Industry Co., Ltd., D1601) was placed in a dryer at 37 ° C. for 2 hours. Two hours later, an anti-hCG antibody (Fitzgelard, # 5014) was added to the Spitz tube so as to be 10 wt% with respect to the grafted colored cellulose fine particles, and further reacted at 37 ° C. for 2 hours. Thereafter, a blocking solution (100 mM borate buffer solution, PH = 8.5) containing 1 wt% casein (manufactured by Wako Pure Chemical Industries, Ltd., 030-01505) was added to a 1200 μL Spitz tube, and 1 in a dryer at 37 ° C. Let stand for hours. After 1 hour, using a centrifuge (manufactured by Kubota Corporation, 6200) and a centrifuge rotor (manufactured by Kubota Corporation, AF-5008C), centrifugation was performed at 14000 g for 30 minutes, and the grafted colored cellulose to which the antibody was bound. After allowing the fine particles to settle, the supernatant was discarded. Subsequently, borate buffer (50 mM, PH = 10.0) was added to a 1200 μL Spitz tube, treated with an ultrasonic disperser (manufactured by SMT Co., UH-50) for 10 seconds, and the grafted colored cellulose to which the antibody was bound. Fine particles were dispersed. After fully dispersing, centrifugation at 14,000 g was performed for 20 minutes, and the supernatant was discarded. A borate buffer solution (50 mM, PH = 10.0) was added so that the concentration of the grafted colored cellulose fine particles to which the antibody was bound was 0.03 wt%, and was sufficiently dispersed by an ultrasonic disperser. By the above method, grafted colored cellulose fine particles (detection reagent) to which an antibody was bound were obtained.

[Impregnation and drying of detection reagent in conjugate pad]
A polyethylene conjugate pad (Ah, 6615) was immersed in 0.05 wt% Tween-20 (Sigma Aldrich, T2700) to remove excess liquid, and then dried at 50 ° C. for 60 minutes. Subsequently, it was cut into a shape having a height of 10 mm and a length of 300 mm. Subsequently, using a micropipette, the detection reagent was evenly applied to 272 μL / 8 cm and dried at 37 ° C. for 30 minutes.

[Pretreatment of sample pad]
A regenerated cellulose continuous long-fiber nonwoven fabric was impregnated in a PBS buffer solution (66 mM, PH 7.4) containing a large excess of 2.0 wt% BSA (Sigma Aldrich, A7906) and 2.0 wt% Tween-20. After removing the excess liquid, it was dried at 50 ° C. for 60 minutes. Subsequently, it was cut into a shape having a height of 20 mm and a length of 300 mm.

[Preparation of capture antibody-coated membrane]
A nitrocellulose membrane (manufactured by Millipore, SHF0900425) was cut into a shape having a width of 25 mm and a length of 300 mm. Using a liquid application apparatus (Musashi Engineering Co., Ltd., 300DS), a PBS solution (66 mM, PH7.4) containing 0.1 wt% anti-hCG-β mouse antibody (MedixBiochemical, 6601) at a rate of 0.1 μl / mm And applied to a portion having a height of 12 mm. Subsequently, it was dried at 37 ° C. for 30 minutes.

[Preparation of immunochromatography diagnostic kit]
The prepared capture antibody-coated membrane, an absorption pad (Millipore, C083), a conjugate pad containing a detection reagent, and a sample pad were attached to a backing card (manufactured by Adhesives Research, AR9020). Subsequently, it was cut into a width of 5 mm by a cutting machine to obtain an immunochromatography diagnostic kit having a width of 5 mm and a height of 60 mm.

[Measurement of detection limit of immunochromatography diagnostic kit]
An immunochromatographic diagnostic kit cut to a width of 5 mm was placed in a plastic housing. The obtained diagnostic kit containing the housing was measured using an immunochromatography reader C10066-10 manufactured by Hamamatsu Photonics. The apparatus was set according to the color of the particles used. Human chorionic gonadotropin (hereinafter referred to as “hCG”) was used as the test substance. hCG was diluted with a phosphate buffer solution (hereinafter referred to as “PBS”) of 66 mM and PH 7.4 containing 1 wt% bovine serum albumin (hereinafter referred to as “BSA”), and the hCG concentration was 3.20 mIU / ml, 1 .60 mIU / ml, 0.80 mIU / ml, 0.40 mIU / ml, 0.20 mIU / ml, 0.10 mIU / ml, 0.05 mIU / ml, 0.025 mIU / ml, 0.0125 mIU / ml, 0.00625 mIU A positive specimen that was gradually diluted to / ml was prepared. 120 ul of this positive specimen was dropped on the sample dropping part of the diagnostic kit, and the chromogenic intensity of TL after 15 minutes was measured with an immunochromatographic reader. This measurement was carried out 5 times at each concentration. When the average of the obtained values was a value obtained by measuring a negative sample + 20 mABS or more, a positive determination was made, and in the following cases, it was considered to be below the detection limit. The lower limit hCG concentration at which this positive determination was obtained was taken as the detection limit.
In the following examples and comparative examples, immunochromatographic evaluation was performed by the above-described method.

[Example 1]
A copper ammonia cellulose solution having a cellulose concentration of 0.37 wt%, a copper concentration of 0.13 wt%, and an ammonia concentration of 1.00 wt% was prepared by a conventionally known method. Further, a coagulation liquid having a tetrahydrofuran concentration of 89.0 wt% and a water concentration of 11.0 wt% was prepared.
500 g of the prepared copper ammonia cellulose solution was added while slowly stirring 5000 g of the coagulation liquid using a magnetic stirrer. After stirring for about 5 seconds, 1000 g of 10 wt% sulfuric acid was added to neutralize and regenerate to obtain 6500 g of a slurry containing cellulose fine particles.
The resulting slurry was centrifuged for 10 minutes at a speed of 10,000 rpm. The precipitate was removed by decantation, poured in deionized water, stirred and centrifuged again. This operation was repeated several times until the pH reached 6.0 to 7.0, and then a dispersion treatment using a high-pressure homogenizer was performed to obtain 150 g of a cellulose fine particle dispersion. As a result of measuring the average particle diameter of the obtained cellulose fine particles, it was 261 nm.

Next, the cellulose fine particles prepared as described above were dyed. To 100 g of cellulose fine particle dispersion adjusted to a fine particle concentration of 1.0 wt%, 30 g of sodium sulfate and 1 g of reactive dye (Levafix Red CA Gr., Manufactured by Dystar) were added and stirred up to 60 ° C. using a thermostatic bath. The temperature rose. After raising the temperature to 60 ° C., 4 g of sodium carbonate was added and dyeing was performed for 2 hours. Subsequently, a series of operations in which the coarsely colored fine particles obtained were washed with a 5% aqueous solution of sodium hydroxide, collected by centrifugation, washed with pure water, and then collected by centrifugation was defined as one cycle. Up to 3 cycles were carried out to obtain colored cellulose fine particles.
Next, the grafting reaction of the colored cellulose fine particles prepared as described above was performed. A free initiator was introduced into the reaction system for GPC measurement together with the colored cellulose fine particles. For all reaction equivalents, the reaction system was designed with 1 equivalent of free initiator. When grafting 1.0 g of colored cellulose fine particles, in a 300 ml eggplant flask, 30 mmol of sodium methacrylate (hereinafter referred to as MAANA. Manufactured by Sigma Aldrich, 408212) (MAANA concentration is 0.1 M), sodium carbonate (Kanto Chemical) 58029-17) 72 mmol, polysorbate 80 (Sigma Aldrich, W291706) as a nonionic surfactant was mixed with 11 mmol, 150 ml of pure water, and after confirming that it was completely dissolved, septum rubber was used. The flask was sealed. Next, another 300 ml eggplant flask was prepared, and 9.0 mmol of potassium peroxodisulfate (hereinafter abbreviated as KPS, manufactured by Wako Pure Chemical Industries, 162-04235) as a free initiator and 1.0 g of colored cellulose fine particles were added. It was mixed with 150 ml of pure water and sealed with a septum rubber. Two eggplant flasks containing the solution were connected with a Teflon (registered trademark) tube, and N ₂ gas was injected from the eggplant flask containing MAANA for 60 minutes to remove oxygen dissolved in the solution. After 60 minutes, the solution in which MAANA was dissolved was injected by cannulation into the solution in which the fine particles were dispersed, and the reaction was started. At this time, the temperature of each eggplant flask was adjusted by placing it in a 30 ° C. water bath. The reaction was complete in 2 hours at 30 ° C. The particle physical properties of the grafted colored cellulose fine particles thus prepared are shown in Table 1 below. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

[Example 2]
Table 1 below shows the particle physical properties of the grafted colored cellulose fine particles prepared in the same manner as in Example 1 except that the MAANA concentration was changed to 1.0M. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 3
Table 1 below shows the particle physical properties of the grafted colored cellulose fine particles prepared in the same manner as in Example 1 except that the MAANA concentration was changed to 3.0M. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 4
Table 1 below shows the particle physical properties of the grafted colored cellulose fine particles prepared by the same method as in Example 1 except that the MAANA concentration was changed to 5.0M. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 5
Table 1 below shows the particle properties of the grafted colored cellulose fine particles prepared in the same manner as in Example 1 except that the MAANA concentration was changed to 8.0M. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 6
Table 1 below shows the particle physical properties of the grafted colored cellulose fine particles prepared in the same manner as in Example 1 except that the MAANA concentration was changed to 10.0M. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 7
It was prepared in the same manner as in Example 1 except that it was changed to a monomer having a phosphate group as a reactive group (Light Ester P-1M, manufactured by Kyoeisha Chemical Co., Ltd.) and the monomer concentration was 1.0M. The particle physical properties of the grafted colored cellulose fine particles are shown in Table 1 below. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as colored particles for immunochromatography and their performance was evaluated. The results are shown in Table 2 below.

Example 8
Grafted colored cellulose fine particles prepared in the same manner as in Example 1 except that the monomer has an amino group as a reactive group (propargylamine, manufactured by Tokyo Chemical Industry Co., Ltd.) and the monomer concentration is 1.0M. The particle physical properties are shown in Table 1 below. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 9
Example 1 except that the monomer has a sulfonic acid group as a reactive group (acrylamide t-butyl sulfonic acid, Nt-butyl acrylamide sulfonic acid, manufactured by Toagosei Co., Ltd.) and the monomer concentration is 1.0 M. Table 1 below shows the particle properties of the grafted colored cellulose fine particles prepared by the same method as described above. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 10
Table 1 below shows the particle physical properties of the grafted colored cellulose fine particles prepared by the same method as in Example 1 except that the dyeing cycle was changed to once. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 11
Table 1 below shows the particle physical properties of the grafted colored cellulose fine particles prepared by the same method as in Example 1 except that the dyeing cycle was changed to 8 times. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 12
Table 1 below shows the particle physical properties of the grafted colored cellulose fine particles prepared by the same method as in Example 1 except that the coagulation liquid was changed to a dimethyl sulfoxide concentration of 50 wt% and a water concentration of 50 wt%. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 13
The particle physical properties of the grafted colored cellulose fine particles prepared in the same manner as in Example 1 except that the coagulation liquid was changed to an acetone concentration of 27 wt%, a water concentration of 0.20 wt%, and an ammonia concentration of 72.8 wt% are shown in the following table. It is shown in 1. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 14
Table 1 below shows the particle properties of the grafted colored cellulose fine particles prepared by the same method as in Example 1 except that the coagulation liquid was changed to a tetrahydrofuran concentration of 90 wt% and a water concentration of 10 wt. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 15
Table 1 below shows the particle physical properties of the grafted colored cellulose fine particles prepared by the same method as in Example 1 except that the coagulation liquid was changed to an ethyl acetate concentration of 94 wt% and a water concentration of 6.0 wt. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

Example 16
The colored cellulose fine particles coagulated and colored by the same method as in Example 1 were grafted by the following method. In a 100 ml eggplant flask, 93 mmol of MAANAa (MAANA concentration is 2.0 M) and 3.7 mmol of 1,1,4,7,10,10-hexamethyltriethylenetetramine (manufactured by Sigma-Aldrich, 366404) in 24 ml of pure After mixing with water and confirming complete dissolution, the flask was sealed with a septum rubber. Then, another 100 ml eggplant flask was prepared, and the catalyst was copper bromide (I) (Wako Pure Chemical Industries, 037-12882) 1.2 mmol, copper bromide (II) (Wako Pure Chemical Industries, Ltd.) Manufactured, 030-10895) 0.69 mmol was added, and the flask was sealed with a septum rubber. Further, in a 200 ml eggplant flask, 1.2 mmol mmol of 2-bromoisobutyric acid (manufactured by Tokyo Chemical Industry Co., Ltd., B0606) and 1.0 g of colored cellulose fine particles were mixed with 24 ml of pure water and sealed with a septum rubber. Three eggplant flasks were connected with a Teflon (registered trademark) tube, and N ₂ gas was injected from the eggplant flask containing MAANA for 60 minutes to remove oxygen dissolved in the solution. After 60 minutes, the MAANA dissolved solution was poured into a flask containing copper bromide by cannulation and stirred for 30 minutes until the copper bromide was completely dissolved. Thereafter, a solution in which MAANA, copper bromide and the like were dissolved was injected into the solution in which the fine particles were dispersed by cannulation to initiate the reaction. At this time, the temperature of each eggplant flask was adjusted by placing it in a 30 ° C. water bath. The reaction was complete in 2 hours at 30 ° C. The particle physical properties of the grafted colored cellulose fine particles thus prepared are shown in Table 1 below. No dye discoloration or fading was observed by this reaction. The grafted colored cellulose fine particles were used as immunochromatographic colored particles, and their performance was evaluated. The results are shown in Table 2 below.

〔result〕
In each of Examples 1 to 16, since many reactive groups were introduced into the microparticles, the antibody and the microparticles were efficiently bound and the directions of the antibodies were aligned. As shown in Table 2 below, the analysis sensitivity was high. A high immunochromatographic diagnostic kit could be obtained.

[Comparative Example 1]
The dyeing operation of Example 1 was carried out for only one cycle, and particles having a dyeing intensity of 0.5 were produced. Grafting was performed in the same manner as in Example 1 to obtain grafted colored cellulose fine particles. This particle was used as a color developing particle, and the performance was evaluated. The results are shown in Table 2. Since the color intensity was weak, the analytical sensitivity was low.

[Comparative Example 2]
Gold colloid (manufactured by Tanaka Kikinzoku Co., Ltd.) having a particle size of 100 nm and having no reactive groups introduced therein was used as colored particles for immunochromatography, and performance was evaluated. The results are shown in Table 2 below. Since the antibody is bound to the particle by physical adsorption, the direction of the antibody is not aligned, and as a result, the antigen cannot be efficiently captured, resulting in low analytical sensitivity.

[Comparative Example 3]
The colloidal gold particle (manufactured by Sigma-Aldrich) having a particle size of 50 nm and having a carboxyl group introduced was used as the immunochromatographic colored particles, and the performance was evaluated. The results are shown in Table 2 below. The sensitivity was low due to the small particle size.

[Comparative Example 4]
Latex particles (manufactured by Bangs) having a particle size of 470 nm, a color intensity of 0.5, and a carboxyl group introduced were used as color particles for immunochromatography, and performance was evaluated. The results are shown in Table 2. The analytical sensitivity was low due to the low color intensity.

[Comparative Example 5]
Colored cellulose fine particles having a particle size of 330 nm, a color development intensity of 4.0, and no reactive active group introduced were used as the immunochromatographic color development particles, and the performance was evaluated. The results are shown in Table 2 below. Although the analytical sensitivity is higher than that of Comparative Example 2, the antibody is bound to the particle by physical adsorption, so that the orientation of the antibody is not aligned, and as a result, the antigen cannot be efficiently captured and the analytical sensitivity is low. became.

[Comparative Example 6]
The colored cellulose fine particles introduced with a carboxyl group having one repeating unit described in Patent Document 4 were used as colored particles for immunochromatography, and the performance was evaluated. The results are shown in Table 2 below. The analytical sensitivity was low due to the small amount of carboxyl group introduced.

[Comparative Example 7]
Grafted colored cellulose fine particles having a dispersity of 8.0 were used as colored particles for immunochromatography, and performance was evaluated. The results are shown in Table 2 below. Due to the high degree of dispersion, the amount of reactive groups varies among particles, resulting in poor reproducibility of immunochromatographic results.

[Comparative Example 8]
Grafted colored cellulose fine particles having a sphericity of 13 were used as colored particles for immunochromatography, and the performance was evaluated. The results are shown in Table 2 below. Since the grain shape is an irregular oval shape, the membrane was clogged and the sensitivity was lowered.

[Comparative Example 9]
Grafted colored cellulose fine particles having a particle size of 1200 nm were used as colored particles for immunochromatography, and performance was evaluated. The results are shown in Table 2 below. Due to the large particle shape, it was clogged in the membrane and did not develop well.

  When the grafted colored cellulose fine particles according to the present invention are used as colored particles in an immunochromatographic diagnostic kit, rapid diagnosis is possible, and the grafted colored cellulose fine particles can be suitably used as a diagnostic agent with high analytical sensitivity.

Claims (7)

  Colored cellulose fine particles having an average particle size of 10 to 850 nm, a color development intensity of 1.0 to 10.0, and a polymer in which a monomer having a reactive group is graft-polymerized with 2 to 1000 repeating units The colored cellulose fine particles, wherein the repeating unit of the monomer having a reactive group has a dispersity of less than 8 and the sphericity of the colored cellulose fine particles is 10 or less.   The colored cellulose fine particles according to claim 1, wherein the amount of the reactive group introduced is 0.50 to 30.0 mmol per 1 g of the colored cellulose fine particles.   The colored cellulose fine particles according to claim 1 or 2, wherein the reactive group is selected from the group consisting of a carboxyl group, an amino group, a phosphoric acid group, and a sulfonic acid group.   The colored cellulose fine particles according to any one of claims 1 to 3, wherein 10 to 80 wt% of the weight of the colored cellulose fine particles is a colored component.   The colored cellulose fine particles according to any one of claims 1 to 4, wherein the colored component is a dye.   The colored cellulose fine particles according to claim 1, wherein a ligand is covalently bonded to the reactive group.   An immunochromatographic diagnostic kit comprising the colored cellulose fine particles according to any one of claims 1 to 6.