JP2020020687A - Immunochromatographic developer - Google Patents

Abstract

To provide an immunochromatographic developer not generating false positive reaction and desensitization following aggregation, to a specimen derived from a biological sample.SOLUTION: An immunochromatographic developer contains both an anionic surfactant and a nonionic surfactant. In the immunochromatographic developer, the concentration of the anionic surfactant is 0.001-1.0 wt%, and the concentration ratio between the nonionic surfactant and the anionic surfactant (nonionic surfactant wt%/anionic surfactant wt%) is 5.0-100.SELECTED DRAWING: None

Description

  The present invention relates to a developing solution constituting a mobile phase in an immunochromatography method, which is one of immunoassay methods.

As an immunoassay for detecting a target substance consisting of a specific antigen or antibody by utilizing a specific reaction of an antigen and an antibody thereto, an antibody or antigen labeled with a labeling substance is detected in a sample by an immunoreaction. And a method for measuring a labeled substance or the like in this bound state, such as a radioimmunoassay using a radioisotope as a labeling substance, an enzyme immunoassay using an enzyme, and a fluorescence immunoassay using a fluorescent substance Laws are also adopted.
These immunoassays require a step of reacting the substance to be detected with an antibody or the like labeled with a labeling substance, and a step of separating a labeling substance bound to the substance to be detected from a labeling substance not bound to the substance. An immunochromatography method is known as a method for performing these steps in a system composed of a stationary phase and a mobile phase that continuously flows in contact with the stationary phase by applying the principle of chromatography.
As a format of the immunoassay, a sandwich format or a competitive format is known, and as a typical example, in a sandwich format by an immunochromatography method, for example, when detecting a target substance consisting of an antigen in a sample, the following method is used. The following operation is performed.
(1) An antibody that specifically binds to an antigen to be detected is used as an immobilization reagent, and the immobilization reagent is applied to a predetermined portion of the chromatographic medium in a predetermined form, for example, so that an arbitrary chromatographic medium can be obtained. A reaction site is formed at the position.
(2) On the other hand, an antibody that specifically binds to the substance to be detected is used as a detection reagent, and the detection reagent is labeled with a labeling substance such as an enzyme, or the detection reagent is sensitized to a labeling substance such as an insoluble carrier. To prepare a labeled detection reagent.
(3) The developing solution constituting the mobile phase is developed on the chromatographic medium as the stationary phase together with the sample containing the substance to be detected and the labeled detection reagent.

  By the above operation, at the reaction site formed in the chromatographic medium, the antigen to be detected is captured by binding to the antibody as the immobilized reagent fixed to the reaction site, and the antigen and the label As a result of an antigen-antibody reaction caused by the antibody which is the immobilized detection reagent, at the reaction site, an immobilized reagent (immobilized antibody), a substance to be detected (antigen), and a detection reagent (labeled antibody) are detected. When a sandwich-type conjugate is generated and a target substance is present in a sample, a predetermined signal appears due to indirect binding of a labeling substance to a reaction site, whereby detection of the target substance can be performed. it can.

  The immunochromatography method described above is characterized in that the operation is simple and the measurement can be performed in about several minutes. For example, when the measurement item is an infectious disease such as influenza, if the presence or absence of infection of a sample collected from a patient is found on the spot, the patient can be treated as soon as possible. If a patient can be treated at an early stage, not only the patient's own illness can be prevented, but also the spread of infection can be prevented. For this reason, the importance of immunochromatographic test kits is increasing year by year in medical practice.

However, when a specimen (sample collected from a patient) is analyzed using an immunoassay method such as an immunochromatography method, there is a problem that false positives occur. That is, there is a problem that a signal is generated at a reaction site due to a non-specific reaction even though the substance to be detected is not present in the sample, and the reaction is determined to be positive. As described above, when a false positive occurs when diagnosing the presence or absence of a pathogen infection, erroneous information about the disease is given. As a result, delays in identifying the cause and improper treatment may be given, which may cause serious problems such as a more serious medical condition. Therefore, suppressing the occurrence of false positives is a very important issue.
Many of the causes of false positives in immunochromatography are aggregation of insoluble carriers and nonspecific adsorption. As a labeling substance for the immunochromatography method, an insoluble carrier that can be visually detected without any special operation is often used from the viewpoint of simplicity. As the insoluble carrier, fine particles such as colored cellulose fine particles, fluorescent cellulose fine particles, gold colloid fine particles and colored latex fine particles are mainly used. If these fine particles form aggregates during development, they are likely to be trapped by the non-specific adsorption by the immobilized reagent at the reaction site, and a signal is considered to be generated at a visually confirmable level.

As a method for suppressing the aggregation of the insoluble carrier, a method of adding a dispersing agent for dispersion stabilization of the detection reagent labeled with the insoluble carrier, in a developing solution in an immunochromatography method or in a sample processing solution for treating a sample, There is.
For example, in Patent Literature 1 below, aggregation of colloidal gold particles is suppressed by including a nonionic surfactant (such as polyoxyethylene lauryl ether) and casein in a developing solution.
Further, in Patent Document 2 below, aggregation of colored latex fine particles is performed by using a sample processing solution containing a nonionic surfactant (such as NP40), a reducing agent (such as 2-mercaptoethylamine hydrochloride), and a chelating agent. We are trying to control it.

As a method for suppressing non-specific adsorption at a reaction site, although the principle thereof has not been clarified, it has been proposed to add various additives to a developing solution or a sample processing solution.
For example, in Patent Document 3 below, for a sample collected with a cotton swab from a nasal suction liquid collected by a suction catheter, a cationic surfactant or an amphoteric surfactant is added to a developing solution, whereby non-specific adsorption is achieved. We are trying to control it.
Further, in Patent Document 4 below, false positive reactions due to non-specific adsorption are reduced by adding a polyoxylen / polyoxypropylene block copolymer to a sample processing solution for a sample from which urine is collected. It has been reported.

  However, none of the conventional techniques is satisfactory as an immunochromatography method that maintains dispersion stability and sufficiently suppresses a false positive reaction due to non-specific adsorption with respect to a specimen derived from a biological sample. In particular, when a highly viscous specimen such as a pharyngeal swab or a nasal discharge is used among biological samples, an environment in which the insoluble carrier is extremely likely to aggregate is generated, and a false positive reaction is amplified. In addition, a specimen collected from a living body may contain metal cations derived from body fluids, specifically, sodium ions, potassium ions, magnesium ions, calcium ions, and the like. It is generally known that the insoluble carrier is negatively charged in the developing solution in any of the colored cellulose fine particles, the gold colloid particles, and the colored latex fine particles. Agglomeration is hindered by electrical repulsion and remains dispersed. However, when a metal cation derived from a specimen is present in the developing solution, its positive charge may attract the negatively charged insoluble carriers to each other, causing aggregation and amplification of a false positive reaction.

  On the other hand, if an additive such as a surfactant is added to suppress the aggregation, the detection sensitivity may be reduced. An increase in detection sensitivity means that a smaller amount of a substance to be detected can be detected. Improving the detection sensitivity enables quick diagnosis, and thus the detection sensitivity is an important factor. That is, a decrease in the detection sensitivity means that an amount of the substance to be detected that should be detectable cannot be detected. This may lead to an erroneous diagnosis because a patient who should be positive will receive a negative determination.

Thus, when using a throat swab or nasal discharge as a sample, simply adding an additive to suppress false positives due to aggregation lowers the detection sensitivity. There is a relationship that a false positive reaction due to agglutination occurs when the value is reduced.
There has been a long-felt need for technical improvements that do not cause false-positive reactions and decrease in sensitivity due to agglutination of biological specimens as described above.

Japanese Patent No. 5567932 JP 2008-164403 A JP-A-2005-291783 International Publication No. 2011/083604

  In view of the above problems of the prior art, the problem to be solved by the present invention is to provide a developing solution for immunochromatography that does not cause a false positive reaction and a decrease in sensitivity due to aggregation for a specimen derived from a biological sample. is there.

  The present inventors have conducted intensive studies and repeated experiments to solve the above problems, and as a result, in the measurement of a substance to be detected in a biological sample using an immunochromatographic method, a nonionic surfactant and an anionic surfactant were used. By including both of the agents in the developing solution at a predetermined concentration and a predetermined ratio, it was found that aggregation and false positive reaction can be effectively suppressed without lowering the sensitivity, and the present invention was completed. It is a thing.

That is, the present invention is as follows.
[1] A developing solution for immunochromatography containing both an anionic surfactant and a nonionic surfactant, wherein the concentration of the anionic surfactant is 0.001 to 1.0% by weight, and The developing solution for immunochromatography, wherein the concentration ratio of the nonionic surfactant to the anionic surfactant (wt% of nonionic surfactant / wt% of anionic surfactant) is 5.0 to 100; .
[2] The developing solution for immunochromatography according to [1], which is used in a detection system using an insoluble carrier as a labeling substance.
[3] The developing solution for immunochromatography according to [1] or [2], wherein the insoluble carrier is selected from the group consisting of colloidal gold, latex fine particles, and cellulose fine particles.
[4] The developing solution for immunochromatography according to the above [3], wherein the cellulose fine particles are colored cellulose fine particles containing a dye.
[5] The developing solution for immunochromatography according to [3], wherein the cellulose fine particles are fluorescent cellulose fine particles containing a fluorescent dye compound.

  The immunochromatography using the developing solution of the present invention suppresses a false positive reaction due to aggregation in a specimen derived from a biological sample, has a good background, and does not lower the detection sensitivity.

It is a top view and a sectional view showing an example of the structure of the immunochromatography kit of this embodiment. It is a photograph instead of a drawing which shows that there is aggregation in the immunochromatography kit. It is a photograph instead of a drawing showing that there is no aggregation in the immunochromatography kit.

Hereinafter, embodiments of the present invention will be described in detail.
The present invention provides a method for measuring a substance to be detected in a biological sample, in which a developing solution in which an anionic surfactant and a nonionic surfactant are mixed at a predetermined concentration and a predetermined ratio is used. The present invention has been found to suppress false positive reactions, to have a good background, and not to lower the detection sensitivity, and to complete the invention based thereon. As described above, metal cations in a sample attract negatively charged insoluble carriers to each other, agglutinating, and amplifying false positive reactions. To solve this problem, when an anionic surfactant is added to the developing solution, it interacts with metal cations in the sample and has the effect of suppressing aggregation, while the anionic surfactant inhibits the antigen-antibody reaction at the reaction site. And the sensitivity is reduced. Further, there is also a problem that a bivalent metal cation (calcium ion, magnesium ion) and an anionic surfactant in a sample form a complex and are insolubilized. Since the insolubilized surfactant causes clogging of the nitrocellulose membrane and causes deterioration of the background, generally, an anionic surfactant cannot be expected to have an aggregation suppressing effect. On the other hand, the nonionic surfactant as described in Patent Document 2 does not show the effect of suppressing aggregation of the insoluble carrier due to the metal cation in the sample. In response to this problem, the present inventors have found that nonionic surfactants inhibit the inhibition of an antigen-antibody reaction by anionic surfactants, and also prevent the anionic surfactants from complexing with metal cations. Thus, it was considered that the aggregation of the insoluble carrier could be suppressed, and the sensitivity could be reduced with good background. Under such a hypothesis, the present inventors have conducted intensive studies and repeated experiments, and as a result, only when anionic surfactant and nonionic surfactant are mixed at a certain ratio, false positive accompanying particle aggregation It has been found that the reaction is suppressed and a decrease in detection sensitivity is suppressed in a good background.

  The developing solution of the present embodiment is a liquid constituting a mobile phase in immunochromatography, and moves on a chromatographic medium as a stationary phase together with a sample containing a substance to be detected and a labeled detection reagent. The developing solution of the present embodiment can be used not only as a developing solution but also as a sample diluting solution. That is, the method of using the developing solution is not limited at all, and may be appropriately selected depending on the purpose of use.

It is preferable that the developing solution of the present embodiment usually contains water as a solvent, and further contains a buffering agent in addition to the anionic surfactant and the nonionic surfactant. Preferred examples of the buffer include, but are not limited to, phosphate, trishydroxymethylaminomethane, and Good's buffer. Further, as other components, a protein component such as bovine serum albumin (BSA) (content is usually 0.01% by weight to 10% by weight) or a disulfide bond reducing agent may be optionally included.
As described above, the developing solution of the present embodiment contains an anionic surfactant. The composition of the anionic surfactant is not particularly limited, and may be used alone or in combination of two or more. As the anionic surfactant, a carboxylate surfactant, a sulfate ester surfactant, a phosphate ester surfactant, a sulfonate surfactant, and the like can be used. It can be preferably used. Specific examples of the sulfonate surfactant include sodium dodecylbenzenesulfonate (SDBS), sodium alkylnaphthalenesulfonate, sodium dialkylsulfosuccinate, sodium alkyldiphenylether disulfonate, sodium alkanesulfonate, and sodium cholate. (CAS), sodium deoxycholate (DCAS) and the like, and particularly preferred is sodium dodecylbenzenesulfonate (SDBS).

  It is important that the amount of the anionic surfactant added is in the range of 0.001 to 1.0% by weight based on the total weight of the developing solution. If the amount of the anionic surfactant is less than 0.001% by weight, the effect of suppressing a false positive reaction accompanying aggregation cannot be exhibited. The lower limit of the addition amount of the anionic surfactant is preferably 0.01% by weight or more, more preferably 0.03% by weight or more. On the other hand, if the amount of the anionic surfactant exceeds 1.0% by weight, the antigen-antibody reaction at the reaction site is inhibited, and the sensitivity is greatly reduced. Further, the complexation between the anionic surfactant and the metal cation is promoted, and the background is deteriorated due to the insoluble material. The upper limit of the amount of the added anionic surfactant is preferably 0.5% by weight, more preferably 0.3% by weight.

  As described above, the developing solution of the present embodiment further includes a nonionic surfactant in addition to the anionic surfactant. The composition of the nonionic surfactant is not particularly limited, and may be used alone or as a mixture of two or more. Examples of the nonionic surfactant include sorbitan trioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene alkyl ether, polyoxyethylene / polyoxypropylene alkyl ether, polyoxyethylene sorbitan fatty acid ester (trade name “Tween "Series (registered trademark)), polyoxyethylene pt-octyl phenyl ether (trade name" Triton "series (registered trademark)), polyoxyethylene pt-nonyl phenyl ether (trade name" Triton N "series (registered trademark) Trademark)), alkyl polyglucoside fatty acid diethanolamide, alkyl monoglyceryl ether and the like.

  The amount of the above-mentioned nonionic surfactant (weight% concentration based on the weight of the entire developing solution) is not particularly limited, but the ratio of the nonionic surfactant to the anionic surfactant is nonionic at the weight% concentration. It is important that the value of (ionic surfactant / anionic surfactant) is in the range of 5.0 to 100. When two or more anionic surfactants or nonionic surfactants are contained as a mixture, the amount added may be the total weight. When the addition amount of the nonionic surfactant is less than 5.0 times the addition amount of the anionic surfactant, the inhibition of the antigen-antibody reaction by the anionic surfactant cannot be suppressed. Since the effect of suppressing the complex formation between the surfactant and the metal cation cannot be exhibited, the background is deteriorated. The lower limit of the addition amount of the nonionic surfactant is preferably at least 5.0 times, more preferably at least 10 times the amount of the anionic surfactant. On the other hand, when the addition amount of the nonionic surfactant exceeds 100 times the addition amount of the anionic surfactant, micelles formed by the nonionic surfactant include the anionic surfactant in inclusion. In addition, the anionic surfactant does not have an aggregation suppressing effect. This results in a false positive reaction associated with the aggregation. The upper limit of the addition amount of the nonionic surfactant is preferably not more than 80 times the amount of the anionic surfactant, more preferably not more than 50 times the amount of the anionic surfactant.

  In the present embodiment, it is more preferable to use a nonionic surfactant having a high HLB value. The “HLB value” is an index indicating the ease of dissolving the nonionic surfactant in water, that is, an index indicating hydrophilicity, and is obtained by (weight% in the molecular weight of hydrophilic group × 0.2). The lower this value is, the higher the lipophilicity is. For example, a nonionic surfactant having an HLB value of about 5.0 is not dispersed in an aqueous medium and is localized on the liquid surface. Complex formation of the surfactant and the metal cation cannot be prevented, and as a result, the insoluble carrier aggregates and a false positive reaction occurs. In the developing solution of the present embodiment, in order to exhibit the above effects, it is necessary to sufficiently disperse in the solution, and it is important that the HLB value of the nonionic surfactant is not less than a certain value, and it is preferable. Is 10.0 or more, more preferably 12.0 or more. Specifically, a polyoxyethylene surfactant can be preferably used. More specifically, in the “Tween” series, in particular, Tween 20 (trade name, HLB value 16.7 (registered trademark)), Tween 40 (trade name, HLB value 15.6 (registered trademark)), Tween 60 (trade name, HLB value 15.0 (registered trademark), Tween 80 (trade name, HLB value 14.9 (registered trademark)), and in the "Triton" series, in particular, Triton X-100 (trade name, HLB value 13.5 (registered trademark)) )), Nonidet P-40 (trade name, HLB value 13.1 (registered trademark)), Triton X-102 (trade name, HLB 14.6 (registered trademark)), Triton X-165 (trade name, HLB 15.8 (registered trademark) )), Triton X-405 (trade name, HLB17.9 (registered trademark)), and in the “Triton N” series, nN-101 (trade name, HLB 13.5 (registered trademark)), Triton N-111 (trade name, HLB 13.8 (registered trademark)), Triton N-150 (trade name, HLB 15.0 (registered trademark)) and the like. be able to. The above nonionic surfactants can be used alone or in combination of two or more.

When the types and contents of the anionic surfactant and the nonionic surfactant in the developing solution or the immunochromatographic medium are unknown, as the analysis method, for example, the surfactant is identified by LC / MS analysis. There is a method of calculating the content from the area of the peak using the absolute calibration method, but the method is not limited to this.
In the present specification, the `` insoluble carrier '' is a labeling substance suitable for visually determining the presence of the substance to be detected, and is preferably colored in order to facilitate visual determination. For example, colloidal gold particles, colored latex fine particles, fluorescent latex fine particles, cellulose fine particles containing a dye (colored cellulose fine particles), cellulose fine particles containing a fluorescent dye compound (fluorescent cellulose fine particles), and the like can be used. You may. It is generally known that these insoluble carriers have a negative surface charge in a medium, and the presence of metal cations in a sample attracts negatively charged insoluble carriers to each other, agglutinates, and false positives. It is considered that the reaction is amplified.

  Further, the insoluble carrier used in the developing solution of the present embodiment preferably has an average particle diameter of 10 nm or more and 700 nm or less. When the average particle diameter is in this range, the sensitivity is good and the composition is suitable for a chromatographic medium. Here, if the average particle size is too small, the sensitivity will decrease. The lower limit of the average particle diameter is preferably at least 10 nm, more preferably at least 20 nm. On the other hand, if the average particle diameter is too large, clogging occurs in the chromatographic medium even if no metal cation is present in the sample, and a problem that the background deteriorates occurs. The upper limit is preferably 600 nm or less, more preferably 500 nm or less. In order to improve the sensitivity as an immunochromatography kit, two or more types of insoluble carriers having an average particle diameter may be mixed and used.

When cellulose fine particles are used as the insoluble carrier, the desired effects are more remarkably observed. Cellulose fine particles have abundant hydroxyl groups on the surface, and when divalent metal cations such as calcium ions and magnesium ions are present in the sample, they form a cross-linked structure with hydroxyl groups, amplifying aggregation and false positive reactions. It is considered to be lost.
When colored cellulose fine particles are used as the cellulose fine particles, the dye content of the colored cellulose fine particles is from 0.01% by weight to 95% by weight. If the amount is less than 0.01% by weight, sufficient coloring properties cannot be obtained as fine particles for detection of a chromatographic medium. On the other hand, if it exceeds 95% by weight, agglomeration of particles occurs, and if the particles are developed with a chromatographic medium, clogging occurs and use becomes impossible. The colored cellulose fine particles can achieve a suitable high sensitivity of an immunochromatography kit when the dye content is around 5.0% by weight. A preferred lower limit of the content of the dye is 6.0% or more, more preferably 7.0% or more. On the other hand, a preferable range of the upper limit of the content of the dye is 90% or less, more preferably 85% or less. The type of dye here is not particularly limited, but direct dyes, gold-containing dyes, acid dyes, reactive dyes, basic dyes, disperse dyes, sulfur dyes, vegetable dyes, naphthol dyes, and various other dyeings And a reactive dye is preferable among them.

  On the other hand, when, for example, fluorescent cellulose fine particles are used as the insoluble carrier, the content of the fluorescent dye compound in the fluorescent cellulose fine particles is from 0.01% by weight to 95% by weight. If the amount is less than 0.01% by weight, sufficient coloring properties cannot be obtained as fine particles for detection in an immunochromatography kit. On the other hand, if it exceeds 95% by weight, agglomeration of particles occurs, and when the particles are developed with a chromatographic medium, clogging occurs and use becomes impossible. Conventional fluorescent nanoparticles often contain a fluorescent dye compound in a range of 0.001% by weight or more and less than 0.01% by weight, but within this range, sufficient sensitivity is not exhibited as an immunochromatography kit. The fluorescent cellulose fine particles can achieve a suitable high sensitivity of the immunochromatography kit when the content of the fluorescent dye compound is from 0.01% by weight to 0.03% by weight. A preferred lower limit of the content of the fluorescent dye compound is 0.04% by weight or more, more preferably 0.05% by weight or more. Further, a preferred range of the upper limit is 90% by weight or less, more preferably 85% by weight or less. The type of the fluorescent dye compound here is not particularly limited, and for example, N-hydroxysuccinimide ester group, ester group, carboxyl group, maleimide group, isocyanate group, isothiocyanate group, cyano group, halogen Group, aldehyde group, paranitrophenyl group, diethoxymethyl group, fluoresceins having an active substituent such as an epoxy group, rhodamines, coumarins, and compounds emitting fluorescence of cyanines. Fluorene-9-acetic acid, fluorene-2-carboxaldehyde, 9-fluorene-1-carboxylic acid, 9-fluorene-4-carboxylic acid, 9-fluorene oxime, 9-fluormethylsuccinimidyl carbonate, 9 -Fluorotriphenylphosphonium bromide, 5-aminofluorescein , Disodium 8-amino-1,3,6-naphthalene trisulfonate hydrate, sulfolo-damine B, ethidium bromide, 6-aminofluorescein, rhodamine B, rhodamine 6G, 8-anilino-1 Ammonium naphthalenesulfonate, sodium 8-anilino-1-naphthalenesulfonate, magnesium 8-anilino-1-naphthalenesulfonate, 2,3-naphthalenedialdehyde, sodium calcein, calcein, coumarin 102, coumarin 314, coumarin 343, AMCA, 5-carboxyfluorescein hydrate, 6-carboxyfluorescein hydrate, fluorescein chloride, 2 ′, 7′-dichlorofluorescein, 2 ′, 7′-dichlorofluorescein sodium, 2,3-diaminonaphthalene, dimidium bromide 2,3-diphenylmale K, fluorescein, uranine, fluorescein diacetate, coumarin-3-carboxylic acid, 7-hydroxycoumarin-3-carboxylic acid, 4-dimethylaminoazobenzene-4′-carboxylic acid, 7-methoxycoumarin -3-carboxylic acid, pinacyanol chloride, pinacyanol iodide, pyranine, N- (1-pyrenyl) maleimide, rhodamine 6G, rhodamine B, sulfonefluorescein, 7-methoxycoumarin-3-carboxylic acid N-succinimidyl, potassium tetrabromofluorescein, Acid Red 87, 2 ', 4', 5 ', 7'-tetrabromo-3,4,5,6-tetrachlorofluorescein, 9H-fluoren-2-yl isocyanate, fluorescein 5-isothiocyanate, Acid Red 92, , 4,5,6-tetrachlorofluorescein, tetraiodofluorescein, erythrosin B, 5- (6-) carboxytetramethylrhodamine-NHS ester, DYLIGHT-405-NHS ester, DY550-NHS ester, DY630-NHS ester, DY-631, DY-633, DY-635, DY-636, DY-650, DY-651-NHS ester, DY-777-NHS ester (Dy is a product of Dyomics) BODYIPY650 / 665, ROX, TAMRA, CFSE, Cyto350, Cyto405, Cyto415, Cyto488, Cyto500LSS, Cyto505, Cyto510SS, Cyto514LSS, Cyto520LSS, Cyto532, Cyto546 S, Cyto 555, Cyto 590, Cyto 610, Cyto 610, Cyto 633, Cyto 647, Cyto 670, Cyto 680, Cyto 700, Cyto 750, Cyto 770, Cyto 780, Cyto 800 (all of which are Cytodiagnos).

  The fluorescence wavelength of these fluorescent dye compounds is preferably in the range of 400 nm or more which does not overlap with the wavelength of water or protein during detection. There is no particular upper limit, and the higher the wavelength, the more preferable, and the more preferable is a fluorescent dye compound in the range of 500 nm or more.

When the content of the dye or the fluorescent dye compound of the cellulose fine particles before the treatment is unknown, the colored cellulose fine particles or the fluorescent cellulose fine particles are subjected to cellulase treatment, acid treatment or base treatment to reduce the degree of polymerization. Thereafter, the sample is dissolved in heavy water, and measured by FT-NMR by ¹³ C-NMR to calculate the degree of substitution. From the substitution degree, the content of the dye or the fluorescent dye compound may be calculated. At this time, the cellulase, acid, and base used are not limited to any one. For example, as cellulase, Onozuka RS (registered trademark, manufactured by Yakult Yakuhin Kogyo Co., Ltd.), Cellsoft (Novo Nordics Co., Ltd.) (Registered trademark)), Meiserase (registered trademark (registered trademark) manufactured by Meiji Seika Co., Ltd.). Examples of the acid include hydrochloric acid, sulfuric acid, and nitric acid. In addition, when the weight of the dye or the fluorescent dye compound of the cellulose fine particles before the treatment is unknown, and the dye and the fluorescent dye compound contain a nitrogen atom, the nitrogen element content is measured by a nitrogen quantifier CHN coder. It may be measured by an analytical method, and the content of the contained dye or fluorescent dye compound may be calculated from the measured nitrogen element content.

  These colored cellulose fine particles and fluorescent cellulose fine particles can be used by supporting components other than cellulose through chemical bonding or physical adsorption. Examples of chemical bonding or physical adsorption include, but are not limited to, covalent bonds, ionic bonds, coordinate bonds, metal bonds, hydrogen bonds, hydrophilic bonds, hydrophobic bonds, van der Waals bonds, and the like. By supporting components other than cellulose on the colored cellulose or the fluorescent cellulose fine particles by the various forces as described above, it is possible to adjust the fine particles having a function not found in the colored cellulose fine particles or the fluorescent cellulose fine particles. Cellulose fine particles in which dyes and fluorescent dyes are not introduced into cellulose can support components other than cellulose, but by changing the type of substituent arbitrarily, it is possible to support more various types of components. Can be.

  The component supported on the insoluble carrier used in the developing solution of the present embodiment refers to various substances other than the insoluble carrier, and the type thereof is not particularly limited. Examples of such include surfactants, inorganic fine particles, organic fine particles, biomaterials, dyes, ionic substances, water-soluble low molecules, water-soluble polymers, blocking agents, and the like, but are not limited thereto. is not. The biomaterial supported on the insoluble carrier refers to various materials obtained from a living body, and the type thereof is not particularly limited. Examples of these include collagen, gelatin, fibroin, heparin, hyaluronic acid, starch, chitin, chitosan, amino acids, peptides, proteins, nucleic acids, carbohydrates, fatty acids, terpenoids, carotenoids, tetrapyrrole, cofactors, steroids, flavonoids, Arkanoids, polyketides, glycosides, enzymes, antibodies, antigens, CMC, CEC, MC and the like. By supporting them on an insoluble carrier, it becomes possible to improve the biocompatibility of the insoluble carrier, and to use it as various bioassays and diagnostic agents.

As one aspect of the present embodiment, an anionic surfactant and a nonionic surfactant are added in advance to the developing solution constituting the moving layer on the chromatographic medium, and then the developing solution is developed on the chromatographic medium. Can be done.
In another embodiment, the anionic surfactant and the nonionic surfactant are added to the mobile phase in the mobile phase in the mobile phase, ie, the end of the mobile phase of the mobile phase to which the mobile phase is applied. It can also be present in the region between the part and the reaction site and dissolved and added when the moving layer is formed by the developing solution.
As still another embodiment, a nonionic surfactant is added to a sample solution containing the substance to be detected, and the sample solution is developed simultaneously with or before the development of the solution containing the anionic surfactant. This makes it possible to add an anionic surfactant and a nonionic surfactant to the developing solution constituting the mobile phase on the mobile phase developing and moving path. In this case, the solution containing the anionic surfactant does not need to contain the nonionic surfactant before moving through the stationary phase, but it contains the nonionic surfactant. There may be.

The substance to be detected is not particularly limited as long as a substance that specifically binds to the substance is present. Proteins, peptides, nucleic acids, and saccharides (particularly, a saccharide part of a glycoprotein, a saccharide part of a glycolipid, etc.) And complex carbohydrates.
In the present specification, “specifically binds” means that the molecules bind based on the affinity of the biomolecule. Examples of such affinity-based binding include antigen-antibody binding, sugar-lectin binding, hormone-receptor binding, enzyme-inhibitor binding, complementary nucleic acids, nucleic acid and nucleic acid binding protein. And the like. Therefore, when the target substance has antigenicity, examples of the substance that specifically binds to the target substance include polyclonal antibodies and monoclonal antibodies. When the substance to be detected is a sugar, a lectin protein can be exemplified as a substance that specifically binds to the substance to be detected. Specific substances to be detected include, for example, carcinoembryonic antigen (CEA), HER2 protein, prostate specific antigen (PSA), CA19-9, α-fetoprotein (AFP), immunosuppressive acidic protein (IPA), CA15- 3, CA125, estrogen receptor, progesterone receptor, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), syphilis antibody, Examples include, but are not limited to, influenza virus, human hemoglobin, chlamydia antigen, group A β-streptococcus antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, glycated albumin, and the like. Among them, an antigen that is solubilized by a nonionic surfactant is preferable, and an antigen that forms a self-assembly such as a viral nucleoprotein is more preferable.

  Examples of the sample containing the substance to be detected include biological samples, for example, whole blood, serum, plasma, urine, saliva, sputum, nasal or pharyngeal swabs, spinal fluid, amniotic fluid, nipple secretions, tears, sweat, and skin Examples include an exudate from the skin, an extract from tissues and cells, and stool, and the developing solution of the present embodiment is particularly effective for a throat swab and nasal discharge. These samples are processed, if necessary, in such a state that the substance to be detected and the detection reagent or the immobilization reagent are liable to cause a specific binding reaction. As the treatment method, any of a chemical treatment method using various chemicals such as an acid, a base and a surfactant, and a physical treatment method using heating, stirring, ultrasonic waves, etc. may be used. You may. Further, these samples may be diluted with a developing solution so that they can be developed in a chromatographic medium as a stationary phase, if necessary.

  In actual implementation of immunochromatography, application of a sample to a reaction site can be performed by developing a sample solution on a chromatographic medium and moving it chromatographically. In this case, the development of the sample solution may be performed simultaneously with the development of the developing solution constituting the mobile phase, or the sample solution may be developed first, and then the developing solution may be developed.

Although the structure of the immunochromatography kit is not particularly limited, it may typically have the following structure. For example, as shown in FIG. 1 , a capillary phenomenon is caused between each member in the order of the sample pad (1), the conjugate pad (2), the reagent-immobilized chromatographic medium (3), and the absorption pad (4). In order to make it easy, it can be manufactured by overlapping (preferably, on a backing sheet (5)) both ends of each member with an adjacent member by about 1 to 5 mm. In addition, the test line (6) refers to a line where the measurement sample reaches the position where the antibody is immobilized on the reagent-immobilized chromatographic medium and is detected, and the control line (7) is It refers to the line where the measurement sample reaches the position where the control capture reagent is immobilized on the medium and is detected.

Hereinafter, members used for the chromatographic medium will be described in detail.
The chromatographic medium (3) used in the immunochromatography method is an inert medium composed of a microporous substance exhibiting a capillary phenomenon, and does not react with a detection reagent, an immobilization reagent, a substance to be detected, and the like used. Yes, the material is not particularly limited as long as it has a developing speed capable of obtaining a sufficient sensitivity in a short time judgment.
Examples of the chromatographic medium (3) include ceramic fine particles such as silica, titania, zirconia, ceria, and alumina or fine particles of an organic polymer, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, nitrocellulose, or cellulose acetate. And fibrous or non-woven fibrous matrices, membranes, filter papers, glass fiber filter papers, cloths, cottons, etc., composed of cellulose derivatives and the like. Even if the microparticles are not porous themselves, voids are generated between the microparticles in a filled state and function as a chromatographic medium. Preferred are cellulose derivative and nylon membranes, filter paper, glass fiber filter paper, and the like, and more preferred are nitrocellulose membranes, mixed nitrocellulose ester (mixture of nitrocellulose and cellulose acetate) membranes, nylon membranes, and filter paper.

  The form and size of the chromatographic medium are not particularly limited as long as they are appropriate in terms of actual operation and observation of reaction results. In order to make the operation easier, it is preferable to provide a support made of plastic or the like on the back surface of the chromatographic medium in which the reaction site is formed on the front surface. The properties of the support are not particularly limited, but when observing the measurement results by visual judgment, the support preferably has a color that is not similar to the color provided by the labeling substance. Usually, it is preferably colorless or white.

  On the chromatographic medium (3), a reaction site in which a substance that specifically binds to the substance to be detected, for example, an antibody is immobilized as an immobilization reagent at an arbitrary position is formed. As a method of immobilizing the immobilized reagent on the chromatographic medium, there is a method of immobilizing the immobilized reagent directly on the chromatographic medium by physical or chemical means. As a method of directly immobilizing, physical adsorption may be used or covalent bonding may be used. Generally, when the chromatographic medium is a nitrocellulose membrane or a mixed nitrocellulose ester membrane, physical adsorption can be performed. The shape of the antibody-immobilized portion (determination portion) in the chromatographic medium (3) is not particularly limited as long as the capturing antibody is locally immobilized, and examples thereof include a line shape, a circular shape, and a band shape. It is preferably a line shape, and more preferably a line shape having a width of 0.5 to 1.5 mm.

  After the immobilization reagent is immobilized, a blocking treatment can be performed on the chromatographic medium by a known method, if necessary, in order to prevent the accuracy of analysis from lowering due to non-specific adsorption. Generally, proteins such as BSA, skim milk, casein, and gelatin are suitably used for the blocking treatment. After the blocking treatment, if necessary, one or two or more surfactants such as Tween-20 (registered trademark), Triton X-100 (registered trademark), and SDS may be washed, or may be washed. Also need not be processed.

  As a method for producing the chromatographic medium (3), the sample pad (1), the conjugate pad (2), the reagent-immobilized chromatographic medium (3), and the absorption pad (4) are arranged in the order of capillary action between the members. In order to facilitate the occurrence of the above-mentioned problem, the respective members can be manufactured by overlapping both ends thereof with the adjacent members by about 1 to 5 mm (preferably on the backing sheet (5)). There is no particular limitation on the materials of these constituent members. In this embodiment, a glass fiber pad of Cellulose Fiber Sample (trade name, manufactured by MILLIPORE) as a sample pad (1) and a glass fiber pad (trade name, manufactured by MILLIPORE) as a conjugate pad (2), and reagent fixing Chromatographic medium (3), that is, a nitrocellulose membrane such as a Hi-Flow Plus membrane (trade name, manufactured by MILLIPORE) as a membrane, and a Cellulose Fiber Sample Pad (trade name, manufactured by MILLIPORE) as an absorption pad (4). AR9020 (trade name, manufactured by Adhesives Research) as a cellulose membrane and a backing sheet with an adhesive (5) And, these are not that most preferred, it can be appropriately selected according to applications. Here, HF090 of the Hi-Flow Plus membrane used as the membrane is used, but any of HF075, HF120, HF135, HF180, and HF240 may be used according to the viscosity of the developing solution of the present embodiment. . In addition, commercially available nitrocellulose membranes are classified according to the time required to move a certain distance called a flow rate, and the higher the flow rate, the larger the pore size. In the present embodiment, in order to maintain a good background and to prevent clogging due to aggregation, it is preferable that the pore size is large, that is, the flow rate is high. Therefore, specifically, a film having a pore size higher than 180 sec / 4 cm, more preferably Films higher than 150 sec / 4 cm are preferred.

  The determination of the degree of color development can be made by visual inspection, for example, immunochromatography reader C10066 (trade name, manufactured by Hamamatsu Photonics), immunochromatography reader TOR-500 (trade name, manufactured by Trust Medical), Pretester RM-405, Pretester RM A urine test paper tester such as -505 (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) may be used, for example, a densitometer or the like, or image analysis after photographing may be used. .

  Hereinafter, the present invention will be described specifically with reference to Examples, but it is needless to say that the present invention is not limited by the Examples and the like. The main measured values in the examples were obtained by measuring at 25 ° C. and 1 atm using the following method.

[Identification of surfactant in developing solution, content (concentration), weight concentration% ratio (ratio)]
The developing solution is diluted with distilled water as needed, and is subjected to LC / MS measurement. The apparatus uses Waters, UPLC (trade name, manufactured by Waters), sets various parameters as follows, and performs measurement.
[LC measurement]
・ LC equipment: Waters, UPLC
・ Column: Waters, ACQUITY UPLC BEH C18 1.7 μm (2.1 mm ID × 50 mm)
-Column temperature: 40 ° C
・ Detection PDA: 210-400nm
・ Flow rate: 0.3 mL / min
Mobile phase A = water (0.1% HCOOH)
-Mobile phase B = acetonitrile (0.1% HCOOH)
・ Gradient Time (min) A% B%
0 90 10
10 0 100
15 0 100
15.1 90 10
20 90 10
・ Injection volume: 10 μL
[MS measurement]
MS device: Waters, Synapt G2
・ Ionization: ESI +
-Scan range: m / z 100-3000

For each peak obtained by the measurement, the type of surfactant is identified from the base peak intensity chromatogram and the mass spectrum.
Next, with respect to the identified surfactants, samples prepared at arbitrary five concentrations were prepared, and LC / MS measurement was performed under the same conditions and methods as above. The peak area is calculated from the obtained base peak intensity chromatogram, and the calibration curve is prepared by plotting the sample concentration on the horizontal axis and the peak area on the vertical axis. A calibration curve is created for each identified surfactant.
The peak area of the base peak intensity chromatogram obtained in the analysis of the developing solution is applied to the calibration curve prepared above, and the content (% by weight) of the surfactant is calculated.
Further, the ratio of the content of the nonionic surfactant to the anionic surfactant is determined by the following formula (1):
Ratio of content (% by weight) of nonionic surfactant to anionic surfactant = A (content of nonionic surfactant) / B (content of anionic surfactant). . . Equation (1)
It is calculated by:

[Average particle size of insoluble carrier]
A dynamic light scattering type Nanotrac particle size distribution analyzer (manufactured by Nikkiso Co., Ltd., UPA-EX150) was used. As a measurement sample, a sample containing 0.01% by weight of an insoluble carrier and 99.99% by weight of pure water is used. As measurement conditions, the number of times of integration is 30 times, the measurement time per measurement is 30 seconds, and the median diameter is defined as the average particle diameter using a volume average particle diameter distribution.

[Dye content]
The ratio of the dye component to the colored cellulose fine particles is calculated from the weight change before and after the dye treatment. Using the weight of the recovered particles after the treatment and the weight of the cellulose fine particles before the treatment after the absolute drying, the following formula (2):
Dye content (%) = 1-{(weight of cellulose fine particles before treatment) / (weight of colored cellulose fine particles after dye treatment)} × 100. . . Equation (2)
To calculate the ratio of the dye component.

(When the weight of cellulose fine particles before treatment is unknown)
After treating the colored cellulose fine particles with a cellulase treatment, an acid treatment or a base treatment, the sample is dissolved in heavy water to prepare a 3 to 5 wt% heavy aqueous solution, and ¹³ C-NMR 400 MHz by FT-NMR (trade name: Avance400 Avance) And the degree of substitution is calculated. The degree of substitution is calculated from the peak area of the dye based on the C1 peak area of cellulose. The content of the dye is calculated from the degree of substitution and the molecular weight of the dye.

(When the weight of cellulose fine particles before treatment is unknown and the dye contains a nitrogen atom)
The nitrogen element content is measured by an emission spectrometry under the following measurement conditions using a nitrogen quantifier CHN coder (trade name, manufactured by Yanaco Analytical Industry Co., Ltd.). The content of the contained dye is calculated from the measured nitrogen element content.
Measurement method: Self-integration method Carrier gas: Helium Supporting gas: High-purity oxygen Supporting method: Helium / oxygen mixture method [Content of fluorescent dye compound]
The ratio of the fluorescent dye compound component to the fluorescent cellulose fine particles can be calculated from the weight change before and after the fluorescent dye compound treatment. The ratio of the fluorescent dye compound component is determined by the following formula (3) using the weight of the collected particles after the treatment and the weight of the cellulose fine particles before the treatment after the absolute drying:
Fluorescent dye compound content (%) = 1-{(weight of cellulose fine particles before treatment) / (weight of fluorescent cellulose fine particles after treatment with fluorescent dye compound)} × 100. . . Equation (3)
It is calculated by:

(When the weight of cellulose fine particles before treatment is unknown)
After subjecting the fluorescent cellulose microparticles to cellulase treatment, acid treatment or base treatment, the sample is dissolved in heavy water to prepare a 3 to 5 wt% heavy aqueous solution, and measured by ¹³ C-NMR (Avance 400 MHz) by FT-NMR. Calculate the replacement degree. The degree of substitution is calculated from the peak area of the fluorescent dye compound based on the C1 peak area of cellulose. The content of the fluorescent dye compound is calculated from the degree of substitution and the molecular weight of the fluorescent dye compound.

(When the weight of the cellulose fine particles before the treatment is unknown and the fluorescent dye compound contains a nitrogen atom)
The nitrogen element content is measured by an emission spectrometry under the following measurement conditions using a nitrogen quantifier CHN coder (trade name, manufactured by Yanaco Analytical Industry Co., Ltd.) The content of the contained fluorescent dye compound is calculated from the measured nitrogen element content.
Measurement method: Self-integration method Carrier gas: Helium Supporting gas: High-purity oxygen Supporting method: Helium and oxygen mixed method

[Example 1]
[Preparation of antibody-sensitized colored cellulose fine particles]
120 μl of colored cellulose fine particles (trade name: NanoAct, manufactured by Asahi Kasei Corporation, average particle size: 352 nm, particle concentration: 1.0%) are put into a 15 ml centrifuge tube, and 240 μl of Tris buffer (50 mM, pH 7.0) is further added to the anti-Flu. 120 μl of a 0.1% aqueous solution of -A mouse antibody (manufactured by BMR, monoclonal antibody) was added, and the mixture was vortexed for 10 seconds. Subsequently, it was placed in a dryer adjusted to 37 ° C. and allowed to stand for 120 minutes. Subsequently, 14.4 ml of a blocking solution (100 mM boric acid, pH 8.5) containing 1.0% by weight of casein (030-01505, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was further dried in a dryer at 37 ° C. for 60 minutes. It was left still. Subsequently, using a centrifugal separator (manufactured by Kubota Shoji Co., Ltd., 6200) and a centrifugal rotor (manufactured by Kubota Shoji Co., Ltd., AF-5008C), centrifugation was performed at 10,000 g for 15 minutes to precipitate the sensitized particles, and then the supernatant was removed. Removed. Subsequently, 14.4 ml of a borate buffer (50 mM boric acid, pH 10.0) was added, and the mixture was treated with an ultrasonic dispersing machine (UH-50, manufactured by SMT Corporation) for 10 seconds. Subsequently, centrifugation was performed at 10,000 g for 15 minutes to precipitate the sensitized particles, and then the supernatant was removed. Subsequently, 0.6 g of sucrose (manufactured by Wako Pure Chemical Industries, Ltd., 196-0015) and 0.8 g of a 1.0% by weight casein blocking solution were added, and a borate buffer (50 mM boric acid, PH 10.0) was used. The weight was adjusted to 4.0 g, and an antibody-sensitized colored cellulose particle dispersion of 0.03% by weight was prepared and treated with an ultrasonic disperser for 10 seconds.

[Impregnating antibody sensitizing particles into conjugate pad and drying]
A polyethylene conjugate pad (Pall, 6613) was immersed in a large excess of 0.05% by weight of Tween-20 (registered trademark, Sigma-Aldrich, T2700) to remove excess liquid and then at 50 ° C. Dried 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, 780 μl of a dispersion of 0.038% by weight of the antibody-sensitized particles was evenly applied, and dried at 50 ° C. for 60 minutes.

[Pretreatment of sample pad]
A cellulose sample pad (Millipore, C083) was washed with a large excess of 2.0 wt% BSA (Sigma Aldrich, A7906) and 2.0 wt% Tween-20® in PBS buffer ( 66 mM, pH 7.4), and dried at 50 ° C. for 120 minutes after removing excess liquid. 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 coating device (Musashi Engineering Co., Ltd., 300DS), a PBS solution (66 mM, pH 7.4) containing 0.1 wt% anti-Flu-A type mouse antibody (6601) was added at 0.1 μL / mm. It was applied to a portion having a height of 12 mm at a ratio (a test line, hereinafter also referred to as TL). At the same time, a PBS solution (66 mM, pH 7.4) containing 0.05 wt% anti-mouse antibody (manufactured by Dako, Z0259) was applied to a portion having a height of 16 mm at a rate of 0.1 μL / mm (control line, hereinafter referred to as control line). , CL). Subsequently, after drying at 37 ° C. for 30 minutes, it was left in a low humidity chamber at 25 ° C. and a humidity of 15% for 12 hours or more.

[Preparation of immunochromatography kit]
The prepared capture antibody-coated membrane, glass fiber absorption pad (PALL, 66211), a conjugate pad containing a detection reagent, and a sample pad were bonded to a backing card (Adhesives Research, AR9020). Then, it was cut to a width of 4 mm by a cutting machine to obtain an immunochromatography kit having a width of 4 mm and a length of 60 mm.

[Preparation of developing solution]
A 100 mM PBS having a pH of 7.4 was prepared, and BSA was added at a concentration of 1.0% by weight. Sodium dodecylbenzenesulfonate (SDBS) which is an anionic surfactant is added at a concentration of 0.05% by weight, and Tween 20 (a nonionic surfactant) is added at a concentration of 1.0% by weight, and the mixture is developed. Liquid.

[Evaluation of presence or absence of aggregation of immunochromatography kit]
400 μL of the developing solution to be evaluated is added to the tube, the pharyngeal swab collected by swab (manufactured by COPAN, FLOQSwabs ^™ ) is sufficiently immersed, and the swab is swirled 10 times from outside the tube while holding the swab ball portion, and then left for 1 minute. To prepare a negative sample. 100 μL was dropped into the kit, and 30 minutes after the start of development, the aggregation state of particles in the overlap portion between the conjugate pad and the membrane was evaluated. "+" When aggregation was confirmed as shown in FIG. 2, and "-" when aggregation was not confirmed as shown in FIG.

[Background evaluation of immunochromatography kit]
For the same kit used for the evaluation of the presence or absence of aggregation, the background intensity at 2 mm upstream and the background intensity at 2 mm downstream of the TL after 15 minutes were measured with an immunochromatograph reader C10066 (trade name, manufactured by Hamamatsu Photonics). did. The average value was defined as the background intensity, and it was determined that the higher the numerical value, the worse the background was.

[Evaluation of the presence or absence of false positives in the immunochromatography kit]
100 μL of the negative sample was dropped into the kit. After 15 minutes, the color intensity of TL was measured with an immunochromatographic reader. This measurement was performed five times, and when the average value of the obtained values was 5 mABS or more, it was determined that there was a false positive (+), and when the average value was 5 mABS or less, there was no false positive (-). Here, the reason why 5 mABS is used is that although there is an individual difference, if it is 5 mABS or less, the presence of TL cannot be confirmed visually.

[Measurement of detection limit of immunochromatography kit]
Flu-A type antigen (Hytest, 8IN74-4) was added to the developing solution to be evaluated, and a positive sample was prepared which was gradually diluted to 50 ng / mL, 5.0 ng / mL, and 0.5 ng / mL. did. 100 μL was dropped on the sample dropping portion of the diagnostic kit, and the color intensity of TL after 15 minutes was measured with an immunochromatographic reader. This measurement was performed five times at each concentration, and when the average value of the obtained values was equal to or higher than the value obtained when the negative sample was measured + 20 mABS, it was determined to be positive, and in the following cases, it was considered to be below the detection limit. The lower limit of the Flu-A type antigen concentration at which a positive determination was obtained was taken as the detection limit.
[Performance evaluation of immunochromatography kit]
The performance of the obtained immunochromatographic diagnostic kit was evaluated. The results are shown in Table 1 below.

[Examples 2 to 5]
Except that the content of Tween 20 (registered trademark) in the developing solution was changed to 0.27% to 4.9%, and the ratio of nonionic surfactant / anionic surfactant was changed to 5.4 to 98. An immunochromatography kit was prepared in the same manner as in Example 1, and its performance was evaluated. The results are shown in Table 1 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Examples 6 to 9]
An immunochromatography kit was prepared in the same manner as in Example 1 except that the content of SDBS in the developing solution was changed from 0.0010% to 0.9%, and its performance was evaluated. The results are shown in Table 1 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Example 10]
An immunochromatography kit was prepared in the same manner as in Example 1 except that SDBS was not added to the developing solution, and instead, sodium cholate (CAS) as an anionic surfactant was added at a concentration of 0.050% by weight. Its performance was evaluated. The results are shown in Table 1 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Example 11]
An immunochromatography kit was prepared in the same manner as in Example 1, except that SDBS was not added to the developing solution, but instead, sodium deoxycholate (DCAS), which is an anionic surfactant, was added at a concentration of 0.050% by weight. And evaluated its performance. The results are shown in Table 1 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Example 12]
A method similar to that of Example 1 was used, except that Tween 20 (registered trademark) was not added to the developing solution, and instead, Triton X-100 (registered trademark), which is a nonionic surfactant, was added at a concentration of 1.0% by weight. An immunochromatography kit was prepared and its performance was evaluated. The results are shown in Table 1 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

Example 13
An immunochromatography kit was prepared in the same manner as in Example 1 except that Tween 20 (registered trademark) was not added to the developing solution, and polysorbate 65, a nonionic surfactant, was added at a concentration of 1.0% by weight instead. And evaluated its performance. The results are shown in Table 1 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Example 14]
An immunochromatography kit was prepared in the same manner as in Example 1 except that colloidal gold particles were used as the insoluble carrier, and its performance was evaluated. The results are shown in Table 1 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Preparation of antibody-sensitized colloidal gold particles]
600 μl of a phosphate buffer (50 mM, pH 7.0) was added to 2500 μl of a gold colloidal fine particle suspension (manufactured by Tanaka Kikinzoku Co., average particle diameter 40 nm, particle concentration 0.006 wt%, average particle diameter 40 nm), and then anti-Flu. Add 200 μl of 0.1% aqueous solution of -A mouse antibody (manufactured by BMR, monoclonal antibody) and vortex. Subsequently, the mixture was stirred at 25 ° C. for 10 minutes while controlling the temperature. 300 μl of a 1% aqueous PEG solution and 600 μl of a 10% aqueous BSA solution (pH 9.0, containing 50 mM boric acid) were added to the above suspension, and the mixture was vortexed. Thereafter, a centrifugation operation (10000 g, 30 minutes) was performed, and the supernatant was removed. To the residue was added 11000 μl of a 1% BSA aqueous solution (0.05% PEG, 150 mM NaCl, pH 8.2, containing 20 mM Tris), and the mixture was vortexed. Thereafter, a centrifugation operation (10000 g, 30 minutes) was performed, and the supernatant was removed. To the residue, 900 μl of a 1% BSA aqueous solution (0.05% PEG, 150 mM NaCl, pH 8.2, containing 20 mM Tris) was added, and sonication was performed for 30 seconds.

[Example 15]
An immunochromatography kit was prepared in the same manner as in Example 1 except that colored latex fine particles were used as the insoluble carrier, and its performance was evaluated. The results are shown in Table 1 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Preparation of antibody-sensitized colored latex fine particles]
To 15 μl of a colored latex fine particle suspension (manufactured by JSR Life Sciences, average particle diameter 400 nm, particle concentration 1.0 wt%), 600 μl of a phosphate buffer (50 mM, pH 7.0) was added, and an anti-Flu-A mouse antibody was further added. 200 μl of a 0.1% aqueous solution (manufactured by BMR, monoclonal antibody) is added thereto, followed by vortexing. Subsequently, the mixture was stirred at 25 ° C. for 10 minutes while controlling the temperature. 300 μl of a 1% aqueous PEG solution and 600 μl of a 10% aqueous BSA solution (pH 9.0, containing 50 mM boric acid) were added to the above suspension, and the mixture was vortexed. Thereafter, a centrifugation operation (10000 g, 30 minutes) was performed, and the supernatant was removed. To the residue was added 11000 μl of a 1% BSA aqueous solution (0.05% PEG, 150 mM NaCl, pH 8.2, containing 20 mM Tris), and the mixture was vortexed. Thereafter, a centrifugation operation (10000 g, 30 minutes) was performed, and the supernatant was removed. To the residue, 900 μl of a 1% BSA aqueous solution (0.05% PEG, 150 mM NaCl, pH 8.2, containing 20 mM Tris) was added, and sonication was performed for 30 seconds.

[Example 16]
An immunochromatography kit was prepared in the same manner as in Example 1 except that fluorescent cellulose fine particles were used as the insoluble carrier, and its performance was evaluated. The results are shown in Table 1 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Preparation of antibody-sensitized fluorescent cellulose fine particles]
120 μl of fluorescent cellulose microparticles (particle concentration: 1.0%) was placed in a 15 ml centrifuge tube, 240 μl of Tris buffer (50 mM, pH 7.0) was added, and 0% of anti-Flu-A mouse antibody (manufactured by BMR, monoclonal antibody) was added. 120% of a 1% aqueous solution was added, and the mixture was vortexed for 10 seconds. Subsequently, it was placed in a dryer adjusted to 37 ° C. and allowed to stand for 120 minutes. Subsequently, 14.4 ml of a blocking solution (100 mM boric acid, pH 8.5) containing 1.0% by weight of casein (030-01505, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was further dried in a dryer at 37 ° C. for 60 minutes. It was left still. Subsequently, using a centrifugal separator (manufactured by Kubota Shoji Co., Ltd., 6200) and a centrifugal rotor (manufactured by Kubota Shoji Co., Ltd., AF-5008C), centrifugation was performed at 10,000 g for 15 minutes to precipitate the sensitized particles, and then the supernatant was removed. Removed. Subsequently, 14.4 ml of a borate buffer (50 mM boric acid, pH 10.0) was added, and the mixture was treated with an ultrasonic dispersing machine (UH-50, manufactured by SMT Corporation) for 10 seconds. Subsequently, centrifugation was performed at 10,000 g for 15 minutes to precipitate the sensitized particles, and then the supernatant was removed. Subsequently, 0.6 g of sucrose (manufactured by Wako Pure Chemical Industries, Ltd., 196-0015) and 0.8 g of a 1.0% by weight casein blocking solution were added, and a borate buffer (50 mM boric acid, PH 10.0) was used. The weight was adjusted to 4.0 g, and an antibody-sensitized colored cellulose particle dispersion of 0.03% by weight was prepared and treated with an ultrasonic disperser for 10 seconds.

[Comparative Examples 1 and 2]
The content of Tween 20 (registered trademark) in the developing solution was changed to 0.15% and 6.5%, and the ratio of nonionic surfactant / anionic surfactant was changed to 3.0, 130, respectively. An immunochromatography kit was prepared in the same manner as in Example 1 except that the performance was evaluated, and its performance was evaluated. The results are shown in Table 1 below. In Comparative Example 1, background coloring and reduction in sensitivity were observed, and in Comparative Example 2, aggregation, false positive reaction, and reduction in sensitivity were observed.

[Comparative Examples 3 and 4]
An immunochromatography kit was prepared in the same manner as in Example 1 except that the content of SDBS in the developing solution was changed to 0.00070% and 1.3%, respectively, and the performance was evaluated. The results are shown in Table 1 below. In Comparative Example 3, aggregation, a false positive reaction, and a decrease in sensitivity were observed, and in Comparative Example 4, a decrease in sensitivity was observed.

[Comparative Example 5]
An immunochromatography kit was prepared in the same manner as in Example 1 except that no surfactant was added to the developing solution, and its performance was evaluated. The results are shown in Table 1 below, where aggregation, false positive reaction, background coloration and decrease in sensitivity were observed.

[Comparative Example 6]
An immunochromatography kit was prepared in the same manner as in Example 1 except that no anionic surfactant was added to the developing solution, and its performance was evaluated. The results are shown in Table 1 below, where aggregation, false positive reaction, and background coloring were observed.

[Comparative Example 7]
An immunochromatography kit was prepared in the same manner as in Example 1 except that no nonionic surfactant was added to the developing solution, and its performance was evaluated. The results are shown in Table 1 below. As a result, the background was colored and the sensitivity was lowered.

[Comparative Example 8]
An immunochromatography kit was prepared in the same manner as in Example 1 except that SDBS was not added to the developing solution and instead of hexadecyltrimethylammonium bromide (CTAB), which was a cationic surfactant, and its performance was evaluated. did. The results are shown in Table 1 below, where aggregation, false positive reactions, and background coloration were observed.

[Comparative Example 9]
An immunochromatography kit was prepared in the same manner as in Comparative Example 5 except that colloidal gold particles were used as the insoluble carrier, and its performance was evaluated. The results are shown in Table 1 below, which showed aggregation, false positive reaction, background coloring, and decreased sensitivity.

[Comparative Example 10]
An immunochromatography kit was prepared in the same manner as in Comparative Example 5 except that colored latex fine particles were used as the insoluble carrier, and its performance was evaluated. The results are shown in Table 1 below, which showed aggregation, false positive reaction, background coloring, and decreased sensitivity.

[Comparative Example 11]
An immunochromatography kit was prepared in the same manner as in Comparative Example 5 except that fluorescent cellulose fine particles were used as the insoluble carrier, and its performance was evaluated. The results are shown in Table 1 below, which showed aggregation, false positive reaction, background coloring, and decreased sensitivity.

[Example 17]
An immunochromatography kit was prepared in the same manner as in Example 1 except that a nasal swab was used as a negative specimen, and its performance was evaluated. The results are shown in Table 2 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Example 18]
An immunochromatography kit was prepared in the same manner as in Example 1 except that a nasal aspirate was used as a negative sample, and its performance was evaluated. The results are shown in Table 2 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Example 19]
An immunochromatography kit was prepared in the same manner as in Example 1 except that nasal bite was used as a negative sample, and its performance was evaluated. The results are shown in Table 2 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Example 20]
An immunochromatography kit was prepared in the same manner as in Example 1 except that saliva was used as a negative sample, and its performance was evaluated. The results are shown in Table 2 below. As intended, the false positive reaction accompanying the aggregation of the particles was suppressed, a favorable background was maintained, and the detection sensitivity did not decrease.

[Comparative Example 12]
An immunochromatography kit was prepared in the same manner as in Comparative Example 5 except that a nasal swab was used as a negative sample, and its performance was evaluated. The results are shown in Table 2 below, where aggregation, false positive reactions, background coloration, and decreased sensitivity were observed.

[Comparative Example 13]
An immunochromatography kit was prepared in the same manner as in Comparative Example 5 except that a nasal aspirate was used as a negative sample, and its performance was evaluated. The results are shown in Table 2 below, where aggregation, false positive reactions, background coloration, and decreased sensitivity were observed.

[Comparative Example 14]
An immunochromatography kit was prepared in the same manner as in Comparative Example 5 except that nasal bite was used as a negative sample, and its performance was evaluated. The results are shown in Table 2 below, where aggregation, false positive reactions, background coloration, and decreased sensitivity were observed.

[Comparative Example 15]
An immunochromatography kit was prepared in the same manner as in Comparative Example 5 except that saliva was used as a negative specimen, and its performance was evaluated. The results are shown in Table 2 below, where aggregation, false positive reactions, background coloration, and decreased sensitivity were observed.

  The immunochromatography using the developing solution of the present invention is preferably used as a developing solution for immunochromatography, since a false positive reaction due to aggregation in a biological sample-derived sample is suppressed, has a good background, and the detection sensitivity does not decrease. It is possible.

1 sample pad 2 conjugate pad,
3 Chromatographic medium immobilized with reagents 4 Absorption pad 5 Backing sheet 6 Test line 7 Control line

Claims (5)

  An immunochromatographic developing solution containing both an anionic surfactant and a nonionic surfactant, wherein the concentration of the anionic surfactant is 0.001 to 1.0% by weight, and The developing solution for immunochromatography, wherein the concentration ratio (wt% of nonionic surfactant / wt% of anionic surfactant) of the nonionic surfactant to the anionic surfactant is 5.0 to 100.   The developing solution for immunochromatography according to claim 1, which is used in a detection system using an insoluble carrier as a labeling substance.   The developing solution for immunochromatography according to claim 1 or 2, wherein the insoluble carrier is selected from the group consisting of colloidal gold, latex fine particles, and cellulose fine particles.   The developing solution for immunochromatography according to claim 3, wherein the cellulose fine particles are colored cellulose fine particles containing a dye.   The developing solution for immunochromatography according to claim 3, wherein the cellulose fine particles are fluorescent cellulose fine particles containing a fluorescent dye compound.