JP2006153523A - Immunochromatograph method using noninvasive specimen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunochromatograph method capable of obtaining sufficient sensitivity even with respect to a specimen to be inspected sampled using a water absorbent material. <P>SOLUTION: The immunochromatograph method includes a process (1) for sampling the specimen having possibility containing a substance to be analyzed by the water absorbent implement material, a precess (2) for bringing the substance to be analyzed extracted from the water absorbent material by an extracting solution into contact with a labelled partner specific to the substance to be analyzed simultaneously with the extraction or continuously from the extraction and a process (3) for developing the extracted liquid obtained in the process (2) on an immunochromatograph development film. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to an immunochromatographic method using a non-invasive specimen (eg, tears, nasal discharge, sputum, or throat swab).
In the present specification, the “immunochromatography method” is not particularly limited. For example, a step of using a chromatographic method (particularly, a thin-layer chromatographic method or a paper chromatographic method) in the process. And an immunological analysis method in which at least a part of one or a plurality of antigen-antibody reactions is carried out on a thin film support for chromatography.

Although methods for immunologically measuring substances that serve as indicators of various diseases have been put into practical use for a long time, the measurement methods have been continuously improved. A number of methods have been commercialized. There is an immunochromatography method as a particularly simple and rapid method, and a method for testing for infectious diseases such as hepatitis and influenza, myocardial infarction such as CK-MB, troponin, etc. has been developed. Has been.
On the other hand, in recent years, allergic patients such as hay fever are increasing, but the point of treatment for allergic diseases is to remove or avoid the antigens that cause them. It is important to know if you are allergic to

There are three types of allergy tests: blood tests, skin tests, and provocation tests, and blood tests and skin tests are mainly performed.
For example, in the case of a blood test, atopic dermatitis or the like is examined by measuring the number of eosinophils in white blood cells by hematology. Eosinophils are involved in the development of late-onset allergies. In addition, IgE, which is a type of immunoglobulin, is involved in one of the main mechanisms of atopic dermatitis caused by allergy, and allergy is determined by a test (so-called RAST method) that measures the total amount of IgE. Estimate if you have a disease. In allergic diseases, since allergen removal (avoidance) is the main treatment, it is important to identify allergens. Allergen-specific IgE detection method (RAST method) is widely used, and if allergic diseases are suspected, the presence of allergen-specific IgE is confirmed even if the total IgE value is within the standard It is effective to do.

There are two main measurement allergen items: inhalation type and food type. Inhalation type (mainly for allergic rhinitis, bronchial asthma) is cedar pollen, garlic leopard mite, camogaya, ragweed, mugwort, dog epithelium, cat epithelium, house dust, mold (fungus), etc., while food type is Specific IgE for egg white, milk, wheat, rice, soybean, shrimp, crab, tuna, pork, beef, etc. has been tested. In addition, drugs and latex are also used as allergens in allergy tests.
The specific IgE antibody is assayed by binding a specific IgE antibody in blood with its partner, that is, an allergen, and reacting the complex with an enzyme-labeled anti-IgE antibody to obtain a signal derived from the labeled enzyme. Depending on the immunological means to detect, it is often used to identify allergy and test its severity from the results.

  In addition, as described above, it is also useful to measure eosinophils. In particular, eosinophile retained protein (hereinafter sometimes abbreviated as ECP) is contained in eosinophils present in the body. And is considered to be a protein associated with type 1 allergic inflammatory reaction. Eosinophils contain proteins such as ECP, eosinophil peroxidase (EPO), eosinophil-derived neurotoxin (EDN), and major basic protein (MBP) in the late phase reaction of type 1 allergy. Release causes tissue damage or inflammation. Therefore, measurement of ECP concentration is considered to be an index of inflammation derived from type 1 allergy. The amount of ECP contained in blood is measured immunologically by many researchers, and there are many reports on the clinical significance of the measurement (Non-patent Document 1).

When measuring a specific IgE antibody (including ECP) in blood, it is easy to obtain a measurement sample by collecting blood from a patient and separating serum or plasma. However, even if a small amount of blood is collected, a considerable burden is placed on the patient. Particularly in infants, it is difficult to collect blood, and it has been examined whether or not samples other than blood can be used.
That is, specific IgE (including ECP) is present not only in blood but also in tears, nasal discharge, saliva, sputum, urine, stool, and the like. In particular, specific IgE antibodies (including ECP) in tears and nasal secretions well reflect their allergic symptoms, and studies using these as specimen samples in place of blood have been conducted for some time.

  However, it is difficult to quantitatively obtain a measurement sample for exocrine fluid such as nasal discharge or tear fluid. For example, as a method for collecting nasal discharge, a method of collecting the secreted fluid itself, or a method of collecting only the newly secreted nasal juice after washing the nasal cavity with a nasal washing solution or the like has been attempted (Non-Patent Document). 2).

  It is more difficult to collect a sufficient amount of tears. Usually, a very small amount (about 2 μL) is collected using a capillary or the like, but this requires advanced techniques, and at the same time causes fear of the patient, and sometimes damages the eyes. It even happened. In addition, for the purpose of research, there is a method of scraping the tissue by rubbing with a metal instrument etc. in order to collect the eye tissue, but this also causes great pain and fear to the patient. Not used. Therefore, regarding the measurement of specific IgE antibody (including ECP) in tear fluid, clinical usefulness has been recognized, but it has not been put into practical use.

  As a means for solving such a problem, it is conceivable that a tear fluid or nasal discharge is once absorbed by a hygroscopic material such as filter paper and extracted from the sample (Non-patent Document 3). This method is a good method that allows sampling without burdening the patient, but the amount of sample that can be collected is smaller than that of blood. When applying such a sampling method to the current RAST method or CAP-RAST method, filter paper is treated (extracted) with a diluting solution (such as physiological saline or phosphate buffer), and a part of it is extracted. Since the reagent configuration (assay system) is used as a specimen, the specimen is diluted several hundred times as a result. Therefore, the weak positive becomes negative, the strong positive becomes weak positive, and especially in terms of sensitivity.

P. Venge et al., “Clinical and Experimental Allergy” (UK), 1999, Vol. 29, p. 1172-1186 D. Wang et al., “European Archives of Otorinolarology”, (Germany), Vol. 252, p. S40-S43 Suzuki Yasunori et al., “Allergy Clinical”, 1990, Vol. 10, p. 409-411

  An object of the present invention is to improve an immunochromatographic method for a test sample collected using a water-absorbing device, which has not been able to obtain sufficient sensitivity until now, ie, using a water-absorbing device. An object of the present invention is to provide an immunochromatographic method capable of obtaining sufficient sensitivity even for a test sample. More specifically, a specific substance contained in a biological fluid other than blood, for example, tears, nasal discharge, sputum, etc., without burdening the patient (so-called non-invasive test), In particular, it is to provide a method for measuring a protein such as a specific IgE antibody or ECP more accurately, simply and rapidly, and a kit therefor. In particular, an object of the present invention is to provide a technique capable of measuring with high sensitivity using a biologically derived liquid other than blood using a simple and rapid assay system called immunochromatography.

The subject is (1) a step of collecting a test sample that may contain a substance to be analyzed with a water-absorbing device according to the present invention;
(2) a step of extracting the analysis target substance from the water-absorbing device with an extraction solution, or a step of contacting with an analyte-specific partner that is continuously labeled from the extraction; and (3) the step ( The extract obtained in 2) can be solved by an immunochromatographic method characterized by including a step of developing the extract on an immunochromatographic developing membrane.

According to a preferred embodiment of the method of the present invention, in the step (2), the extraction target solution containing the labeled analysis target substance-specific partner is used as the extraction solution, whereby the analysis target substance is changed from the water-absorbing material. Is extracted and simultaneously contacted with a labeled analyte-specific partner.
According to another preferred embodiment of the method of the present invention, in the step (2), the water-absorbing device is brought into contact with the immunochromatographic sample addition part impregnated with the labeled analyte-specific partner. In addition, the extraction solution is added to the water-absorbing device and developed from the water-absorbing device to the sample addition section, so that the analyte can be extracted from the water-absorbing device, or continuously from the extraction. Contact with the specific analyte-specific partner.
According to still another preferred embodiment of the present invention, the extraction solution contains a surfactant.
According to still another preferred embodiment of the present invention, the extraction solution contains a viscosity reducing substance.

In addition, the present invention provides (1) an immunochromatographic strip and (2) an extraction for extracting an analyte from a water-absorbing material for collecting a sample to be examined, which contains a labeled analyte-specific partner. The present invention relates to an immunochromatographic kit, characterized in that it contains a working solution.
In addition, the present invention provides an analysis from (1) an immunochromatographic strip including an immunochromatographic sample addition part impregnated with a labeled analyte-specific partner, and (2) a water-absorbing material for collecting a test sample. The present invention relates to an immunochromatographic kit comprising an extraction solution for extracting a target substance.

  According to the present invention, sufficient sensitivity can be obtained even for a test sample collected using a water-absorbing device, which has not been able to obtain sufficient sensitivity until now. The reason why the above-mentioned effect is obtained has not been determined at present, but for example, improvement in extraction efficiency from a water-absorbing device, improvement in formation efficiency of a complex between a substance to be analyzed and a labeling partner can be considered. .

  Further, in the method of the present invention, the collected specimen can be subjected to measurement without dilution as it is or at a low dilution factor. This method is more sensitive, simple, and quicker than the case where the absorbent material containing the measurement object is extracted with a buffer solution and a part of the sample is measured as a sample as previously reported. Can be measured. Furthermore, in the present invention, by reacting with the labeled antibody while extracting, the reaction proceeds quickly, so that rapid measurement is possible and the operability of the measurement becomes simpler. Even with a quick and simple assay system called immunochromatography, it is possible to achieve high sensitivity equal to or higher than the result obtained by a conventional automatic analyzer.

  The present invention is an improved immunochromatographic method, in which a test sample is collected by a water-absorbing device, and then the test sample absorbed by the water-absorbing device is extracted by an appropriate method described in detail below. After that, analysis is performed by an immunochromatography method, and conventionally known immunochromatography methods can be applied as they are in other respects.

The method of the present invention includes (1) a step of collecting a test sample that may contain a substance to be analyzed with a water-absorbing device (hereinafter referred to as a collection step);
(2) The step of contacting the labeled analyte-specific partner simultaneously with the extraction of the analyte from the water-absorbing device with the extraction solution or continuously from the extraction (hereinafter referred to as the extraction step) And (3) a step of developing the extract obtained in the extraction step (2) on an immunochromatographic development membrane (hereinafter referred to as a development step).
including.

According to the method of the present invention, due to the difference in means of extraction from the water-absorbing device of the substance to be analyzed in the extraction process,
(A) an embodiment using an extraction solution containing a labeling partner as the extraction solution,
(B) With the water-absorbing material in contact with the immunochromatographic sample addition part impregnated with the labeling partner, the extraction solution is added to the water-absorbing tool material and developed from the water-absorbing material to the sample addition part. The aspect to be made can be mentioned.

In the said aspect (a), it can be made to contact with a labeling partner simultaneously with extraction from the water-absorbing device of the substance to be analyzed.
Moreover, in the said aspect (b), the extraction solution which does not contain a labeling partner can also be used as an extraction solution [aspect (b1)], or the extraction solution containing a labeling partner is used. [Aspect (b2)]. In the said aspect (b1), it can be made to contact with a labeling partner continuously from extraction from the water absorptive material of a to-be-analyzed substance. On the other hand, in the said aspect (b2), after making it contact with a labeling partner simultaneously with extraction from a water absorbing tool material, it can be made to contact with a labeling partner also immediately after extraction.

In the collection step in the method of the present invention, a test sample that may contain an analysis target substance is collected using a water-absorbing device.
The test sample is not particularly limited as long as it is a sample that can be collected with a water-absorbing device, but other than a sample that does not require the use of a water-absorbing device (for example, blood, serum, plasma). A sample is preferred. For example, exocrine fluid, more specifically, tear fluid (preferably, tear fluid between the conjunctiva and the eyeball surface), nasal discharge (eg, nasal aspirate), sputum, pharyngeal wipe, mucosal tissue, saliva Or lymph. These biological samples are collected using a water-absorbing device, thereby greatly reducing the burden on the patient.

  The substance to be analyzed contained in the test sample is not particularly limited as long as it is a substance that can be analyzed by immunochromatography. For example, an antibody (particularly, specific IgE) or an antigen (eg, ECP, allergen) ). For example, in the case of tears, it can be suitably used to detect infectious substances such as adenovirus. It can also be applied to various cancer marker tests such as a test for influenza virus using nasal discharge (for example, nasal aspirate), and a lung cancer test using sputum as a specimen.

As the water absorbent material, any material capable of absorbing moisture can be used. For example, paper [for example, filter paper (cellulose)], fabric (for example, woven fabric, knitted fabric, cotton swab, non-woven fabric), fiber (for example, Cotton, nylon), membranes [for example, cellulose derivative membranes (for example, nitrocellulose membrane)], and hydrophilic resins.
Filter paper (cellulose) is the most common because it is inexpensive and easily available. In addition, in the case of tears, in particular, the amount of absorption of the test sample can be controlled quantitatively. It is preferable to use Menicon Inc.). In the case of a throat swab, a cotton swab is preferably used.

  The labeled partner used in the extraction step in the method of the present invention (that is, the labeled analyte-specific partner) is appropriately selected according to the type of analyte and the signal detection means, as in the usual immunochromatographic method. You can choose.

As the analyte-specific partner, a substance that specifically captures the analyte, such as an antibody or an antigen, is used. The antibody can be either a monoclonal antibody or a polyclonal antibody, and can also be used as an antibody fragment, for example, a fragment of Fab, Fab ′, F (ab ′) ₂ , or Fv.
Examples of the labeling substance include enzymes (eg, alkaline phosphatase, peroxidase, β-galactosidase), colored particles (eg, gold colloid, color latex), fluorescent substances, and luminescent substances.

In the aspect (a), that is, in the aspect in which the extraction solution containing the labeling partner is used as the extraction solution, for example, the water-absorbing device that has absorbed the test sample is appropriately extracted including the extraction solution. After being transferred into a container for extraction and performing extraction in the container, the obtained extract (preferably, the whole amount or most of it) can be used in the next development step.
The amount of the extraction solution used for the extraction can be appropriately determined according to the size of the water-absorbing device, but can usually be 10 to 100 μL. Moreover, the temperature at the time of extraction can be normally implemented at 20-40 degreeC. Although extraction time can be suitably determined according to conditions, such as liquid amount or temperature, Usually, it can implement within 1 minute-10 minutes.

In the state (b), that is, with the water-absorbing device in contact with the immunochromatographic sample addition part impregnated with the labeling partner, the extraction solution is added to the water-absorbing device, and In an embodiment where the sample is added to the sample addition portion, the sample addition portion can be, for example, a sample addition pad or a sample dropping port. In this aspect, when the extraction solution is added to the water-absorbing device that has absorbed the test sample, the extract containing the component (for example, the analysis target substance) in the test sample is used as the sample addition unit. In the next development step, it is developed on the immunochromatographic development film. In this process, the analyte is labeled with the analyte-specific partner by extracting the analyte from the water-absorbing device or immediately after the extraction (continuously) by contacting with the labeled analyte-specific partner. The reaction with the analyte-specific partner can be performed almost simultaneously with the extraction process. In addition, there is also an aspect in which a water-absorbing material is brought into contact with an immunochromatographic sample addition part not impregnated with a labeling partner, an extract containing the labeling partner is added, the reaction is performed simultaneously with the extraction, and the development is performed simultaneously. Is possible.
The amount of the extraction solution to be added to the absorbent device can be appropriately determined according to the volume of the water-absorbent device, but is usually 10 to 100 μL. Moreover, after adding, time until it moves to the next expansion | deployment process can be suitably determined by conditions, such as a liquid quantity or temperature (usually room temperature), but it is usually within 1 minute-10 minutes. it can.

The extraction solution used in the extraction step can contain a surfactant and / or a viscosity reducing substance as desired.
As the surfactant, for example, any of a nonionic surfactant, an anionic surfactant, a cationic surfactant, or an amphoteric surfactant can be used. By including a surfactant in the extraction solution, the extraction efficiency from the water-absorbing material can be improved. In addition, even when a solution containing a surfactant is used in the extraction solution without containing a labeling partner, the extraction efficiency from the water-absorbing material can be improved. include. That is, the present invention includes
(1) A step of collecting a test sample that may contain a substance to be analyzed with a water-absorbing device;
(2) a step of extracting the water-absorbing device with an extraction solution containing a surfactant; and (3) a step of developing the extract obtained in the step (2) on an immunochromatographic development film. And an immunochromatographic method characterized in that

As the nonionic surfactant, specifically, for example, polyoxyethylene alkyl ether, polyoxyethylene alkyl allyl ether, polyoxyethylene derivative, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbitol fatty acid ester, Examples thereof include glycerin fatty acid ester, polyoxyethylene alkylamine, and alkyl alkanolamide.
Specific examples of the anionic surfactant include fatty acid salts, alkyl sulfate esters, alkylbenzene sulfonates, alkyl naphthalene sulfonates, and alkyl sulfosuccinates.
Specific examples of the cationic surfactant include alkylamine salts and quaternary ammonium salts.
Specific examples of amphoteric surfactants include alkyl betaines, amine oxides, and dimethylammoniopropane sulfonic acid.
Further, in addition to the above-mentioned classifications, for example, sodium cholate or sodium deoxycholate having a steroid skeleton known as a membrane protein solubilizer is a surfactant that can be used in the present invention.
In the method of the present invention, these surfactants can be used alone or in combination of two or more. Moreover, these addition density | concentrations can be suitably selected and used between 0.01 to 30%.

  The viscosity-reducing substance is a substance used to reduce the viscosity for the purpose of improving handling when handling a test sample having viscosity, for example, nasal discharge or sputum. For example, it is possible to treat sputum or nasal discharge with a viscosity reducing substance, absorb a specimen having a low viscosity with a filter paper, cut the impregnated portion, and treat with an extraction solution. Further, the extract may contain a viscosity reducing substance. Examples of the viscosity reducing substance include surfactants (for example, Triton X-100), quaternary ammonium salts (for example, cetylpyridinium salts), disulfide bond reducing agents (for example, dithiothreitol), chaotropic substances (for example, urea) , Guanidine), proteolytic enzymes or polysaccharide-degrading enzymes that degrade mucin causing viscosity, etc. are known (for example, Japanese Patent Application Laid-Open No. 2003-287529, Japanese Patent Application Laid-Open No. Hei 2-273197). By including a viscosity reducing substance in the extraction solution, the extraction efficiency from the water-absorbing material can be improved.

The development step of the present invention can be carried out in the same manner as a normal immunochromatographic method, except that the extract obtained in the extraction step is used.
For example, development on an immunochromatographic development membrane can be carried out on a membrane on which an analyte-specific partner different from the labeling partner is immobilized. As the membrane used in this case, for example, any membrane used in the immunochromatography method such as nitrocellulose or polyamide can be used. In addition, when the analyte is an antigen, an antibody or binding protein that binds to the antigen can be used as an immobilization partner, and when the analyte is a specific IgE antibody, an allergen is used. be able to.

  In addition, when the labeling substance is an enzyme, a washing solution containing a substrate substance is subsequently developed on the immunochromatographic development film, so that contaminants on the immunochromatographic development film are removed and at the same time supplemented by the immobilization partner. Immune complexes can be detected. When the labeling substance is colored particles, the colored particles bound on the film can be detected visually or with a measuring instrument.

The immunochromatographic kit of the present invention is
(1) an immunochromatographic strip, and (2) an extraction solution for extracting the analyte from the sample-absorbing water-absorbing material containing the labeled analyte-specific partner. it can. This immunochromatographic kit can be used for the said aspect (a) of the method of this invention, for example.

The immunochromatographic kit of the present invention is
(1) To extract an analyte from an immunochromatographic strip including an immunochromatographic sample addition part impregnated with a labeled analyte-specific partner, and (2) a water-absorbing material for collecting a test sample Solution for extraction. This immunochromatographic kit can be used, for example, in the embodiment (b) of the method of the present invention.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1
(1) Production of strip for enzyme method immunochromatography (1-1) Production of allergen-immobilized membrane A nitrocellulose membrane (HF180; pore size 5-8 μm; manufactured by Millipore) was cut into a size of 5 mm x 25 mm. At a position of 15 mm from one end (upstream direction) of the membrane, a mite allergen-extracted protein aqueous solution (Dark mushroom mite extract; manufactured by Glia Inc.) [concentration 1 mg / mL; 5 mmol / L borate buffer solution (pH 8.5), followed by dialysis Was applied in a straight line having a width of 1 mm.
The mixture was allowed to stand at room temperature for 1 hour and then allowed to stand at 37 ° C. for 30 minutes to immobilize the mite extract protein. A mite allergen-immobilized membrane was prepared by storing in a silica gel desiccator and drying overnight at room temperature.

(1-2) Preparation of enzyme-labeled anti-IgE antibody Anti-human IgE mouse monoclonal antibody was treated with pepsin, then reduced with 2-mercaptoethylamine, and 1 mg of purified Fab ′ fragment was prepared by gel filtration.
Separately, 2 mg of bovine small intestine alkaline phosphatase was diluted with a phosphate buffer so as to be 2 mg / mL, dialyzed, and N-succinimidyl 3- (2-pyridylthio) prepared to 7.5 mmol / L. ) 100 μL of propionate (SPDP) was added. After reacting at 4 ° C. for 5 hours, dialyzed with a phosphate buffer, pyridyldithiopropionate (PDP) -introduced alkaline phosphatase was prepared.
The anti-IgE antibody Fab ′ and the PDP-introduced alkaline phosphatase were mixed in an equal volume of 1 mL, and 40 μL of 1 mol / L hydroxylamine was added. After reacting at 4 ° C. for 3 days, gel filtration was performed using a gel filtration column (TSKgel G3000SW; manufactured by Tosoh Corporation) to remove unreacted anti-IgE antibody Fab ′ and PDP-introduced alkaline phosphatase, and enzyme-labeled anti-IgE. An antibody aqueous solution was prepared.

(1-3) Production of Absorbent Pad A cellulose pad (AP25; manufactured by Millipore) was cut into a size of 5 mm × 20 mm to obtain an absorbent pad.

(1-4) Production of Development Pad A cellulose pad (AP25; manufactured by Millipore) was cut into a size of 5 mm × 17 mm to obtain a development pad.

(1-5) Preparation of developing solution A 100 μg / mL aqueous solution of 5-bromo-4-chloro-3-indolyl acid (BCIP; manufactured by Boehringer Mannheim) was prepared and used as a developing solution.

(1-6) Preparation of strip for immunochromatograph In Example 1 (1-4), a plastic adhesive sheet (manufactured by BioDot) cut to a size of 5 mm × 60 mm was applied from the upstream side to the downstream side in the development direction. In order of the produced spreading pad, the mite allergen-immobilized membrane produced in Example 1 (1-1), and the absorbent pad produced in Example 1 (1-3), each end thereof is overlapped with an adjacent member by about 1 mm. The strip for immunochromatography was produced by sticking together. This was put into an immunochromatographic case to obtain an immunochromatographic device.

(2) Sample preparation and immunochromatographic measurement (2-1) Preparation of extraction solution Deionized water, physiological saline (150 mmol / L sodium chloride solution), and 10 mmol / L phosphate buffer (pH 7.5, 150 mmol) / L sodium chloride), and Triton X-100 (Wako Pure Chemical Industries, Ltd.), Tween 20 (Sigma), sodium dodecyl sulfate, or Emulgen 108 (Kao) with the phosphate buffer. The concentration was adjusted to 5.0%. To this, the enzyme-labeled anti-IgE antibody prepared in Example 1 (1-2) was dissolved to 1 μg / mL to obtain an extraction solution.

(2-2) Sample collection Samples were collected from patients with allergic symptoms. When collecting tears, the tip of Silmel paper (made by Showa Yakuhin Kako) is sandwiched between the conjunctiva and the eyeball for several minutes to infiltrate a sufficient amount of tears, and a certain amount is cut by cutting the tip 5 mm. Tears were collected. In the case of collecting nasal discharge, it was performed in the same manner except that Silmer paper was inserted into the deep nostril. In the case of a spear, spout was spouted into a container, and water was absorbed by immersing Silmer paper there. Six specimens were prepared for each.

(2-3) Measurement The tip of the cut sirmel paper was reacted with the labeled antibody while extracting the specific IgE antibody for 1 minute in 20 μL of the extraction solution prepared in Example 1 (2-1). 20 μL of the obtained extract was dropped onto the specimen dropping part of the immunochromatographic device produced in Example 1 (1-6). Thereafter, 200 μL of the developing solution prepared in Example 1 (1-5) was dropped onto the developing pad prepared in Example 1 (1-4), and the development was performed. After 20 minutes, the color development by BCIP of the allergen-coated portion was visually detected.

  Similarly, tear fluid, nasal discharge, and sputum are collected using sirmel paper, and the tip is immersed and extracted in 100 μL of physiological saline. The obtained extract is used as a sample, and a commercially available allergy measurement reagent (product name : DPC / Imrise Alastat IgE Yakuhyo Hyakutani (manufactured by Mitsubishi Chemical Yatron). The measurement was performed with Imrise 2000 (DPC).

(2-4) Measurement results Table 1 shows the results of visual detection. In the table, each symbol “−”, “±”, “+” and “++” of the method of the present invention means no coloring, weak coloring, clear coloring, and strong coloring, respectively. The stronger the degree of coloring, the higher the degree of allergy. The result by the conventional method is displayed by replacing class 0 as “−”, class 1 as “±”, classes 2 to 4 as “+”, and classes 4 and higher as “++”. The larger the class, the stronger the allergy. In addition, although the result of this invention method is a result at the time of using Triton X-100 as surfactant, the same tendency was recognized also with other surfactant.
As shown in Table 1, there was a sufficient correlation between the conventional method and the method of the present invention. In particular, in terms of sensitivity, the conventional method uses a light-emitting method with high sensitivity, but the method of the present invention is better than the conventional method.

<Table 1>
Sample Method of the present invention Conventional method
No. tears nasal discharge 痰 tears nasal discharge 痰
1 ++ ++ ++ + + +
2 + + + ± ± ±
3 ++ + + + + +
4 ± ± ± − − −
5 + ++ ++ + + +
6 ++ ++ + + + +

Example 2
An immunochromatographic device was prepared in the same manner as in Example 1, and a sample was collected in the same manner. However, the tip of the silmel paper was placed on the specimen dropping part of the device, and 30 μL of the extraction solution was dropped. The results are shown in Table 2.
As shown in Table 1, there was a sufficient correlation between the conventional method and the method of the present invention, and particularly in the case of tears, sufficient sensitivity comparable to the dropping operation of the extract of Example 1 was obtained. In addition, although the result of this invention method is a result at the time of using Triton X-100 as surfactant, the same tendency was recognized also with other surfactant. The same tendency was observed when cetylpyridinium salt or guanidine was further added.

<Table 2>
Sample Method of the present invention Conventional method
No. tears nasal discharge 痰 tears nasal discharge 痰
1 ++ ++ ++ + + +
2 + + ± ± ± ±
3 ++ + ++ + + +
4 ± + ± − − −
5 + ++ ++ + + +
6 ++ ++ ++ + + +

Example 3
(1) Preparation of colloidal gold immunochromatography strip (1-1) Preparation of allergen-immobilized membrane A nitrocellulose membrane (HF180; pore size 5-8 μm; manufactured by Millipore) was cut into a size of 5 mm × 25 mm. At a position of 15 mm from one end (upstream direction) of the membrane, a mite allergen-extracted protein aqueous solution (Dark mushroom mite extract; manufactured by Glia Inc.) [concentration 1 mg / mL; 5 mmol / L borate buffer solution (pH 8.5), followed by dialysis Was applied in a straight line having a width of 1 mm.
The mixture was allowed to stand at room temperature for 1 hour and then allowed to stand at 37 ° C. for 30 minutes to immobilize the mite extract protein. A mite allergen-immobilized membrane was prepared by storing in a silica gel desiccator and drying overnight at room temperature.

(1-2) Preparation of colloidal gold-labeled anti-IgE antibody After 1 mg of anti-human IgE mouse monoclonal antibody was diluted with a phosphate buffer (2 mmol / L, pH 7.0) so as to be 0.1 mg / mL, Dialyzed. Subsequently, while stirring 100 mL of gold colloid suspension (GOLD COLLLOID 20; manufactured by British BioCell International), 10 mL of the anti-human IgE mouse monoclonal antibody aqueous solution was slowly added dropwise and stirred at room temperature for 10 minutes. Further, 10 mL of 10% bovine serum albumin (A-7888; manufactured by Sigma; hereinafter referred to as BSA) aqueous solution was slowly added dropwise with stirring, and the mixture was further stirred at room temperature for 10 minutes. To this aqueous solution, 15 mL of an aqueous solution containing 5% sucrose and 0.2% Tween 20 was added and mixed, and then centrifuged at 16,000 × g for 1 hour at 10 ° C. to remove the supernatant.

The precipitated colloidal gold labeled antibody is resuspended in 100 mL of borax buffer (pH 8.0) containing 0.5% sucrose and 0.02% Tween 20, and 16,000 × g for 1 hour at 10 ° C. Centrifuge and remove the supernatant. Further, the precipitated colloidal gold-labeled antibody was resuspended in 100 mL of borax buffer (pH 8.0) containing 0.5% sucrose and 0.02% Tween 20, and 16,000 × g at 10 ° C. Centrifugation was performed for 1 hour, and the supernatant was removed. Finally, after resuspending the precipitated colloidal gold labeled antibody in borax buffer (pH 8.0) containing 0.5% sucrose and 0.02% Tween 20, the particle density is about 10 ¹³ / mL. The concentration was adjusted so that (absorbance at a wavelength of 520 nm = about 14) to obtain a gold colloid-labeled anti-IgE antibody suspension.

(1-3) Preparation of colloidal gold-labeled antibody pad The colloidal gold-labeled anti-IgE antibody suspension prepared in Example 3 (1-2) was mixed with 5.0% sucrose so that the amount of antibody was 1 μg. After diluting with a 5 mmol / L phosphate buffer solution (pH 7.2) containing 10 μL of the diluted solution, it was applied to an absorption pad (PREM1420; manufactured by Pall) cut to 5 mm × 5 mm, and at room temperature in a silica gel desiccator. Then, the mixture was dried overnight under reduced pressure (≦ 100 mmHg) to prepare a colloidal gold labeled antibody pad.

(1-4) Preparation of sample addition pad A cellulose pad (AP25; manufactured by Millipore) was cut into a size of 5 mm × 17 mm to obtain a sample addition pad.

(1-5) Preparation of Absorption Pad A cellulose pad (AP25; manufactured by Millipore) was cut into a size of 5 mm × 20 mm to obtain an absorption pad.

(1-6) Production of strip for immunochromatograph In Example 3 (1-4), the plastic adhesive sheet (manufactured by BioDot) cut to a size of 5 mm × 60 mm was applied from the upstream side to the downstream side in the developing direction. The prepared sample addition pad, the colloidal gold labeled antibody pad prepared in Example 3 (1-3), the mite allergen-immobilized membrane prepared in Example 3 (1-1), and Example 3 (1-5) The strips for immunochromatography were prepared by stacking and adhering both ends of the absorbent pads in the order of 1 mm with adjacent members in the order of the absorption pads prepared in 1. This was put into an immunochromatographic case to obtain an immunochromatographic device.

(2) Sample preparation and immunochromatographic measurement (2-1) Sample collection Samples of tears, nasal discharge, and sputum were collected in the same manner as in Example 1 (2-2).

(2-2) Measurement The cut sirmel paper tip was placed on the specimen dropping part of the immunochromatographic device produced in Example 3 (1-6) and brought into contact with a colloidal gold labeled antibody pad. Thereafter, 200 μL of the developing solution prepared in Example 1 (1-5) was dropped from the top of Silmel paper, and extraction and reaction of the specific IgE antibody were performed simultaneously. After 20 minutes, the color development due to the gold colloid in the allergen-coated portion was visually detected.

(2-3) Measurement results Table 3 shows the results. The same tendency was observed when cetylpyridinium salt or guanidine was further added.
<< Table 3 >>
Sample Method of the present invention Conventional method
No. tears nasal discharge 痰 tears nasal discharge 痰
1 ++ + + + + +
2 + + ± ± ± ±
3 + + + + + +
4 ++ ++ + + + +
5 + + +----
6 ++ ++ + + + +

  The present invention can be applied to the use of non-invasive specimen (eg, tear, nasal discharge, or sputum) analysis.

Claims (7)

(1) A step of collecting a test sample that may contain a substance to be analyzed with a water-absorbing device;
(2) a step of extracting the analysis target substance from the water-absorbing device with an extraction solution, or a step of contacting with an analyte-specific partner that is continuously labeled from the extraction; and (3) the step ( An immunochromatographic method comprising a step of developing the extract obtained in 2) on an immunochromatographic developing membrane.   In the step (2), by using an extraction solution containing a labeled analyte-specific partner as the extraction solution, the analyte is extracted from the water-absorbing material and at the same time the labeled analyte The immunochromatographic method according to claim 1, which is brought into contact with a substance-specific partner.   In the step (2), the extraction solution is added to the water-absorbing device in a state where the water-absorbing device is brought into contact with the immunochromatographic sample addition section impregnated with the labeled analyte-specific partner. The analytical substance is extracted from the water-absorbing material by being developed from the water-absorbing material to the sample addition part, or is contacted with the labeled analyte-specific partner at the same time or continuously from the extraction. The immunochromatographic method according to 1 or 2.   The immunochromatographic method according to any one of claims 1 to 3, wherein the extraction solution contains a surfactant.   The immunochromatographic method according to any one of claims 1 to 4, wherein the extraction solution contains a viscosity reducing substance. (1) an immunochromatographic strip, and (2) an extraction solution for extracting the analyte from the sample-absorbing water-absorbing material containing the labeled analyte-specific partner. A feature of the immunochromatography kit. (1) To extract an analyte from an immunochromatographic strip including an immunochromatographic sample addition part impregnated with a labeled analyte-specific partner, and (2) a sample-absorbing water-absorbing material. An immunochromatographic kit comprising a solution for extraction.