WO2002088737A1

WO2002088737A1 - Immunochromatographic test piece and diagnosis kit

Info

Publication number: WO2002088737A1
Application number: PCT/JP2002/004011
Authority: WO
Inventors: Seigo Nakaya; Masami Sato; Hirofumi Kajiyama; Haruhisa Hirata
Original assignee: Wakamoto Pharmaceutical Co., Ltd.
Priority date: 2001-04-23
Filing date: 2002-04-23
Publication date: 2002-11-07
Also published as: JPWO2002088737A1; JP4443117B2

Abstract

It is intended to provide an immunochromatographic test piece and a diagnosis kit whereby infection with Helicobacter pylori can be judged at a high sensitivity with the use of feces as specimens. An immunochromatographic test piece comprising a laminate composed of a rectangular antibody immobilized substrate which has, on its bottom end, a support holding a colored latex labeled-material and a liquid sample-absorbing support made of filter paper laminated thereon in the order from the bottom to the top, and on its top end, a water-absorbing support made of filter paper laminated thereon. In the antibody immobilized substrate, a monoclonal antibody undergoing an antigen-antibody reaction with native catalase of H. pylori is immobilized on a nitrocellulsoe sheet. In the support holding the colored latex particle labeled-material, nonwoven fabric is impregnated with a colored latex particle-labeled anti-H. pylori antibody wherein a monoclonal antibody undergoing an antigen-antibody reaction with native catalase of H. pylori is immobilized on colored latex particles.

Description

Specification

Immunochromatography-one test strip and diagnostic kit

Technical field

TECHNICAL FIELD The present invention relates to an oral matography test strip and a diagnostic kit.

Helicobacter pylori is a bacterium found in human gastric mucosa. The infection rate of Helicobacter pylori is closely related to the socio-economic status, with the infection rate tending to be higher in developing countries and lower in developed countries, but is lower in Japanese people. It is by far the highest among developed countries, with 80% of those over the age of 40 reportedly infected. In recent years, it has become clear that Helicobacter pylori can cause various gastric and duodenal diseases such as gastric ulcer, duodenal ulcer, chronic gastritis, and even gastric cancer.

Helicobacter pylori eradication has been shown to reduce the likelihood of developing these diseases, and there has been international debate on the possible eradication of Helicobacter pylori. In addition, duodenal ulcer, gastric malignant lymphoma, and stomach after early gastric cancer resection are recognized as applicable indications for eradication. With the recognition and recognition of Helicobacter pylori eradication therapy as a new treatment for gastric and duodenal diseases, the Japanese Society of Gastroenterology has created clinical trial guidelines in Japan and diagnosed the presence of Helicobacter pylori infection. (Journal of the Japanese Society of Gastroenterology, Vol. 96, 199—207, 199). According to the above guidelines, the diagnosis of presence is performed by culture, microscopy, and urease tests of gastric biopsy tissue, which are invasive tests, and eradication is determined by culture and microscopy of gastric biopsy tissue. It has been shown that a urea breath test, a non-invasive test, is required. In addition, it has been shown that in special cases, such as when the subject is a child, the determination is made using blood anti-Helicobacter pylori antibody test and presence diagnosis in combination.

However, these methods for detecting Helicobacter pylori infection have the following problems. Invasive testing is a method in which the subject suffers a great deal of pain due to insertion of a gastroscope and biopsy. I will be able to. Non-invasive testing significantly improves the patient's pain, whereas urea breath testing requires fasting before testing. In addition, the urea breath test requires devices such as a mass spectrum and an infrared spectrometer, and can be performed only in a specific facility, and has the disadvantage of increasing costs. Antibody testing is not suitable for eradication determination because the blood antibody titer remains high for a long time even after eradication. Therefore, there is a demand for a non-invasive and a method for directly and specifically detecting Helicobacter pylori infection that can accurately and accurately detect Helicobacter pylori infection.

Conventionally, as a direct test method for infectious bacteria in the gastrointestinal tract, separation culture using a selective medium for infectious bacteria from gastrointestinal excreta, particularly feces, has been performed. However, there have been few reports of Helicobacter pylori isolated and cultured from feces, despite numerous attempts. The reason for this is that Helicobacter pylori is in vitro, and when environmental conditions such as low temperature, nutrient deficiency, and oxygen deficiency deteriorate, the morphology changes from a normal spiral to a non-cultureable spheroid, and It is also conceivable that the spheres have changed to spheres that cannot be separated and cultured.

On the other hand, regarding the direct detection of Helicobacter pylori from feces by an immunological method based on an antigen-antibody reaction, helicobacter pylori in feces and other excrement samples was analyzed by immunoassay using a polyclonal antibody against Helicobacter pylori. A detection method has been reported (J. C. inn. Microbiol., Vol. 33, 2162-2165, 1995, JP-A-10-10128 (Patent No. 3043999)).

However, polyclonal antibodies generally have cross-reactivity, are poor in specificity, and have the disadvantage that the antibody titer and specificity fluctuate with each antiserum. Manufacturing has the problem that quality control is inherently difficult. In fact, the stool Helicobacter pylori antigen detection kit `` Hp SA '' using polyclonal antibodies manufactured by Meridian, Inc., the patent holder of Patent No. 3043999, has false negatives and low specificity. (Medical Tribune, 4-5, June 3, 1999; Am. J. Gastroenterol., Vol. 94, 1830-1833, 1999). Summary of the Invention

In view of the above situation, an object of the present invention is to provide an immunochromatographic test strip and a diagnostic kit which can determine infection to Helicobacter pylori with high sensitivity using feces as a specimen. is there.

In the present invention, a carrier for holding a colored latex label and a carrier for absorbing a liquid sample composed of filter paper are laminated in order from the bottom on the lower end of the strip-shaped antibody-immobilized support, A test piece for immunochromatography comprising a laminate in which a water-absorbing carrier made of filter paper is laminated on the upper end of a body, wherein the antibody-immobilized support is a helicobacter pylori on a dinitrocellulose sheet. A monoclonal antibody that performs an antigen-antibody reaction with the native force tarase of the present invention is immobilized. The carrier holding the labeled latex particle label is a helicopter. Immobilized non-woven fabric with colored latex particle-labeled anti-Helicobacter pylori antibody immobilized with monoclonal antibody A chromatography specimen. Detailed Disclosure of the Invention

Hereinafter, the present invention will be described in detail.

The immunochromatography one test piece of the present invention comprises, in order from the bottom, a carrier for holding a colored latex label, and a carrier for absorbing a liquid sample composed of filter paper on the lower end of the strip-shaped antibody-immobilized support, On the other hand, it comprises a laminate in which a water-absorbing carrier made of filter paper is laminated on the upper end of the antibody-immobilized support.

The antibody-immobilized support is obtained by immobilizing a monoclonal antibody (hereinafter also referred to as an anti-Hp monoclonal antibody) that performs an antigen-antibody reaction with a native catalase of Helicobacter pylori on a nitrocellulose sheet. Is what it is.

Examples of the anti-Hp monoclonal antibody include Hypridoma 21G2 (FERM BP-7337), 41A5 (FERM BP-7337), or 82B9 (FERM BP-7338), 31A3 (FERM P-18329) Or a monoclonal antibody produced from 82A3 (FERM P-18328). The immobilized anti-Hp monoclonal antibody recognizes Helicobacter pylori catalase in the sample and can capture a complex of catalase and colored latex particle-labeled anti-Helicobacter pylori antibody. Therefore, if the anti-Hp monoclonal antibody is immobilized on a line on the antibody-immobilized support, the line will be raised by the colored latex particles.

The antibody-immobilized support can be obtained by applying a buffer in which an anti-Hp monoclonal antibody is dispersed to a nitrocellulose sheet and drying.

The buffer is not particularly limited, but sodium acetate buffer, ammonium acetate buffer, sodium phosphate buffer and the like are preferable from the viewpoint of the detection sensitivity of Hemobacter pylori in the obtained immunochromatographic test piece.

Further, an antibody against the anti-Hp monoclonal antibody, a catalase of Helicobacter pylori, and a substance that specifically binds to the labeled latex particles for control lines are immobilized on the antibody-immobilized support. It may be. This makes it possible to determine whether or not the immunochromatography test has been performed normally.

The colored latex particle-labeled substance holding carrier includes a colored latex particle-labeled anti-Helicobacter pylori antibody formed by immobilizing a monoclonal antibody that performs an antigen-antibody reaction with the native catalase of Helicobacter pylori on the colored latex particles. It is impregnated in nonwoven fabric.

The nonwoven fabric is not particularly limited, and examples thereof include those made of polyester, rayon, polypropylene, cellulose, pulp, and the like.

The latex particles are not particularly limited. For example, homopolymers or copolymers of vinyl / vinyl monomers such as styrene, butyl chloride, acrylonitrile, butyl acetate, acrylates and methacrylates; styrene-butadiene copolymer Fine particles obtained by coloring a polymer, a butadiene-based copolymer such as a methyl methacrylate-butadiene copolymer, and the like can be given. Of these, polystyrene-based latex particles are preferably used because they have excellent adsorptivity for antibodies or antigens and can maintain biological activity stably for a long period of time.

A method for supporting an anti-Hp monoclonal antibody on the above colored latex particles is as follows. There is no particular limitation, and for example, it can be carried out by a method of physically or chemically adsorbing.

The colored latex particle labeled substance holding carrier may further be impregnated with a second colored latex particle labeled substance in which an antibody or the like is immobilized on a colored latex colored in a different color. Such a labeled second colored latex particle can be used as a control line tool.

The colored latex particle labeled substance holding carrier is preferably blocked with skim milk or pepsin albumin. This makes it possible to suppress non-specific reactions and eliminate false positives in which a negative sample is determined to be positive. The liquid sample-absorbing carrier preferably has an acidic substance-adsorbing ability. . Pigments and impurities in feces are often acidic. By retaining the ability to adsorb acidic substances, it is possible to exhibit excellent detection sensitivity and specificity for Helicobacter and H. pylori. As the liquid sample-absorbing carrier having such an acidic substance-adsorbing ability, a commercially available product can be used, and for example, DE81 (manufactured by Whatman) and the like can be mentioned.

The immunochromatographic test piece of the present invention may be further fixed to a substrate made of plastic or the like to have strength, or may be provided with a transparent tape or the like on the surface for protection. .

The method for producing the immunochromatographic test strip of the present invention is not particularly limited, and can be obtained, for example, by the following method.

(1) Preparation of antibody-immobilized support

In order to prepare an antibody-immobilized support in which an anti-Hp monoclonal antibody and an anti-magpie IgG antibody were linearly immobilized, a nitrocellulose sheet (manufactured by Whatman) was cut into 5 mm X 2 Omm, and A solution of monoclonal antibody 2 1G2 was applied at a position 10 mm from the lower end, and a solution of anti-Egret IgG goat polyclonal antibody (Cappe 1) was applied at a position 15 mm using Biojet Q3000 (Biodot). . After drying at room temperature for 2 hours, masking was performed by immersing in 1% skim milk (manufactured by Difco) -0.1% Tween 20-PBS for 10 minutes. Then, it was sufficiently dried. (2) Preparation of carrier holding colored latex particle label

a. Red latex particle labeled anti-Helicobacter pylori antibody

Red latex particle dispersion (PL-Latex, 10%, 450 nm, manufactured by Polymer Laboratories) 1.2 mL of PBS was added to 300 juL, and centrifuged at 13000 rpm for 5 minutes. . 1.5 mL of a monoclonal antibody 21 G2 solution (5 mg ZmL) was added to the precipitate, mixed well, and reacted at room temperature for 1 hour. To remove unreacted monoclonal antibody, centrifugation was performed at 13000 rpm for 5 minutes, and the precipitate was suspended in 1.5 mL of PBS and centrifuged again. Masking was performed by adding 1 mL of 1% skim milk and reacting at room temperature for 1 hour. Thereafter, the mixture was centrifuged at 13000 rpm for 5 minutes, and the precipitate was suspended in 1.5 mL of PBS containing 1% skim mill Cou 0.01% sodium azide.

b. Blue latex particle labeling egret IgG

Same as above using blue latex particle dispersion (PL-Late III, 10%, 450 nm, manufactured by Polymer Laboratories) and egret IgG (0.5 mg / ml, manufactured by Cappel) By using a simple operation, blue latex particle-labeled rabbits IgG was prepared.

c Colored latex particle label carrier

Equal amounts of the above two types of colored latex particle-labeled antibodies were mixed, impregnated with 10 μL of a 5 mm × 5 mm nonwoven fabric (registered trademark) (trademark), and dried by ventilation.

(3) Preparation of test piece for immunochromatography

The carrier holding the colored latex label was stacked up to 2.5 mm from the lower end of the antibody-immobilized support. Furthermore, a liquid sample absorbing carrier (3 MM Chr, manufactured by Whatman) was placed on the colored latex label holding carrier to a position 2.5 mm from the lower end. In addition, a water-absorbing carrier (3 MM Chr, manufactured by Whatman) is placed up to 2 mm from the upper end of the antibody-immobilized support, and finally a transparent tape is attached to the top and fixed, and the immunochromatography test piece is fixed. And

A diagnostic kit including the immunochromatographic test strip of the present invention is also one of the present invention.

The diagnostic kit includes, in addition to the immunochromatographic test strip of the present invention, For example, a well, a positive control, a negative control, a stool suspension buffer, a concentrated washing solution, and the like may be included.

The stool suspension buffer is not particularly limited, but PBS and sodium or sodium borate buffer are preferred in view of excellent detection sensitivity. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to only these Examples.

(Example 1) Examination of buffer solution for immobilizing antibody

(1) Preparation of antibody-immobilized support

1 OmM buffer containing 3% methanol (sodium phosphate (pH 5-8), sodium acetate (pH 4-6), ammonium acetate ( _PH 4-6), sodium tenoate (pH 4-6), tris-hydrochloride (PH7 ~ 9), sodium borate (pH8-10), ammonium chloride (pH8 ~ ll), ammonium carbonate (pH8 ~ 10)), lmg of anti-Helicobacter pylori (Hp) monoclonal antibody 21G2 / mL. A nitrocellulose sheet (manufactured by Whatman) was cut into 5 mm x 2 Omm, and an anti-Hp monoclonal antibody 21G2 solution prepared with each buffer for test lines was placed at a position 10 mm from the lower end, and a position 15 mm from the bottom. Then, an anti-mouse IgG polyclonal antibody (Cappe 1) (lmg / mL) was applied linearly using Biojet Q3000 (Biodot) as a control line to immobilize it. After drying at room temperature for 2 hours, masking was performed by immersing in 1% skim milk (manufactured by Difco) -0.1% Tween 20-PBS for 10 minutes. Then, it was sufficiently dried.

(2) Preparation of colored latex particle labeled antibody

100 / zL of a red latex particle dispersion (PL-Latex, 10%, particle diameter 450 nm, manufactured by Polymer Laboratories) was centrifuged at 10,000 rpm for 5 minutes. An anti-Hp monoclonal antibody 21 G 2 solution (lmg / mL) was added to the precipitate, and the mixture was mixed well and reacted at room temperature for 1 hour. Unreacted monoclonal anti To remove the body, centrifugation was performed at 10,000 rpm for 5 minutes, the precipitate was suspended in 1 mL of PBS, and centrifuged again. Masking was performed by adding 1 mL of 1% skim milk and reacting at room temperature for 1 hour. Thereafter, centrifugation was performed at 10,000 rpm for 5 minutes, and the precipitate was suspended in PBSlmL containing 1% skim milk and 0.01% sodium azide.

(3) Preparation of test piece for immunochromatography

Glass filters (manufactured by Whatman) cut to 5 mm × 1 Omm were overlapped to a position 2 mm from the lower end of each antibody-immobilized support prepared in (1). In addition, a liquid sample absorbing carrier (C / P-30, manufactured by Whatman) was stacked on the glass filter to a position 2.5 mm from the lower end. In addition, a water-absorbing carrier (3 MM Chr, manufactured by Whatman) is stacked up to 2 mm from the upper end of the antibody-immobilized support. It was a piece. (4) Preparation of Hp standard antigen

Helicobacter Pylori ATCC43504 strain was microaerobically cultured for 4 days at 37 ° C on 150 plates of Brain Heart Infusion Agar Medium (Diff Kone Soil) containing 5% permeabilized blood. After the culture, the cells were collected, suspended in 40 mL of PBS, and centrifuged at 300 rpm for 20 minutes. The precipitate was suspended in 4 OmL of PBS containing 0.5% formalin and inactivated by leaving it at 4 ° (:, 1 晚. The inactivated cells were PBS

After washing three times with 4 OmL to remove formalin, the cells were resuspended in the same amount of PBS. After confirming the turbidity of the bacterial solution (absorbance at 540 nm) and the total number of bacteria (microscopic observation using a calculator), the cells were disrupted by sonication. The disrupted cells were centrifuged at 3,000 rpm for 20 minutes, and the supernatant was further ultracentrifuged at 30,000 rpm for 30 minutes. The supernatant was filtered through a 0.2 μιη filter to obtain an Hp standard antigen.

(5) Measurement of Hp antigen standard solution

0.5% BSA-0.02% Tween 20 _PBS diluted Hp antigen standard solution

Mix 50 μL with 10 μL of the colored latex particle-labeled antibody solution prepared in (2). The carrier for absorbing the liquid sample of the test piece of the immunochromatography ^ "obtained in (3) was immersed and developed in the liquid thus obtained, and was visually judged after 10 minutes. The results are shown in Table 1. "Ten" indicates that the test line red line can be confirmed, "Soil" indicates that the test line red color can be confirmed but is very light, and "One" indicates that the test line red line 'cannot be confirmed. By immobilizing the test line using sodium acetate buffer (pH 5) and ammonium acetate buffer (pH 5), 0.5 μg / mL of Hp antigen standard solution was obtained. It was possible to judge up to. table 1

Hp antigen standard solution ^ g / ml)

Antibody immobilization

0 0.2 0.5 1 2 5 10 Na phosphate (pH5) + + + Na phosphate (pH6) +

Na phosphate (pH7) +

Na phosphate (pH8) +

Na acetate (pH4) Soil + + + Na acetate (pH5) Soil + + + + Na acetate (pH6) ± + + + Ammonium acetate (pH4) Soil + + + Ammonium acetate (pH5) Soil + + + + Ammonium acetate ( pH6) M + + + Na citrate (pH4) ± + + + Na citrate (pH5) Sat + + + Na citrate (pH6) Sat + + + Na borate (pH 8) Sat + Na borate (pH 9) Sat + Na borate (pH 10) M Tris hydrochloric acid (pH 7) + Tris hydrochloric acid (pH 8) + + Tris hydrochloric acid (pH 9) + +

NH ₄ OH-NH ₄ Cl (pH8) + +

NH OH-NH C 1 (pH9) + +

NH ₄ OH-NH ₄ Cl (pH10) Sat +

NH ₄ OH-NH ₄ Cl (pHll) ± + Ammonium carbonate (pH8) + + Ammonium carbonate (pH9) + + Ammonium carbonate (ρΗΙΟ) (Example 2) Comparison of blocking solution of carrier holding colored latex label

(1) Preparation of antibody-immobilized support

A nitrocellulose sheet (made by Petman) was cut into a size of 5 mm x 20 mm, and an anti-Hp prepared with 10 mM ammonium acetate (pH 5) buffer containing 3% methanol for the test line at a position 1 Omm from the lower end. A monoclonal antibody solution (1 mg / mL) and anti-mouse IgG polyclonal antibody (Cappe 1) (lmg / mL) (lmg / mL) as a control line at the position of 15 mni were added to BioDiet Q3000 (Biodot). It was applied linearly and solidified. After drying at room temperature for 2 hours, masking was performed by immersing in 1% skim milk (manufactured by Difco) -0.1% Tween 20-PBS for 10 minutes. Then, it was sufficiently dried.

(2) Comparison of blocking solution when preparing a carrier holding colored latex labeled substance Polyvinyl alcohol (hereinafter referred to as PVA, manufactured by Wako Pure Chemical Industries), polyvinylinolepyrrolidone K15 (hereinafter, referred to as PVP, manufactured by Tokyo Chemical Industry), Using Tween 20, Triton X-100, sodium dodecyl sulfate (hereinafter referred to as SDS), skim milk, and serum albumin (hereinafter referred to as BSA) for blocking a glass fiber filter which is a carrier for holding colored latex label. The effects were examined. The glass filter cut to 5 mm × 10 mm was immersed in PVA, PVP, Tween 20, Triton X-100, SDS, skim milk, and BSA adjusted to a concentration of 1% for 5 minutes each to perform blocking. After drying at room temperature for 2 hours, the colored latex particle-labeled antibody prepared in Example 1 was applied in an amount of 10 μL. After that, it was sufficiently dried to obtain a colored latex labeled substance holding carrier.

(3) Preparation of test piece for immunochromatography

The respective colored latex-labeled antibody holding carriers prepared in (2) were overlapped to a position 2 mm from the lower end of the antibody-immobilized support prepared in (1). In addition, a liquid sample absorption carrier (CZP_30, manufactured by Whatman) was placed on the colored latex holding carrier to a position 2.5 mm from the lower end. In addition, a water-absorbent carrier (3 MM Chr, manufactured by Whatman) is placed 2 mm from the upper end of the antibody-immobilized support. And fix it on the top to make a chromatographic test piece.

(4) Detection of Helicobacter pylori in fecal samples

Fecal samples of 0.15 g of 5 Hp-positive and 5 Hp-negative were suspended in 2 mL of 0.5% BSA—0.02% Tween 20—PBS. The contaminants were removed by centrifugation at 3000 rpm for 1 minute, and 50 L of the supernatant was dropped onto the liquid sample absorption carrier part of the immunochromatography one test piece prepared in (4). After 10 minutes, it was visually judged. Table 2 shows the results. "Ten" indicates that the red line of the test line can be confirmed, "Sat" indicates that the red color of the test line can be confirmed but is very light, "One" indicates that the red line of the test line cannot be confirmed, "*" Indicates that the latex-labeled antibody has aggregated and cannot be expanded to the test line. Glass fiber filters blocked with BSA or skim milk could be determined as positive in all positive samples and negative in all negative samples. Table 2

(Example 3) Comparison of carriers for liquid sample absorption

(1) Preparation of carrier holding colored latex label

The glass filter ′ cut to 5 mm × 10 mm was immersed in skim milk adjusted to 1% concentration for 5 minutes to perform blocking. After drying at room temperature for 2 hours, Example 1 10 μL of the colored latex particle-labeled antibody prepared in 1) was applied. Thereafter, it was sufficiently dried to obtain a colored latex labeled substance holding carrier.

(2) Comparison of carriers for liquid sample absorption

The carrier holding the colored latex label produced in (1) was overlapped with a position 2 mm from the lower end of the antibody-immobilized support produced in Example 2. Further, CZP-30 (manufactured by Petman), P81 (manufactured by Petman), C / DE 30 (manufactured by Petman), DE81 (manufactured by Whatman) are used as carriers for absorbing liquid samples on the colored latex-labeled carrier. ), GF / QA30 (manufactured by Whatman) was placed up to 2.5 mm from the lower end. In addition, a water-absorbent carrier (3MM Chr, manufactured by Petman) is placed up to 2 mm from the top of the antibody-immobilized support. did.

(3) Measurement of Hp antigen standard solution

0.5% B SA-O. 02% Tween 20-50 L of Hp antigen standard solution diluted with PBS is dropped and spread on the liquid sample absorption support part of the immunono chromatography one test piece obtained above. After 10 minutes, a visual judgment was made. Table 3 shows the results. “Ten” indicates that the red line of the test line can be confirmed, “Sat” indicates that the red color of the test line can be confirmed but is very light, and “One” indicates that the red line of the test line cannot be confirmed. When DE 81 was used as a carrier for liquid sample absorption, determination was possible up to a Hp antigen concentration of 0.1 μg / mL.

Table 3

(Example 4) Effect of composition of stool suspension buffer on Hp detection

(1) Preparation of test piece for immunochromatography

The colored latex-labeled substance holding carrier prepared in Example 3 was overlapped with a position 2 mm from the lower end of the antibody-immobilized support prepared in Example 2. Further, DE 81 (manufactured by Whatman) was superimposed on the carrier holding the colored latex label as a liquid sample absorbing carrier to a position 2.5 mm from the lower end. In addition, a water-absorbing carrier (3 MM Chr, manufactured by Whatman) is placed up to 2 mm from the upper end of the antibody-immobilized support, and finally a transparent tape is stuck on top and immobilized. It was a piece. (2) Effect of stool suspension buffer composition on Hp standard antigen detection

0.5% BS A-0. 02% Tween 20—PBS and 0.5% BSA—0.02% Tween 20 in 15 OmM buffer (sodium phosphate (pH 5-8), boric acid 50 μL of the Hp antigen standard solution diluted with sodium (pH 8: L 0)) was dropped on the carrier for liquid sample absorption of the immunochromatography-one test piece obtained above, and developed. Judged. Table 4 shows the results. "+ J indicates that the red line of the test line can be confirmed," Soil "indicates that the red color of the test line can be confirmed but is very pale, and" one "indicates that the red line of the test line cannot be confirmed. When PBS and sodium borate buffer were used as the stool suspension buffer, accurate determination was possible up to an Hp antigen concentration of 0.1 μgZmL. Table 4

(Example 5) Preparation of immunochromatographic test piece using anti-Hp monoclonal antibody 82B9

(1) Preparation of antibody-immobilized support

Prepare 1 OmM buffer solution containing 3% methanol (sodium phosphate (pH 5-8), ammonium acetate (pH 4-6), sodium borate (pH 8-10)) and prepare anti-Helicobacter pylori (Hp) Monoclonal antibody 82B9 was added to a concentration of lmgZ mL. An Etrocellulose sheet (manufactured by Whatman) was cut into 5 mm X 2 Omm, and the anti-Hp monoclonal antibody 82B9 solution prepared with each buffer for the test line was used at a position 1 Omm from the lower end, and the solution was placed at a position 15 mm away. An anti-mouse IgG polyclonal antibody (Cappe 1) (lmg / mL) was applied linearly to a solid line by using Biojet Q3000 (Biodot) for a troll line. After drying at room temperature for 2 hours, it was immersed in 1% skim milk (manufactured by Difco) -0.1% Tween 20-PBS for 10 minutes to perform masking. Then, it was sufficiently dried. (2) Preparation of colored latex particle labeled antibody

Red latex particle dispersion (PL-Latex, 10%, particle diameter 450 nm, manufactured by Polymer Laboratories) lO O ^ L was centrifuged at 10,000 rpm for 5 minutes. 1 mL of anti-Hp monoclonal antibody 82B9 solution (Img / mL) was added to the precipitate, mixed well, and reacted at room temperature for 1 hour. To remove unreacted monoclonal antibody, centrifugation was performed at 10,000 rpm for 5 minutes, and the precipitate was suspended in 1 mL of PBS and centrifuged again. Masking was performed by adding 1 mL of 1% skim milk and reacting at room temperature for 1 hour. Thereafter, centrifugation was performed at 10,000 rpm for 5 minutes, and the precipitate was suspended in PBSlmL containing 1% skim milk and 0.01% sodium azide.

(3) Preparation of carrier holding colored latex label

The glass filter cut to 5 mm × 10 mm was immersed in skim milk adjusted to a concentration of 1% for 5 minutes to perform blocking. After drying at room temperature for 2 hours, one of the colored latex particle-labeled antibodies prepared in (2) was applied. Thereafter, it was sufficiently dried to obtain a colored latex labeled substance holding carrier.

(4) Preparation of test piece for immunochromatography

The colored latex label holding material prepared in (3) was superimposed on the antibody-immobilized support prepared in (1) to a position 2 mm from the lower end of the support. Further, DE81 (made by Petman) as a carrier for absorbing a liquid sample was stacked on the carrier holding the colored latex label to a position 2.5 mm from the lower end. In addition, a water-absorbing carrier (3MM Chr, manufactured by Whatman) is placed up to 2 mm from the upper end of the antibody-immobilized support, and finally a transparent tape is attached to the top and fixed, and the immobilized chromatographic test piece is attached. did.

(5) Measurement of Hp antigen standard solution

Liquid of 50 μL of Hp antigen standard solution diluted with 15 OmM sodium borate buffer (PH8) containing 0.5% BS A-0.02% Tween 20 Drop on the sample-absorbing carrier and let it develop, and after 10 minutes Was visually determined. Table 5 shows the results. "+" Indicates that the test line red line can be confirmed and can be confirmed. "'Soil" indicates that the test line red color can be confirmed but is very light. "I" indicates that the test line red line cannot be confirmed. Show. By immobilizing the test line using sodium phosphate buffered saline (pH 6), it was possible to accurately determine the Hp antigen concentration up to 0.25 μgZmL. Table 5

(Example 6) Preparation of immunochromatographic test piece using anti-Hp monoclonal antibody 41A5

(1) Preparation of antibody-immobilized support

Prepare 1 OmM buffer (sodium phosphate (pH 5-8), ammonium acetate (pH 4-6), sodium borate (pH 8-10)) containing 3% methanol, and prepare anti-Helicobacter pylori (H) monoclonal Antibody 41 A5 was added to a concentration of lmgZ mL. A nitrocellulose sheet (manufactured by Whatman) was cut into 5 mm X 2 Omm, and an anti-Hp monoclonal antibody 41A5 solution prepared with each buffer solution for a test line was used at a position 10 mm from the lower end, and a solution was added at a position 15 mm from the buffer. Using anti-mouse IgG polyclonal antibody (C appe 1) (lmg / mL) and BioJet Q3000 (Biodot) for troll line To form a solid phase. After 2 hours drying at room temperature, 1% skim milk (D ifco Co.) Single 0.1% Tween 20- PB KoTsuta _c then a 10-minute immersion was masked S, and thoroughly dried. (2) Preparation of colored latex particle labeled antibody

100 L of a red latex particle dispersion (PL-Latex, 10%, particle diameter 450 nm, manufactured by Polymer Laboratories) was centrifuged at 10,000 rpm for 5 minutes. 1 mL of anti-Hp monoclonal antibody 41A5 solution (lmg / mL) was added to the precipitate, mixed well, and reacted at room temperature for 1 hour. To remove unreacted monoclonal antibodies, centrifugation was performed at 10,000 rpm for 5 minutes, and the precipitate was suspended in 1 mL of PBS and centrifuged again. Masking was performed by adding 1 mL of 1% skim milk and reacting at room temperature for 1 hour. Thereafter, centrifugation was performed at 10,000 rpm for 5 minutes, and the precipitate was suspended in PBS B containing 1% skim milk and 0.01% sodium azide.

(3) Preparation of carrier holding colored latex label

The glass filter cut to 5 mm × 10 mm was immersed in skim milk adjusted to 1% concentration for 5 minutes to perform blocking. After drying at room temperature for 2 hours, 10 L of the colored latex particle-labeled antibody prepared in (2) was applied. Thereafter, it was sufficiently dried to obtain a colored latex labeled substance holding carrier.

(4) Preparation of test piece for immunochromatography

The colored latex label holding material prepared in (3) was superimposed on the antibody-immobilized support prepared in (1) to a position 2 mm from the lower end of the support. Furthermore, DE81 (made by Petman) as a carrier for absorbing a liquid sample was superimposed on the carrier holding the colored latex label to a position 2.5 mm from the lower end. In addition, a water-absorbent carrier (3 MM Chr, manufactured by Whatman) is placed up to 2 mm from the upper end of the antibody-immobilized support. And (5) Measurement of Hp antigen standard solution

0.5% BS A- 0.02% Tween 20 Hp antigen standard solution 50 / L diluted with 15 OmM sodium borate buffer (pH 8) The sample was dropped on the carrier for sample absorption and developed, and a visual judgment was made 10 minutes later. Table 5 shows the results. “10” indicates that the red line on the test line can be confirmed, and “1” indicates that the red line on the test line cannot be confirmed. By immobilizing the test line with ammonium acetate buffer (pH 4 and pH 5), it was possible to accurately determine the Hp antigen concentration up to 0.25 μg / mL. Table 6

(Example 7) Preparation of immunochromatographic test piece using anti-Hp monoclonal antibody 31A3 and antibody 21G2

(1) Preparation of antibody-immobilized support

Prepare 1 OmM buffer solution containing 3% methanol (sodium phosphate (pH 5-8), ammonium acetate (pH 4-6), sodium borate (pH 8-10)) and prepare anti-helicopter pylori (H) monoclonal Antibody 31A3 was added to a concentration of lmgZ ml. Nitrocellulose sheet (made by Whatman) is cut into 5mm x 2 Omm, and each buffer is placed at 1 Omm from the lower end for the test line. The anti-Hp monoclonal antibody solution prepared in the above solution was placed at a position of 15 mm, and an anti-mouse IgG polyclonal antibody (manufactured by Cappe 1) (1 mg / m 1) was used as a control line for Biojet Q3000 (Biodot). Was applied linearly to make a solid phase. After drying at room temperature for 2 hours, masking was performed by immersing in 1% skim milk (manufactured by Difco) -0.1% Tween 20-; PBS for 10 minutes. Then, it was sufficiently dried.

(2) Preparation of test piece for immunochromatography

The carrier holding the antibody 21G2 colored latex labeling substance prepared in Example 3 (1) was overlapped to a position 2 mm from the lower end of each of the antibody-immobilized supports prepared in (1). Further, DE 81 (manufactured by Whatman) as a carrier for absorbing a liquid sample was overlaid on the carrier holding the colored latex label to a position 2.5 mm from the lower end. In addition, a water-absorbing carrier (3 MM Chr, manufactured by Whatman) is placed up to 2 mm from the upper end of the antibody-immobilized support. It was a piece.

(3) Measurement of Hp antigen standard solution

0.5% BS A- 0.02% Tween 20 containing 15 OmM sodium borate buffer (pH 8) diluted with 50 / L Hp antigen standard solution The sample was dropped on the carrier for sample absorption and developed, and a visual judgment was made 10 minutes later. Table 7 shows the results. “Ten” indicates that the red line of the test line can be confirmed, “Sat” indicates that the red color of the test line can be confirmed but is very pale, and “One” indicates that the red line of the test line cannot be confirmed. By immobilizing the test line using sodium phosphate buffer (pH 7), it was possible to accurately determine the Hp antigen concentration up to 0.25 μg / ml. Table 7

(Example 8) Preparation of immunochromatographic test piece using anti-Hp monoclonal antibody 82A3

(1) Preparation of antibody-immobilized support

Prepare 1 OmM buffer solution containing 3% methanol (sodium phosphate (pH 5-8), ammonium acetate (pH 4-6), sodium borate (pH 8-10)) and prepare anti-helicopter pylori (H) monoclonal Antibody 82A3 was added to a concentration of Img, in 1. A nitrocellulose sheet (manufactured by Whatman) is cut into 5 mm x 2 Omm, and the anti-Hp monoclonal antibody solution prepared with each buffer for the test line is placed at 1 Omm from the lower end, and the control line is placed at 15 mm from the lower end. An anti-mouse IgG polyclonal antibody (C appe 1) (lm g / m 1) was applied linearly using a BioJet Q3000 (Biodot) to solidify it. After drying at room temperature for 2 hours, 1% skim milk (Difco ) —0.1% Tween 20—Passed for 10 minutes and masked. Then, it was sufficiently dried.

(2) Preparation of colored latex particle labeled antibody

Red latex particle dispersion (PL-Late: 10%, particle diameter 450 nm, manufactured by Polymer Laboratories) lO O ^ L was centrifuged at 10,000 rpm for 5 minutes. 1 ml of anti-Hp monoclonal antibody 82A3 solution (lmg / ml) was added to the precipitate, mixed well, and reacted at room temperature for 1 hour. To remove unreacted monoclonal antibodies, centrifugation was performed at 10,000 rpm for 5 minutes, and the precipitate was suspended in 1 ml of PBS and centrifuged again. Masking was performed by adding 1 ml of 1% skim milk and reacting at room temperature for 1 hour. Thereafter, centrifugation was performed at 10,000 rpm for 5 minutes, and the precipitate was suspended in PBS Ira 1 containing 1% skim milk and 0.01% sodium azide. (3) Preparation of carrier holding colored latex label

The glass filter cut to 5 mm 10 mm was immersed in skim milk adjusted to 1% concentration for 5 minutes to perform blocking. After drying at room temperature for 2 hours, the colored latex particle-labeled antibody prepared in (2) was applied in an amount of 10 ZL. Thereafter, it was sufficiently dried to obtain a colored latex labeled substance holding carrier.

(4) Preparation of test piece for immunochromatography

The colored latex label holding material prepared in (3) was superimposed on the antibody-immobilized support prepared in (1) to a position 2 mm from the lower end of the support. Furthermore, DE81 (manufactured by Wattman) as a carrier for absorbing a liquid sample was overlaid on the carrier holding the colored latex label to a position 2.5 mm from the lower end. In addition, a water-absorbent carrier (3 MM Chr, manufactured by Whatman) is placed up to 2 mm from the upper end of the antibody-immobilized support. And

(5) Measurement of Hp antigen standard solution 0.5% B SA-0.02% Tween 20 containing 15 L of Hp antigen standard solution diluted with 15 OmM sodium borate buffer (pH 8) The sample was dropped on the carrier for sample absorption and developed, and a visual judgment was made 10 minutes later. Table 8 shows the results. “Ten” indicates that the red line on the test line can be confirmed, “Shi” indicates that the red color of the test line can be confirmed but is very light, and “One” indicates that the red line of the test line cannot be confirmed. By immobilizing the test line with ammonium acetate buffer (pH 4), it was possible to accurately determine the Hp antigen concentration up to 0.5 μg / ml. Table 8

(Example 9) Detection of Hp in fecal samples by immunochromatography

A stool suspension using 15 OmM sodium borate (pH 8) shown in Example 4 was obtained by using 5 Omg of stool specimens of each of 10 subjects determined as Hp positive and negative by the urea breath test. The suspension was suspended in 1 mL of a turbidity buffer. The stool suspension 50 was used as the liquid for each test specimen prepared in Example 3 (using DE 81 as a carrier for liquid sample absorption), Example 5, Example 6, Example 7, and Example 8. The solution was dropped onto the sample absorbing carrier. Ten minutes later, the presence or absence of a red line on the test line was visually determined. As a result, in all of the test specimens, a red line was confirmed in all of the 10 positive specimens, and the specimen was determined to be positive. On the other hand, in all of the negative samples, no red line could be confirmed in all 10 cases, and it was determined to be negative. The control line was confirmed for all samples. These samples were tested using Meridian's HpSAELISA (fecal Hp antigen detection enzyme immunoassay kit using polyclonal antibodies) according to the package insert, and all positive samples were determined to be positive. . However, of the 10 negative samples, 2 were determined to be positive, with a false positive rate of 20%. Example 10 Detection of Hp in fecal samples by immunochromatography

A fecal sample (5 Omg) of 66 Hp-positive persons and 12 negative persons determined by the urea breath test was suspended in 1 mL of the stool suspension buffer using PBS shown in Example 4. 50 L of the stool suspension was dropped onto the liquid sample-absorbing carrier of the immunochromatography one test piece (DE 81 was used as the liquid sample-absorbing carrier) prepared in Example 3. After 10 minutes, the presence or absence of a red line on the test line was visually determined. As a result, a red line was confirmed in all 66 positive samples, and it was determined to be positive. On the other hand, in the negative samples, no red line was observed in all of the 12 cases, and the sample was judged to be negative and the concordance rate was 100%. The control line was confirmed for all samples. Example 11 Investigation of reactivity to clinically isolated Hp strain

The reactivity with 120 Hp strains isolated from the clinic was examined. Each strain was cultured in the same manner as described in Example 1 to collect cells, and the cells were sonicated to prepare Hp antigen. 0.5% BSA—50 μL of Hp antigen solution diluted to 0.25 ZmL with 15 OmM sodium borate buffer (pH 8) containing 0.02% Tween 20 was prepared in Example 3. A drop was dropped onto a liquid sample absorbing carrier of a munochromatographic test piece (DE81 was used as a liquid sample absorbing carrier). Ten After one minute, the presence or absence of a red line on the test line was visually determined. As a result, a red line was confirmed in all of the 120 clinically isolated Hp strains, and it was determined to be positive. The control line was confirmed for all samples. Example 1 2 Reactivity to other strains

Helicobacter hepaticus ATCC51448, Helicobacter felis ATCC49179, Helicobacter mustelae ATCC43772, Helicobacter cinaedi ATCC35683, Escheric hia coli ATCC25922, Campylobacter jejuni ATCC29428, Bacteroides vulga tus IFO 14291, Bifidobacterium Josten The reactivity to wm bre ve JCM1192 strain was examined. The cells were sonicated to obtain crushed cells. 0.5% BSA — 0.02% Tween 20 15 μL of each cell lysate diluted with 15 OmM sodium borate buffer (pH 8) to a protein concentration of 1 Og Zin L Was dropped onto the liquid sample absorption carrier of the immunochromatography one test piece (DE81 used as a liquid sample absorption carrier) prepared in Example 3. After 10 minutes, the presence or absence of a red line on the test line was visually determined. As a result, no red line was confirmed in all strains, and the strain was determined to be negative. The control line was confirmed for all samples. Industrial applicability

Since the present invention has the above-described configuration, it is possible to provide an immunochromatography-one test strip and a diagnostic kit that can determine infection to Helicobacter pylori with high sensitivity using feces as a specimen.

Claims

The scope of the claims

1. A carrier for holding a colored latex label and a carrier for absorbing a liquid sample composed of filter paper are laminated in order from the bottom on the lower end of the strip-shaped antibody-immobilized support, while the upper end of the antibody-immobilized support is An immunochromatographic test piece consisting of a laminate obtained by laminating a water-absorbing carrier made of filter paper thereon,

The antibody-immobilized support is obtained by immobilizing a monoclonal antibody that performs an antigen-antibody reaction with native Helicobacter pylori catalase on a nitrocellulose sheet,

The colored latex particle-labeled substance holding carrier is a non-woven cloth containing a colored latex particle-labeled anti-Helicobacter pylori antibody obtained by immobilizing a monoclonal antibody that performs an antigen-antibody reaction with a native helicobacter 1 'pylori force tarase on the colored latex particles. Become impregnated

An immunochromatographic test strip, characterized in that:

2. Immobilization of monoclonal antibody that performs antigen-antibody reaction with native Helicobacter pylori catalase on an antibody-immobilized support consists of sodium acetate buffer, ammonium acetate buffer, and sodium phosphate buffer. A monoclonal antibody that performs an antigen-antibody reaction with Helicobacter pylori's native catalase is dispersed in at least one buffer selected from the group consisting of the following components, and the resulting dispersion is applied to a nitrocellulose sheet. The test specimen according to claim 1, wherein

3. An immunochromatographic test strip characterized in that the carrier for holding labeled colored latex particles is blocked with skim milk or pepsin albumin.

4. A test piece for immunochromatography, wherein the carrier for absorbing a liquid sample retains the ability to adsorb acidic substances.

5. A diagnostic kit comprising the immunochromatographic test strip according to claim 1, 2, 3, or 4.

6. The diagnostic kit according to claim 5, further comprising a stool suspension buffer consisting of PBS and Z or sodium borate buffer.