JP2001289852A - Immunochromatographic apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunochromatographic apparatus by which a substance, to be tested, in a sample can be detected or quantitatively determined precisely and quickly by a method wherein a background coloring operation, a nonspecific color development and a prozone phenomenon which cause false positivity and false negativity in an analysis are suppressed. SOLUTION: The immunochromatographic apparatus has a structure in which the substance to be tested can be moved inside a porous carrier by a mobile phase which contains basic amino acid or amino sugar.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an immunochromatographic analysis method and analyzer, and a stabilizer for the analyzer.

[0002]

2. Description of the Related Art As an immunological analysis method for immunologically detecting or quantifying a test substance in a sample utilizing the specificity of an antigen-antibody reaction (immune reaction), a radioimmunoassay, an enzyme immunoassay, Various types such as a latex agglutination method have been put to practical use. Recently, immunochromatography methods with high detection sensitivity and simple operation have been actively performed. The basic measurement format of the immunochromatography method is described in, for example, Proposals for Clinical Laboratory Methods (31st edition, p. 61, 1997,
Kinbara Publishing).

[0003] The structure of the simplest immunochromatography apparatus used for immunochromatography and the analysis method using this apparatus will be described below. The immunochromatography apparatus includes a sample addition site, a labeling site containing a labeling substance labeled with a capture substance (antibody or antigen) corresponding to the antigen or antibody as the test substance in the sample, the antigen or the test substance in the sample, It consists of a porous carrier on which a capture substance (antibody or antigen) corresponding to the antibody is immobilized (having a function as a development site and a detection site), and an excess sample absorption site, and the liquid containing the sample is added to the sample addition site. When added, the liquid is used as the mobile phase, the labeling site, the porous carrier,
The structures are interconnected so that the test substance can move by capillary action in the order of the excess sample absorption sites (FIG. 1).

[0004] The analysis method will be described with reference to the detection of human hemoglobin in stool used for screening for colorectal cancer. The stool suspension, which is a sample, is placed at a sample addition site made of a water-absorbing material.
When the drop is added, if the test substance, hemoglobin, is present, it reacts with the first antibody (labeled substance) against hemoglobin labeled with colored particles (colloidal gold or latex particles) impregnated at the labeled site at the labeled site. Produces a colored immune complex. Thereafter, the colored immune complex moves on the porous carrier in accordance with the movement of the liquid, and is captured by the second antibody against hemoglobin immobilized on the detection site. Excess liquid, substances other than the label and the test substance move together with the liquid to the excess sample absorption site and are absorbed. The colored immune complex captured by the second antibody provided as a determination site can be visually determined to be positive. As described above, the immunochromatography method has been widely used because it has high sensitivity, the measurement method is simple, and does not require a large-scale apparatus.

[0005]

In the above-mentioned immunochromatography apparatus, coloring (background coloring) of portions other than the detection site occurs, and in some cases, non-specific coloring of the detection site occurs when no test substance is present. Problem. Background coloring and nonspecific coloring not only lower the S / N ratio at the time of detection, but can also cause false positives and false negatives. The main cause of the background coloration is thought to be the hydrophobic binding between the label and the colored immune complex and the porous carrier. The main cause of non-specific color development is considered to be the electrostatic interaction between the label and the immobilized antibody (antigen).

Another problem in the immunochromatography apparatus is that when a large excess of a test substance is used as a sample, a false negative (prozone) phenomenon occurs in which the amount of the test substance is apparently small. It is considered that this is because the excess test substance that failed to react with the label reacts with the detection site and the colored immune complex cannot react with the detection site.

[0007] In view of the above, the present invention provides a method of false-positive analysis,
An object of the present invention is to provide an immunochromatographic analysis method and apparatus capable of suppressing background coloring, non-specific coloring, and prozone phenomenon, which can be a cause of false negatives, and capable of accurately and quickly detecting or quantifying a test substance in a sample. And Furthermore, it is an object of the present invention to provide a stabilizer which is added to a solution containing a test substance at the time of analysis in immunochromatographic analysis to suppress background coloring, nonspecific coloring, and prozone phenomenon which may cause false positive and false negative. It is.

[0008]

The present invention is an immunochromatographic analysis method characterized by developing a test substance with a mobile phase containing a basic amino acid or an amino sugar.
Furthermore, the present invention is an immunochromatographic analysis method characterized by adding a basic amino acid or amino sugar to a solution containing a test substance and analyzing the solution. The present invention also includes an immunochromatography analyzer characterized by containing a basic amino acid or amino sugar and a stabilizer for an immunochromatography analyzer containing a basic amino acid or amino sugar as an active ingredient. Hereinafter, the present invention will be described in detail.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The structure of the immunochromatography apparatus of the present invention is as follows: a test substance comprises a mobile phase containing a basic amino acid or an amino sugar (hereinafter referred to as a base).
The structure is not particularly limited as long as the structure can move in the porous carrier. A structure in which a carrier containing a base is provided at the sample addition site and / or the labeling site is preferable. In the case of this structure, a mobile phase in which a base is eluted is formed by the sample solution introduced into the carrier, and the reaction starts in a state where the test substance is dissolved in the mobile phase containing the base, and can move in the porous carrier. Further, a kit-type structure in which a sample preparation container containing a liquid containing a base (a solution for dissolving a soluble sample or a solution for suspending an insoluble sample) is attached to a conventional immunochromatography apparatus is preferable. In the case of the kit type structure, the test substance is added to the sample preparation container, and a liquid appropriately diluted or suspended with a base-containing liquid is added to the sample addition site, so that the test substance is prepared in advance in the mobile phase containing the base. The reaction starts in a state of dissolution in the porous carrier and can move in the porous carrier.

The base used in the present invention is not particularly limited, but is preferably a basic amino acid such as arginine, lysine, histidine, ornithine or citrulline, or an amino sugar such as glucosamine. Basic amino acids are L-form,
Both the DL and D optical isomers are included in the present invention. The base is included in the present invention even if it is an acid addition salt.
The bases can be used alone or in combination.

The concentration of the base used in the present invention varies depending on the type of the sample, the type of the test substance, etc., but the final concentration is preferably in the range of about 0.5 to 20%.
More preferably, it is in the range of about 5-10%.

Conventionally, basic substances to be contained in the mobile phase of an immunochromatography apparatus are disclosed in JP-A-11-1.
No. 53601 discloses a tetramethylammonium salt. However, in this publication, it is described that a positively charged tetramethylammonium salt was able to cancel the negative charge of the dye of the dye-labeled antibody and remove non-specific coloration. No effect of the frequently used colored particles (colloidal gold or latex particles) on the label is shown. Therefore, it cannot be expected that the immunochromatography apparatus of the present invention can suppress background coloring, nonspecific coloring, and prozone phenomenon in immunological analysis using colored particle labels having no negative charge. Was.

[0013] The immunochromatography apparatus of the present invention can be produced by the same method as the conventional method using the raw materials used in the production of the conventional immunochromatography apparatus. That is, according to a conventional method, a sample dropping site, a labeling site, a labeling site, a porous carrier, and a carrier serving as an excess sample absorption site are produced, and the carriers can be connected to each other. In order to make a carrier used for a sample addition site or a labeling site contain a base, the carrier can be produced by impregnating the carrier with a solution in which the base is dissolved and drying it by air drying or the like.

The method of using the immunochromatography apparatus of the present invention is exactly the same as the method of using a conventional immunochromatography apparatus. That is, in the case of a structure in which a sample addition site or a labeling site containing a base is provided, the reaction is started by adding an appropriate amount of the sample directly to the sample addition site. In the case of a kit-type structure with a sample preparation container, add an appropriate amount of sample to the container to prepare a sample diluent or sample suspension.Add this sample diluent or sample suspension to the sample addition site in an appropriate amount To start the reaction.
Regardless of the structure, the test substance is detected or quantified by visually observing the determination site or measuring with a chromaticity measuring device after an appropriate time has elapsed since the start of the reaction.

[0015]

The present invention will be described in more detail with reference to examples and test examples below, but the present invention is not limited to these examples.

Example 1 Preparation of an immunochromatography apparatus for detecting human hemoglobin (Hb) An immunochromatography apparatus for detecting Hb was prepared as a typical example of the present invention. 1) Preparation of a porous carrier for immunochromatography on which an anti-Hb secondary antibody was immobilized A nitrocellulose sheet (BAS-85, manufactured by Schleicher-Schuell) was cut into 5 mm x 30 mm, and 10 mm from its end was cut. Anti-Hb monoclonal antibody solution 0.5 mg /
mL (manufactured by Nippon Biotest) was applied linearly using an airbrush (manufactured by Olympos) to prepare an immobilization line (detection site) of the anti-human Hb antibody. After drying at room temperature for 2 hours, 1% skim milk (Difco), 0.1% Tween
After blocking by immersion in 37% phosphoric acid saline (PBS) containing 20 at 37 ° C. for 2 hours, the plate was sufficiently dried to prepare a porous carrier for immunochromatography.

2) Anti-H labeled with base-containing red latex particles
b. Preparation of primary antibody holding carrier (labeled site) a. Preparation of red latex particle labeled anti-Hb primary antibody suspension Red latex particle dispersion (PL-Latex, 10%, particle size
450 nm, manufactured by Polymer Laboratories) 300 μL of PBS
1.2 mL was added, and centrifuged at 13,000 rpm for 5 minutes.
1 mL of an anti-Hb monoclonal antibody solution (0.5 mg / mL) (manufactured by Nippon Biotest) was added, mixed well, and reacted at room temperature for 1 hour. To remove the unreacted anti-Hb monoclonal antibody, centrifugation was performed at 13,000 rpm for 5 minutes, and the precipitate was suspended in 1.5 mL of PBS and centrifuged again. PB containing 1% skim milk-0.01% sodium azide
S The suspension was suspended in 1.5 mL and stored refrigerated. b. Preparation of carrier holding anti-Hb primary antibody labeled with red latex particles 5 μm suspension of anti-Hb primary antibody labeled with red latex particles
L and 5 μL of a 10% aqueous solution of various bases were impregnated in a 5 mm × 5 mm Bemliese nonwoven fabric (manufactured by Asahi Kasei Corporation) and air-dried to prepare a carrier containing a base-containing red latex particle-labeled anti-Hb first antibody.

3) Preparation of a test strip type immunochromatography apparatus (comprising a porous carrier) Labeling of red latex particles up to a position 2.5 mm from one end of a porous carrier for immunochromatography on which an anti-Hb secondary antibody is immobilized. The carrier holding the anti-human Hb primary antibody was overlaid.
Further, a liquid sample absorption site carrier (filter paper No. 526, manufactured by Advantech Toyo Co., Ltd.) 5 × 15 mm was stacked on the carrier holding the red latex particle-labeled anti-human Hb primary antibody to a position 5 mm from the end. In addition, the carrier for the excess sample absorption site 5 mm × 5 mm from the other end of the immunochromatographic support to the position
20 mm (filter paper No. 526) was stacked. Finally, a tape was stuck on the back side, and the whole was fixed to prepare a test piece type immunochromatography apparatus. As a result of the test using the standard Hb solution, the minimum measurement sensitivity of Hb of this apparatus was 50 ng / mL.

Comparative Example 1 The same procedure as in Example 1 was carried out except that a carrier containing a base-free red latex particle-labeled anti-Hb primary antibody was prepared by using purified water in place of the various bases in 2) b of Example 1. A test piece type immunochromatography apparatus was prepared.

Test Example 1 Inhibitory effect of base on background coloration Fecal suspension in which Hb was dissolved at 50 ng / mL at the sample addition site of the test strip type immunochromatography apparatus prepared in Example 1 and Comparative Example 1. Buffer (PBS + 2 mM)
50 μL of ethylenediamine-N, N, N ′, N′-tetraacetic acid, 0.1% sodium azide, 0.01% Tween 20) was added dropwise, and 10 minutes later, the coloration of the detection site and the background color around the detection site Was visually observed. As a result, as shown in Table 1, no background coloring was observed in any of the test piece immunochromatography apparatuses prepared using the base-containing labeled sites.
On the other hand, in the device of Comparative Example 1, background coloring was observed.

[0021]

[Table 1]

Test Example 2 Non-Specific Coloring Inhibition Effect of Base The Hb-negative stool sample was added to the sample addition site of the test strip type immunochromatography apparatus prepared in Example 1 and Comparative Example 1 so that the Hb-negative stool sample became 10 mg / mL. 50 μL of the suspension suspended in the turbidity buffer was dropped, and 10 minutes later, the presence or absence of nonspecific coloring at the detection site was visually observed. As a result of the test using 10 stool samples, as shown in Table 2, no non-specific coloring was observed in the test piece type immunochromatography apparatus prepared using the base-containing labeled sites. On the other hand, in the device of Comparative Example 1, non-specific coloring was observed at 10%.

[0023]

[Table 2]

Example 2 Preparation of a kit-type immunochromatography apparatus In the same manner as in Comparative Example 1, a test piece-type immunochromatography apparatus was prepared using a base-free labeling site.
A sample preparation solution was prepared by adding various bases or various amino acids to the stool suspension buffer. The sample preparation liquid was dispensed into plastic test tubes with lids in an amount of 1 mL each to prepare a sample preparation container. A kit type immunochromatography apparatus was prepared by combining the test piece type immunochromatography apparatus and the sample preparation container.

Test Example 3 Background Suppressing Effect of Base A sample solution was prepared by dissolving Hb at 50 ng / mL in each sample preparation solution of the kit-type immunochromatography apparatus prepared in Example 2. 50 μL of each sample solution was dropped at the sample addition site, and after 10 minutes, the color of the detection site and the presence or absence of background coloring around the detection site were visually observed. As a result, as shown in Table 3, no background coloring was observed in any of the sample solutions prepared using the base-containing sample preparation solutions. On the other hand, the background coloring was observed in all of the sample solutions prepared using the base-free sample preparation solutions, and the background coloring was particularly remarkable in the glutamic acid-containing sample preparation solutions. Furthermore, it was found that the sample preparation containing glutamic acid or glycine had a fatal defect that no coloration was observed at the detection site.

[0026]

[Table 3]

Test Example 4 Nonspecific Color Inhibitory Effect of Base A Hb-negative stool sample was added to each sample preparation of the kit-type immunochromatography apparatus prepared in Example 2 so as to be 10 mg / mL and suspended. 50 μL is dropped and 10
After a minute, the presence or absence of nonspecific coloring at the detection site was visually observed.
As a result of a test using 10 stool samples, as shown in Table 4, no non-specific coloring was observed in any of the sample solutions prepared using the base-containing sample preparation solutions. On the other hand, non-specific color development was observed in any of the sample solutions prepared using the base-free sample preparation solution, and particularly, the sample preparation solution containing ammonium chloride had a fatal defect of showing 100% non-specific color development. understood.

[0028]

[Table 4]

Test Example 5 Background effect of base on base A sample solution was prepared by dissolving Hb at 50 ng / mL in each sample preparation solution of the kit-type immunochromatography apparatus prepared in Example 2. 50 μL of each sample solution was dropped at the sample addition site, and after 10 minutes, the color of the detection site and the presence or absence of background coloring around the detection site were visually observed. As a result, as shown in Table 5, in the sample solution prepared using the sample preparation solution containing 0.5% or more of the base,
In any case, no background coloring was observed.

[0030]

[Table 5]

Test Example 6 Inhibitory Effect of Base on Prozone Phenomenon Hb was added to the sample-prepared solution containing L-arginine hydrochloride (5%) and the base-free sample prepared by the kit-type immunochromatography apparatus prepared in Example 2. , 100, 250,
A sample solution was prepared so as to be dissolved at 1000 and 2500 ng / mL. 50 μL of each sample solution is dropped onto the sample addition site, and after 10 minutes, the red line coloration degree of the detection site is measured by an immunoscanner (Microtech scan maker).
x6 and an immunochromatography chromaticity digitizing device comprising image analysis software manufactured by Sanko Junyaku Co., Ltd.) and expressed as pixel values. As a result, as shown in Table 6, in the sample solution prepared using the arginine-containing sample preparation solution, H
b Up to 2500 ng / mL, the color degree increased in a concentration-dependent manner. On the other hand, in the base-free sample preparation solution, Hb1000n
In the high concentration range of g / mL or more, a decrease in coloration (prozone phenomenon) was observed.

[0032]

[Table 6]

[0033]

According to the immunochromatography apparatus of the present invention, in the field utilizing immunoassay,
Background coloring, non-specific coloring, and prozone phenomena, which can cause false positives and false negatives during immunochromatographic analysis, can be suppressed, and a test substance in a sample can be accurately and quickly detected or quantified.

[0034]

[Brief description of the drawings]

FIG. 1 is an explanatory view (top view and cross-sectional view) of the structure of a general immunochromatography apparatus.

[Explanation of symbols]

 a. Sample addition site b. Labeled site (labeled capture substance containing site) c. Detection site (captured substance immobilized site) d. Porous carrier for immunochromatography e. Excess sample absorption site

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Haruhisa Hirata 1-3-5 Nihonbashi Muromachi, Chuo-ku, Tokyo Wakamoto Pharmaceutical Co., Ltd.

Claims (12)

[Claims] 1. An immunochromatographic analysis method comprising developing a test substance in a mobile phase containing a basic amino acid or an amino sugar. 2. An immunochromatographic analysis method comprising adding a basic amino acid or amino sugar to a solution containing a test substance and analyzing the solution. 3. The method according to claim 1, wherein the basic amino acid is arginine, lysine, histidine, ornithine or citrulline. 4. The immunochromatographic analysis method according to claim 1, wherein the amino sugar is glucosamine. 5. The immunochromatographic analysis method according to claim 1, further comprising a label labeled with colored particles having no negative charge. 6. An immunochromatography analyzer comprising a basic amino acid or an amino sugar. 7. The immunochromatographic analyzer according to claim 6, wherein the immunochromatographic analyzer includes a label labeled with a colored particle having no negative charge. 8. The immunochromatographic analyzer according to claim 7, wherein the colored particles are colloidal gold or latex particles. 9. The immunochromatographic analyzer according to claim 6, wherein the site containing a basic amino acid or amino sugar is a labeling site. 10. The method according to claim 10, wherein the basic amino acid is arginine, lysine,
10. Histidine, ornithine or citrulline.
The immunochromatographic analyzer according to the above. 11. The method according to claim 1, wherein the amino sugar is glucosamine.
0. The immunochromatographic analyzer according to 0. 12. A stabilizer for an immunochromatography analyzer comprising a basic amino acid or amino sugar as an active ingredient.