WO2003044534A1

WO2003044534A1 - Immunochromatographic test strip for measuring analysis subject in specimen in trace amount

Info

Publication number: WO2003044534A1
Application number: PCT/JP2002/011973
Authority: WO
Inventors: Hidekazu Matsuoka; Fujiko Kanezawa; Toshio Watanabe
Original assignee: Wakunaga Pharmaceutical Co.,Ltd.
Priority date: 2001-11-22
Filing date: 2002-11-15
Publication date: 2003-05-30
Also published as: AU2002343815A1; JPWO2003044534A1

Abstract

It is intended to provide an immunochromatographic test strip comprising a porous support wherein the porous support has a detection area (1) having an immobilized capturing reagent for detecting the subject to be analyzed in the downstream and a developing solution receiver area (2) for receiving a developing reagent in the upstream, and, between these areas, a reagent area (3) containing a labeling reagent having an affinity for the subject to be analyzed and a separation area (4) for separating substances inhibiting the measurement from the specimen.

Description

Description Immunochromatography test strip for measuring analytes in trace samples Field of the invention

The present invention relates to the field of immunodiagnosis, particularly to a simple and accurate test strip for a small amount of a sample using immunochromatography. Background art

In the case of a test strip in the form of a strip using immunochromatography, analysis results can be obtained simply by applying a sample. Widely used in general households. In particular, a device in which a strip using immunochromatography is cascaded with a moisture-impermeable solid material, such as a pregnancy test drug, is the mainstream in the market. In such test strips intended for use in ordinary households, there is no need to consider the limitation of the sample volume because urine is used for the sample.

However, in the field of clinical testing, urine is rarely used as a sample, and most samples are whole blood, serum, plasma, cerebrospinal fluid, tissue extract, and the like. When these are used as samples, there is essentially no difference from a test strip that uses urine as a sample, but when whole blood is used as a sample, there are some concerns. For example, the presence of red blood cells can scatter and absorb light, which can interfere with the assay method for measuring reflected or transmitted light.

In addition, whole blood samples have a deep red color that substantially interferes with dye or other visible tests, thus inhibiting red blood cells, hemoglobin, and other measurements. Harmful substances must be separated from plasma or serum by incorporating a blood cell separation membrane or the like into the test strip before the blood sample is analyzed for a particular analyte. In addition, even when other samples are used, they often contain measurement inhibitory substances, so it is necessary to separate these substances in order to perform accurate measurement.

There are many prior literatures on blood cell separation membranes having a function of separating plasma from a whole blood sample. Patent No. 2,940,990 describes a test strip such as a test strip for a blood cell separation membrane using a hydrophilic sintered porous matrix incorporating hemagglutinin. Discloses that it can be used as a component of In addition, Japanese Patent Publication No. 7-85083 discloses a device characterized by containing glass fibers coated with polyvinyl alcohol or polyvinyl alcohol / polyvinyl acetate. JP-T-7-504747 describes a method of applying a matrix containing an antibody against erythrocytes to Depis.

These methods are excellent in their ability to separate blood cells from whole blood. However, the amount of whole blood is a problem in test strips using immunochromatography. When incorporated into a test strip using immunochromatography, plasma components of at least about 50 μL and complete blood volume of about 100 μL are required to complete the reaction completely. This is unsuitable for home use because blood samples are required, and blood collection using a blood collection tube and syringe for injection is required to secure a certain amount of blood.

There is also a method using a developing solution to reduce the amount of the sample such as whole blood to be used.However, since the separation step of the measurement inhibitor such as blood cells and the developing step are performed continuously on the same strip, However, the blood cells trapped by the separation membrane may flow out, which may affect the determination. There are also ways to reduce the size of a separate test strip. However, downsizing of this test strip is not always appropriate in terms of operability and the ease of observing the results.

As a method for improving these methods, Japanese Patent Application Laid-Open No. 2000-321278 discloses a method in which a whole blood sample is applied to a non-porous liquid receiving member, and a detection unit detects an analyte while sequentially absorbing the reagent. The method is presented. This method has excellent blood cell separation ability because the whole blood sample is absorbed from the end of the test strip, and it is considered that the reaction is completed with a smaller amount of the whole blood sample.

However, this method also requires a certain amount of plasma sample, depending on the other components of the test strip, the size of the binding pad and the cross-linking pad, etc. There was a limit to reducing the amount of whole blood.

For this reason, there has been a demand for a test strip that can easily and accurately measure even a small amount of sample. Disclosure of the invention

An object of the present invention is to provide a simple and accurate test strip using immunochromatography, which can be measured with a small amount of a sample.

The present inventors have conducted intensive studies in order to solve the above-described problems, and as a result, have found that the immunoclotting chromatography test strip of the present invention can accurately measure a small amount of a sample. Accordingly, the present invention provides an immunocytomatography test strip having a porous carrier for qualitatively or quantitatively determining an analyte in a sample. The detection area (1) where the capture reagent for detecting the analyte is fixed downstream of the carrier (1), A reagent area (2) containing a developing solution receiving area (2) for receiving a drug and containing a labeled reagent having an affinity for the analyte between the two areas, and a substance that inhibits measurement from the sample are separated. Another object of the present invention is to provide an immunochromatography test strip comprising a separation region (4). BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows one embodiment of the immunochromatography test strip of the present invention. Embodiment of the Invention

FIG. 1 shows an example of a typical embodiment of the present invention. (1) is a detection area in which a capture reagent for detecting an analyte is fixed, (2) is a developing liquid receiving area for receiving the developed reagent, and (3) is an affinity with the analyte. The reagent region containing the labeling reagent, and (4) shows the separation region for separating the substance that inhibits the measurement from the sample, respectively, and the positional relationship between (3) and (4) may be reversed. Hereinafter, description will be made in accordance with this, but the present invention is not limited to this mode.

In the immunochromatography test strip of the present invention, the developing solution moves by capillary action from the developing solution receiving region (2) made of a fibrous material, and the reagent region (4) located in the downstream region thereof. When the developing solution reaches the developing solution, the labeling reagent having an affinity for the analyte is dissolved and further developed together with the developing solution. On the downstream side, there is a contact point (5) with a separation region (4) for separating a substance that inhibits measurement from the sample, and contains a sample purified in the separation region and a labeled reagent developed from the upstream. The developing solution merges here and reaches the detection area (1) where the capture reagent is immobilized while the analyte and the labeling reagent form a complex. If the analyte is present in the sample, the complex of the analyte and the labeling reagent is captured by the supplementary reagent and observed as an arbitrary signal.

Each of these regions may be on the same porous matrix, or may be on a different matrix, even if the reagents and samples are in contact so that they can move. good.

The porous carrier used for the test strip is not particularly limited as long as a developing solution, a reagent, an object to be analyzed, and the like can flow from upstream to downstream by capillary action. Examples include glass fiber, celluloses (filter paper, nitrocellulose, etc.), porous plastics such as polyethylene, polypropylene, and the like, and nylon. More preferred are nitrocellulose, nylon, cellulose and glass fiber.

As a means for separating a substance that inhibits measurement from a sample, an arbitrary filter, a resin, a hemagglutinating agent, or the like can be used, although it differs depending on the sample. For example, when the specimen is blood, a blood cell separation membrane such as a glass fiber membrane or a hemagglutinating agent such as antibody lectin or sugar alcohol can be used.

The separation region (4) has a structure branched from the test slip, and portions other than the branch point (5) are prevented from directly contacting the slip. As the method, for example, using a moisture-impermeable material such as a film which does not affect the measurement, a portion other than the branch point of the separation region or a portion corresponding to the separation region of the strip ( 6) can be coated.

The labeling reagent used in the present invention refers to a reagent that has an affinity for the analyte to be labeled with an arbitrary labeling reagent, and for example, labeled antibodies, antigens, haptens, and the like can be used. The labeling method includes direct labeling and indirect labeling (enzymes such as alkaline phosphatase and the like, and biotin labeling). Direct labeling that does not require a substrate is preferred. Direct labels include, for example, metals such as platinum, gold, silver and copper, silver iodide, silver bromide, silver hydroxide, iron oxide, iron hydroxide or iron oxide hydrate, aluminum hydroxide or water. Aluminum oxide, chromium hydroxide or hydrated chromium hydroxide, copper sulfate, mercury sulfate, barium sulfate, and the like, selected from the group consisting of metal compounds such as titanium dioxide, and preferably gold colloid. It can be prepared using particles.

Examples of the capture reagent immobilized on the detection region used in the present invention include an antibody, an antigen, a hapten, an avidin, a receptor, etc., which specifically bind to an analyte.

Examples of the sample that can be used in the present invention include blood, urine, saliva, mucus, and the like. Blood is particularly preferable, and components to be measured include, for example, proteins, viral antigens, and tumor markers. And inflammation markers, hormones or drugs.

Examples of the immunologically specific binding assay used in the present invention include a competitive binding assay and a sandwich assay (including a sandwich assay using the binding of a biotinylated antibody and avidin). Although it is applicable, the sandwich method is particularly preferable.

Further, since the present invention does not require any special skills, it can be widely applied from a clinical site to home use as a simple and accurate test strip. Example

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. Example 1

Preparation of film for detection area

The anti-HCG antibody was applied to the imminodyne membrane ABC in a line using a dispenser, dried, blocked with a casein solution, washed with a PBS solution, and then dried at 25 ° C. Preparation of gold colloid labeling reagent finally cut into 5 mm x 25 mm strip

The gold colloid solution was labeled with anti-HCG antibody using Bldg. Gold-sol G40 according to the protocol. Gold colloid labeling reagent was applied to the conjugate pad with a dispenser and lyophilized. A 5 mm x 5 mm knot was used for the final 0 shank.

Preparation of test strip

The cellulose membrane was cut to a width of 5 mm × 10 mm to provide a mixed region of the plasma component and the labeling reagent. The conduit gate pad was cut into 5mmxl0nnn, which was used as a developer receiving part. An immunodyne membrane with a detection area is pasted on an adhesive-coated polyethylene film, and a cellulose membrane, a pad containing a gold colloid labeling reagent, and a developer receiving section are sequentially attached to one end of the membrane. And one strip. In addition, one end of a blood cell separation membrane, Hemasep (5mmxl0mm), is brought into contact with the contact part of the cellulose membrane and the pad containing the gold colloid labeling reagent, and this contact part is applied to an adhesive-coated film. More bonded. A part of the surface of the developing solution receiving part was covered with an impermeable film coated with an adhesive so that a part other than the contact part of the blood cell separation membrane did not contact the developing reagent receiving area.

HCG was added to the whole blood sample so that it became 100 IU / L, and 10 L was added to the blood cell separation area of the test strip prepared as described above. After 5 minutes, add the developing solution ΙΟΟμί to the developing solution receptor, and lower it so that it reaches the detection area. After developing for 15 minutes in the flow direction, the colored line in the detection area was measured with 520 nm reflected light. On the other hand, a general test strip using a blood cell separation membrane in the developing solution receiving part was prepared and used as a control. The control samples were measured by adding 10 μL of whole blood sample, adding 10 / XL of whole blood sample, and then adding 100 developing solution.

Table 1 shows a comparison between the measurement results of the test strip of the present invention and the measurement results of the control product.

table 1

Based on the results shown in Table 1, when analyzing a very small amount of 10 μL of whole blood sample by immunochromatographic test strip, the volume of the liquid can be reduced in a typical test strip. It is evident that the lack of deployment is not enough to make the analysis impossible. Also, if the developing solution is added to the same site after dropping the whole blood sample, the amount of the solution is sufficient, but the blood cells are not sufficiently separated and the blood cells leak to the detection area. It was shown that detection became impossible.

It has been proved that by clearly distinguishing the dropping site of the whole blood sample and the developing solution receiving site as in the present invention, it is possible to measure an analyte using a small amount of a whole blood sample. Industrial applicability

The immunochromatographic test strip of the present invention is simple and convenient. Since accurate measurement is possible, it can be widely applied from clinical sites to home use.

Claims

The scope of the claims

1. In an immunochromatography test strip with a porous carrier for qualitatively or quantitatively determining an analyte in a sample, the porous carrier captures the analyte downstream of the test. The detection area (1) where the reagent is fixed, the developing solution receiving area (2) that receives the developing reagent upstream, and a labeling reagent that has an affinity for the analyte between both areas An immunochromatography test strip comprising a reagent region (3) and a separation region (4) for separating a substance that inhibits measurement from a sample.

2. The separation region (4) for separating the substance that inhibits the measurement from the sample has a structure branched from the strip, and is directly connected to the strip at a portion other than the branch point. The test strip according to claim 1, wherein the test strip does not contact.

3. The test strip according to claim 1, wherein the capture reagent has a property of specifically binding to an analyte.

4. The immunochromatography test strip according to any one of claims 1 to 3, wherein the porous carrier includes any of nitrocellulose, nylon, cellulose, and glass fiber.

5. The immunochromatographic phytotest strip according to any one of claims 1 to 4, wherein the specimen is blood.