JPH04254761A - Instrument for immunological measurement - Google Patents

Abstract

PURPOSE:To obtain an instrument for immunological measurement which can make measurement in a short time and give accurate results. CONSTITUTION:This instrument for immunological measurement is constituted of a supporting body 2 having a hygroscopic property, holding member stuck to the rear surface of the body 2, and analyte absorbing material 7 mounted on the top of the body 2. The bottom section of the supporting body 2 constitutes an analyte supplying section 3 which is dipped in an analyte and a detecting section 4 is provided at a prescribed distance from the section 3. In the detecting section 4, the first material which can be specifically linked with a material to be measured contained in the analyte is fixed. The water permeability of this supporting body 2 under AMMI Standard is 0.04-20l/ min.cm<2> at 30OmmHG differential pressure.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay device used in an immunoassay method for detecting a specific substance in a sample using an immune reaction.

0002

[Prior Art] Immunoassay is a method for detecting or quantifying the presence of specific substances (antigens, antibodies, etc.) in specimens such as blood or urine, and is used for determining pregnancy, diagnosing various diseases, etc. It is widely used in

[0003] One of such immunoassay methods is affinity chromatography. This affinity chromatography technology is known, for example, from Japanese Patent Application Laid-open No. 6
No. 0-133368, No. 61-142463, No. 62-169054, No. 62-169055
No. 63-25551, No. 63-2555
It is described in Publication No. 3, Publication No. 64-32169, etc., and the outline thereof is as follows.

For example, when detecting chorionic gonadotropin (hCG) in female urine by the sandwich method, hCG specifically binds to the recognition site α of hCG at a predetermined part of a water-absorbing band-shaped support. Anti-hCG antibody A is immobilized, and a urine sample to which labeled anti-hCG antibody B, which specifically binds to other recognition site β of hCG, is added to urine is supplied to one end of the support.

[0005] When hCG is present in urine, this hCG
A complex between CG and labeled anti-hCG antibody B is formed. The urine sample containing this complex moves on the support by capillary action and comes into contact with anti-hCG antibody A to form an anti-hCG antibody A-hCG-anti-hCG antibody B-label complex. For example, if the label is a colored particulate,
The support is colored and the color density is examined visually or optically to detect the presence of hCG in the urine and determine pregnancy.

[0006] In the absence of hCG in the urine, no complex is formed and therefore no coloration is produced by the label.

By the way, the support in such affinity chromatography is composed of a material having a large number of pores, and includes filter paper, membrane filter, etc.
Thin-layer plates were used.

However, these materials cannot obtain sufficient capillary flow due to discontinuous pores or inadequate pore volume, and the sample (urine sample) is The disadvantage is that the moving speed is slow and the measurement time is long.

In particular, when colored fine particles are used as a label, the fine particles clog the pores of the support during the movement of the sample, further slowing down the movement of the sample and preventing accurate measurement results from being obtained. There are drawbacks.

[0010] Also, due to the characteristics of the material of the support, anti-hC
There is also a drawback that the specimen cannot be uniformly supplied to the part where G antibody A is immobilized, causing color unevenness in the colored part, resulting in variations in measurement results.

[0011]

SUMMARY OF THE INVENTION An object of the present invention is to provide an immunoassay instrument that takes a short measurement time and provides accurate measurement results.

0012

[Means for Solving the Problems] Such objects are achieved by the present invention as described in (1) to (8) below.

(1) A water-absorbing support having a sample supply section and a detection section provided at a predetermined distance from the sample supply section; an immunoassay device supporting a first substance capable of binding to
20 l/min·cm2. An immunoassay device characterized by:

(2) A label-carrying part is provided between the sample supply part and the detection part of the support, and a recognition site different from the recognition site that binds to the first substance is provided in the label-carrying part. The immunoassay device according to (1) above, which carries a labeled substance in which a second substance capable of specifically binding to the specific substance is labeled.

(3) A label-carrying part is provided between the sample supply part and the detection part of the support, and a label-carrying part is provided with a label that competes with the specific substance to specifically target the first substance. The immunoassay device according to (1) above, which carries a label in which a third substance capable of binding is labeled.

(4) The immunolabel according to (2) or (3) above, wherein the label is formed by immobilizing the second substance or the third substance on fine particles having a different color from the support. measurement tool.

(5) The immunoassay device according to any one of (1) to (4) above, wherein the support is a woven fabric, a knitted fabric, or a nonwoven fabric of fibers.

(6) The support body is a strip-shaped chromatographic medium whose one end is a sample supply section.
The immunoassay device according to any one of (5).

(7) The immunoassay device according to (6) above, which has a sample absorbing portion at the other end of the support for absorbing and retaining excess sample.

(8) The immunoassay device according to any one of (1) to (7) above, which has a holding member that holds the support.

[0021]

[Function] The immunoassay device of the present invention has a water permeability of the support according to the AMMI standard of 0.0 when the differential pressure is 300 mmHg.
04 to 20 l/min・cm2, sufficient capillary flow is obtained when moving the sample, the movement speed is fast, and the support is not clogged with labels, etc., thus shortening the measurement time. and accurate measurement results.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The immunoassay device of the present invention will be described in detail below based on preferred embodiments shown in the accompanying drawings.

FIG. 1 is a front view showing a configuration example of the immunoassay instrument of the present invention, and FIG. 2 is a sectional view taken along the line A--A in FIG. As shown in these figures, the immunoassay device 1A includes a support 2 which is a strip-shaped chromatographic medium having water absorption properties.
has. This support 2 will be explained in detail later.

A plate-shaped holding member 6 for holding the support 2 is installed on the back surface of the support 2 (see FIG. 2). This holding member 6 is, for example, a plate-shaped member with a thickness of approximately 0.05 to 1 mm, and the support body 2 and the holding member 6 are fixed by, for example, adhesive bonding or fusion (such as ultrasonic fusion). has been done.

The constituent material of the holding member 6 is preferably one that is inert to the specimen, such as polystyrene, polyethylene, polypropylene, polyvinyl chloride, P.
Examples include resins such as polyesters such as ET and PBT, various glasses, and ceramics such as alumina and silica.

Further, the holding member is not limited to a plate-like member as shown in the drawings, but may also be a tubular member, a frame-like member, a member having a curved surface such as a spherical or cylindrical shape, a case, a holder, or the like.

Note that if the support body 2 itself has sufficient rigidity, it is not necessary to provide such a holding member.

A specimen supply section 3 for supplying a specimen is provided at the lower end of the support 2 in the figure, and the specimen is supplied by, for example, dipping this specimen supply section 3 into a specimen (liquid).

A detection section 4 for detecting a specific substance (hereinafter referred to as a target substance) in a sample to be measured is provided at a position of the support 2 at a predetermined distance from the sample supply section 3. The distance from the lower end of the support 2 in the figure to the detection part 4 is 5 to 1
It is preferably about 0.00 mm, particularly about 20 to 70 mm.

[0030] The detection section 4 carries a first substance that can specifically bind to the target substance. For example, when the target substance is an antigen or a hapten, the first substance is an antibody. This antibody includes immunoglobulin (IgG,
IgM, IgA, IgD, IgE) are preferred. This antibody may be a monoclonal antibody, a polyclonal antibody, or a mixture thereof.

Conversely, when the target substance is an antibody, the first substance is an antigen or a hapten.

The contour shape (shape of the detection part 4) of the region where the first substance is immobilized (hereinafter referred to as the first substance immobilization region) is not particularly limited, and may be band-shaped, linear, rectangular, or triangular. , an oval, a circle, or a specific character shape.

The area of the first substance fixing region is not particularly limited, but is usually about 0.001 to 1 cm2, particularly about 0.001 to 1 cm2.
It is preferable to set it to about 0.01 to 0.2 cm2.

The amount of the first substance supported on the detection section 4 is not particularly limited, but it is usually about 0.05 to 200 μg, particularly preferably about 0.5 to 10 μg.

The method of supporting the first substance on the detection section 4 is, for example, impregnating the detection section 4 with a liquid containing the first substance and drying it (air-drying, freeze-drying, etc.), or loading the detection section 4 with a liquid containing the first substance. This can be done by spraying directly on the surface and drying it.

The support 2 has a water permeability (hereinafter simply referred to as water permeability) according to the AMMI standard at a differential pressure of 300 mm.
When Hg, 0.04 to 20 l/min·cm2, preferably 0.1 to 20 l/min·cm2
is used. Water permeability at 300 mmHg differential pressure is 0.04 l/m
If it is less than 20 l/min·cm2, sufficient capillary flow will not be obtained and clogging will occur, resulting in a slow measurement speed. This is because the adhesion stability of substances and labels such as colloidal particles described below is poor, and color unevenness is likely to occur in the detection section 4.

[0037] Such water permeability is achieved by the material of the support 2 itself, or by subjecting the support 2 to a hydrophilic treatment (for example, by applying a predetermined water permeability adjusting agent). . In this case, the water permeability regulator is
For example, sodium dodecyl sulfate, sodium deoxycholate, surfactants such as Triton etc.

The material for the support 2 may be fibrous or non-fibrous, such as a porous material such as a foam or a honeycomb structure, or a thin layer of fine granules. It is preferably fibrous, especially woven, knitted or non-woven fabrics.

[0039] When the support 2 is a woven fabric, the weaving method thereof may be any one such as plain weave, twill weave, satin weave, etc.

When the support 2 is knitted, the knitting method may be any method such as weft knitting (flat knitting), warp knitting (tricot knitting), circular knitting, flat knitting, stockinette knitting, etc.

[0041] When the support 2 is a woven or knitted fabric, the weaving method or knitting method is preferably regular, and when the support 2 is a nonwoven fabric, the fiber arrangement density (porosity) is uniform. Preferably. This allows the specimen to move uniformly, prevents color unevenness in the detection section 4, and improves measurement accuracy.

The fibers in the fibrous support 2 are as follows:
Chemical fibers such as rayon, cupro, polyester (PET, PBT, etc.), polypropylene, nylon, Tetron, acetate, vinylon, acrylic, natural fibers such as cotton, silk, flax, hemp, ramie, wool, etc., or two of these. A combination of two or more types can be used, and among these, rayon, cupro, polyester, polypropylene, nylon, tetron, or a combination of two or more of these are preferred. When these fibers are used, labels such as colloidal particles can be smoothly moved and separated (released) from the label carrier 5 (described later), improving measurement sensitivity and shortening measurement time. can be achieved.

[0043] Examples of combinations of two or more types of fibers include blends of two or more types of fibers, two types of fibers each used as warp and weft, split yarns that are a combination of two or more types of materials, etc. The ratio is not particularly limited.

[0044] The diameter of the fiber is not particularly limited;
．． It is preferably about 1 to 100 μm, particularly about 2 to 30 μm.

The support 2 is not limited to a one-layer structure as shown in the drawings, but may be a laminate of two or more layers of the same or different structures.

The dimensions of the support 2 are not particularly limited, but the length of the support 2 is preferably about 10 to 150 mm, particularly about 30 to 100 mm, and the width is about 3 to 20 mm.
The thickness is preferably about 5 mm, especially about 5 to 10 mm, and the thickness (
In the case of a laminate, the total thickness) is approximately 0.01 to 1 mm,
In particular, about 0.05 to 0.4 mm is preferable.

The color of the support 2 is preferably an achromatic color, particularly white or a color similar to white, but other colors may be used.

A specimen absorbing material 7 for absorbing and retaining excess specimen is bonded to the upper end of the support 2 in the figure. Examples of the sample absorbing material 7 include filter paper, paper towel,
Examples include those made of absorbent paper, superabsorbent polymers, hydrophilic porous plastics, and the like.

[0049] By installing such a sample absorbing material 7, excess sample will not remain in the detection section 4, and a certain amount of sample can be supplied to the detection section 4 in a short time. , enabling more accurate measurements.

Next, the immunoassay method using the immunoassay device 1A will be described for detecting chorionic gonadotropin (hCG) in urine by a sandwich method and a competitive method, respectively.

A-1) An anti-hCG antibody X that specifically binds to the recognition site α of hCG is immobilized on the detection portion 4 of the sandwich support 2 as a first substance.

On the other hand, the container 10 contains a urine sample (specimen).
8 is included. This urine sample 8 is obtained by adding a labeled anti-hCG antibody Y (hereinafter referred to as labeled antibody Y) that specifically binds to another recognition site β of hCG to urine as a measurement target. Note that this labeled antibody Y is an example of a label described later.

When the specimen supply section 3 at the lower end of the support 2 is immersed in the urine sample 8, the urine sample 8 moves upwards of the support 2 due to capillary action.

[0054] When hCG is present in the urine, this hCG
A complex is formed between CG and the labeled antibody Y, and this complex also moves as the urine sample 8 moves. When the urine sample 8 reaches the detection part 4, this complex contacts and reacts with the anti-hCG antibody X immobilized on the detection part 4, and the complex of anti-hCG antibody X-hCG-labeled antibody Y is formed. formed and fixed.

[0055] When the label is, for example, a colored fine particle, the detection part 4 is colored, and the presence of hCG in urine can be detected or quantified by checking the color density visually or by optical measurement. Ru.

[0056] When hCG is not present in the urine, no complex is formed and therefore no coloring is caused by the label in the detection part 4.

The urine sample 8 that has reached the detection section 4 further rises on the support 4, and the excess urine sample 8 is absorbed and retained by the specimen absorbent material 7.

A-2) Competitive method An anti-hCG antibody X that specifically binds to hCG is immobilized on the detection part 4 of the competitive support 2 as a first substance.

On the other hand, the container 10 contains a urine sample (specimen).
8 is included. This urine sample 8 is prepared by adding a labeled hCG-like substance Z (hereinafter referred to as labeled hCG-like substance Z) that competes with hCG and specifically binds to the anti-hCG antibody X to the urine to be measured. This is what I did. Note that this labeled hCG-like substance Z is an example of a labeled substance that will be described later.

When the specimen supply section 3 at the lower end of the support 2 is immersed in the urine sample 8, the urine sample 8 moves upwards of the support 2 due to capillary action, and along with this, the labeled hCG
The similar substance Z also moves.

[0061] When hCG is present in urine, when the urine sample 8 reaches the detection section 4, hCG and the labeled hCG-like substance Z compete and bind to the anti-hCG antibody X, and the anti-hCG antibody X-hCG and anti-hCG antibody X-labeled hCG-like substance Z are formed at a predetermined ratio and fixed.

[0062] When the label is, for example, colored fine particles, the detection part 4 is colored, and the color density becomes darker as the amount of hCG in the urine is smaller. Therefore, the presence of hCG in urine can be detected or quantified by visually or optically measuring its color density.

[0063] When hCG is not present in the urine, only a complex of anti-hCG antibody be done.

The urine sample 8 that has reached the detection section 4 further rises on the support 4, and the excess urine sample 8 is absorbed and retained by the specimen absorbent material 7.

In such measurements, the water permeability of the support 2 is 0.04 to 20 when the differential pressure is 300 mmHg.
1/min cm2, sufficient capillary flow is obtained during the movement of the urine sample 8, the movement speed is fast, and the support is not clogged with the above-mentioned complexes, labeled substances, etc. , measurement time is shortened and accurate measurement results are obtained.

FIG. 3 is a front view showing another example of the configuration of the immunoassay instrument of the present invention, and FIG. 4 is a sectional view taken along the line BB in FIG. The immunoassay instrument 1B shown in these figures has a label carrier 5 between the sample supply section 3 and the detection section 4, and the other configurations are the same as the immunoassay instrument 1A.

The following markers are supported on the marker carrier 5. This label differs depending on whether the immunoassay device 1B measures by a sandwich method or a competitive method.

[0068] In the case of the sandwich method, the label is a label attached to a second substance that can specifically bind to the target substance at a recognition site different from the recognition site that binds to the first substance. be.

For example, when the target substance is an antigen or a hapten, the second substance is an antibody. This antibody includes immunoglobulin (IgG, IgM, IgA, IgD
, IgE) are preferred. This antibody may be a monoclonal antibody, a polyclonal antibody, or a mixture thereof.

Conversely, when the target substance is an antibody, the second substance is an antigen or a hapten.

On the other hand, when the competitive method is used, the labeled substance is a third substance that can compete with the target substance and specifically bind to the first substance, and is labeled.

For example, when the target substance is an antigen or hapten, the third substance is an antigen or hapten that is similar or similar to the target substance. Furthermore, when the target substance is an antibody, the third substance is an antibody similar to or similar to the target substance.

Examples of the label include fine particles with a color different from that of the support 2 (herein referred to as colored fine particles), enzymes, radioactive substances, fluorescent substances, etc., but it is said that measurements can be easily performed. In this respect, colored fine particles are preferred.

Colloidal particles are preferably used as the colored fine particles. The colloid particles may be either metal particles or non-metal particles.

The colloidal metal particles are not limited to those consisting of pure metals or metal compounds, but may also be those in which a polymer core is coated with a metal or a metal compound. Examples of such metals include platinum, gold, silver, etc., and examples of metal compounds include silver nitrate, silver iodide, silver bromide, silver hydroxide, iron oxide, iron hydroxide, hydrated iron oxide, and hydroxide. Examples include aluminum, hydrated aluminum oxide, chromium hydroxide, hydrated chromium oxide, copper sulfate, mercury sulfate, barium sulfate, titanium dioxide, ruthenium chloride, rhodium chloride, palladium chloride, chloroplatinic acid, and the like. Among these, particularly preferred is colloidal gold.

[0076] Examples of colloidal nonmetallic particles include:
Examples include those made of polymers such as polystyrene (polystyrene latex), Sepharose, cellulose, Sephadex, and dye polymers, and particles of selenium, tellurium, sulfur, and the like.

The average particle size of such colored fine particles is 0.
．． The thickness is preferably about 1 to 100 μm, particularly about 10 to 30 μm. In the present invention, the water permeability of the support 2 is set as described above, so even if particles with a particle size of about 50 μm or more are used, the support 2 will be clogged with these fine particles and the movement of the specimen will be inhibited. There is no such thing.

The color of such colored fine particles on the support 2 varies depending on the type and particle size of the fine particles. For example, colloidal gold produces colors ranging from orange to red to blue.

[0079] The second substance or third substance may be bound to the label by a method generally known in the technical field of the present invention.

[0080] The contour shape of the area where such a sign is supported (hereinafter referred to as the sign support area) (the sign support section 5
The shape (shape) is not particularly limited, and may be any shape such as a strip, a line, a rectangle, a triangle, an ellipse, a circle, or a specific character shape. Further, the label carrying region may be divided into two or more in the longitudinal direction and/or width direction of the support 2.

[0081] The area of the label-carrying region is not particularly limited, but in both the sandwich method and the competitive method, the total area is usually 0.
It is preferably about 0.05 to 5 cm2, particularly about 0.1 to 3 cm2.

[0082]Although the amount of the labeled substance supported on the labeled substance supporting portion 5 is not particularly limited, both the sandwich method and the competitive method
Usually, it is preferably about 1 to 100 μl, particularly about 5 to 20 μl.

[0083] Such a method for supporting the label on the label support 5 includes, for example, impregnating the label support 5 with a liquid containing the label and drying (air drying, freeze drying, etc.), or This can be done by directly spraying and drying the object holding part 5, or the like.

[0084] In the immunoassay instrument 1B, the label carrier 5 is installed at a position where the average distance between the label carrier 5 and the detection unit 4 is approximately 5 to 70 mm, particularly 10 to 50 mm.
It is preferable to set it to about mm.

Next, the immunoassay method using the immunoassay device 1B will be described for detecting hCG in urine by the sandwich method and the competitive method, respectively.

B-1) Anti-hCG antibody 5 carries a labeled antibody Y that specifically binds to another recognition site β of hCG.

On the other hand, the container 10 contains a urine sample (specimen) 9 containing urine to be measured.

When the specimen supply section 3 at the lower end of the support 2 is immersed in the urine sample 9, the urine sample 9 moves upward of the support 2 due to capillary action and first reaches the label support section 5. When hCG is present in the urine, the label support portion 5
In this step, a complex between hCG in the urine sample 9 and labeled antibody Y is formed.

Further, the urine sample 9 moves above the support 2, but at this time, the formed hCG-labeled antibody Y complex separates from the label support 5, and the urine sample 9 moves upward. Move as you move.

[0090] When the urine sample 9 reaches the detection part 4, this complex contacts and reacts with the anti-hCG antibody A complex is formed and fixed.

[0091] When the label is, for example, a colored fine particle, the detection part 4 is colored, and the presence of hCG in urine can be detected or quantified by checking the color density visually or by optical measurement. Ru.

[0092] If hCG is not present in the urine, no complex is formed and therefore no coloring is caused by the label in the detection part 4.

[0093] The urine sample 9 that has reached the detection section 4 further rises on the support 4, and the excess urine sample 9 is absorbed and retained by the specimen absorbing material 7.

B-2) Anti-hCG antibody X that specifically binds to hCG is immobilized as a first substance on the detection part 4 of the competitive method support 2, and the label-carrying part 5 has A labeled hCG-like substance Z that competes with hCG and specifically binds to the anti-hCG antibody X is supported.

On the other hand, the container 10 contains a urine sample (specimen) 9 containing urine to be measured.

When the specimen supply section 3 at the lower end of the support 2 is immersed in the urine sample 9, the urine sample 9 moves upwards of the support 2 due to capillary action, first reaches the label support section 5, and then Move above the support 2. When the urine sample 9 passes through the label support part 5, the labeled hCG-like substance Z separates from the label support part 5 and moves along with the movement of the urine sample 9.

When hCG is present in the urine, when the urine sample 9 reaches the detection unit 4, hCG and the labeled hCG-like substance Z compete with each other and bind to the anti-hCG antibody X, resulting in the formation of anti-hCG antibody and anti-hCG antibody X-labeled hCG-like substance Z are formed at a predetermined ratio and fixed.

[0098] When the label is, for example, colored fine particles, the detection part 4 is colored, and the color density becomes darker as the amount of hCG in the urine is smaller. Therefore, the presence of hCG in urine can be detected or quantified by visually or optically measuring its color density.

[0099] When hCG is not present in urine, only a complex of anti-hCG antibody be done.

[0100] The urine sample 9 that has reached the detection section 4 further rises on the support 4, and the excess urine sample 9 is absorbed and retained by the specimen absorbing material 7.

In such measurements, the water permeability of the support 2 is 0.04 to 20 when the differential pressure is 300 mmHg.
1/min cm2, sufficient capillary flow is obtained during the movement of the urine sample 9, the movement speed is fast, and the support is not clogged with the above-mentioned complexes, labeled substances, etc. , measurement time is shortened and accurate measurement results are obtained.

Although the immunoassay instrument of the present invention has been described above with reference to the configuration example shown in the drawings, the present invention is not limited thereto. For example, the shape of the support is not limited to a belt-like shape, but may be a disc-like shape, or a shape having a curved surface such as a spherical shape (hollow or solid), a cylindrical shape, a cylindrical shape, a curved plate shape, or the like.

[0103] The present invention is also disclosed in Japanese Patent Application Laid-Open No. 64-32169.
As described in the above publication, a structure may be adopted in which a chromatographic solvent is supplied to one end of the support to transfer the target substance or label. In this case, in the immunoassay instrument of the present invention corresponding to the immunoassay instrument 1B, a sample supply section is installed between the detection section and the label support section. Further, a configuration may be adopted in which a plurality of paths through which the specimen and the chromatographic solvent move are formed on the support.

The immunoassay instrument of the present invention is widely used in the field of immunoassay, and in addition to the above-mentioned detection of hCG in urine (determination of pregnancy), the immunoassay instrument of the present invention is used for detecting various types of urine such as albumin, globulin, and LH in urine. It can also be applied to the detection of various blood components such as hormones and drugs in blood and plasma.

[0105]

EXAMPLES Specific examples of the immunoassay instrument of the present invention will be described below.

(Example 1) An immunoassay device (configuration shown in FIGS. 1 and 2) consisting of a support, a holding member, and a sample absorbing material under the following conditions was manufactured.

[0107] Fabric (thickness 0
．． 3 mm) was cut into a width of 10 mm x length of 70 mm, and this was used as a support. When the water permeability of this support was measured, it was found that the water permeability according to the AMMI standard was 300mmH
g, it was 7.2 l/min·cm2.

Holding member A holding member made of a polyethylene terephthalate film having a thickness of 0.05 mm cut to the same size as the support was adhered to the back surface of the support.

[0109] A sample absorbent made of hydrophilic porous plastic having a width of 10 mm and a length of 20 mm was adhesively fixed to one end of the sample absorbent support.

[0110] A detection part is set at a position 20 mm from the other end (sample supply part) of the first substance immobilization support, and human chorionic gonadotropin (hCG) and human chorionic gonadotropin (hCG) are placed in this detection part as the first substance. specifically binding anti-h
CG antibody (rabbit-derived IgG) was immobilized by the following method.

[0111] A solution with an anti-hCG antibody concentration adjusted to 10 mg/ml is delivered at a constant speed using an extremely thin needle, and the anti-hCG antibody is transferred to the detection area at a constant speed in the width direction on the support 2. Fixed.

[0112] The shape of the region on which the anti-hCG antibody is immobilized is as follows:
It was a thin line, and its area was 0.02 cm2.

[0113] Furthermore, the amount of anti-hCG antibody supported was 1 μg.
Met.

[0114] Next, the sample was prepared as follows,
Immunoassay was performed on this sample by the sandwich method using the immunoassay device described above.

Specimen Preparation Literature G. Frens, Nature Physics
al Science vol. 241, 1990 (1)
An anti-hCG antibody that specifically binds to the hCG at a recognition site different from the recognition site that binds to the hCG to colloidal gold (labeled) with an average particle size of 20 μm prepared by the method described in P20-22. A labeled substance to which (mouse-derived IgG) was physically adsorbed was prepared by the following method.

[0116] First, the above immunoglobulin was dissolved in a phosphate buffer (3 mM) at pH 8, and the above colloidal gold suspension was added thereto, followed by stirring at 4°C for 24 hours. after that,
Immunoglobulin was physically adsorbed onto colloidal gold. Next, this labeled substance was collected by centrifugation, and the pH was adjusted to 7.
The label suspension was prepared by resuspending the sample in 0.0 phosphate buffer (20 mM) and adding 1% bovine serum albumin.

[0117] The labeled substance suspension prepared in this way is
It was added to human urine so that the absorbance at 530 nm was 1 (cell length 1 cm), and hCG was added to 0, 50, and 50, respectively.
Urine sample No. adjusted to 0 mU/ml.
1, 2 and 3 were produced.

Measurement method Urine sample No. Samples 1, 2, and 3 were placed in containers, and the other end of the support (sample supply section) was immersed in them. Each urine sample moved on the support and reached the detection part in about 35 seconds. Furthermore, the excess urine sample moved beyond the detection part and was absorbed and retained by the specimen absorbent material.

In this state, the color density of the detection area was optically measured. This measurement was performed by measuring the reflected absorbance at 560 nm using a flying spot device model CS-9000 manufactured by Shimadzu Corporation. By applying this measurement value to a pre-prepared calibration curve, we can calculate the amount of hCG in the urine sample.
Determine the concentration and change the symbols -, +, + in Table 1 according to this concentration.
It was distinguished by + and +++. The results are shown in Table 1 below along with the values of the calibration curve.

(Example 2) The hCG concentration in a urine sample was determined in the same manner as in Example 1, except that the following support was used. The results are shown in Table 1 below.

[0121] Fabric (thickness 0
．． 2 mm) was cut into a width of 10 mm x length of 70 mm, and this was used as a support. When the water permeability of this support was measured, it was found that the water permeability according to the AMMI standard was 300mmH
g, it was 9.8 l/min·cm2.

(Example 3) The hCG concentration in a urine sample was determined in the same manner as in Example 1, except that the following support was used. The results are shown in Table 1 below.

[0123] A knitted fabric made by warp knitting polyester fibers with a support diameter of 10 μm (
(thickness: 0.25 mm) was cut into a width of 10 mm x length of 70 mm, and this was used as a support. When the water permeability of this support was measured, it was found that the water permeability according to the AMMI standard was 30
At 0 mmHg, it was 5.1 l/min·cm2.

(Example 4) The hCG concentration in a urine sample was determined in the same manner as in Example 1, except that the following support was used. The results are shown in Table 1 below.

Support A woven fabric (thickness 0.3 mm) made of a blend of 60% Tetoron fibers with a diameter of 5 μm and 40% nylon fibers with a diameter of 5 μm was cut into a width of 10 mm x length of 70 mm, and this was used as a support. . When the water permeability of this support was measured, AMMI
When the water permeability in the standard is 300 mmHg differential pressure, 3
．． It was 0 l/min·cm2.

(Comparative Example 1) The hCG concentration in a urine sample was determined in the same manner as in Example 1, except that the support shown below was used. The results are shown in Table 1 below.

[0127] Support membrane filter (manufactured by Fuji Photo Film Co., Ltd., Fuji Microfilter FM-500) was 5 mm wide x 70 mm long.
It was cut into mm pieces and used as a support. When the water permeability of this support was measured, the water permeability according to the AMMI standard was
When the differential pressure is 300mmHg, 0.03 l/min・c
It was m2.

[0128]

[Table 1]

[0129] As shown in Table 1, Examples 1 to 4 of the present invention
In both cases, accurate measurement results similar to those of the calibration curve can be obtained, and the judgment time is also shortened. On the other hand, comparative example 1
In this case, the support was clogged with the labeled substance, resulting in errors in the measurement results, and furthermore, it took a long time to make the determination.

(Example 5) Other end of support (sample supply section)
An immunoassay device similar to that of Example 1 was used except that a labeled substance carrying part was provided at a position 20 mm apart from the detecting part between the detection part and the detecting part, and the labeled substance was carried on this labeled substance carrying part (Fig. 3 and 4) were manufactured.

[0131] The label is described in the literature G. Frens, Na
ture Physical Science vol.
．． 241, 1990 (1) Colloidal gold (label) with an average particle size of 20 μm prepared by the method described in P20-22 was specifically labeled with the hCG at a recognition site different from the recognition site that binds to the hCG. The anti-hCG antibody (mouse-derived IgG) that binds to is physically adsorbed. The manufacturing method is as follows.

[0132] First, a phosphate buffer solution (3mM
), the above colloidal gold suspension was added thereto, and the mixture was stirred at 4°C for 24 hours. Immunoglobulins were then physically adsorbed onto the colloidal gold. Next, this labeled substance was collected by centrifugation, and the pH was adjusted to 7.
The label suspension was prepared by resuspending the sample in 0.0 phosphate buffer (20 mM) and adding 1% bovine serum albumin.

[0133] 5μ of the labeled substance suspension prepared in this manner
1 (an amount that shows absorbance of 60 (cell length 1 cm) at 530 nm) was dropped onto the labeled substance-supporting part to impregnate it, and this was dried at room temperature for 30 minutes to support the labeled substance.

[0134] The shape of the area carrying the label was a rectangle, and the area was 0.5 cm2.

[0135] Next, the sample was prepared as follows,
Immunoassay was performed on this sample by the sandwich method using the immunoassay device described above.

[0136] Preparation of specimen Human urine was tested with hCG of 0, 50 and 500, respectively.
Urine sample No. adjusted to mU/ml.
4, 5 and 6 were produced.

Measurement method Urine sample No. Samples 4, 5, and 6 were placed in containers, and the other end (sample supply section) of the support was immersed in them. Each urine sample moved on the support, passed through the label-carrying part, and then reached the detection part. This time was approximately 30 seconds. Furthermore, the excess urine sample moved beyond the detection part and was absorbed and retained by the specimen absorbent material.

[0138] In this state, the color density of the detection part was optically measured. This measurement was performed by measuring the reflected absorbance at 560 nm using a flying spot device model CS-9000 manufactured by Shimadzu Corporation. By applying this measurement value to a pre-prepared calibration curve, we can calculate the amount of hCG in the urine sample.
The concentration was determined, and the symbols -, +, and ++ in Table 1 were used to determine the concentration. The results are shown in Table 2 below along with the values of the calibration curve.

(Example 6) The hCG concentration in a urine sample was determined in the same manner as in Example 5, except that the following support was used. The results are shown in Table 2 below.

[0140] A woven fabric (thickness 0.3 mm) made of satin weave cupra fibers with a support diameter of 30 μm was cut into a width of 10 mm x length of 70 mm.
This was used as a support. When the water permeability of this support was measured, it was found that the water permeability according to the AMMI standard was 300mm differential pressure.
In the case of Hg, it was 6.5 l/min·cm2.

(Example 7) The hCG concentration in a urine sample was determined in the same manner as in Example 5, except that the support shown below was used. The results are shown in Table 2 below.

Support A knitted fabric (thickness: 0.25 mm) made by weft-knitting polypropylene fibers having a diameter of 10 μm was cut into a size of 10 mm in width and 70 mm in length, and this was used as a support. When the water permeability of this support was measured, it was found that the water permeability according to the AMMI standard was 3
At 00 mmHg, it was 4.3 l/min·cm2.

(Example 8) The hCG concentration in a urine sample was determined in the same manner as in Example 5, except that the following support was used. The results are shown in Table 2 below.

[0144] Support: A stockinette knitted fabric (thickness 0.23 mm) made of a blend of 60% Tetoron fibers with a diameter of 3 μm and 40% nylon fibers with a diameter of 3 μm is cut into 10 mm width x 70 mm length, and supported. As a body. When the water permeability of this support was measured, it was found that the water permeability according to the AMMI standard was 300 m
At mHg, it was 4.7 l/min·cm2.

(Comparative Example 2) The hCG concentration in a urine sample was determined in the same manner as in Example 5, except that the support shown below was used. The results are shown in Table 2 below.

[0146] Support membrane filter (manufactured by Fuji Photo Film Co., Ltd., Fuji Micro Filter FM-500) was 5 mm wide x 70 mm long.
It was cut into mm pieces and used as a support. When the water permeability of this support was measured, the water permeability according to the AMMI standard was
When the differential pressure is 300mmHg, 0.03 l/min・c
It was m2.

[0147]

[Table 2]

[0148] As shown in Table 2, Examples 5 to 8 of the present invention
In both cases, accurate measurement results similar to those of the calibration curve can be obtained, and the judgment time is also shortened. On the other hand, comparative example 2
In this case, the support was clogged with the labeled substance, resulting in errors in the measurement results, and furthermore, it took a long time to make the determination.

(Example 9) As a label, reference G. F
rens, Nature Physical Sci.
ence vol. 241, 1990 (1) P20-
Average particle size 20 μm adjusted by the method described in 22
Immunoassay was performed by the competitive method in the same manner as in Examples 1 to 8, except that hCG, which competitively binds to the hCG, was physically adsorbed to colloidal gold (label). As a result, results similar to those shown in Tables 1 and 2 above were obtained, and accurate and rapid measurements were possible.

[0150]

Effects of the Invention As described above, according to the immunoassay device of the present invention, the water permeability of the support according to the AMMI standard is
When the differential pressure is 300 mmHg, 0.04 to 20 l/mi
Since it is n·cm2, a sufficient capillary flow is ensured and no clogging occurs, so that it is possible to improve measurement accuracy and shorten measurement time.

[Brief explanation of the drawing]

FIG. 1 is a front view showing an example of the configuration of an immunoassay instrument of the present invention.

FIG. 2 is a sectional view taken along line AA in FIG. 1.

FIG. 3 is a front view showing another example of the configuration of the immunoassay instrument of the present invention.

FIG. 4 is a sectional view taken along line BB in FIG. 3;

[Explanation of symbols]

1A, 1B Immunoassay instrument 2 Support body 3 Sample supply section 4
Detection part 5 Labeled object carrying part 6
Holding member 7 Sample absorbing material 8, 9
Urine sample 10 container

Claims (8)

[Claims] 1. A water-absorbing support having a sample supply section and a detection section provided at a predetermined distance from the sample supply section; The support has a water permeability of 0.04 to 20 liters per hour at a differential pressure of 300 mmHg according to the AMMI standard.
An immunoassay device characterized by a measurement of min.cm2. 2. A label-carrying part is provided between the sample supply part and the detection part of the support, and a label-carrying part is provided with a recognition site different from the recognition site that binds to the first substance. 2. The immunoassay device according to claim 1, wherein the immunoassay device carries a labeled substance in which a second substance capable of specifically binding to the specific substance is labeled. 3. A label-carrying part is provided between the sample supply part and the detection part of the support, and the label-carrying part is provided with a label that competes with the specific substance to specifically bind to the first substance. Claim 1 in which a labeled substance is supported, which is a third substance labeled with a label.
The immunoassay device described in . 4. The immunoassay device according to claim 2, wherein the label is formed by immobilizing the second substance or the third substance on fine particles having a color different from that of the support. 5. The immunoassay device according to claim 1, wherein the support is a woven fabric, a knitted fabric, or a nonwoven fabric of fibers. 6. The immunoassay device according to claim 1, wherein the support is a strip-shaped chromatographic medium whose one end is a sample supply section. 7. The immunoassay device according to claim 6, further comprising a sample absorbing portion at the other end of the support for absorbing and retaining excess sample. 8. The immunoassay device according to claim 1, further comprising a holding member that holds the support.