JP2714855B2 - Simple immunological diagnostic device - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a simple immunological diagnostic device, and more specifically, typically, to perform an immunological diagnostic utilizing an antigen-antibody reaction simply, quickly and with high precision. The present invention relates to a simplified immunological diagnostic device obtained.

2. Description of the Related Art Detection and measurement of various components and drugs contained in body fluids such as serum and urine are extremely important clinically, and conventionally performed by various methods. As such a method, typically, an immunoassay using an antigen or an antibody which is a test substance in a body fluid and an antibody or an antigen having specific binding properties to the antigen or antibody is known. However, enzyme immunoassays using enzyme-labeled immunity, such as enzyme-labeled antibodies and antigens, are widely used because of their high sensitivity and measurement stability in order to know whether or not there is a reaction between antigens and antibodies. I have.

As the enzyme immunoassay, various methods are known, for example, as described in "Enzyme immunoassay" by Eiji Ishikawa et al. (Published by Medical Shoin Co., Ltd., 1987). Generally, for example, an antibody for performing an antibody-antigen reaction with a test substance is immobilized on the surface of a water-insoluble solid support such as a polystyrene bead, an inner wall surface of a test tube, a plate, a membrane, and the like. A test solution containing an antigen to be tested is added, and the antigen is bound to the solid support via the antibody by an antigen-antibody reaction. Next, the unreacted antigen is washed and removed with a buffer solution or the like, and the enzyme-labeled antibody is reacted with the antigen, and the unreacted enzyme-labeled antibody is bound to the antigen. After washing and removing,
The amount of the antigen as the test substance to be measured is determined from the amount of the labeled enzyme bound on the solid support. The amount of the labeled enzyme is, for example, a substrate corresponding to the enzyme, or reacting the substrate with a coloring agent, and detecting the antigen by measuring the color change of the liquid phase based on the production of the reaction product with the naked eye, or Measure optically.

Problems to be Solved by the Invention However, with the recent development of the medical system, there has been a strong demand for rapid and highly sensitive measurement of trace components in a large number of body fluids such as serum and urine. The conventional enzyme immunoassay as described above is complicated in operation and lacks in operability and quickness.

Therefore, for example, JP-A-61-173160 and RFZu
k et al., Clin. Chem., 31, 1144-1150 (1985), etc.
Various methods have been proposed for easily performing an immunoassay, but are still extremely insufficient in terms of practicality, accuracy, and the like.

The present invention has been made to solve the above-mentioned problems in immunodiagnosis represented by a conventional enzyme immunoassay, and it is an object of the present invention to carry out an immunodiagnosis simply, quickly and with high sensitivity. It is an object of the present invention to provide a simple immunological diagnostic device which enables the diagnostic device.

Means for Solving the Problems A simplified immunological diagnostic device according to the present invention is formed by an upper wall, a side wall, and a lower wall, and includes a test solution and reagent inlet and an observation window at intervals on the upper wall. In the container, a continuous band-shaped water-absorbing base material layer is provided with a solid phase immobilized portion in which an immune body that can bind to the immune body that is the test substance is immobilized at intervals from the side wall, At least housed in close contact with the upper wall, and the solidified portion is positioned corresponding to the observation window, at an interval from the solidified portion, the development layer facing the injection port, further The injection port is formed in a groove shape extending in a direction perpendicular to the longitudinal direction of the developing layer.

Hereinafter, a simplified immunological diagnostic device according to the present invention will be described with reference to the drawings showing examples.

As shown in FIG. 1, FIG. 2 and FIG. 3, the diagnostic device according to the present invention is formed by an upper wall 1, a side wall 2 and a lower wall 3; In a container 6 having a reagent solution inlet 4 and an observation window 5, a later-described developing layer 7 is housed at a distance from the side wall 2 and in close contact with the upper and lower walls 1 and 3. The immobilized part 8 in which the immunity that can bind to the immunity which is the test substance is immobilized on the layer 7,
The developing layer 7 is positioned corresponding to the observation window 5 and spaced from the solid-phased portion 8 so that the spreading layer 7 faces the injection port.

The spreading layer 7 is formed of a continuous band-shaped water-absorbing base material layer, and the base material layer is formed of a test solution containing a test substance, such as serum, blood, urine, or a buffer solution thereof. It is made of a material that can be made to penetrate a diluent or various reagent solutions at an appropriate speed, move it in a longitudinal manner, and reach the solid-phased portion 8. When the water-absorbing base material layer is poor in water absorption, it takes a long time for the test solution or the reagent solution to reach the solid-phased portion, and thus it is not possible to perform a quick measurement. However, when the water absorption of the water-absorbing base material layer is too high, the test substance or the reagent in the test liquid is insufficient in time for performing a sufficient reaction with the profit and loss body of the solid-phased part,
It is difficult to make accurate measurements.

Therefore, in the present invention, it is necessary to use a water-absorbing substrate layer that can secure time for the test substance in the test solution to sufficiently react with the immunizing body in the solid-phased portion. However, the selection of such a substrate is within a range that can be easily achieved if one is familiar with enzyme immunoassay. However, particularly preferred examples include, for example, nonwoven fabric, filter paper, glass fiber cloth, and porous material.

Further, in order to adjust the water absorption of these substrates, the substrates can be coated with or impregnated with a hydrophilic polymer. Further, in the present invention, it is not necessary to use the same absorbent material as the water-absorbing substrate, and it is also possible to join the substrates made of different materials to form a continuous substrate.

The method for immobilizing an immunizing body such as an antibody or an antigen on the water-absorbing substrate layer is not particularly limited, but is preferably a conventional adsorption method or a covalent bonding method conventionally known. In particular, it is preferable to use a covalent bonding method in which the immune body is not detached from the substrate layer. When the water-absorbing substrate does not have a functional group for the covalent bonding method, for example, a polymer having an appropriate functional group may be adhered to the substrate to such an extent that its water absorption is not hindered.

In the diagnostic device according to the present invention, the required amount of the test solution and other reagents is reduced as much as possible,
These test liquids and reagents injected into the developing layer from the injection port penetrate in the longitudinal direction of the developing layer at each point in the width direction of the developing layer at a uniform speed as much as possible, move, and move to the solid-phased portion. The spreading layer and the upper wall of the container are in close contact with each other so as to reach a uniform position, but are spaced from the side wall. In particular, it is necessary that the developing layer be in close contact with the entire periphery of the test liquid inlet of the container.

When there is a gap between the developing layer and the upper wall, the test liquid and the reagent injected from the inlet are preferentially sucked into the gap. When the developing layer is in contact with the side wall of the container, the test liquid or cormorant injected from the inlet easily flows along the contact portion with the side wall, and a velocity gradient is generated in the width direction of the developing layer. Occurs. However, according to the present invention, the developing layer is brought into close contact with the upper wall of the container, and in particular, the developing layer is brought into close contact with the entire periphery of the test liquid inlet of the container, and is separated from the side wall of the container. The test solution or reagent injected from the inlet substantially penetrates only into the developing layer, and does not cause a velocity gradient in the width direction of the developing layer. Move in the direction.

The developing layer preferably has a thickness of 3 mm or less, and particularly preferably 1.5 mm or less, in order to ensure a uniform flow of the test solution or the reagent solution.
Further, the width is usually 2 mm so as to secure the flow of the test solution or the reagent solution and to ensure that the reaction result can be visually confirmed.
It is preferable that it is above.

Further, in the diagnostic device according to the present invention, the injection port is formed in a groove shape extending in a direction perpendicular to the longitudinal direction of the developing layer. As described above, by forming the injection port into a groove shape extending in a direction orthogonal to the longitudinal direction of the spreading layer,
The test liquid or reagent injected into the injection port uniformly penetrates in the width direction of the developing layer, and moves in the longitudinal direction at a uniform speed without generating a velocity gradient in the width direction. Although not particularly limited, usually, the width of the injection port, that is, the distance in the longitudinal direction of the spreading layer is preferably 4 mm or less, and particularly preferably 2 mm or less.

As shown in the figure, the solidified portion 8 in the spreading layer 7 is arranged at a distance from a desired portion of the spreading layer 7 at the injection port 4. The test liquid or reagent injected into the injection port penetrates the developing layer 7 from the periphery of the injection port in the direction perpendicular to the tangential contact point at that position, and moves. If the extended line of the perpendicular line intersects with the solid-phased portion at as high a density as possible, the test solution and reagent do not stay in the developing layer and remain substantially constant. Since it quickly reaches the solid-phased portion at a high speed, highly sensitive immunological diagnosis can be reliably performed in a short time.

In the diagnostic device according to the present invention, the test liquid and the reagent solution are sequentially injected from the injection port. In order to absorb and store the liquid, it is desirable to connect the liquid absorption storage layer 9. Such a liquid absorption storage layer is made of, for example, a thick filter paper. Further, if necessary, such a liquid absorption storage layer (not shown) may be connected to the periphery where the spreading layer desires the injection port.

Using the diagnostic device according to the present invention, for example, to perform diagnosis by enzyme immunoassay, first, a test solution containing an antigen as a test substance is injected into the developing layer from the inlet of the container, and the developing layer is washed. It is moved to reach the solid-phased part, where it is bound to the antibody. Next, the enzyme-labeled antibody solution is injected into the developing layer from the injection port, and similarly, the developing layer is moved to reach the solid-phased portion and bind to the antibody. Thereafter, a washing solution is injected into the developing layer, and then a substrate-color former solution is injected into the developing layer, and color development in the solid-phased portion is observed through an observation window.

Effect of the Invention As described above, according to the simplified immunological diagnostic apparatus of the present invention, when the test solution and the reagent are sequentially injected into the injection port, they are subjected to a velocity gradient in the width direction of the continuous band-shaped developing layer. Without penetrating, it penetrates and moves in the longitudinal direction sequentially at a uniform speed, reaches the solid-phased part, and performs the required immunological reaction. Therefore, if an enzyme immunoassay is performed using such a diagnostic device, the reaction will occur uniformly in the solid-phased part at each reaction stage, and finally a chromogenic substrate will be given to the enzyme. The reaction result can be clearly and easily known by coloring the phased portion.

Further, according to the diagnostic device of the present invention, a small amount of each reagent can be used, and almost the entire amount of the reagent can be sequentially passed through the solid-phased part, so that the reaction efficiency is high and the enzyme immunoassay is high. The original benefits and sensitivity of the method are maintained. Also, B /
Since F separation is also easy, non-specific reactions such as false positives due to the remaining labeled antibody and enzyme substrate hardly occur.

An example in which human chorionic gonadotropin (HGC) was measured by an enzyme-linked immunosorbent assay using a diagnostic device as shown in the figure is shown below.

The developing layer is made of glass fiber filter paper (GA10 manufactured by Toyo Roshi Kaisha, Ltd.).
0, thickness 0.65mm (actual measurement value, retention particle diameter 0.1μm) 7mm
Cut to a size of × 50 mm, one end is the part where the injection port is desired,
In the vicinity of the other end, an anti-HCG antibody was uniformly immobilized on a square having a length of 5 mm and a width of 3 mm. The solidified portion was provided such that the edge on the injection port side had a distance of 15 mm from the edge on the solidified portion side of the injection port. Furthermore, at the end of the spreading layer on the solid-phased portion side,
A thick filter paper was connected for liquid absorption storage.

The immobilization of the antibody was performed as follows. Particle size 0.18
An anti-HCG antibody (rabbit antiserum) was bound to the surface of a μm styrene-acrylic acid copolymer latex by a carbodiimide method to obtain an aqueous solution having a solid content of 0.5% by weight. The glass fiber filter was previously treated with a 0.01% solution of polyethyleneimine for 30 minutes, dried, and then treated with a 0.1% solution of glyoxal in methanol for 30 minutes. 5 μl of the anti-HCG antibody-binding latex was applied to a predetermined portion of the glass fiber filter paper and dried. Thereafter, the glass fiber filter was treated with 0.5% milk casein and dried.

An alkaline phosphatase-labeled monoclonal antibody was used as the enzyme-labeled antibody, and a 10 mM solution (pH 9) of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) was used as a substrate-color former of alkaline phosphatase. .8) was used.

250 μl of HCG aqueous solution with saline at the inlet of the diagnostic device
And 100 μl of the enzyme-labeled antibody solution were sequentially injected, and allowed to react for 1 minute each. Then, 100 μl of the washing solution was injected, 100 μl of the substrate-color former solution was further injected, and the mixture was reacted for 2 minutes. Was injected, and color development was visually observed.

As a result, when observed from the observation window at a distance of lm, the sensitivity that can be clearly recognized by the color development is 20 to 200 mIU / m.
l Also, when given an HCG concentration of 1000 mIU / ml,
No color shading was observed in the solid-phased portion. Furthermore, almost no nonspecific reaction was observed.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view showing the appearance of a simplified immunological diagnostic device according to the present invention, FIG. 2 is a perspective view showing the diagnostic device with the upper wall of the container removed, FIG. FIG. 3 is a cross-sectional view of FIG.
It is sectional drawing which follows the II line. 2 ... top wall, 2 ... side wall, 3 ... bottom wall, 4 ... injection port of test solution and reagent solution, 5 ... window for observation, 6 ... container, 7 ... development layer, 8 ...
Solid-phased part, 9 ... liquid absorption storage layer.

Continuation of the front page (72) Inventor Yoko Takamatsu 1-1-2 Shimohozumi, Ibaraki-shi, Osaka Nitto Denko Corporation (56) References JP-A-64-72066 (JP, A) JP-A-61- 277059 (JP, A)

Claims (1)

(57) [Claims] 1. A continuous band-shaped water-absorbing substrate formed in an upper wall, a side wall, and a lower wall, and provided in a container provided with a test liquid and reagent inlet and an observation window at intervals on the upper wall. A developing layer provided with a solid phase immobilizing section on which an immune body capable of binding to an immune substance as a test substance is immobilized is provided at a distance from the side wall, at least in close contact with the upper wall, and accommodated therein. While the phased portion is positioned corresponding to the observation window, at a distance from the solidified portion, the developing layer faces the injection port, and the injection port is orthogonal to the longitudinal direction of the development layer. A simple immunological diagnostic device characterized by being formed in a groove extending in a direction.