JP2008164520A - Detection device utilizing antigen-antibody reaction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection device capable of detecting a test material by a simple method by utilizing antigen-antibody reaction. <P>SOLUTION: This invention is completed from such a conception that a labor for collecting test solution can be omitted by being brought into direct contact with the test solution instead of collection of the test solution by washing from a specimen. Namely, this detection device has an antibody holding film permeable by the test solution existing on the surface of the specimen, for holding a test material bonding antibody bonding specifically to the test material in the test solution; and an antibody immobilization film having an immobilizing antibody bonding specifically to the test material and/or the test material bonding antibody, and laminated on the antibody holding film, and is characterized by being used by bringing the antibody holding film side into contact with the surface of the specimen. Namely, the test solution on the surface of the specimen penetrates successively from the antibody holding film side of the detection device, to thereby progress the antigen-antibody reaction, and the test material is detected when being included in the test solution. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a detection apparatus for detecting a test substance contained in a test liquid using an antigen-antibody reaction.

  Conventionally, attempts have been made to detect bacteria, physiologically active substances, and the like present on the surface of a subject. Examples of subjects include the surface of kitchen equipment at restaurants, suspected food poisoning such as foods such as seafood, suspected pathogenic bacteria such as wounds, sweaty skin and secretions. Examples include those in which the presence of physiologically active substances such as attached mucous membranes is a problem.

  Bacteria and the like cannot be seen with the naked eye. Bacteria can be obtained by culturing an agar medium pressed against the surface of the subject or a filter that filters the liquid obtained by washing the surface of the subject with physiological saline. There is a method of detecting the colony by forming a colony.

  However, since these techniques are complicated, after the pressure-sensitive adhesive layer of the pressure-sensitive adhesive sheet for microorganism testing having a pressure-sensitive adhesive layer mainly composed of a water-insoluble polymer compound is pressure-bonded to the surface of the specimen and peeled to accumulate the microorganisms, A method for testing microorganisms present in the specimen has been proposed, comprising detecting the microorganisms by bringing an aqueous solution containing one or more chromogenic substances capable of staining the microorganisms into contact with the surface of the adhesive layer. (Patent Document 1).

  In addition, methods for detecting microorganisms using antigen-antibody reaction have been disclosed (Patent Documents 2, 3, etc.). The method disclosed in Patent Document 2 is a so-called flow-through immunoassay method, and the method disclosed in Patent Document 3 is a so-called lateral flow immunoassay method.

For the purpose of improving the storability and ease of handling of the immunomembranes used in these immunoassay methods, a transparent layer is provided on the back surface of the backing sheet to which the reagent is fixed, and a release liner is laminated on the adhesive layer. A test paper in which a protective sheet made of a thermoplastic resin is folded is disclosed (Patent Document 4).
JP 2002-142797 A Japanese Patent No. 2818191 JP-A-6-167497 JP 2001-56339 A

  However, the conventional technology has not been able to perform a detection operation simply as described below. That is, although the method disclosed in Patent Document 1 can be detected in a short time, it is difficult to say that it is easy to measure because it requires a color developing substance to be brought into contact with the adhesive sheet. In the methods described in Patent Documents 2 and 3, it is necessary to collect the test liquid by washing the surface of the test object, and it is difficult to say that this is also a simple method. The method disclosed in Patent Document 4 is a technique for preventing the reaction portion from being contaminated with bare hands and the like, and does not simplify the measurement method.

  The present invention has been completed in view of the above circumstances, and provides a detection apparatus and method capable of detecting a test substance using an antigen-antibody reaction by a simple technique regardless of the skill of the examiner. Is a problem to be solved.

In order to solve the above-mentioned problems, the present inventors have conducted intensive studies, and instead of collecting the test liquid from the surface of the specimen by washing or the like, they directly contact the test liquid existing on the surface of the specimen. As a result, the inventors have conceived the idea of reducing the number of steps for collecting the test liquid, and completed the following invention. That is, the detection apparatus using the antigen-antibody reaction of the present invention that solves the above-described problems allows the test solution on the surface of the specimen to pass in the thickness direction and is contained in the test solution 1 An antibody-holding membrane that is held so that it can be dissolved in the test solution through which the test substance-binding antibody that specifically binds to the test substance passes,
An antibody-immobilized membrane that has one or more immobilized antibodies that specifically bind to the test substance and / or the test substance-binding antibody, and is laminated so that one side is in contact with one side of the antibody-holding film;
Have
The antibody holding film side is used in contact with the surface of the subject.

In addition, the detection method of the present invention that solves the above problems includes a step of wetting the surface of the subject when the surface of the subject is not wet;
Contacting the surface of the analyte with the antibody-holding membrane side of the detection apparatus using the antigen-antibody reaction of the present invention;
Leaving the test solution up to the antibody-immobilized membrane of the detection device;
And a step of detecting the test substance with the amount of the test substance appearing on the other surface side of the antibody-immobilized membrane.

  In other words, when the test liquid on the surface of the specimen permeates sequentially from the antibody holding film side of the detection apparatus of the present invention, the antigen-antibody reaction proceeds, and the test substance is contained in the test liquid Will be detected. Specifically, first, the test substance-bound antibody is dissolved in the process in which the test solution permeates the antibody holding membrane. The dissolved test substance-binding antibody specifically binds to the test substance when the test substance contains the test substance in the test solution. Thereafter, the test solution reaches the antibody-immobilized membrane. In the antibody-immobilized membrane, the test substance (and / or test substance-binding antibody) in the test solution is captured by the immobilized antibody. Since the amount captured by the immobilized antibody varies depending on the amount of the test substance in the test solution, the presence / absence and amount of the test substance can be detected from the amount captured by the immobilized antibody.

  And it is desirable to have a 1st porous film | membrane laminated | stacked between the said antibody holding | maintenance film | membrane and the said antibody fixed film | membrane, and the said test liquid can permeate | transmit in the thickness direction. By interposing the first porous membrane between the antibody holding membrane and the antibody-immobilized membrane, the backflow of the test solution between them can be suppressed. Moreover, it is desirable to have a second porous membrane that is laminated on the antibody holding membrane side and that allows the test solution to permeate in the thickness direction. By contacting the test solution through the second porous membrane, the test substance-bound antibody that has once permeated the antibody holding membrane can be prevented from flowing back to the surface of the test sample.

  The antibody-immobilized membrane preferably has an absorption membrane that allows the test solution to pass through in the thickness direction and absorbs the test solution on the other side of the antibody-immobilized membrane. By having an absorption membrane that can absorb the test solution on the other side of the antibody-immobilized membrane, a large amount of test solution can be permeated through both the antibody holding membrane and the antibody-immobilized membrane. Substance detection sensitivity can be improved. It is desirable that the absorption film becomes transparent or translucent after absorbing the test liquid so that the absorption film does not interfere with detection of the test substance.

  Furthermore, the test solution has a test solution rectifying film made of a material that cannot pass the test solution, laminated on the other surface side of the antibody holding film, and having a notch of a predetermined size. It is possible to control the site where the water penetrates to flow uniformly. In particular, two or more of the cutouts of the test solution rectifying membrane are formed, and the test substance-bound antibody and the immobilized antibody in the antibody holding membrane and the antibody-immobilized membrane have holes formed therein. By adopting a configuration corresponding to different types or combinations of the test substances for each position corresponding to the position, it becomes possible to detect a plurality of test substances independently with one detection device.

  And by having the protective film which the said test liquid arrange | positioned on the outermost surface of the other surface side of the said antibody fixed film cannot permeate | transmit, there is less possibility of touching a test liquid and handling property improves.

  Furthermore, the test substance binding antibody is labeled, and the antibody-immobilized membrane detects the test substance by the label of the test substance binding antibody captured by the immobilized antibody. It becomes possible to detect the test substance also by optical means.

  Since the detection apparatus of the present invention has the above-described configuration, it is possible to save labor for collecting the test liquid from the surface of the test object and to easily detect the test substance.

  Hereinafter, the detection apparatus and method of the present invention will be described in detail based on embodiments. The detection apparatus of this embodiment is a method for detecting a test substance on the surface of a test object. The test substance is not particularly limited as long as it can act as an antigen in an antigen-antibody reaction. For example, bacteria (pathogenic fungi, etc.), specific proteins (allergens in food, physiologically active substances exuded from the skin of a living body, etc.), etc.

  These test substances are applied to this detection apparatus in a state of being dissolved in a liquid. If the test substance is dissolved in water from the beginning and is liquid, it can be applied as it is as the test liquid, and if the liquid volume is insufficient or dry, it does not contain the test substance. A test liquid can be obtained by moistening the surface of the subject with water spray or the like.

(Constitution)
The detection device of this embodiment is a laminate in which an antibody holding film and an antibody immobilization film are laminated in this order. Other members may be included as necessary.

  The antibody-holding membrane is a membrane-like and porous member, and the test solution can penetrate in the thickness direction, and the test solution can penetrate in the thickness direction by capillary action or the like. For example, a nonwoven fabric is illustrated. A test substance binding antibody that is an antibody corresponding to the test substance is held on the antibody holding film. The analyte-bound antibody can be retained on the antibody retention membrane by physical adsorption, or can be retained by binding to the analyte immobilized on the antibody retention membrane using an antigen-antibody reaction. it can. The test substance-bound antibody dissolves and elutes in the permeated test solution. In the case of holding using an antigen-antibody reaction, the test substance-bound antibody is eluted by binding competitively with the test substance in the test solution.

  It is desirable that the analyte-bound antibody is labeled in some way for the purpose of facilitating later detection. Examples of the label include colloidal gold, a fluorescent substance, and an enzyme. Colloidal gold and fluorescent materials can detect color development and fluorescence, and enzymes can be detected by adding a substrate that promotes the reaction and changing the substrate.

  The antibody-immobilized membrane can be penetrated in the thickness direction by a capillary phenomenon or the like. It is also possible to make the test solution permeate in the thickness direction. Nonwoven fabric is exemplified as the antibody-immobilized membrane. The antibody-immobilized membrane has at least one type of immobilized antibody that specifically binds to the test substance and / or test substance-binding antibody. The immobilized antibody is bound on the antibody-immobilized membrane. The immobilization (binding) of the immobilized antibody here is sufficient as long as it does not elute in the test solution.

  After the test substance (and / or test substance binding antibody) is bound to the immobilized antibody, the test is performed by detecting the presence of the test substance binding antibody (and / or test substance) that is bound simultaneously. Detect the presence of a substance.

  Immobilized antibodies are immobilized on an antibody-immobilized membrane so as to show various shapes (+,-, ◯, x, positive, negative, etc.), and the shape is revealed when a test substance is detected. be able to.

  Other members that can be included as necessary include a second porous film, an absorption film, a test liquid rectifying film, and a protective film.

  The first porous membrane is a membrane-like member through which the test solution can permeate in the thickness direction. The test solution penetrates in the thickness direction due to capillary action. The first porous membrane can prevent the flow of the test solution from the antibody holding membrane to the antibody-immobilized membrane from flowing backward.

  The second porous membrane is a member having substantially the same configuration as the first porous membrane. The second porous membrane is laminated on the other side of the antibody holding membrane. It may be in contact with the antibody holding film, or may be laminated via another member. The second porous membrane prevents the antibody-holding membrane from directly contacting the surface of the specimen, thereby preventing the backflow of the flow of the test liquid and preventing contamination of the surface of the specimen by the test substance binding antibody. .

  The absorption membrane is a member laminated on the other side of the antibody-immobilized membrane. It is formed from a porous body or the like. The absorption membrane absorbs the test solution, so that a large amount of the test solution can flow in the order of the antibody holding membrane, the first porous membrane, and the antibody-immobilized membrane, and the test substance in the test solution The detection sensitivity can be improved.

  For the purpose of facilitating optical detection of the test substance on the antibody-immobilized film, it is desirable that the absorption film becomes transparent or translucent when the test solution permeates. For example, a nonwoven fabric formed from transparent fibers (cellulose, chemically treated cellulose, polyvinyl alcohol, acrylic resin, PET, polyamide, etc.) can be employed.

  The test liquid rectifying film is formed of a material that does not allow the test liquid to pass therethrough. The test liquid rectifying film is laminated on the antibody holding film side of the detection apparatus. It may be in contact with the antibody holding film, or may be laminated via another member. The test solution rectifying film has a notch of a predetermined size. The test solution penetrates only in the notched portion. Two or more cutouts are provided, and a test substance binding antibody and an immobilized antibody corresponding to different types of test substances or test substances at different concentrations are held and fixed at each notch position. It is possible to detect two or more test substances and the concentration of the test substance in one measurement.

  The protective film is made of a material that does not pass through the test liquid. It is desirable to be formed from a transparent or translucent material so as not to hinder optical detection. The possibility that the operator of the detection apparatus of the present embodiment touches the test liquid is reduced.

(Function and effect)
Hereinafter, a method for detecting a test substance using the detection apparatus of the present embodiment will be described. The detection apparatus of this embodiment is used with the antibody holding film side attached to the surface of the subject. The surface of the subject is required to be wet. If it is not moistened, it is moistened by spraying water (such as an appropriate buffer or pure water) that does not contain the test substance.

  When the antibody holding film side is attached to the surface of the subject, the test solution on the surface of the subject soaks into the antibody holding membrane. Here, when the test liquid rectifying film is present, it penetrates only from the cutout portion of the test liquid rectifying film. When the second porous membrane is provided, the test solution is rapidly permeated.

  The test solution reaching the antibody holding film dissolves the held test substance-bound antibody. The test substance binding antibody specifically binds to the test substance contained in the test solution. The test solution permeates the antibody-immobilized membrane as it is through the first porous membrane. When the absorption membrane is employed, the test solution can be absorbed and a large amount of the test solution can be permeated in the order of the antibody holding membrane, the first porous membrane, and the antibody-immobilized membrane.

  In the antibody-immobilized membrane, the immobilized antibody binds to a complex in which the test substance and the test substance-bound antibody are bound. As a result, the complex of the test substance and the test substance binding antibody is concentrated at the site where the immobilized antibody is immobilized.

  The test substance can be detected by binding the test substance or the like to the immobilized antibody. When a labeled compound is employed as the test substance binding antibody, the test substance can be detected by detecting the labeled portion.

  Therefore, the detection apparatus of this embodiment can omit the labor of collecting the test sample from the surface of the subject, and anyone can easily detect the test substance.

(Test 1)
As shown in FIG. 1, a polyethylene sheet 11 as a test solution rectifying membrane, and three nitrocellulose membranes 12 to 14 (Millipore # HABG04700) corresponding to the antibody holding membrane, the first porous membrane, and the antibody-immobilized membrane, respectively. ) To form a model of the detection device. The polyethylene sheet 11 was formed by opening 3 pairs of holes (notches) each having a diameter of 6 mm.

  A 150 mM phosphate buffer solution (0.5 mL) was hung on a table 90 as an object, and the detection device was put on the polyethylene sheet 11 side from above. The buffer solution permeated from the hole formed in the polyethylene sheet 11 and was able to be infiltrated into the three membranes 12 to 14 while maintaining the shape of the circular notch.

  By adopting the test liquid rectifying membrane and the test liquid, it was possible to infiltrate the test liquid into the necessary part without strictly selecting the measurement part.

(Test 2)
As shown in FIG. 2, from the bottom, an antibody holding membrane 21 made of a glass fiber sheet (Millipore # GFCP103000), an antibody-immobilized membrane 22 made of a nitrocellulose membrane (Millipore Durapore membrane filter, 0.1 mmVVPP), and Kimwipe (trademark) The detection device was manufactured by laminating an absorption film 23 and a protective film 24 made of a polyethylene sheet in this order.

  The antibody holding film 21 is a film in which Rabbit polyclonal to Ovalbumin (abcam) as a test substance binding antibody is held on a glass fiber sheet.

  First, Rabbit polyclonal to Ovalbumin (abcam) was mixed with colloidal gold (British-biocell International) having a diameter of 40 nm. Two types of buffers were used, one using 10% NaCl and the other using 10% Sucrose.

  An antibody holding film 21 was obtained by dropping 25 μL of a gold colloid solution using 10% Sucrose and 25 μL of a gold colloid solution using 10% NaCl two times onto a glass fiber sheet and air drying.

  The antibody-immobilized membrane 22 was fixed to four locations 222 by dropping Anti OVA (Rabbit Polyclonal to Ovaalbumin (abcam)) and Anti Rabbit (GtxRabbitIgG # AP132 (CHEMICON)) on the nitrocellulose membrane and air-drying. In the portion 222 where the immobilized antibody is immobilized, the anti OVA is immobilized on the circular portion 222a surrounding the anti-OVA, and the anti-rabbit is immobilized on the central linear portion 222b. Therefore, when the test solution contains OVA as the test substance, the central linear part 222b is colored by the gold colloid, and the circular part 222a around it is the test substance in the test liquid. Regardless of the presence or absence, color is developed by permeation of the test solution.

  On a slide glass 90 as an object, 25 μL of 100 μg / mL OVA (Wako, Albumin from Chicken Egg # 010-17071) was dropped in two places, and 25 μL of 150 mM phosphate buffer was further dropped in two places.

  When the manufactured detection device was placed on the slide glass 90, the test solution permeated, the absorption film 23 became transparent, and a signal could be observed through the protective film 24. Circular and rod-shaped signals were observed in the combination of colloidal gold solution prepared using Sucrose buffer and OVA. In the combination of gold colloid solution prepared using NaCl buffer and OVA, a circular signal was observed, and a linear signal was slightly observed. Only a circular signal was observed in the phosphate buffer.

  In other words, when OVA is included as a test solution, the presence of the test substance (OVA) is detected although there is a difference in reactivity depending on the type of buffer used for the preparation, and when OVA is not included, color development occurs. It was found that it was possible to detect the absence of OVA.

3 is a schematic exploded perspective view showing a detection device used in Test 1. FIG. 6 is a schematic exploded perspective view showing a detection device used in Test 2. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 ... Test liquid rectification film | membrane 12-14 ... Nitrocellulose membrane 21 ... Antibody holding film | membrane 22 ... Antibody fixed film | membrane 222 ... The site | part to which the fixed antibody was fixed 23 ... Absorption film | membrane 24 ... Protective film 90 ... Test object

Claims (10)

The test solution present on the surface of the test sample is permeable in the thickness direction, and the test substance binding antibody that specifically binds to one or more test substances contained in the test solution is transmitted therethrough. An antibody-holding membrane that is held so as to be soluble in the test solution;
An antibody-immobilized membrane that has one or more immobilized antibodies that specifically bind to the test substance and / or the test substance-binding antibody, and is laminated so that one side is in contact with one side of the antibody-holding film;
Have
A detection apparatus using an antigen-antibody reaction, wherein the antibody holding film side is used in contact with the surface of the subject.   The detection apparatus according to claim 1, further comprising a first porous film that is laminated between the antibody holding film and the antibody-immobilized film so that the test solution can permeate in a thickness direction.   The detection apparatus according to claim 1 or 2, further comprising a second porous film that is laminated on the other surface side of the antibody holding film and allows the test solution to pass in the thickness direction. The antibody-immobilized membrane allows the test solution to penetrate in the thickness direction,
The detection apparatus according to claim 1, further comprising an absorption film that absorbs the test solution on the other surface side of the antibody-immobilized film.   The detection apparatus according to claim 4, wherein the absorption film becomes transparent or translucent after absorbing the test liquid.   The detection according to any one of claims 1 to 5, further comprising a test liquid rectifying film made of a material that cannot pass the test liquid, laminated on the antibody holding film side, and having a notch of a predetermined size. apparatus. The notch of the test liquid rectifying film is formed in two or more,
The test substance binding antibody and the immobilized antibody in the antibody holding film and the antibody-immobilized film correspond to different types or combinations of the test substances for each position corresponding to the position where the hole is formed. The detection device according to claim 6.   The detection apparatus according to any one of claims 1 to 7, further comprising a protective film that is not allowed to pass through the test solution disposed on the outermost surface on the other surface side of the antibody-immobilized film. The analyte binding antibody is labeled,
The detection apparatus according to claim 1, wherein the antibody-immobilized membrane detects the test substance by the label of the test substance-bound antibody captured by the immobilized antibody. Wetting the surface of the subject when the surface of the subject is not wet;
Contacting the antibody holding film side of the detection device according to any one of claims 1 to 9 with the surface of the subject;
Leaving the test solution up to the antibody-immobilized membrane of the detection device;
And a step of detecting the test substance with the amount of the test substance appearing on the other surface side of the antibody-immobilized membrane.

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