JP2007278883A - Detection method of antigen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection method of an antigen capable of simply reducing non-specific dyeing in an immunochemical dyeing method. <P>SOLUTION: In the detection method of the antigen for detecting the antigen in the specimen using the immunochemical dyeing method, an antibody liquid is dripped on the specimen placed on a flat plate and subsequently covered with a resin film having no reactivity with an antibody to perform antigen-antibody reaction. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for detecting an antigen in a specimen.

  The immunochemical staining method is a method of detecting antigens in tissues and cells using antigen-antibody reaction and visualizing and identifying them. Only HE staining and special staining commonly used for tissue and cell searches are used. This is a very useful method in that it can detect various substances such as enzymes, hormones, tumor markers, immunoglobulins, pathogens, oncogene-related antigens, etc. that cannot be detected by (Non-patent Document 1).

  On the other hand, in the immunochemical staining method, non-specific staining is likely to occur during the staining process, and in order to discriminate between non-specific staining and specific staining, the characteristics of the intracellular or tissue distribution of the antigen should be known in advance. Furthermore, there is a large portion based on experience such as comparison with a HE-stained specimen (Non-patent Document 2). For this reason, there has been a problem that it is difficult to accurately discriminate changes in cell or tissue distribution and non-specific staining.

  Conventionally, as a method for improving the staining of an antigen, an apparatus (Patent Document 1) that promotes penetration of an antibody into a specimen by using ultrasonic waves, shortens the antibody reaction time, and improves the reactivity with the antibody (Patent Document 1) A method in which a specimen is pretreated with a proteolytic enzyme and the staining property is improved by using an evaporation inhibitor (Patent Document 2), and when the antigen is inactivated during the preparation process of the sample, the method is activated to improve the staining property ( Non-patent document 3, Non-patent document 4) and the like have been reported.

However, such a technique can improve antigen staining, but cannot reduce non-specific staining. On the other hand, there is a problem that the operation becomes complicated and the specimen is easily detached. It was.
JP-A-8-304388 JP 2001-231552 A Medical technology Separate volume All new dyeing methods p200 ~ 211 Pathology and Clinical Medicine, 23 (3), p309-316, 2005 Inspection and Technology, 28 (11), p1331-1336, 2000, Inspection and Technology, 29 (7), p910-915, 2001

  It is an object of the present invention to provide an antigen detection method that can easily reduce non-specific staining in an immunochemical staining method.

  In the immunochemical staining method, the present inventors added an antibody solution to an antigen in a specimen, and then covered the antibody solution with a resin film that is not reactive with the antibody, thereby uniformizing the antibody concentration in the antibody solution. It was found that the sample could be prevented from drying out and non-specific staining could be reduced.

  That is, the present invention relates to an antigen detection method for detecting an antigen in a specimen using an immunochemical staining method, wherein the antibody liquid is dropped onto an analyte placed on a flat plate, and then the antibody liquid is combined with an antibody. The present invention relates to a method for detecting an antigen, wherein the antigen-antibody reaction is performed by coating with a non-reactive resin film.

  The present invention also relates to a resin film for coating an antibody solution used in the antigen detection method.

  The present invention also relates to an antigen detection kit for detecting an antigen in a sample using an immunochemical staining method, a plate for mounting the sample, an antibody solution, a resin film that is not reactive with an antibody, and a color development The present invention relates to an antigen detection kit containing a reagent.

  The present invention also relates to a method for detecting an antigen in a specimen using an immunochemical staining method, wherein an antibody solution is dropped onto a specimen placed on a flat plate, and then the antibody liquid is a resin that is not reactive with an antibody. The present invention relates to a method for preparing a stained specimen, which is covered with a film, subjected to an antigen-antibody reaction, and then stained.

  According to the method of the present invention, non-specific staining can be reduced by a simple operation regardless of the type of antigen or antibody in various methods of immunochemical staining. Since this method can be applied regardless of the nature of the specimen, not only tissue search and pathological tissue search that require immunochemical staining, but also confirmation of the expression of various antigens in cultured cells, blood cells, and biopsy materials. It is also useful when examining distributions.

The antigen detection method of the present invention is an antigen detection method for detecting an antigen in a specimen using an immunochemical staining method, wherein the antibody liquid is dropped onto a specimen placed on a flat plate, and then the antibody liquid Is coated with a resin film that is not reactive with an antibody, and an antigen-antibody reaction is performed.
The immunochemical staining method is a staining method for visualizing a specific antigen using an antigen-antibody reaction on a tissue or cell. The immunoassay method including the immunochemical staining method includes a direct method in which a labeled antibody (primary antibody) obtained by binding a labeling substance to an antibody against an antigen to be detected is directly reacted with the antigen and then visualized. There is an indirect method in which an antibody against the antigen (primary antibody) is reacted with the antigen and then visualized by reacting a labeled antibody (secondary antibody) in which a labeling substance is bound to the antibody that recognizes the antibody, Any of them may be used in the present invention, and may be appropriately selected for each examination according to the types of antigens and antibodies in the specimen.
As the labeling substance, a fluorescent substance, an enzyme, or the like can be used, but it is preferable to use an enzyme.

As a specimen used in the antigen detection method of the present invention, any tissue or cell can be used, and any of biological materials such as cells collected from biological tissues and ecology, and cultured cells can be used.
Among these, for biological tissue, it is preferable to use a tissue section (paraffin section) embedded in paraffin and sliced, or a tissue section (frozen section) sliced after freezing.
The specimen can be appropriately subjected to known antigen activation treatment within a range that does not cause peeling or damage of the specimen according to the type of the detected antigen. Examples of such treatment include heating with an autoclave or a microwave or a proteolytic enzyme treatment.

The specimen is placed on a flat plate, but for the presence or absence of fixation and the fixing method, a known method may be appropriately selected for each test depending on the type of specimen and antibody. It may be placed or smeared on a flat plate.
The material of the flat plate is not limited as long as it can place a specimen and can hold a resin film, but it is made of plastic or glass because it is easy to observe the specimen under an optical microscope. The flat plate etc. are mentioned. It is preferable to use a silane-coated slide glass for preparing a tissue specimen, particularly from the viewpoint of less specimen peeling.

The resin film used for this invention should just be a sheet | seat which has no reactivity with an antibody and can cover an antibody liquid.
Specifically, the specific gravity is sufficiently lighter than the antibody solution and has water repellency, and it can hold a sufficient antibody solution on the specimen while floating on the antibody solution, and has sufficient flexibility. A resin film that can be peeled off without damaging the specimen during peeling is preferable.

Such a resin film preferably has a specific gravity of 0.7 to 0.9, more preferably 0.75 to 0.85, particularly 0.80 ± 0.10 mg / cm ³ , and an antibody diluted 100 times It is desirable to have water repellency such that the contact angle between the liquid and the film surface is 80 ° to 120 °, more preferably 85 ° to 115 °, particularly 98.0 ± 10.5 °.
Here, the contact angle between the antibody solution and the film surface refers to the angle formed by the film surface and the tangent to the film of the antibody solution (when a 100-fold diluted antibody is used).

  The maximum point weight when the buckling test is performed by the bulk softness method (JIS standard) is 3.0N to 6.0N, more preferably 3.3N to 5.6N, particularly 4.6 ± 0. More preferred is 9N.

  The thickness of the resin film is more preferably 0.08 mm to 0.14 mm, further 0.9 mm to 0.13 mm, and particularly 0.12 ± 0.07 mm.

The film is more preferably transparent or translucent from the viewpoint of increasing the certainty and simplicity of the experimental operation, such as preventing bubbles from entering the antibody solution.
Examples of such resin films include polyester resin films such as PET (polyethylene terephthalate) and PTT (polytriethylene terephthalate), polyolefin resin films such as polypropylene, and polyamide resin films PVC (polychlorinated) such as nylon films. Vinyl) resin film, polyvinylidene chloride resin film, and the like. Commercially available films such as “Saran Wrap ^™ ” can also be used, and “Parafilm ^™ ” is particularly preferable.

An antigen-antibody reaction is performed in a state where the antibody solution is covered with a resin film. As a result, the reaction can be performed in a state where the antibody concentration in the antibody solution is uniformized. At the same time, it is possible to prevent the specimen from drying.
The method of the present invention can be applied regardless of the type of antigen or antibody, and the concentration of the antibody in the antibody solution should be optimized for each test so that the staining sensitivity is increased due to the activity and specificity of the antibody. What is necessary is just to determine suitably. The reaction time of the antibody is closely related to the reaction temperature, but an optimal reaction time and temperature may be set for each test. For commercially available antibodies, the reaction may be performed at the recommended reaction time or temperature. .

An antigen detection procedure using the method of the present invention will be described below (see FIG. 1).
1) Place the specimen on a flat plate.
2) The antibody solution is dropped on the specimen.
The dropping of the antibody solution may be performed with a micropipette or the like.
3) Cover the antibody solution with a resin film, and cover the entire antibody solution with the resin film.
The resin film is placed on the antibody solution so that dust does not enter. At that time, it is preferable to remove air bubbles between the film and the antibody solution by gently covering with tweezers or the like.
4) Incubate under specific conditions for antigen / antibody reaction.
5) After the reaction is completed, the resin film is removed, a coloring reagent for washing and staining is added to visualize the antigen, and a stained specimen for measurement is prepared.
When an indirect method is used for the measurement, an antibody solution containing a primary antibody is added, the antibody solution is coated with a resin film, and then an antigen-antibody reaction is performed. After completion of the reaction, the resin film is removed, the primary antibody is washed and removed, an antibody solution containing a labeled antibody (secondary antibody) is added, and the reaction is carried out by coating the antibody solution with a resin film. What is necessary is just to dye.
6) The visualized antigen (stained specimen) is measured.
Examples of the measuring means include observation under an optical microscope.

  The kit of the present invention is a kit for carrying out the above-described antigen measurement method of the present invention, and contains at least a plate for mounting a specimen, an antibody solution, a resin film that is not reactive with the antibody, and a coloring reagent. Is.

  Thus, by using the method of the present invention, as shown in the examples described later, nonspecific staining can be reduced regardless of the type of antigen or antibody, staining unevenness can be reduced, and more accurately. Antigens can be visualized for identification and verification.

Example 1 TUNEL staining by avidin biotin method for detecting apoptosis In order to detect apoptosis in rat tissue, immunostaining was performed by direct method using Apoptosis in situ detection kit (Wako Pure Chemicals, No. 295-5301). Carried out. Deparaffinized (xylene, 5 minutes x 3 times), and washed with water (Milli-Q water, 5 minutes x 3 times). Antigen was activated by proteolytic treatment (37 ° C / 30 minutes) with an enzyme (diluted 2000 times with Milli-Q). Subsequently, the section was washed with PBS (pH 7.4 (−)) in a staining vat (2 minutes × 4 times). Biotin-labeled deoxyuridine triphosphate (hereinafter referred to as b-dUTP) was added (50 μl / 37 ° C./30 minutes) to the 3 ′ end of DNA fragmented by apoptosis in the specimen using the attached TdT. After adding B-dUTP, the sections were washed with PBS in a staining vat (5 minutes × 3 times). The sections were inactivated with endogenous peroxidase with 3% hydrogen peroxide (room temperature / 5 minutes) and washed with PBS in a staining vat (5 minutes × 2 times). Next, add a peroxidase-labeled primary antibody (100 μl per slice: preferably 200 μl, but the amount of antibody solution can be halved by using a resin film) to the b-dUTP extended chain on the slide. Then, the antibody solution was coated (a schematic diagram of this operation process is shown in FIG. 1). After removing air bubbles between the film and the antibody solution, the sections were incubated (37 ° C / 30 minutes or 4 ° C / overnight). The section was washed with PBS in a staining vat (5 minutes × 3 times) to remove excess antibody, and then DAB reaction solution (200 μl) was added onto the section under a microscope to develop color. For sections, DAB color development was stopped in tap water, and nuclear staining was performed with 5% methyl green (room temperature / 5 minutes), followed by dehydration (ethanol, 10 seconds x 3 times), clearing (xylene, 10 seconds) × 3 times), enclosed (violite), and used for microscopic examination. A result is shown in FIG. 2 with the case where it dye | stains by the method which does not use the conventional sheet | seat.

    As shown in FIG. 2, in the sample (A) immunostained by the method of the present invention using a resin film, compared to the sample (B) stained by the conventional method without using the method of the present invention, non-specific It became clear that the specific staining, especially the non-specific staining observed around the specimen, can be reduced. This was thought to be due to the fact that in the method of the present invention, the reaction was carried out in a state where the antibody concentration in the antibody solution was made uniform, and at the same time, drying of the specimen was prevented.

FIG. 1 is a diagram showing an experimental process when an antibody solution is coated with a resin film. FIG. 2 is a graph showing the results of TUNEL staining of formalin-fixed and paraffin-embedded specimens of rat liver. (A): When a resin film is used, (B): When a resin film is not used

Claims (6)

  An antigen detection method for detecting an antigen in a specimen using an immunochemical staining method, wherein the antibody liquid is dropped onto a specimen placed on a flat plate, and then the antibody liquid is not reactive with an antibody. A method for detecting an antigen, characterized in that an antigen-antibody reaction is carried out.   The detection method according to claim 1, wherein the resin film has a specific gravity of 0.7 to 0.9.   The detection method according to claim 1 or 2, wherein the resin film has a contact angle of 80 ° to 120 ° between the antibody solution diluted 100 times and the film surface.   The resin film for coat | covering the antibody liquid used in the detection method of the antigen of any one of Claims 1-3.   An antigen detection kit for detecting an antigen in a sample using an immunochemical staining method, comprising a plate for mounting the sample, an antibody solution, a resin film that is not reactive with the antibody, and a color detection reagent kit.   In a method for detecting an antigen in a specimen using an immunochemical staining method, an antibody solution is dropped onto a specimen placed on a flat plate, and the antibody solution is then coated with a resin film that is not reactive with the antibody. A method for preparing a stained specimen, comprising performing an antibody reaction and then staining.