JP2009121906A - Antigen activating method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a more effective antigen activating method for a target antigen substance, when conducting an immunohistochemical staining by using an antibody recognizing the target antigen substance present in a tissue or a cell about a sample being previously paraffin-embedded. <P>SOLUTION: In an analysis of the paraffin-embedded sample using the immunohistochemical staining, where the sample is to be processed with a reducing agent prior to the immunohistochemical staining. In a protein structure, an intrinsically present S-S link and an S-S link formed when being fixed are cut off, whereby antigenicity can be recovered due to the linearization of the target protein. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an antigen activation method. More specifically, in the field of pathology or the like, when immunohistochemical staining is performed for a specific antigen present in a tissue or cell using a formalin-fixed, paraffin-embedded specimen of a tissue or cell that is routinely processed And a method for recovering the antigenicity of the antigenic substance.

  Immunohistochemistry (IHC) is a method for analyzing specific proteins in individual cases using an antibody that recognizes the expressed protein, and can be analyzed with a paraffin block. Immunohistochemical staining is a staining method widely used pathologically, visualized by using an antibody that recognizes a specific antigen present in a tissue or cell, and observed under an optical microscope or an electron microscope. This is a staining method devised for this purpose. In specimens fixed and embedded with aldehyde or normal formalin / paraffin, cross-linking occurs that masks antigens present in tissues and cells during the preparation of the specimen, resulting in loss of immunogenicity of the antigen. It is often experienced that recognition is impaired. Some antibodies can be recognized by Western blotting but cannot be recognized by immunohistochemistry.

  In order to prevent a decrease in immunoreactivity due to formalin fixation, 1) proteolytic enzyme treatment such as pepsin and trypsin, 2) heat treatment, pressure treatment and the like are performed. In proteolytic enzyme treatment, a part of the protein is digested and extracted, and the antibody easily comes into contact with the antigen. However, dramatic activation of antigenicity cannot often be expected. For heating and pressurization, a method using a microwave oven or an autoclave can be used. In the heat treatment, the cross-linking of the protein by formalin is cut and a part of the protein and nucleic acid is extracted, so that the antibody is likely to come into contact with the antigen, but it is still not sufficient.

  In addition, a method of pretreatment using CCA (system name citraconic anhydride (2-methylmaleic anhydride)) at the time of pretreatment of immunostaining is disclosed (Patent Document 1).

  On the other hand, frozen sections may be used in order to prevent a decrease in immunoreactivity due to specimen sections with formalin and paraffin. However, since frozen sections have poor tissue morphology compared to paraffin sections, the current amount of information obtained from histological and cytological observations is poor. Therefore, a specimen preparation method useful for immunohistochemical and enzyme histochemical analysis has been demanded.

  As a preparation method useful for morphological, immunohistochemical and enzymatic histochemistry of various organs or tissues (excluding hard tissues), it is fixed with PLP (periodate-lysine-paraformaldehyde) solution, and AMeX (aceton, A method using a paraffin embedding method by a methyl benzonate and xylene-method) method is also disclosed (Patent Document 2).

However, neither method is sufficient, and a more effective method is required.
Japanese Patent No. 3797418 Special Table No. 2002-82026

  The present invention is more effective when immunohistochemical staining is performed on a specimen after embedding in paraffin using an antibody (primary antibody) that recognizes the target antigen substance present in the tissue or cells. It is an object to provide a method for performing activation.

  The present inventors focused on the structure of the protein in order to solve the above problems, and as a result of intensive studies, the objective is to cut the SS bond originally present and the SS bond formed at the time of fixation. The present inventors have found that antigenicity can be recovered by protein linearization, thereby completing the present invention.

That is, this invention consists of the following.
1. A method for activating an antigenic substance of interest, characterized by treating a specimen with a reducing agent before immunohistological chemical staining when analyzing the specimen after embedding in paraffin by immunohistological chemical staining.
2. 2. The activation method according to item 1 above, wherein the reducing agent is a mercapto-based reducing agent, an aromatic thiol-based reducing agent, an organic phosphate compound and / or a pyruvate amide-based reducing agent.
3. 3. The activation method according to item 2, wherein the mercapto-based reducing agent is 2-mercaptoethanol and / or 2-mercaptoethanolamine.
4). 3. The activation method according to item 2 above, wherein the aromatic thiol-based reducing agent is dithiothreitol.
5). 3. The activation method according to item 2 above, wherein the organophosphate compound is TCEP (Tris [2-carboxyethyl] phosphine).
6). 6. The activation method according to any one of 1 to 5 above, wherein the reducing agent is added in an amount of 0.01 to 50 times the molar amount of the target molecule in the specimen.
7). A method for activating an antigenic substance of interest characterized by combining the activation method according to any one of items 1 to 6 above and heat-treating the specimen after embedding with paraffin at 30 to 130 ° C.
8). 8. A method for activating a target antigenic substance, comprising performing the activation method according to any one of items 1 to 7 in combination with a proteolytic enzyme treatment.
9. A tissue or cell analysis method, characterized in that the paraffin-embedded specimen treated by the activation method according to any one of items 1 to 8 is analyzed by immunohistochemical staining.
10. A kit for analyzing tissue or cells by immunohistochemical staining comprising at least a reducing agent.

  According to the method for activating a target antigenic substance in which a paraffin-embedded specimen according to the present invention is treated with a reducing agent, the SS bond originally present in the protein and the SS bond formed at the time of tissue or cell fixation are cleaved. When the target antigen protein is linearized, the antigenicity is restored, and stronger expression of the target protein is observed. By this method, more effective immunohistochemical staining can be performed, and pathological analysis of tissues or cells can be accurately performed.

In the present invention, a method known per se can be applied as a method for preparing a paraffin-embedded specimen.
The fixation of tissues and cells is as follows: 1) Immediately immobilize tissues and cells and keep them close to the living body 2) Prevent autolysis and decay, minimize post-mortem changes 3) Reproducible This is done for the purpose of maintaining the tissue and cell image, 4) giving contrast to the structure of the target tissue and cells, and the like. There are chemical fixation and physical fixation. Examples of the fixing agent used for chemical fixation include formaldehyde (formalin), glutaraldehyde, osmium tetroxide and the like. Formaldehyde and glutaraldehyde mainly fix proteins. Formaldehyde penetrates quickly into large pieces of tissue. Examples of physical fixation include 1) denaturation by boiling or microwave irradiation (thermal fixation), 2) precipitation with an organic solvent such as ethanol or acetone, 3) denaturation with acetic acid or trichloroacetic acid, precipitation, or 4) freezing.

  After fixation, a degreasing operation or a deashing operation can be further performed according to the properties of the specimen such as tissue or cells. For example, when embedding tissues with a high fat content in the specimen, such as mammary gland, subcutaneous tissue, bone marrow, etc., defatting by fixation with acetone, chloroform, methanol / chloroform mixture (1: 1or1: 2), etc. The operation can be performed. Also, if the specimen contains lime, such as bones, teeth, calcified lesions, kidneys or gallbladder containing stones, it is difficult to slice the specimen. Prior to embedding, a decalcification operation can be performed to remove lime in advance in order to facilitate slicing with a microtome from the calcified tissue. In general, a method of treating with acids such as nitric acid, formic acid, and trichloroacetic acid can be mentioned.

  Next, ethanol is usually used for dehydration. If the tissue is suddenly put into a concentrated ethanol, the tissue will be strongly shrunk and hardened. For example, 70% → 80% → 90% → 100%. it can. Furthermore, in order to infiltrate the paraffin into the specimen, it can be treated with, for example, xylene. First, the specimen can be immersed in a 1: 1 xylene / paraffin mixture, and then in a paraffin liquid.

  The above-treated specimen can be put into a mold, and melted paraffin can be poured and solidified and embedded in paraffin to prepare a paraffin-embedded specimen. The processing temperature of paraffin can be appropriately selected according to the type of paraffin, specimen, and purpose. When observing a tissue embedded in paraffin under a microscope, it is necessary to cut a thick tissue piece thinly and allow a transmitted light to pass through sufficiently. As described above, a specimen such as a tissue piece can be cut into thin pieces using, for example, a microtome after applying fixation, dehydration, and embedding operations to give a certain hardness.

  The sliced section of the formalin-fixed paraffin-embedded tissue obtained as described above is deparaffinized with, for example, xylene, and then subjected to an antigen-antibody reaction with an antibody that recognizes the target antigen for immunohistochemical staining.

  Immunohistochemical staining is a method of visualizing the localization using an antibody against a target antigen substance. Since an antibody recognizes a target antigenic substance, that is, a small part of a peptide of a target protein as an epitope, an SS bond that is inherent to the protein or formed by immobilization treatment has a disadvantageous structure for antibody recognition. sell. When immunohistochemical staining is performed, the target protein present in the fixed tissue or cell is not returned to the state of the living body, but is brought closer to the state of a linear peptide recognized when the antibody is produced, The antigenicity of the target antigen substance can be activated.

  The activation treatment of the target antigenic substance of the present invention can be performed before the antigen-antibody reaction with the primary antibody. The activation treatment of the target antigenic substance of the present invention is not particularly limited as long as the antibody against the target antigenic substance is applied, but after considering the effect of the reducing agent, the amount used, etc. It is desirable to process. Specifically, it may be after the sliced slice is placed on a slide and deparaffinized.

  Here, the reducing agent used is not particularly limited as long as it can cleave the SS bond in the protein, and specifically, a mercapto reducing agent, an aromatic thiol reducing agent, an organic phosphate compound, and And / or pyruvamide reducing agent. Examples of the mercapto-based reducing agent include mercaptoethanol, and more specifically, 2-mercaptoethanol and / or 2-mercaptoethanolamine. Examples of the aromatic thiol-based reducing agent include dithiothreitol (DTT), and examples of the organic phosphate compound include TCEP (Tris [2-carboxyethyl] phosphine). Further, an alkaline agent such as ammonia, monoethanolamine, ammonium hydrogen carbonate, sodium hydroxide or the like may be used in combination with a reducing agent such as thioglycolic acid and salts thereof, cysteine and salts thereof, and sulfite.

  The amount of reducing agent to be used is 0.01 to 50-fold mol, preferably 15 to 30-fold mol, more preferably about 20-fold mol amount relative to the amount of the target molecule in the sample, that is, the target antigenic substance. be able to. In order to use at the above-mentioned concentration, the reducing agent can be dissolved using a buffer or the like, and the concentration is 1 to 50 (v / v%), preferably 2 to 10 (v / v%). can do. As the buffer solution, one not mixed with metal ions or an oxidizing agent can be used. Specifically, a citrate buffer solution, an EDTA solution, phosphate buffered saline (PBS), or the like can be used.

  The treatment time of the specimen after paraffin embedding with the reducing agent is not particularly limited as long as the SS bond of the target antigen substance can be cleaved, but for example, about 1 to 60 minutes, preferably about 5 to 20 minutes, More preferably, it can be about 15 minutes.

  The activation process of the target antigen substance may be performed in combination with a method known per se. The specimen after embedding in paraffin can be processed by combining, for example, the above activation treatment and heat treatment at 30 to 130 ° C., preferably 50 to 125 ° C., more preferably 95 to 121 ° C. Said activation process and heat processing may be performed simultaneously, and may be performed in a different order. When the heat treatment and the reducing agent treatment are performed in different orders, any of them may be performed first. For example, with the reducing agent added, the heat treatment can be performed for a necessary time within the above temperature range. Further, a proteolytic enzyme treatment may be combined. The proteolytic enzyme is not particularly limited as long as it is an enzyme that can be used in the immunostimulation method. For example, a known enzyme such as pepsin or trypsin can be used.

  After the activation treatment of the target antigen substance, an antibody (primary antibody) that recognizes the target antigen substance is allowed to act on the target antigen substance to cause an antigen-antibody reaction. The primary antibody can be used according to the target antigenic substance, and a primary antibody known per se, in particular, an antibody already on the market can be used.

  The present invention also extends to a tissue or cell analysis method in which a paraffin-embedded specimen treated by the activation method is analyzed by immunohistochemical staining. Further, the present invention extends to a kit for analyzing tissue or cells by immunohistochemical staining, which contains at least the above-described reducing agent. In addition to the reducing agent, the kit may contain a solvent for the reducing agent, a primary antibody, a labeled antibody, a labeled primary antibody, and the like.

  Examples of the present invention will be described below in more detail, but the present invention is not limited to these, and various applications are possible without departing from the technical idea of the present invention. It is.

(Example 1) Liver cancer lung metastases (confirmation of RFP)
1) About the liver cancer lung metastasis | tissue, the sample by paraffin embedding was produced in accordance with the general method, and the thin section was produced using the microtome.
2) Treat the sliced slices with xylene 100% -5 minutes, 100% -5 minutes, 100% -5 minutes, methanol 100% -1 minutes, 100% -1 minutes, 100% -1 minutes, Deparaffinization operation was performed.
3) Thereafter, the sample was washed with running water for 5 minutes to obtain a pretreated sample.
4) For the pretreated sample obtained as described above, antigen activation treatment was performed by the following method a) or b).

a) With reducing agent Pretreatment sample (slide glass) was added to 0.01M citric acid solution (179 mL) pH 7.0 and 0.5% (v / v) polyoxyethylene alkylphenol (Igepal ^(R) Ca-630) (1 mL ) And 10% (v / v) -2 mercaptoethanol (20 mL), and then autoclaved at 121 ° C. for 15 minutes.
b) No reducing agent The pretreated sample (slide glass) was placed in a vat containing a solution containing 0.01M citric acid solution (199 mL) pH 7.0 and 0.5% (v / v) polyoxyethylene alkylphenol (1 mL). And then autoclaved at 121 ° C. for 15 minutes.

5) Immunohistochemical staining Each antigen-activated sample of a) or b) above was washed 3 times for 5 minutes each using phosphate buffered saline (PBS). In order to suppress endogenous peroxidase, it was left in a vat containing 150 mL of 3% H ₂ O ₂ (15 mL of 30% H ₂ O ₂ +135 mL of methanol) for 15 minutes. Again, it was washed 3 times for 5 minutes each with PBS. Care was taken not to dry the tissue, and the surrounding PBS was thoroughly wiped with tissue paper.

  A blocking solution contained in an immunohistochemical staining kit (UltraTech HRP Streptavidin-Biotin Detection System (Beckman Coulter)) was dropped onto the sample and allowed to stand at room temperature for 10 minutes. Thereafter, the blocking agent was removed and the primary antibody was added to the sample. As the primary antibody, an anti-RFP rabbit polyclonal antibody (concentration: 1 μg / mL) was used, and 100 μL of the antibody solution was applied to the sample and allowed to stand at room temperature for 60 minutes.

  Thereafter, the plate was washed three times for 5 minutes each with PBS, the biotin-labeled secondary antibody contained in the kit for immunohistochemical staining was added, and the mixture was allowed to stand at room temperature for 10 minutes. The plate was washed 3 times for 5 minutes each with PBS, enzyme-labeled streptavidin solution was added, and the mixture was allowed to stand at room temperature for 10 minutes. The plate was washed 3 times for 5 minutes each with PBS, and diaminobenzidine (DAB) coloring solution was added to confirm color development at room temperature for 1 to 5 minutes. After color development was stopped with PBS, the plate was washed with running water for 5 minutes and stained with hematoxylin for 20 seconds. Colored at 50 ° C for 1 minute, washed with running water for 1 minute, and dehydrated (100% -1 minute methanol, 100% -1 minute, 100% -1 minute, xylene 100% -1 minute, 100% -1 minute, 100 % -1 min) and enclosed.

  The tissue was observed with an optical microscope.

(Example 2) Breast cancer tissue (confirmation of androgen receptor)
The breast cancer tissue was subjected to the operations 1) to 3) in the same manner as in Example 1 to obtain a pretreated sample. For the pretreated sample obtained as described above, the antigen activation treatment was performed by the following method a) or b).
a) With reducing agent Pre-treated sample (slide glass), 0.01 M citric acid solution (179 mL) pH 7.0 and 0.5% (v / v) polyoxyethylene alkylphenol (1 mL) and 10% (v / v)- It was immersed in a vat containing a solution containing 2 mercaptoethanol (20 mL) and autoclaved at 121 ° C. for 15 minutes.
b) Without reducing agent Pre-treated sample (slide glass) is placed in a vat containing a solution containing 0.01 M citric acid solution (199 mL) pH 7.0 and 0.5% (v / v) polyoxyethylene alkylphenol (1 mL). It was immersed and autoclaved at 121 ° C. for 15 minutes.

  The primary antibody for immunohistochemical staining was stained by the same method as in Example 1 except that an anti-androgen receptor mouse monoclonal antibody (Dako) (concentration: 11 μg / mL) was used. Enclosed. The structure was observed by the same method as in Example 1.

(Example 3) Breast cancer tissue (confirmation of CD24)
The breast cancer tissue was subjected to the operations 1) to 3) in the same manner as in Example 1 to obtain a pretreated sample. For the pretreated sample obtained as described above, the antigen activation treatment was performed by the following method a) or b).
a) With reducing agent Pre-treated sample (slide glass) is placed in a vat containing a solution containing 0.01 M citric acid solution (180 mL) pH 7.0 and 10% (v / v) -2 mercaptoethanol (20 mL). It was immersed and autoclaved at 121 ° C. for 15 minutes.
b) No reducing agent The pretreated sample (slide glass) was immersed in a vat containing a solution containing 0.01 molar citric acid solution (200 mL) pH 7.0 and autoclaved at 121 ° C for 15 minutes.

  Staining was performed in the same manner as in Example 1 except that an anti-CD24 mouse monoclonal antibody (NeoMarkers) (concentration: 10 μg / mL) was used as the primary antibody for immunohistochemical staining. The structure was observed by the same method as in Example 1.

  As described in detail above, according to the method for activating a target antigenic substance in which a specimen after embedding in paraffin according to the present invention is treated with a reducing agent, it is formed at the time of fixation of tissue or cells with SS bonds originally present in proteins. The SS bond was cleaved, the antigenicity was restored by linearization of the target protein, and stronger expression of the target protein was observed. By this method, more effective immunohistochemical staining can be performed, and pathological analysis of tissues or cells can be accurately performed. Therefore, the immunostimulation method of the present invention can be used in pathological analysis institutions and medical basic research fields. Furthermore, when analysis is performed using the tissue or cell analysis kit of the present invention for immunohistochemical staining, analysis can be performed accurately and easily, and therefore, the minimum necessary specimen may be prepared. It is also economically superior.

It is a figure which shows the analysis result in the immunohistochemistry by the anti-RET finger protein (FRP) antibody of a liver cancer lung metastasis | tissue (thin slice tissue). In both cases, tissue sections that had passed about one year after the preparation of the sliced sections were used. In general, it is known that in the case of a nuclear antigen, color development by immunostaining decreases with time after preparation of a sliced section. a) The antigen activation treatment of the present invention in which treatment is performed using a reducing agent. Strong expression of the antigen is observed in the nucleus of the cancer cell. b) An antigen activation treatment was performed by heat treatment containing a surfactant without using a reducing agent. Slight staining is observed in the nuclei of some cancer cells. Example 1 It is a figure which shows the analysis result in the immunohistochemistry by the anti-androgen receptor antibody of a breast cancer tissue. a) The antigen activation treatment of the present invention in which treatment is performed using a reducing agent. Staining is observed in almost the entire nucleus of cancer cells. b) An antigen activation treatment was performed by heat treatment containing a surfactant without using a reducing agent. Slight staining is observed in the nuclei of some cancer cells. (Example 2) It is a figure which shows the analysis result in the immunohistochemistry by the anti-CD24 antibody of a breast cancer tissue. a) The antigen activation treatment of the present invention in which treatment is performed using a reducing agent. Strong staining is observed in the cytoplasm and cell membrane of almost all cancer cells. b) Antigen activation treatment by heat treatment without using a reducing agent. Staining is observed in the cytoplasm and membrane of only a few cancer cells. (Example 3)

Claims (10)

A method for activating an antigenic substance of interest, characterized by treating a specimen with a reducing agent before immunohistological chemical staining when analyzing the specimen after embedding in paraffin by immunohistological chemical staining. The activation method according to claim 1, wherein the reducing agent is a mercapto-based reducing agent, an aromatic thiol-based reducing agent, an organic phosphate compound, and / or a pyruvate amide-based reducing agent. The activation method according to claim 2, wherein the mercapto-based reducing agent is 2-mercaptoethanol and / or 2-mercaptoethanolamine. The activation method according to claim 2, wherein the aromatic thiol-based reducing agent is dithiothreitol. The activation method according to claim 2, wherein the organic phosphate compound is TCEP (Tris [2-carboxyethyl] phosphine). The activation method according to claim 1, wherein the reducing agent is added in an amount of 0.01 to 50 times the molar amount of the target molecule in the specimen. A method for activating an antigenic substance of interest, comprising performing the activation method according to any one of claims 1 to 6 and a heat treatment at 30 to 130 ° C on a specimen after embedding in paraffin. A method for activating a target antigenic substance, comprising performing the activation method according to any one of claims 1 to 7 in combination with a proteolytic enzyme treatment. A tissue or cell analysis method comprising analyzing a paraffin-embedded specimen treated by the activation method according to claim 1 by immunohistochemical staining. A kit for analyzing tissue or cells by immunohistochemical staining comprising at least a reducing agent.