JPS62274260A - Novel multiimmunohistochemical dyeing method and kit for dyeing cell using said method - Google Patents

Abstract

PURPOSE:To obtain a multiimmunohistochemical dyeing method which permits easy and simultaneous dyeing of the antigen of cells or tissue by bringing a labeling antibody and the antigen of the cell of tissue and immunochemically dyeing the respective markers. CONSTITUTION:The antigen of the cell or tissue such as the surface antigen of the lymphocyte and the labeling antimonoclonal antibody are brought into reaction. A labeling method by peroxidase or biotin is adequate for the labeling. Dyeing by a PAP method, etc., is then executed. The biotinated monoclonal antibody is subjected to the dyeing by a glucose oxidase dyeing reagent by executing a composite adipin/biotin method using the glucose oxidase as indicating enzyme via the labeled biotin and repeating said method. The above- mentioned reactions are simultaneously progressed for the plural antigens.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a multiplex immunohistochemical staining method and a cell staining kit using the same.

[Conventional technology]

Tissue staining methods in the clinical field have mainly been used to observe the structure of a living body at the cellular level using a microscope and to detect abnormalities in living tissue that occur at that location. In this group-m staining method, immunofluorescence has traditionally been used for antigen identification and local immunological diagnosis. Fluorescently labeled antibodies against antigens in cellular tissues are replacing enzyme-linked antibodies in recent years, and with advances in these immunohistochemical staining techniques,
Cell functions became visible. however,
Conventionally used antiserum is a polyclonal antibody and contains antibodies that react with other tissue antigens than the target tissue antigen, which has the disadvantage of causing non-specific reactions. Recently, three monoclonal antibodies have been used for immunohistochemistry staining, and the useful information obtained from clinical tests such as tumor diagnosis has increased significantly, and the fields of application have rapidly expanded.

Up until now, much useful knowledge has been accumulated through antigen recognition using a single monoclonal antibody, and it has become useful for disease diagnosis, but if it were possible to simultaneously stain multiple antigens on the same Mi tissue section, it would be possible. It becomes possible to know the relationship in which antigens are distributed on cells or within tissues, and dramatically superior information can be obtained.

Double immunostaining techniques include: (1) Fluorescent antibody method that labels different fluorescent dyes (Masaaki Abe et al., Pathology and Subjects 1)
984.2:1512-1514), (21) immunoenzymatic histological techniques using different enzymes, and (3) methods of differential staining using the same enzyme but changing the chromogenic substrate.

[Problem that the invention seeks to solve]

In particular, there are many problems that must be solved when performing double staining using two types of mouse monoclonal antibodies. That is, in order to increase the staining intensity, the avidin-biotin complex (A B C) method and the peroxidase anti-peroxidase (PAP) method are used, but in this case, multiple anti-mouse Ig antibodies used as secondary antibodies are used. Conventionally, it has not been possible to use two types of mouse monoclonal antibodies simultaneously as primary antibodies because they do not discriminate between mouse monoclonal antibodies (secondary antibodies). In addition, in all conventional double staining methods, one type of mouse monoclonal antibody is reacted to develop color, and then another mouse monoclonal antibody is reacted. In this method, the second anti-mouse Ig antibody (secondary antibody) recognizes not only the second mouse monoclonal antibody (-second antibody) but also the -th mouse monoclonal antibody, resulting in cross-reactivity. It was inevitable that it would happen. In addition, although this cross-reactivity can theoretically be blocked with excess normal mouse serum, it has been difficult to completely block it in practice.

A first object of the present invention is to provide a novel and simple multiplex immunohistochemical staining method for cell or tissue antigens and a kit for cell staining using the same.

A second object of the present invention is a novel multiplex immunohistochemical staining method, which is a multiplex immunohistochemical staining method characterized by simultaneously performing reactions between each labeled antibody and an antibody of cells/tissues. The present invention also provides a cell staining kit using the same.

A third object of the present invention is to provide a method for diagnosing T lymphocyte subsets using a novel multiplex immunohistochemical staining method and a kit for the same.

[Means for Solving the Problems] The means of the present invention for solving the above-mentioned problems is to use two or more different types of monoclonal antibodies so that each of the two or more types of monoclonal antibodies has one marker. A novel multiplex immunohistochemical staining method characterized by labeling a marker to produce a plurality of labeled antibodies, reacting the labeled antibody with a cell or tissue antigen, and immunochemically staining each marker, and To provide a kit for cell staining using the same.

Furthermore, the present invention provides a novel multiplex immunohistochemical method characterized in that surface antigens of T lymphocyte subsets are simultaneously reacted with peroxidase-labeled monoclonal antibodies and biotinylated monoclonal antibodies, and each labeled antibody is immunochemically stained. The present invention provides a staining method and a cell staining kit.

Furthermore, the present invention involves simultaneously reacting a peroxidase-labeled monoclonal antibody and a biotinylated monoclonal antibody with a surface antigen of a T lymphocyte subset, reacting the ζ-peroxidase-labeled monoclonal antibody with peroxidase anti-peroxidase antibody method, and reacting the biotinylated antibody with avidin-biotin-glucose oxidase. The object of the present invention is to provide a novel multiplex immunohistochemical staining method for diagnosing T lymphocyte subsets, which is characterized by staining with a method, and a cell staining kit.

Another object of the present invention is to provide a T lymphocyte subset diagnostic kit containing a peroxidase-labeled monoclonal antibody and a biotinylated monoclonal antibody as active ingredients.

In the present invention, first, a labeled monoclonal antibody is prepared. The monoclonal antibodies used are based on lymphocyte surface antigens, especially human T cells (
Examples include monoclonal antibodies against surface antigens of T lymphocytes. Such examples include monoclonal antibodies that recognize human tumor cell antigens or specific surface antigens of human 'r cells, which are prepared using conventional monoclonal antibody production methods, as well as commercially available monoclonal antibodies, such as OK series monoclonal antibodies (USA, 0rtho), Leu series monoclonal antibodies (USA, Bec
Kton Dickinson) and NU series monoclonal antibodies (Nichirei, Japan) can also be used. These monoclonal antibodies are then used for two or more types of antibodies depending on the purpose.

These monoclonal antibodies are then labeled with a labeling compound. Although various labeling methods are possible for labeling, in the present invention, labeling methods using peroxidase or biotin are preferred. It goes without saying that other known labeling methods are also applicable.

Biotinylation of monoclonal antibodies was performed by the method of Guesdon et al., and peroxidase labeling was performed by Wilson & Na.
Directly labeled monoclonal antibodies are prepared according to the method of Ka-ne. In the case of biotinylated antibodies, the degree of labeling is determined by the amount of free amino groups using the TNBS method, and
In the case of peroxidase-labeled antibodies, spectroscopically 0.0
The values of O403n and 0.0280 nm are calculated. For the peroxidase-labeled antibody, an anti-peroxidase antibody that identifies peroxidase is used as a secondary antibody, and after reacting with an anti-rabbit antibody using this rabbit antibody as an antigen, staining is performed by the PAP method. Furthermore, biotinylated monoclonal antibodies can be obtained using the avidin-biotin complex method (ABC-G), which uses glucose oxidase (Go) as an indicator enzyme via labeled biotin.

method), repeat the same operation to further intensify this reaction, and develop color with a glucose oxidase staining reagent. These reactions proceed simultaneously against multiple antigens.

Next, multiple staining of human 'r-cell subsets by the multiple staining method of the present invention will be described.

For the purpose of simultaneous color development of lymphocyte T subset, NU-Tll/■(
A peroxidase-labeled antibody for NU-TS/C (which recognizes helper T cells) and a biotinylated antibody for NU-TS/C (which recognizes suppressor T cells) are prepared. The production method is as described above, and labeled monoclonal antibodies are prepared according to each method. It is important to determine the degree of labeling based on protein chemistry, as there is a binding amount suitable for immunohistological staining.

When lymph nodes are stained with these antibodies under optimal conditions, NLJ-TH/T-positive cells turn red, while NU-TS/T-positive cells turn red.
C-positive cells are dyed blue. Both cells are easy to distinguish, and there is almost no overlap in staining.

The results of staining with these antibodies are influenced by the concentration of the antibody, the order of color development, and the quality of the PAP. Among these, the optimal concentration range for the antibody concentration is narrow, but it can be easily achieved by determining conditions for slightly dilute staining using the same method used to determine the optimal dilution in normal immunohistological reactions. be overcome.

Furthermore, another multiplex staining will be described.

NU-TH/I, NU-T using frozen sections of H.
Double staining of lymphocyte subsets with Sac is performed. For NU-TH/I, the immunostaining procedure used a rabbit anti-peroxidase antibody that identifies peroxidase labeled as a secondary antibody, and then reacted with an anti-rabbit antibody that uses this rabbit antibody (immunoglobulin) as an antigen. Later, PAP method is performed. For N U -T Sac, the avidin-biotin method using glucose oxidase (C, O) as an indicator enzyme is performed via the biotin labeled thereon, and the same manipulation is repeated to further strengthen this reaction. These two reactions are allowed to proceed simultaneously. The immunoreaction is carried out in four stages, and the reaction solution used in each stage contains a peroxidase-based staining reagent and a glucose oxidase-based staining reagent, each adjusted to an optimal concentration and mixed. The color reaction is preferably performed first with peroxidase and then with glucose oxidase. Regarding coloration, better results can be obtained if peroxidase coloration is performed first.

As an application field of the multiplex immunostaining method described above, measurement of T subsets is useful in diagnosing diseases in which various abnormalities such as tumors and infections exhibit immunodeficiency. With the method of the present invention, it is possible to visually confirm abnormalities in T subsets, which are of immunological interest, at the individual cell level from a perspective different from flow cytometry. ll! !
+ As an application to tumor diagnosis, by using the method of the present invention, it has been observed that lymphocytes come to 't+ K8 around the tumor site where necrosis has occurred, and in the early stage of the lesion, 'rS/C positive However, as the disease progresses, TS/C-positive cells decrease and most of them change to TH/I-positive cells.
It can provide information useful for prognosis.

In addition, although this is a very special example, the immunomultiplex staining method of the present invention can be used to observe cells that have markers for both TI (/T-positive cells and T S/C-positive cells) in human T-cell lymphoma. successful.

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

Example 1 A peroxidase-labeled antibody of NU-TH/I and a biotinylated antibody of NU-TS/C were prepared in the NU series monoclonal antibody for the purpose of simultaneous staining of double-infected negative lymphocyte T subset in Hentou group. did. The manufacturing method was as described above.

5 μm sections of the frozen hematopoietic tissue were prepared using a cryostat. This was fixed with acetone at -20°C for 5 minutes, and washed with 0.01M phosphate buffer (pH 7°4) (PBS washing). Add this to the blocking serum (
Approximately 100 μβ of 0.1% horse serum) was applied to each section and allowed to react for 10 minutes.

Peroxidase labeled N U -TH/I (X300)
100 μl of a mixed solution of NU-TS/C and biotinylated NU-TS/C (X40) was poured onto the plate, and the mixture was reacted at 37° C. for 20 minutes. After the reaction, the rabbit anti-peroxidase antibody (X
500) and avidin/biotin glucose oxidase was poured onto the plate, and the mixture was reacted at 37°C for 20 minutes. After the reaction, wash with PBS and add goat anti-rabbit Igr),
(X3000) and biotinylated glucose at 100. cr# was applied and the reaction was carried out at 37°C for 20 minutes. After the reaction, the plate was washed with PBS, and 100 μl of a mixed solution of rabbit peroxidase anti-peroxidase antibody and avidin/biotin/glucose oxidase was poured onto the plate and allowed to react at 37°C for 20 minutes. After the reaction, it was washed with PBS. Each reaction solution was diluted with PBS to the optimal concentration of each antibody. Approximately 100 μ per section! It was necessary.

Care must be taken not to allow the sections to dry out during the reaction.

Peroxidase color reaction is performed using ethylcarbazole (O
rtho's kit) was used to develop a red color.

After washing with distilled water, a glucose oxidase coloring reaction was performed. That is, 10 ml of Tris-HC1 buffer at pH 8.3
0mj? 670 mg of β-d-glucose and 67 mg of nitro blue tetrazolium (NBT) were dissolved in the solution and warmed to 37°C in advance. Immediately before use, add 1.67 mg of phenazine methosulfate (PMS) to block light.
The rack was immersed in this solution and allowed to react for 1 hour. After washing with distilled water, it was encapsulated in polyvinylpyrrolidone at 30°C.

As a result, NU-TH/I positive cells turn red and NU-TH/I positive cells turn red.
TS/C positive cells were stained blue.

Example 2 Double staining method for human tissue Double immunostaining of human tissue was carried out in the same manner as in Example 1, except that bundle-fixed frozen human tissue was used. As a result, they were able to differentiate lymphocytes infiltrating the dermis.

Example 3 Tissue from a necrotic tumor site was collected from a patient with a T-lymph tumor and subjected to double immunostaining in accordance with Example 1. As a result, lymphocyte infiltration was observed around the tumor site, and NU-TS/C-positive cells were observed at the early stage of the lesion, and as the lesion progressed, the number of NU-TS/C-positive cells decreased, and almost all NU-TS/C-positive cells were observed. - It was observed that the cells changed to TO/I positive cells.

Therefore, this information could be useful for the treatment and prevention of this disease.

Bottle 1: peroxidase labeled NU-TH/1 (0,
17μg/ml) Biotin-labeled NU-TS/C (1.25μg/ml Stabilizer (bovine albumin) Bottle 2: Rabbit anti-peroxidase antibody avidin stabilizer (bovine albumin) Bottle 3: Biotinylated glucose stabilization Agent (bovine albumin) Bottle 4: Goat anti-rabbit 1.gG biotinylated glucose stabilizer (bovine albumin) Bottle 5: Rabbit peroxidase anti-peroxidase antibody avidin stabilizer (bovine albumin) The protocol is illustrated using the example of hentou tissue.

5 μm sections were prepared from unfixed frozen hematopoietic tissue using a cryostat. This was fixed with acetone at -20°C for 5 minutes and washed with PBS.

100 μl of the block serum was added to this and allowed to react at room temperature for 10 minutes. After the reaction, discard the block serum and refill bottle 1 with 100μ! The mixture was heated and reacted at 37°C for 20 minutes.

After the reaction, the plate was washed with PBS, and 100 μl of a mixture of equal amounts of the solutions in bottles 2 and 3 was added to the plate, and the plate was reacted at 37° C. for 20 minutes. After the reaction, wash with PBS and bottle 4
0 μl was applied and the reaction was carried out at 37° C. for 20 minutes.

After the reaction, wash with PBS and mix equal amounts of the solutions in bottles 3 and 5 to 100μ! After the reaction, the mixture was washed with PBS and the peroxidase color reaction shown in Example 1 and then the glucose oxidase color reaction were performed. The solutions in bottles 2 and 3 and bottles 3 and 5 were mixed in equal amounts 30 minutes before use. Using this kit and protocol, several dozen Hentou tissues and Hif tissues were double-stained, and the differential staining of lymphocyte T subsets was observed very clearly and easily.

Claims (8)

[Claims] (1) Two or more different types of markers are labeled to form a plurality of labeled antibodies so that each of the two or more types of monoclonal antibodies becomes one marker, and the labeled antibodies are reacted with a cell or tissue antigen. A novel multiplex immunohistochemical staining method characterized by immunochemically staining each marker, and a kit for cell staining using the same. (2) A novel multiplex immunohistochemical staining method and a cell staining kit using the same according to claim 1, wherein the labeled antibody is a peroxidase-labeled monoclonal antibody and a biotinylated monoclonal antibody. (3) The staining means is peroxidase-antiperoxidase antibody (PAP method) for peroxidase-labeled monoclonal antibodies, and avidin-biotin-glucose oxidase for biotinylated monoclonal antibodies, according to claim 1 A new multiplex immunohistochemical staining method and a cell staining kit using it. (4) A novel multiplex immunohistochemical staining method and a kit for cell staining using the same according to claim 1, characterized in that a plurality of labeled monoclonal antibodies and an antigen are reacted simultaneously. (5) A novel multiplex immunohistochemical staining method and a kit for cell staining using the same according to claim 1, wherein the cells are T lymphocytes and a T lymphocyte subset is stained. (6) A novel multiplex immunohistochemical staining method characterized by simultaneously reacting surface antigens of T lymphocyte subsets with peroxidase-labeled monoclonal antibodies and biotinylated monoclonal antibodies, and immunochemically staining each labeled antibody; A kit for cell staining using it. (7) A peroxidase-labeled monoclonal antibody and a biotinylated monoclonal antibody are simultaneously reacted with the surface antigen of a T lymphocyte subset, and the peroxidase-labeled monoclonal antibody is stained with the peroxidase anti-peroxidase antibody method, and the biotinylated antibody is stained with the avidin-biotin glucose oxidase method. T characterized by
A novel multiplex immunohistochemical staining method for lymphocyte subset diagnosis and a kit for cell staining using the same. (8) A T lymphocyte subset diagnostic kit containing a peroxidase-labeled monoclonal antibody and a biotinylated monoclonal antibody as active ingredients.